@article{bonyak_vann_ye_lewis_gorny_2024, title={A 2-year, multi-county survey of plant-parasitic nematodes in North Carolina flue-cured tobacco}, volume={3}, ISSN={["1435-0645"]}, url={https://doi.org/10.1002/agj2.21565}, DOI={10.1002/agj2.21565}, abstractNote={AbstractNorth Carolina is the leading producer of flue‐cured tobacco (Nicotiana tabacum L.) in the United States. Production in the state is threatened by numerous plant‐parasitic nematodes, including Meloidogyne spp. (root‐knot), Globodera tabacum (tobacco cyst), Pratylenchus spp. (lesion), Rotylenchulus reniformis (reniform), and Tylenchorhynchus spp. (stunt) nematodes. The invasive Meloidogyne enterolobii is also a major threat as it can infect tobacco varieties carrying the Rk gene, which conditions resistance to other Meloidogyne species. To gain an updated understanding of which nematode taxa are present in tobacco production and investigate distribution of M. enterolobii in North Carolina, a 2‐year survey was conducted. From 2021 to 2022, 188 fields were sampled across 24 tobacco‐growing counties. Samples were assayed for identification and quantification of nematodes at the genus level; where Meloidogyne spp. was identified, molecular testing was used to determine the species. Data were also collected on the previous crop and tobacco variety grown. In both years, lesion, root‐knot, dagger, and stunt nematodes were found in the highest incidence among samples. In 2022, M. enterolobii was identified in a single sample each from Granville and Pitt counties; this is the first report of M. enterolobii from Granville County. Tobacco variety NC 196 and soybean were the most common variety and previous crop grown, respectively, in both years of the survey. Tobacco variety did not have an effect on nematode population density. The previous crop had a significant effect on root‐knot and spiral nematodes in 2021, and lesion and stubby root nematodes in 2022.}, journal={AGRONOMY JOURNAL}, author={Bonyak, Hannah C. and Vann, Matthew C. and Ye, Weimin and Lewis, Ramsey S. and Gorny, Adrienne M.}, year={2024}, month={Mar} }
 @article{fraher_watson_nguyen_moore_lewis_kudenov_yencho_gorny_2024, title={A Comparison of Three Automated Root-Knot Nematode Egg Counting Approaches Using Machine Learning, Image Analysis, and a Hybrid Model}, volume={9}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-01-24-0217-SR}, abstractNote={spp. (root-knot nematodes [RKNs]) are a major threat to a wide range of agricultural crops worldwide. Breeding crops for RKN resistance is an effective management strategy, yet assaying large numbers of breeding lines requires laborious bioassays that are time-consuming and require experienced researchers. In these bioassays, quantifying nematode eggs through manual counting is considered the current standard for quantifying establishing resistance in plant genotypes. Counting RKN eggs is highly laborious, and even experienced researchers are subject to fatigue or misclassification, leading to potential errors in phenotyping. Here, we present three automated egg counting models that rely on machine learning and image analysis to quantify RKN eggs extracted from tobacco and sweet potato plants. The first method relied on convolutional neural networks trained using annotated images to identify eggs (}, journal={PLANT DISEASE}, author={Fraher, Simon P. and Watson, Mark and Nguyen, Hoang and Moore, Savannah and Lewis, Ramsey S. and Kudenov, Michael and Yencho, G. Craig and Gorny, Adrienne M.}, year={2024}, month={Sep} }
 @article{ma_steede_dewey_lewis_2024, title={Engineering Sclareol Production on the Leaf Surface of <i>Nicotiana tabacum</i>}, ISSN={["1520-5118"]}, DOI={10.1021/acs.jafc.4c02442}, abstractNote={Sclareol, a diterpene alcohol, is the most common starting material for the synthesis of ambrox, which serves as a sustainable substitute for ambergris, a valuable fragrance secreted by sperm whales. Sclareol has also been proposed to possess antibacterial, antifungal, and anticancer activities. However, in nature, sclareol is only produced by a few plant species, including}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Ma, Hong and Steede, Tyler and Dewey, Ralph E. and Lewis, Ramsey S.}, year={2024}, month={Jun} }
 @article{castillo_acosta_hodge_vann_lewis_2023, title={Analysis of alkaloids and reducing sugars in processed and unprocessed tobacco leaves using a handheld near infrared spectrometer}, volume={1}, ISSN={["1751-6552"]}, DOI={10.1177/09670335221148594}, abstractNote={ Near infrared (NIR) spectroscopy calibration models were developed to predict chemical properties of flue-cured tobacco ( Nicotiana tabacum L.) leaf samples using a microPHAZIRTM handheld NIR spectrometer. The sample data set consisted of 348 leaf-bundled samples of upper-stalk flue-cured tobacco leaves collected from an array of cultivars evaluated in multiple locations. Unprocessed leaf samples were intact whole unground leaves collected from curing barns. Processed leaf samples were further dried and ground before scanning. The NIR prediction models for percent reducing sugars, percent total alkaloids, and percent nicotine were very good for processed leaves [r2 (SEP in %) values = 0.98 (0.82), 0.92 (0.17), and 0.92 (0.14), respectively]. The models for the same three variables for unprocessed leaves were also very good, with only slightly lower fit statistics [r2 (SEP) = 0.93 (1.58), 0.87 (0.22), and 0.88 (0.18), respectively). Fit statistics for anabasine NIR models were intermediate with r2 (SEP in %) values ranging from 0.73 (0.003) to 0.76 (0.003), while the lowest fit statistics were observed for anatabine and norticotine with r2 (SEP in %) ranging from 0.49 (0.005) to 0.55 (0.017), respectively, for both unprocessed and processed leaves. Hence, use of a handheld NIR spectrometer would be of more limited value for these variables. The chemical composition of flue-cured tobacco leaf samples for some chemical traits can be directly assessed at the point when the leaves exit the curing barns, thus minimizing the need to dry and grind samples for colorimetric and chromatographic analyses. }, journal={JOURNAL OF NEAR INFRARED SPECTROSCOPY}, author={Castillo, Miguel S. and Acosta, Juan J. and Hodge, Gary R. and Vann, Matthew C. and Lewis, Ramsey S.}, year={2023}, month={Jan} }
 @article{bukan_mandic_kozumplik_lewis_simic_sarcevic_liu_2024, title={Effects of Recurrent Selection on Population Structure and Allele Frequencies in the M3S Maize Population}, volume={14}, ISSN={["2077-0472"]}, DOI={10.3390/agriculture14010049}, abstractNote={The effects of four cycles of recurrent selection on the allele frequencies of simple sequence repeat (SSR) markers and population structure were examined in the Maksimir 3 Synthetic (M3S) maize population (Zea mays L.). Genotyping of 32 plants from each selection cycle at 38 SSR loci revealed that the mean number of alleles per locus and the mean expected heterozygosity were preserved across selection cycles, indicating the maintenance of sufficient genetic variability in the population required for future genetic gain. The Waples test of selective neutrality revealed that genetic drift was the main force in changing allele frequencies in the population. The proportion of selectively non-neutral loci in single cycles of selection varied between 16% and 37%. Some non-neutral loci shared the same genomic locations with previously published QTLs controlling important agronomic traits. An analysis of molecular variance revealed that 5.6% of the genetic variation occurred among and 94.4% within cycle populations. Between 5% and 29% of loci were found to be in a significant Hardy–Weinberg (HW) disequilibrium, with the majority showing an excess of homozygosity. The excess of homozygosity at several loci was highly consistent across cycle populations, suggesting positive assortative mating as a possible cause of the observed HW disequilibrium. Linkage disequilibrium (LD) tests revealed that the M3S population was essentially in linkage equilibrium. The proportion of pairs of loci in significant LD varied from 0.1% to 1.8% across selection cycles, probably due to the effects of genetic drift and epistatic selection.}, number={1}, journal={AGRICULTURE-BASEL}, author={Bukan, Miroslav and Mandic, Ana and Kozumplik, Vinko and Lewis, Ramsey S. and Simic, Domagoj and Sarcevic, Hrvoje and Liu, Yunjun}, year={2024}, month={Jan} }
 @article{shi_kernodle_steede_lewis_2023, title={Modified physiology of burley tobacco plants genetically engineered to express Yb<sub>1</sub>, a functional EGY enzyme}, volume={258}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-023-04235-8}, number={4}, journal={PLANTA}, author={Shi, Rui and Kernodle, Sheri P. and Steede, Tyler M. and Lewis, Ramsey S.}, year={2023}, month={Oct} }
 @article{lewis_2023, title={Use of exotic Nicotiana tabacum germplasm for confronting an inverse genetic correlation in flue-cured tobacco}, ISSN={["1435-0653"]}, DOI={10.1002/csc2.20954}, abstractNote={AbstractInverse genetic correlations between cured leaf yields and alkaloid accumulation in flue‐cured tobacco (Nicotiana tabacum L.) complicate the development of higher yielding cultivars while maintaining alkaloids at commercially acceptable levels. Introgression of genomic regions from exotic source material that positively affect alkaloid levels, but with less of a corresponding negative influence on yielding ability, could enhance long‐term efforts to develop improved cultivars. Germplasm accessions “TI 464” and “TI 959” were previously identified as being potentially useful for this purpose. In this study, the relationships between yield and alkaloid accumulation were investigated in BC1F1 populations derived from crosses with these materials and found to be slightly negative or slightly positive. Derived BC1F2 families were used to calculate heritability estimates for alkaloid accumulation on a single plant basis that were low to intermediate in magnitude. The relationships between alkaloid levels of leaves from individual stalk positions and alkaloid levels averaged over all stalk positions were determined. The third stalk position was found to be the most predictive of average alkaloid levels. Selection for alkaloid levels using a single leaf from this stalk position could increase breeding efficiency. BC4F6 lines carrying low genomic contributions from either TI 464 or TI 959 were generated and evaluated as lines per se and in F1 hybrid combinations with a very high‐yielding inbred line with increased leaf number. Performance of these materials indicated that TI 464 and, to a lesser extent TI 959, may be useful for developing higher yielding tobacco cultivars with acceptable alkaloid levels.}, journal={CROP SCIENCE}, author={Lewis, Ramsey S. S.}, year={2023}, month={Apr} }
 @article{carvalho_lewis_bruzi_padua_patto ramalho_2022, title={Adding genome-wide genotypic information to a tobacco (Nicotiana tabacum) breeding programme}, ISSN={["1439-0523"]}, DOI={10.1111/pbr.12979}, abstractNote={AbstractLarge‐scale genotypic information can be used to increase genetic gain in plant breeding programmes. In this research, we evaluated the following: (i) statistical models that could be useful in selection of superior tobacco genotypes in absence of phenotypic information; (ii) the applicability of genome‐wide selection (GWS) for predicting tobacco hybrid performance, and (iii) correlations between genetic divergence of parental lines and F1 hybrid performance. We crossed 13 inbred lines of flue‐cured Virginia tobacco crossed in a diallel scheme to generate 72 hybrid combinations and evaluated them in two field environments. Genotype by sequencing was used for single nucleotide polymorphism (SNP) marker generation, and prediction model validation was performed with different levels of missing information. Hybrid performance was predicted using only the genotypic and phenotypic information. We found genetic divergence among lines to be uncorrelated with hybrid performance or heterosis. Genotype × environment interaction affects GWS efficiency, however, and an index that incorporates both genotypic and phenotypic information improves selection accuracy. Tobacco hybrid prediction utilizing GWS data can be used as additional information to increase the response to selection.}, journal={PLANT BREEDING}, author={Carvalho, Bruna Line and Lewis, Ramsey and Bruzi, Adriano Teodoro and Padua, Jose Maria Villela and Patto Ramalho, Magno Antonio}, year={2022}, month={Jan} }
 @article{burner_mccauley_pramod_frederick_steede_kernodle_lewis_2022, title={Analyses of diverse low alkaloid tobacco germplasm identify naturally occurring nucleotide variability contributing to reduced leaf nicotine accumulation}, volume={42}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-021-01274-5}, abstractNote={Recent suggestions for mandated lowering of nicotine content in cigarettes have prompted tobacco breeders to search for N. tabacum germplasm with allelic variability contributing to low alkaloid accumulation. In this research, we phenotyped a series of 81 selected diverse tobacco introductions (TIs) to identify a sub-group with authentic low alkaloid phenotypes. We also genotyped these materials for sequences associated with the Nic1 and Nic2 loci previously reported to influence tobacco alkaloid biosynthesis. Only five low alkaloid TIs possessed previously described deletions of Ethylene Response Factor (ERF) genes at the Nic2 locus that contribute to lower alkaloid accumulation. Eleven TIs possessed an apparent deletion of ERF199, a gene recently reported to underlie the effect at the Nic1 locus. Quantitative trait locus (QTL) mapping was performed using populations derived from three selected low alkaloid TIs to possibly identify new genomic regions affecting alkaloid accumulation. A major QTL was identified on linkage group 7 in all three populations that aligned with the Nic1 locus. A newly discovered 5 bp deletion in the gene MYC2a on linkage group 5 was found to likely partially underlie the ultra-low alkaloid phenotype of TI 313. This new information is useful for tobacco breeders attempting to assemble novel genetic combinations with the potential for meeting future levels of tolerance for nicotine concentration in cigarette tobacco.}, number={1}, journal={MOLECULAR BREEDING}, author={Burner, Nathaniel and McCauley, Abigail and Pramod, Sreepriya and Frederick, Jesse and Steede, Tyler and Kernodle, Sheri P. and Lewis, Ramsey S.}, year={2022}, month={Jan} }
 @article{bovet_campanoni_lu_hilfiker_kleinhans_laparra_schwaar_lewis_matsuba_ma_et al._2022, title={CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves}, volume={13}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2022.741078}, abstractNote={Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Bovet, Lucien and Campanoni, Prisca and Lu, Jian and Hilfiker, Aurore and Kleinhans, Samuel and Laparra, Helene and Schwaar, Joanne and Lewis, Ramsey S. and Matsuba, Yuki and Ma, Hong and et al.}, year={2022}, month={Feb} }
 @article{kernodle_webb_steede_lewis_2022, title={Combined reduced expression of two gene families lowers nicotine content to ultra-low levels in cultivated tobacco}, ISSN={["1432-203X"]}, DOI={10.1007/s00299-022-02895-6}, abstractNote={Reduced expression of two gene families results in ultra-low nicotine accumulation in Nicotiana tabacum. The potential for mandated lowering of tobacco cigarette filler nicotine levels to below 0.4 mg g -1  is currently being discussed by regulatory and public health organizations. Commercial tobacco cultivars that would routinely meet this proposed standard do not currently exist. Inactivation or silencing of gene families corresponding to single enzymatic steps in the nicotine biosynthetic pathways have not resulted in tobacco genotypes that would meet this standard under conventional agronomic management. Here, we produced and evaluated under field conditions tobacco genotypes expressing an RNAi construct designed to reduce expression of the Methyl Putrescine Oxidase (MPO) gene family associated with nicotine biosynthesis. In a standard flue-cured genetic background, cured leaf nicotine levels were reduced to only 1.08 to 1.65 mg g -1 . When MPO RNAi was combined with reduced Berberine Bridge Like (BBL) activity conferred by induced mutations, genotypes producing cured leaf nicotine levels slightly lower than 0.4 mg g -1  were generated. Past research has suggested that MPO activity may contribute to the biosynthesis of nornicotine in a route that does not involve nicotine. However, nornicotine was not reduced to zero in MPO-silenced plants that were also homozygous for induced mutations in known Nicotine Demethylase genes that are responsible for the vast majority of nornicotine accumulation.}, journal={PLANT CELL REPORTS}, author={Kernodle, Sheri P. and Webb, Sydney and Steede, Tyler M. and Lewis, Ramsey S.}, year={2022}, month={Jul} }
 @article{burner_kernodle_steede_lewis_2022, title={Editing of A622 genes results in ultra-low nicotine whole tobacco plants at the expense of dramatically reduced growth and development}, volume={42}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-022-01293-w}, abstractNote={Due to potential regulations that could affect nicotine levels in some tobacco products, there is interest in using genetic modification to reduce levels of this pyridine alkaloid in tobacco leaves. Enzymes coded by A622 genes have previously been indicated to be involved in one of the latter steps of tobacco alkaloid biosynthesis. Whole tobacco plants with reduced A622 activity have never been evaluated, however. We utilized CRISPR/Cas9-based editing to introduce deleterious mutations into the two A622 genes present in the Nicotiana tabacum genome. Double homozygous A622 mutant genotypes established in four recipient genotypes varying for the presence/absence of mutations in other alkaloid biosynthetic genes exhibited severely reduced nicotine accumulation in field and greenhouse experiments. A622 knockout lines exhibited lower nicotine levels than previously created genotypes with deleterious mutations in BBL genes also associated with one of the latter steps in tobacco alkaloid biosynthesis. Reduced A622 activity resulted in plants with drastically reduced growth and development, however. A622 mutant lines were later flowering and produced green leaf yields that were 60.6% lower, on average, than those for non-A622-mutated control lines.The online version contains supplementary material available at 10.1007/s11032-022-01293-w.}, number={4}, journal={MOLECULAR BREEDING}, author={Burner, Nathaniel and Kernodle, Sheri P. and Steede, Tyler and Lewis, Ramsey S.}, year={2022}, month={Apr} }
 @article{mir_kudapa_pramod_lewis_2022, title={Editorial: Biotechnological and genomic approaches for enhancing agronomic performance of crops}, volume={13}, ISSN={["1664-8021"]}, DOI={10.3389/fgene.2022.991630}, abstractNote={dandelion plants its and regulatory activities. Transcriptome widetissue-speci fi c expression analyses its contribution to abiotic stress tolerance in soybean. The gene is known to play an important role in salt tolerance in A. thaliana . Transcriptome sequencing and subsequent differential expression analysis of transgenic soybean overexpressing AtARA6 and wild-type, “ Shen Nong 9 ” (SN9) under salt treatment and water treatment indicate several key categories of genes, including transcription factors such as WRKY86 that are signi fi cantly impacted after salt treatment. The results suggest the role of AtARA6 gene in salt tolerance in transgenic soybean via regulation of soybean SNARE complexes. Thus, these investigations provide the gene expression changes mediated by abiotic stresses in transgenic soybean overexpressing AtARA6 gene, and some insights into the mechanism mediating salt tolerance in soybean.}, journal={FRONTIERS IN GENETICS}, author={Mir, Reyazul R. and Kudapa, Himabindu and Pramod, Sreepriya and Lewis, Ramsey S.}, year={2022}, month={Oct} }
 @article{carvalho_bruzi_lewis_villela padua_patto ramalho_2022, title={Exploitation of heterosis in tobacco breeding in Brazil}, volume={22}, ISSN={["1984-7033"]}, DOI={10.1590/1984-70332022v22n2a13}, abstractNote={: This study estimated the general and specific combining abilities and mean heterosis of tobacco lines of the varietal groups Flue-Cured Virginia (FCV) and Air-Cured Burley (BY). In addition, possible advantages and an improved strategy of using hybrids in tobacco breeding in the tropics were discussed. Ten BY and 13 FCV lines were crossed in a diallel mating design. The hybrids and parental lines were evaluated at two locations in Southern Brazil. The mean heterosis was 5.37% for FCV and zero for BY lines, although higher than 15% in some combinations. The occurrence of heterosis indicates dominance in the control of tobacco yield. Tobacco breeding programs must focus primarily on improving the performance of lines per se and then on the identification of the best hybrid by testing combinations.}, number={2}, journal={CROP BREEDING AND APPLIED BIOTECHNOLOGY}, author={Carvalho, Bruna Line and Bruzi, Adriano Teodoro and Lewis, Ramsey and Villela Padua, Jose Maria and Patto Ramalho, Magno Antonio}, year={2022} }
 @article{jin_mccorkle_cornish_carbone_lewis_shew_2022, title={Adaptation of Phytophthora nicotianae to Multiple Sources of Partial Resistance in Tobacco}, volume={106}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-06-21-1241-RE}, abstractNote={ Host resistance is an important tool in the management of black shank disease of tobacco. Race development leads to rapid loss of single-gene resistance, but the adaptation by Phytophthora nicotianae to sources of partial resistance from Beinhart 1000, Florida 301, and the Wz gene region introgressed from Nicotiana rustica is poorly characterized. In greenhouse environments, host genotypes with quantitative trait loci (QTLs) conferring resistance from multiple sources were initially inoculated with an aggressive isolate of race 0 or race 1 of P. nicotianae. The most aggressive isolate was selected after each of six host generations to inoculate the next generation of plants. The race 0 isolate demonstrated a continuous gradual increase in disease severity and percentage root rot on all sources of resistance except the genotype K 326 Wz/–, where a large increase in both was observed between generations 2 and 3. Adaptation by the race 0 isolate on Beinhart 1000 represents the first report of adaptation to this genotype by P. nicotianae. The race 1 isolate did not exhibit significant increases in aggressiveness over generations but exhibited a large increase in aggressiveness on K 326 Wz/– between generations 3 and 4. Molecular characterization of isolates recovered during selection was completed via double digest restriction-site associated DNA sequencing, but no polymorphisms were associated with the observed changes in aggressiveness. The rapid adaptation to Wz resistance and the gradual adaptation to other QTLs highlights the need to study the nature of Wz resistance and to conduct field studies on the efficacy of resistance gene rotation for disease management. }, number={3}, journal={PLANT DISEASE}, author={Jin, Jing and McCorkle, Kestrel L. and Cornish, Vicki and Carbone, Ignazio and Lewis, Ramsey S. and Shew, H. David}, year={2022}, month={Mar}, pages={906–917} }
 @article{carvalho_lewis_villela padua_bruzi_patto ramalho_2021, title={Combining ability of standardized indices for multi-trait selection in tobacco}, volume={45}, ISSN={["1981-1829"]}, DOI={10.1590/1413-7054202145005521}, abstractNote={ABSTRACT Several traits must be simultaneously considered in selection of tobacco cultivars for meeting the requirements of farmers, manufacturers and consumers. Breeders should have an understanding of associations between economically-important traits in order to devise the best strategy to have response to selection for all them. Utilization of selection indices has been shown to be one of the most efficient strategy for selecting for correlated characters. In tobacco, no reports can be found within the academic literature for multiple trait selection. In this study we demonstrate the efficiency of the Sum of the Standardized Variables Index (SSV) for multi-trait selection in tobacco. The method was evaluated using a collection of thirteen inbred lines and hybrids derived using a diallel scheme. The hybrids, parental lines, and checks were evaluated in two field locations. The traits assessed were yield (YLD), general quality index (GQI), leaf stem to lamina ratio, percent total alkaloids, and percent total sugars. We considered weights according to the economic importance of each trait: 0.4 for YLD, 0.3 for GQI and 0.1 for the remaining characters. The SSV index proved to be efficient and easy to interpret, and could be used to generate genetic gain for almost all characters in the desired direction. General combining ability (GCA) explained the larger part of the variation of the index in the diallel analysis. The mean heterosis estimate was 2.02%. Some hybrid combinations exhibited heterosis levels greater than 10%, indicating that increased attention might need to be given to commercialization of F1 hybrids.}, journal={CIENCIA E AGROTECNOLOGIA}, author={Carvalho, Bruna Line and Lewis, Ramsey and Villela Padua, Jose Maria and Bruzi, Adriano Teodoro and Patto Ramalho, Magno Antonio}, year={2021} }
 @article{lu_chandrakanth_lewis_andres_bovet_goepfert_dewey_2021, title={Constitutive activation of nitrate reductase in tobacco alters flowering time and plant biomass}, volume={11}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-021-83797-7}, abstractNote={AbstractPyridine alkaloids produced in tobacco can react with nitrosating agents such as nitrite to form tobacco-specific nitrosamines (TSNA), which are among the most notable toxicants present in tobacco smoke. The market type known as burley tobacco is particularly susceptible to TSNA formation because its corresponding cultivars exhibit a nitrogen-use-deficiency phenotype which results in high accumulation of nitrate, which, in turn, is converted to nitrite by leaf surface microbes. We have previously shown that expression of a constitutively activated nitrate reductase (NR) enzyme dramatically decreases leaf nitrate levels in burley tobacco, resulting in substantial TSNA reductions without altering the alkaloid profile. Here, we show that plants expressing a constitutively active NR construct, designated 35S:S523D-NR, display an early-flowering phenotype that is also associated with a substantial reduction in plant biomass. We hypothesized that crossing 35S:S523D-NR tobaccos with burley cultivars that flower later than normal would help mitigate the undesirable early-flowering/reduced-biomass traits while maintaining the desirable low-nitrate/TSNA phenotype. To test this, 35S:S523D-NR plants were crossed with two late-flowering cultivars, NC 775 and NC 645WZ. In both cases, the plant biomass at harvest was restored to levels similar to those in the original cultivar used for transformation while the low-nitrate/TSNA trait was maintained. Interestingly, the mechanism by which yield was restored differed markedly between the two crosses. Biomass restoration in F1 hybrids using NC 645WZ as a parent was associated with delayed flowering, as originally hypothesized. Unexpectedly, however, crosses with NC 775 displayed enhanced biomass despite maintaining the early-flowering trait of the 35S:S523D-NR parent.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Lu, Jianli and Chandrakanth, Niharika N. and Lewis, Ramsey S. and Andres, Karen and Bovet, Lucien and Goepfert, Simon and Dewey, Ralph E.}, year={2021}, month={Feb} }
 @article{heim_ma_willard_shelton_lewis_2021, title={Evaluation of tobacco lines and hybrids carrying Beinhart-1000 alleles at the Phn15.1 locus for agronomic and disease resistance characteristics}, volume={61}, ISSN={["1435-0653"]}, DOI={10.1002/csc2.20520}, abstractNote={AbstractIntrogression of novel components of partial resistance to P. nicotianae can be used to increase the range, level, and durability of resistance to the black shank disease of tobacco. Here we describe the development and evaluation of BC9F3 sub‐sub‐NILs of elite flue‐cured tobacco cultivar ‘K326’ carrying a favorable allele(s) for resistance at the Phn15.1 quantitative trait locus derived from cigar cultivar ‘Beinhart 1000.’ We were able to identify a series of genotypes with significantly improved black shank resistance relative to K326 and, importantly, that possessed a mutant allele of NtCPS2, a closely linked gene involved in the biosynthesis of Z‐abienol (an undesirable flavor and aroma compound for flue‐cured tobacco). Some evidence of a potential influence of Phn15.1 on resistance to bacterial wilt caused by Ralstonia solanacearum was also observed. The Beinhart 1000 allele(s) at Phn15.1 acted in an additive manner to affect resistance to black shank. No significant reductions in cured leaf yields were found to be associated with Phn15.1‐mediated black shank resistance. Although not statistically significant, trends of slightly reduced physical cured leaf quality were observed as the zygosity of the Beinhart 1000 Phn15.1 allele(s) increased. Increases in percent total alkaloids and reductions in percent reducing sugars were also found as the zygosity at Phn15.1 increased. These changes probably did not result in cured leaf chemistry being outside of the bounds of commercial acceptability, however. The derived genetic materials enhanced for partial genetic resistance to black shank should be of value for future flue‐cured tobacco cultivar development efforts.}, number={4}, journal={CROP SCIENCE}, author={Heim, Crystal and Ma, Justin and Willard, Eric and Shelton, Cameron and Lewis, Ramsey S.}, year={2021}, month={Jul}, pages={2456–2466} }
 @article{shi_jin_nifong_shew_lewis_2021, title={Homoeologous chromosome exchange explains the creation of a QTL affecting soil-borne pathogen resistance in tobacco}, ISSN={["1467-7652"]}, DOI={10.1111/pbi.13693}, abstractNote={SummaryCrop plant partial resistance to plant pathogens controlled by quantitative trait loci (QTL) is desirable in cultivar development programmes because of its increased durability. Mechanisms underlying such resistance are difficult to study. We performed RNA‐seq analyses for tobacco (Nicotiana tabacum) nearly isogenic lines (NILs) with and without favourable allele(s) at Phn7.1, a major QTL influencing partial resistance to the soil‐borne pathogens Phytophthora nicotianae and Ralstonia solanacearum. Based upon combined analyses of transcriptome‐based sequence variation and gene expression profiles, we concluded that allelic variability at the Phn7.1 locus was likely generated from homoeologous exchange, which led to deletion of low‐expressing members of the SAR8.2 gene family and duplication of high‐expressing SAR8.2 genes from a different subgenome of allotetraploid tobacco. The high expression of endogenous Phn7.1‐associated SAR8.2 genes was correlated with observed resistance to P. nicotianae. Our findings suggest a role for genomic rearrangements in the generation of favourable genetic variability affecting resistance to pathogens in plants.}, journal={PLANT BIOTECHNOLOGY JOURNAL}, author={Shi, Rui and Jin, Jing and Nifong, Jessica M. and Shew, David and Lewis, Ramsey S.}, year={2021}, month={Oct} }
 @article{agacka-moldoch_rehman arif_lohwasser_doroszewska_lewis_boerner_2021, title={QTL analysis of seed germination traits in tobacco (Nicotiana tabacum L.)}, volume={62}, ISSN={["2190-3883"]}, DOI={10.1007/s13353-021-00623-6}, abstractNote={AbstractGenetic mapping of seed germination traits has been performed with many plant species. In tobacco, however, investigations are rare. In the present study, a bi-parental mapping population consisting of 118 doubled haploid lines and derived from a cross between ‘Beinhart-1000’ and ‘Hicks’ was investigated. Four germination-related traits, total germination (TG), normal germination (NG), time to reach 50% of total germination (T50), and the area under the curve after 200 h of germination (AUC) were considered by examining seeds either untreated or after a moderate controlled deterioration (CD). Quantitative trait loci were found for all traits distributed on 11 out of the 24 linkage groups. It was demonstrated that, as in many other species, germination-related traits are very complex and under polygenic control.}, number={3}, journal={JOURNAL OF APPLIED GENETICS}, author={Agacka-Moldoch, Monika and Rehman Arif, Mian Abdur and Lohwasser, Ulrike and Doroszewska, Teresa and Lewis, Ramsey S. and Boerner, Andreas}, year={2021}, month={Sep}, pages={441–444} }
 @article{jin_shi_lewis_shew_2021, title={RNAseq Reveals Differential Gene Expression Contributing to Phytophthora nicotianae Adaptation to Partial Resistance in Tobacco}, volume={11}, ISSN={["2073-4395"]}, DOI={10.3390/agronomy11040656}, abstractNote={Phytophthora nicotianae is a devastating oomycete plant pathogen with a wide host range. On tobacco, it causes black shank, a disease that can result in severe economic losses. Deployment of host resistance is one of the most effective means of controlling tobacco black shank, but adaptation to complete and partial resistance by P. nicotianae can limit the long-term effectiveness of the resistance. The molecular basis of adaptation to partial resistance is largely unknown. RNAseq was performed on two isolates of P. nicotianae (adapted to either the susceptible tobacco genotype Hicks or the partially resistant genotype K 326 Wz/Wz) to identify differentially expressed genes (DEGs) during their pathogenic interactions with K 326 Wz/Wz and Hicks. Approximately 69% of the up-regulated DEGs were associated with pathogenicity in the K 326 Wz/Wz-adapted isolate when sampled following infection of its adapted host K 326 Wz/Wz. Thirty-one percent of the up-regulated DEGs were associated with pathogenicity in the Hicks-adapted isolate on K 326 Wz/Wz. A broad spectrum of over-represented gene ontology (GO) terms were assigned to down-regulated genes in the Hicks-adapted isolate. In the host, a series of GO terms involved in nuclear biosynthesis processes were assigned to the down-regulated genes in K 326 Wz/Wz inoculated with K 326 Wz/Wz-adapted isolate. This study enhances our understanding of the molecular mechanisms of P. nicotianae adaptation to partial resistance in tobacco by elucidating how the pathogen recruits pathogenicity-associated genes that impact host biological activities.}, number={4}, journal={AGRONOMY-BASEL}, author={Jin, Jing and Shi, Rui and Lewis, Ramsey Steven and Shew, Howard David}, year={2021}, month={Apr} }
 @article{ma_heim_humphry_nifong_lewis_2020, title={Characterization of Phn15.1, a Newly Identified Phytophthora nicotianae Resistance QTL in Nicotiana tabacum}, volume={104}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-10-19-2257-RE}, abstractNote={ Phytophthora nicotianae is an oomycete that causes black shank, one of the most economically important diseases affecting tobacco production worldwide. Identification and introgression of novel genetic variability affecting partial genetic resistance to this pathogen is important because of the increased durability of partial resistance over time as compared with genes conferring immunity. A previous mapping study identified a quantitative trait locus (QTL), hereafter designated as Phn15.1, with a major effect on P. nicotianae resistance in tobacco. In this research, we describe significantly improved resistance of nearly isogenic lines (NILs) of flue-cured tobacco carrying the introgressed Phn15.1 region derived from highly resistant cigar tobacco cultivar Beinhart 1000. The Phn15.1 region appeared to act in an additive or partially dominant manner to positively affect resistance. To more finely resolve the position of the gene or genes underlying the Phn15.1 effect, the QTL was mapped with an increased number of molecular markers (single-nucleotide polymorphisms) identified to reside within the region. Development and evaluation of subNILs containing varying amounts of Beinhart 1000-derived Phn15.1-associated genetic material permitted the localization of the QTL to a genetic interval of approximately 2.7 centimorgans. Importantly, we were able to disassociate the Beinhart 1000 Phn15.1 resistance alleles from a functional NtCPS2 allele(s) which contributes to the accumulation of a diterpene leaf surface exudate considered undesirable for flue-cured and burley tobacco. Information from this research should be of value for marker-assisted introgression of Beinhart 1000-derived partial black shank resistance into flue-cured and burley tobacco breeding programs. }, number={6}, journal={PLANT DISEASE}, author={Ma, Justin M. and Heim, Crystal B. and Humphry, Matt and Nifong, Jessica M. and Lewis, Ramsey S.}, year={2020}, month={Jun}, pages={1638–1646} }
 @article{lewis_drake-stowe_heim_steede_smith_dewey_2020, title={Genetic and Agronomic Analysis of Tobacco Genotypes Exhibiting Reduced Nicotine Accumulation Due to Induced Mutations in Berberine Bridge Like (BBL) Genes}, volume={11}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2020.00368}, abstractNote={Genetic methodologies for reducing nicotine accumulation in the tobacco plant (Nicotiana tabacum L.) are of interest because of potential future regulations that could mandate lowering of this alkaloid in conventional cigarettes. Inactivation of tobacco genes such as the Berberine Bridge Like (BBL) gene family believed to encode for enzymes involved in one of the latter steps of nicotine biosynthesis could be a viable strategy for producing new tobacco cultivars with ultra-low leaf nicotine accumulation. We introduced deleterious mutations generated via ethyl methanesulfonate treatment of seed or gene editing into six known members of the BBL gene family and assembled them in different combinations to assess their relative contribution to nicotine accumulation. Significant reductions (up to 17-fold) in percent leaf nicotine were observed in genotypes homozygous for combined mutations in BBL-a, BBL-b, and BBL-c. The addition of mutations in BBL-d1, BBL-d2, and BBL-e had no additional significant effect on lowering of nicotine levels in the genetic background studied. Reduced nicotine levels were associated with reductions in cured leaf yields (up to 29%) and cured leaf quality (up to 15%), evidence of physiological complexities within the tobacco plant related to the nicotine biosynthetic pathway. Further nicotine reductions were observed for a BBL mutant line cultivated under a modified production regime in which apical inflorescences were not removed, but at the expense of further yield reductions. Plants in which BBL mutations were combined with naturally occurring recessive alleles at the Nic1 and Nic2 loci exhibited further reductions in percent nicotine, but no plant produced immeasurable levels of this alkaloid. Findings may suggest the existence of a minor, alternative pathway for nicotine biosynthesis in N. tabacum. The described genetic materials may be of value for the manufacture of cigarettes with reduced nicotine levels and for future studies to better understand the molecular biology of alkaloid accumulation in tobacco.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Lewis, Ramsey S. and Drake-Stowe, Katherine E. and Heim, Crystal and Steede, Tyler and Smith, William and Dewey, Ralph E.}, year={2020}, month={Apr} }
 @article{cheek_vann_lewis_fisher_2021, title={Genetics influence postharvest measurements of flue-cured tobacco more than nitrogen application rate}, volume={113}, ISSN={["1435-0645"]}, url={https://doi.org/10.1002/agj2.20565}, DOI={10.1002/agj2.20565}, abstractNote={AbstractRegulations under consideration by the U.S. Food and Drug Administration and the World Health Organization propose that nicotine concentration in tobacco (Nicotiana tabacum L.) should be lowered to non‐addictive levels (0.3 to 0.5 mg g−1). The proposed standards are 90 to 95% lower than the nicotine concentration typically documented in commercially available cultivars. Research was conducted in six environments to evaluate two cultivars with normal alkaloid levels (K326 and NC95) and four genotypes with low alkaloid levels (DH16A, DH22A, DH32, and LAFC53). Each cultivar and genotype was paired with three N application rates: 70, 85, and 100% of the recommended rate. As N application declined, so too did cured leaf yield and nicotine, anabasine, and anatabine concentration in K326 and NC95. These factors were generally not affected by N application in the low alkaloid genotypes. In contrast, LAFC53 consistently produced the lowest cured leaf quality, value, and reducing sugar concentration when compared to all other cultivars. This observation demonstrates that K326 isolines are agronomically superior to LAFC53. Despite reductions in nicotine, the lowest documented concentration was still 10‐fold greater than the proposed minimum (LAFC53). Nitrogen did not influence the measured parameters as much as genetics; therefore, additional research that involves other agronomic practices is warranted. In addition, further genetic manipulation will be required to meet the standards proposed by regulatory groups.}, number={2}, journal={AGRONOMY JOURNAL}, publisher={Wiley}, author={Cheek, Joseph A. and Vann, Matthew C. and Lewis, Ramsey S. and Fisher, Loren R.}, year={2021}, month={Mar}, pages={1020–1028} }
 @article{ma_hancock_nifong_kernodle_lewis_2020, title={Identification and editing of a hybrid lethality gene expands the range of interspecific hybridization potential in Nicotiana}, volume={133}, ISSN={["1432-2242"]}, DOI={10.1007/s00122-020-03641-w}, abstractNote={Identification and inactivation of hybrid lethality genes can be used to expand the available gene pool for improvement of a cultivated crop species. Hybrid lethality is one genetic mechanism that contributes to reproductive isolation in plants and serves as a barrier to use of diverse germplasm for improvement of cultivated species. A classic example is the seedling lethality exhibited by progeny from the Nicotiana tabacum × N. africana interspecific cross. In order to increase the body of knowledge on mechanisms of hybrid lethality in plants, and to potentially develop tools to circumvent them, we utilized a transposon tagging strategy to identify a candidate gene involved in the control of this reaction. N. tabacum gene Nt6549g30 was identified to code for a class of coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) proteins, the largest class of plant defense proteins. Gene editing, along with other experiments, was used to verify that Nt6549g30 is the gene at the N. tabacum Hybrid Lethality 1 (NtHL1) locus controlling the hybrid lethality reaction in crosses with N. africana. Gene editing of Nt6549g30 was also used to reverse interspecific seedling lethality in crosses between N. tabacum and eight of nine additional tested species from section Suaveolentes. Results further implicate the role of disease resistance-like genes in the evolution of plant species and demonstrate the possibility of expanding the gene pool for a crop species through gene editing.}, number={10}, journal={THEORETICAL AND APPLIED GENETICS}, author={Ma, Justin and Hancock, Wesley G. and Nifong, Jessica M. and Kernodle, Sheri P. and Lewis, Ramsey S.}, year={2020}, month={Oct}, pages={2915–2925} }
 @misc{henry_vann_lewis_2019, title={Agronomic Practices Affecting Nicotine Concentration in Flue-Cured Tobacco: A Review}, volume={111}, ISSN={["1435-0645"]}, DOI={10.2134/agronj2019.04.0268}, abstractNote={Proposed regulations mandating lower nicotine concentrations in tobacco (Nicotiana tabacum L.) products will likely require changes in tobacco production to reduce nicotine while maintaining yield and quality. The agronomic practices used for tobacco production have a significant impact on the synthesis and accumulation of nicotine in flue‐cured tobacco. Nicotine is the primary alkaloid in flue‐cured tobacco and is one of the main reasons for its commercial production. Most agronomic practices that improve plant health and yield have a positive effect on nicotine production and accumulation. Some of the most important factors that affect nicotine concentrations are N fertilization, planting density, topping practices, sucker control, and harvesting practices. The amount of N available to the plant has a substantial effect on nicotine, as N is a primary component of the nicotine molecule. Factors leading to higher N uptake lead to higher nicotine concentrations. Plant and leaf densities within the field also have a significant effect on nicotine, where increasing densities leads to lower nicotine concentrations. Flowering and sucker production are both significant sinks of energy and other resources. Eliminating the inflorescence via topping and controlling suckers lead to higher nicotine concentrations. In fact, substantial nicotine synthesis and accumulation occurs in the days and weeks following topping. This comprehensive review discusses the agronomic factors affecting alkaloid production in flue‐cured tobacco, and how these factors can be adjusted to manipulate the ultimate nicotine concentration.Core Ideas

Proposed regulations may require lower tobacco nicotine concentrations.
Production practices and timing significantly influence nicotine and leaf quality.
Nitrogen fertility, crop density, growth regulation, and harvesting are paramount.
Low density, high N, and increased maturity enhance nicotine accumulation.
Flowers and axillary shoots are sinks that limit foliar nicotine concentrations.
}, number={6}, journal={AGRONOMY JOURNAL}, author={Henry, Josh B. and Vann, Matthew C. and Lewis, Ramsey S.}, year={2019}, pages={3067–3075} }
 @article{zeng_nifong_liu_huang_fang_lewis_li_2019, title={Evaluating diverse systems of tobacco genetic resistance to Phytophthora nicotianae in Yunnan, China}, volume={68}, ISSN={["1365-3059"]}, DOI={10.1111/ppa.13091}, abstractNote={Black shank, caused by the soilborne pathogen Phytophthora nicotianae, is one of the most devastating diseases affecting tobacco production in China. The most effective strategy for reducing economic loss from this pathogen is development and use of resistant tobacco varieties. Multiple sources and systems of resistance have been developed in the Western Hemisphere; however, populations of P. nicotianae are variable around the world, including the predominance of different races. Different P. nicotianae isolates may react differently on tobacco plants with different systems of resistance, a possibility that could complicate the breeding of cultivars with resistance that is effective in different tobacco production regions worldwide. The objective of this research was to evaluate an array of tobacco germplasm possessing different systems of genetic resistance to black shank disease in tobacco‐growing regions of Yunnan, China. Resistance types included simply inherited resistance mechanisms introgressed from wild Nicotiana relatives and polygenic partial resistance systems of N. tabacum origin. The loci of Wz exhibited high level resistance to black shank in the five diverse disease environments in Yunnan, China. K326 Php/−Wz/− genotype and Beinhart 1000 exhibited the greatest levels of resistance in both 2015 and 2016. Field observed results for 13 tobacco genotypes were highly correlated with those tested in growth chamber evaluation. These findings suggest that both Wz− and Beinhart 1000‐mediated resistance have important commercial value in flue‐cured tobacco breeding programmes in China. Cultivars developed for black shank resistance in China may also have utility in other tobacco‐growing areas.}, number={9}, journal={PLANT PATHOLOGY}, author={Zeng, J. M. and Nifong, J. and Liu, Y. and Huang, C. J. and Fang, D. H. and Lewis, R. S. and Li, Y. P.}, year={2019}, month={Dec}, pages={1616–1623} }
 @article{dexter-boone_humphry_shi_lewis_2019, title={Genetic Control of Facultative Parthenocarpy in Nicotiana tabacum L.}, volume={110}, ISSN={["1465-7333"]}, DOI={10.1093/jhered/esz025}, abstractNote={AbstractInvestigation of parthenocarpy, the production of fruit without fertilization, in multiple plant species could result in development of technologies for conferring seedless fruits and increased stability of fruit formation in economically important plants. We studied parthenocarpy in the model species Nicotiana tabacum L., and observed variability for expression of the trait among diverse genetic materials. Parthenocarpy was found to be partially dominant, and a single major quantitative trait locus on linkage group 22 was found to control the trait in a doubled haploid mapping population derived from a cross between parthenocarpic cigar tobacco cultivar “Beinhart 1000” and nonparthenocarpic flue-cured tobacco cultivar, “Hicks.” The same genomic region was found to be involved with control of the trait in the important flue-cured tobacco cultivar, “K326.” We also investigated the potential for the production of maternal haploids due to parthenogenesis in parthenocarpic tobacco seed capsules. Maternal haploids were not observed in parthenocarpic capsules, suggesting a requirement of fertilization for maternal haploid production due to parthenogenesis in N. tabacum.}, number={5}, journal={JOURNAL OF HEREDITY}, author={Dexter-Boone, Abigail and Humphry, Matt and Shi, Rui and Lewis, Ramsey S.}, year={2019}, month={Jul}, pages={610–617} }
 @article{ma_heim_humphry_nifong_lewis_2019, title={Genetic analysis of Phn7.1, a major QTL conferring partial resistance to Phytophthora nicotianae in Nicotiana tabacum}, volume={39}, DOI={10.1007/s11032-018-0923-x}, number={1}, journal={MOLECULAR BREEDING}, author={Ma, Justin M. and Heim, Crystal and Humphry, Matt and Nifong, J. M. and Lewis, Ramsey S.}, year={2019} }
 @article{dexter-boone_lewis_2019, title={Heterosis in Flue-Cured Tobacco and Its Utility in Predicting Transgressive Segregation within Derived Populations of Inbred Lines}, volume={59}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2018.08.0486}, abstractNote={Heterosis might be used in a strategic way to increase yields of flue‐cured tobacco (Nicotiana tabacum L.), but previous reports have suggested levels of heterosis to be low for F1 hybrids of this market class. Based on analysis of a 14‐parent diallel, we found average levels of F1 midparent heterosis to be 10.3%, a level that is substantially higher than previous estimates from the 1960s. Thirteen of ninety‐one tested hybrids significantly outyielded the highest yielding parental lines. Heterosis might also be an indicator of superior allelic complementation by parental lines that, in principle, could be fixed in derived inbred lines. We compared F3:4 families derived from three high heterotic crosses and three low heterotic crosses and observed positive correlations between F1 heterosis and desirable transgressive segregation. Hence, the data support the use of F1 heterosis as a predictor of breeding crosses with increased potential for desired outcomes. Several derived F3:4 families exhibited yields that were comparable with, or significantly better than, corresponding heterotic F1 hybrids, indicating that heterotic effects could be fixed within inbred lines. Results suggest a reconsideration of heterosis for increasing yields of flue‐cured tobacco.}, number={3}, journal={CROP SCIENCE}, author={Dexter-Boone, Abigail and Lewis, Ramsey S.}, year={2019}, pages={957–967} }
 @article{shi_hubert_dexter-boone_zeng_kernodle_lewis_2019, title={Identification and validation of SNP markers associated with Wz-mediated Phytophthora nicotianae resistance in Nicotiana tabacum L.}, volume={39}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-019-1015-2}, number={7}, journal={MOLECULAR BREEDING}, author={Shi, Rui and Hubert, Hannah and Dexter-Boone, Abigail and Zeng, Jianmin and Kernodle, Sheri P. and Lewis, Ramsey S.}, year={2019}, month={Jul} }
 @misc{sinclair_rufty_lewis_2019, title={Increasing Photosynthesis: Unlikely Solution For World Food Problem}, volume={24}, ISSN={["1878-4372"]}, DOI={10.1016/j.tplants.2019.07.008}, abstractNote={Increasing the photosynthesis rate of plants has been recently revitalized as an approach for increasing grain crop yields and solving world food crises. The idea that photosynthesis is the key to increasing grain crop yields is not new. Considerable research in the 1970s and 1980s showed that carbon input was not limiting for crop growth and yield. Instead, the availability and uptake of water and nutrients were found to be critical for increasing grain yield, and that conclusion still applies today. In this Opinion article, nitrogen limitation is given particular attention because of its quantitative linkage with vegetative and reproductive growth and its essential role as a quantitative component of seeds.}, number={11}, journal={TRENDS IN PLANT SCIENCE}, author={Sinclair, Thomas R. and Rufty, Thomas W. and Lewis, Ramsey S.}, year={2019}, month={Nov}, pages={1032–1039} }
 @article{shi_lewis_panthee_2018, title={Filter paper-based spin column method for cost-efficient DNA or RNA purification}, volume={13}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0203011}, abstractNote={We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented.}, number={12}, journal={PLOS ONE}, author={Shi, Rui and Lewis, Ramsey S. and Panthee, Dilip R.}, year={2018}, month={Dec} }
 @article{lewis_2019, title={Potential Mandated Lowering of Nicotine Levels in Cigarettes: A Plant Perspective}, volume={21}, ISSN={["1469-994X"]}, DOI={10.1093/ntr/nty022}, abstractNote={The Food and Drug Administration has announced the potential for mandated lowering of nicotine levels in combustible cigarettes. The World Health Organization has recommended a lowering of cigarette filler nicotine levels to below 0.4 mg/g. To devise appropriate nicotine control strategies, regulators must consider technical feasibility, timelines for compliance, and potential impediments to implementation. Outlined here is previously unsummarized information on genetic approaches that might be used to reduce nicotine levels in cured tobacco leaves. For the benefit of regulators, altered alkaloid or toxicant profiles that might result by implementation of some of these methodologies are discussed. Also mentioned are potential licensing or regulatory impediments to use of some of the technologies per se. Implications: An understanding of technical feasibility of plant-based nicotine reduction technologies, along with the potential for corresponding alterations in alkaloid or toxicant profiles, is needed by regulators to develop effective nicotine control strategies with minimal impediments or undesirable consequences.}, number={7}, journal={NICOTINE & TOBACCO RESEARCH}, author={Lewis, Ramsey S.}, year={2019}, month={Jul}, pages={991–995} }
 @article{edwards_fernandez-pozo_drake-stowe_humphry_evans_bombarely_allen_hurst_white_kernodle_et al._2017, title={A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency}, volume={18}, ISSN={["1471-2164"]}, DOI={10.1186/s12864-017-3791-6}, abstractNote={Tobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation. Here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N50 size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production. Development of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.}, journal={BMC GENOMICS}, author={Edwards, K. D. and Fernandez-Pozo, N. and Drake-Stowe, K. and Humphry, M. and Evans, A. D. and Bombarely, A. and Allen, F. and Hurst, R. and White, B. and Kernodle, S. P. and et al.}, year={2017}, month={Jun} }
 @article{mccorkle_drake-stowe_lewis_shew_2018, title={Characterization of Phytophthora nicotianae Resistance Conferred by the Introgressed Nicotiana rustica Region, Wz, in Flue-Cured Tobacco}, volume={102}, ISSN={["1943-7692"]}, DOI={10.1094/pdis-03-17-0339-re}, abstractNote={ Black shank, caused by Phytophthora nicotianae, is one of the most important diseases affecting tobacco worldwide and is primarily managed through use of host resistance. An additional source of resistance to P. nicotianae, designated as Wz, has been introgressed into Nicotiana tabacum from N. rustica. The Wz gene region confers high levels of resistance to all races, but has not been characterized. Our study found Wz-mediated resistance is most highly expressed in the roots, with only a slight reduction in stem-lesion size in Wz genotypes compared with susceptible controls. No substantial relationships were observed between initial inoculum levels and disease development on Wz genotypes, which is generally consistent with qualitative or complete resistance. Isolates of P. nicotianae adapted for five host generations on plants with the Wz gene caused higher disease severity than isolates adapted on Wz plants for only one host generation. Wz-adapted isolates did not exhibit increased aggressiveness on genotypes with other sources of partial resistance, suggesting pathogen adaptation was specific to the Wz gene. To reduce potential for pathogen population shifts with virulence on Wz genotypes, Wz should be combined with other resistance sources and rotation of varying black shank resistance mechanisms is also recommended. }, number={2}, journal={PLANT DISEASE}, author={McCorkle, Kestrel L. and Drake-Stowe, Katherine and Lewis, Ramsey S. and Shew, David}, year={2018}, month={Feb}, pages={309–317} }
 @article{hancock_lewis_2017, title={Heterosis, transmission genetics, and selection for increased growth rate in a N. tabacum x synthetic tobacco cross}, volume={37}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-017-0654-4}, number={4}, journal={MOLECULAR BREEDING}, author={Hancock, Wesley G. and Lewis, Ramsey S.}, year={2017}, month={Apr} }
 @article{drake-stowe_bakaher_goepfert_philippon_mark_peterson_lewis_2017, title={Multiple Disease Resistance Loci Affect Soilborne Disease Resistance in Tobacco (Nicotiana tabacum)}, volume={107}, ISSN={["1943-7684"]}, DOI={10.1094/phyto-03-17-0118-r}, abstractNote={ Phytophthora nicotianae and Ralstonia solanacearum are two of the most important pathogens affecting tobacco worldwide. Greater insight regarding genetic systems controlling resistance to these two soilborne pathogens, as well as identification of DNA markers associated with genomic regions controlling this resistance, could aid in variety development. An evaluation of 50 historical tobacco lines revealed a high positive correlation between resistances to the two pathogens, preliminarily suggesting that some genomic regions may confer resistance to both pathogens. A quantitative trait loci (QTL) mapping experiment designed to investigate the genetic control of soilborne disease resistance of highly resistant ‘K346’ tobacco identified four QTL significantly associated with resistance to P. nicotianae (explaining 60.0% of the observed phenotypic variation) and three QTL to be associated with R. solanacearum resistance (explaining 50.3% of the observed variation). The two QTL with the largest effect on Phytophthora resistance were also found to be the QTL with the greatest effects on resistance to Ralstonia. This finding partially explains previously observed associations between resistances to these two pathogens among U.S. current cultivars and within breeding populations. Further study is needed to determine whether these relationships are due to the same genes (i.e., pleiotropy) or favorable coupling-phase linkages that have been established over time. }, number={9}, journal={PHYTOPATHOLOGY}, author={Drake-Stowe, Katherine and Bakaher, Nicolas and Goepfert, Simon and Philippon, Berangere and Mark, Regis and Peterson, Paul and Lewis, Ramsey S.}, year={2017}, month={Sep}, pages={1055–1061} }
 @article{steede_ma_eickholt_drake-stowe_kernodle_shew_danehower_lewis_2017, title={The Tobacco Trichome Exudate Z-abienol and Its Relationship With Plant Resistance to Phytophthora nicotianae}, volume={101}, ISSN={["1943-7692"]}, DOI={10.1094/pdis-10-16-1512-re}, abstractNote={ In previous research, we discovered a favorable quantitative trait locus (QTL) in cigar tobacco cultivar ‘Beinhart 1000’ designated as Phn15.1, which provides a high level of partial resistance to the black shank disease caused by Phytophthora nicotianae. A very close genetic association was also found between Phn15.1 and the ability to biosynthesize Z-abienol, a labdanoid diterpene exuded by the trichomes onto above-ground plant parts, and that imparts flavor and aroma characteristics to Oriental and some cigar tobacco types. Because accumulation of Z-abienol is considered to be undesirable for cultivars of other tobacco types, we herein describe a series of experiments to gain insight on whether this close association is due to genetic linkage or pleiotropy. First, in an in vitro bioassay, we observed Z-abienol and related diterpenes to inhibit hyphal growth of P. nicotianae at concentrations between 0.01 and 100 ppm. Secondly, we field-tested transgenic versions of Beinhart 1000 carrying RNAi constructs for downregulating NtCPS2 or NtABS, two genes involved in the biosynthesis of Z-abienol. Thirdly, we also field tested a recombinant inbred line population segregating for a truncation mutation in NtCPS2 leading to an interrupted Z-abienol pathway. We observed no correlation between field resistance to P. nicotianae and the ability to accumulate Z-abienol in either the transgenic materials or the mapping population. Results suggest that, although Z-abienol may affect P. nicotianae when applied at high concentrations in in vitro assays, the compound has little effect on black shank disease development under natural field conditions. Thus, it should be possible to disassociate Phn15.1-mediated black shank resistance identified in cigar tobacco cultivar Beinhart 1000 from the ability to accumulate Z-abienol, an undesirable secondary metabolite for burley and flue-cured tobacco cultivars. }, number={7}, journal={PLANT DISEASE}, author={Steede, William T. and Ma, Justin M. and Eickholt, David P. and Drake-Stowe, Katherine E. and Kernodle, Sheri P. and Shew, H. David and Danehower, David A. and Lewis, Ramsey S.}, year={2017}, month={Jul}, pages={1214–1221} }
 @article{lu_zhang_lewis_bovet_goepfert_jack_crutchfield_ji_dewey_2016, title={Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke}, volume={14}, ISSN={["1467-7652"]}, DOI={10.1111/pbi.12510}, abstractNote={SummaryBurley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.}, number={7}, journal={PLANT BIOTECHNOLOGY JOURNAL}, author={Lu, Jianli and Zhang, Leichen and Lewis, Ramsey S. and Bovet, Lucien and Goepfert, Simon and Jack, Anne M. and Crutchfield, James D. and Ji, Huihua and Dewey, Ralph E.}, year={2016}, month={Jul}, pages={1500–1510} }
 @article{wells_eickholt_lewis_vann_fisher_2016, title={Heat Unit Accumulation and Days to Anthesis Relationship in Tobacco Genotypes with an Introgressed QTL Affecting Leaf Number}, volume={56}, ISSN={["1435-0653"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84990194504&partnerID=MN8TOARS}, DOI={10.2135/cropsci2016.04.0278}, abstractNote={Plant breeders and crop managers would benefit from an increased ability to predict the requirements for onset of anthesis in tobacco (Nicotiana tabacum L.). This study was performed to determine the relationship in the field between heat unit (HU) accumulation and the onset of anthesis in tobacco genotypes varying for the zygosity of an introgressed quantitative trait loci (QTL) designated as Many Leaves (Ml) affecting flowering team and leaf number. Three commercially important cultivars or breeding lines (Speight 168, K326, and NCTG‐61), their nearly isogenic homozygous (MlMl) and heterozygous (Mlml) counterparts, and three BC6F3 null‐segregant (mlml) lines, were evaluated at three locations and over 2 yr. Days to anthesis (DTA) and HU accumulation were determined. In each environment, the MlMl, Mlml, and mlml genotypes were found to be grouped according to the zygosity of the Ml trait, with the mlml genotypes flowering first followed by Mlml and MlMl genotypes, respectively. An exception to this trend was Speight 168 MlMl, which consistently fell within the Mlml grouping for DTA. No consistent relationships were observed between HU and DTA, which was highly variable among environments. Integrated HU (area under the HU curve) during the 28 d after transplanting (DAT) was positively associated (R2 = 0.98; P = 0.0001) with the range of DTA among all genotypes. Integrated HU was a better indicator of the pattern of HU attainment than merely the amount. Early HU exposure seemed important in determining the range in DTA amongst the genotypes examined.}, number={6}, journal={CROP SCIENCE}, author={Wells, Randy and Eickholt, David P. J. and Lewis, Ramsey and Vann, Matthew C. and Fisher, Loren R.}, year={2016}, pages={3228–3236} }
 @article{wang_lewis_shi_song_gao_li_chen_qu_2015, title={Genetic Factors for Enhancement of Nicotine Levels in Cultivated Tobacco}, volume={5}, ISSN={["2045-2322"]}, DOI={10.1038/srep17360}, abstractNote={AbstractNicotine has practical applications relating to smoking cessation devices and alternative nicotine products. Genetic manipulation for increasing nicotine content in cultivated tobacco (Nicotiana tabacum L.) may be of value for industrial purposes, including the possibility of enhancing the efficiency of nicotine extraction. Biotechnological approaches have been evaluated in connection with this objective, but field-based results are few. Here, we report characterization of two genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs), NtMYC2a and NtMYC2b from tobacco. Overexpression of NtMYC2a increased leaf nicotine levels in T1 transgenic lines approximately 2.3-fold in greenhouse-grown plants of tobacco cultivar ‘NC 95′. Subsequent field testing of T2 and T3 generations of transgenic NtMYC2a overexpression lines showed nicotine concentrations were 76% and 58% higher than control lines, respectively. These results demonstrated that the increased nicotine trait was stably inherited to the T2 and T3 generations, indicating the important role that NtMYC2a plays in regulating nicotine accumulation in N. tabacum and the great potential of NtMYC2a overexpression in tobacco plants for industrial nicotine production. Collected data in this study also indicated a negative feedback inhibition of nicotine biosynthesis. Further enhancement of nicotine accumulation in tobacco leaf may require modification of the processes of nicotine transport and deposition.}, journal={SCIENTIFIC REPORTS}, author={Wang, Bingwu and Lewis, Ramsey S. and Shi, Junli and Song, Zhongbang and Gao, Yulong and Li, Wenzheng and Chen, Hongxia and Qu, Rongda}, year={2015}, month={Dec} }
 @article{hancock_kuraparthy_kernodle_lewis_2015, title={Identification of maternal haploids of Nicotiana tabacum aided by transgenic expression of green fluorescent protein: evidence for chromosome elimination in the N-tabacum x N-africana interspecific cross}, volume={35}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-015-0372-8}, number={9}, journal={MOLECULAR BREEDING}, author={Hancock, Wesley G. and Kuraparthy, Vasu and Kernodle, Sheri P. and Lewis, Ramsey S.}, year={2015}, month={Sep} }
 @article{agacka-modoch_nagel_doroszewska_lewis_boerner_2015, title={Mapping quantitative trait loci determining seed longevity in tobacco (Nicotiana tabacum L.)}, volume={202}, ISSN={["1573-5060"]}, DOI={10.1007/s10681-015-1355-x}, number={3}, journal={EUPHYTICA}, author={Agacka-Modoch, M. and Nagel, M. and Doroszewska, T. and Lewis, R. S. and Boerner, A.}, year={2015}, month={Apr}, pages={479–486} }
 @article{lewis_lopez_bowen_andres_steede_dewey_2015, title={Transgenic and Mutation-Based Suppression of a Berberine Bridge Enzyme-Like (BBL) Gene Family Reduces Alkaloid Content in Field-Grown Tobacco}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0117273}, abstractNote={Motivation exists to develop tobacco cultivars with reduced nicotine content for the purpose of facilitating compliance with expected tobacco product regulations that could mandate the lowering of nicotine levels per se, or the reduction of carcinogenic alkaloid-derived tobacco specific nitrosamines (TSNAs). A berberine bridge enzyme-like (BBL) gene family was recently characterized for N. tabacum and found to catalyze one of the final steps in pyridine alkaloid synthesis for this species. Because this gene family acts downstream in the nicotine biosynthetic pathway, it may represent an attractive target for genetic strategies with the objective of reducing alkaloid content in field-grown tobacco. In this research, we produced transgenic doubled haploid lines of tobacco cultivar K326 carrying an RNAi construct designed to reduce expression of the BBL gene family. Field-grown transgenic lines carrying functional RNAi constructs exhibited average cured leaf nicotine levels of 0.684%, in comparison to 2.454% for the untransformed control. Since numerous barriers would need to be overcome to commercialize transgenic tobacco cultivars, we subsequently pursued a mutation breeding approach to identify EMS-induced mutations in the three most highly expressed isoforms of the BBL gene family. Field evaluation of individuals possessing different homozygous combinations of truncation mutations in BBLa, BBLb, and BBLc indicated that a range of alkaloid phenotypes could be produced, with the triple homozygous knockout genotype exhibiting greater than a 13-fold reduction in percent total alkaloids. The novel source of genetic variability described here may be useful in future tobacco breeding for varied alkaloid levels.}, number={2}, journal={PLOS ONE}, author={Lewis, Ramsey S. and Lopez, Harry O. and Bowen, Steve W. and Andres, Karen R. and Steede, William T. and Dewey, Ralph E.}, year={2015}, month={Feb} }
 @article{gottula_lewis_saito_fuchs_2014, title={Allopolyploidy and the evolution of plant virus resistance}, volume={14}, ISSN={["1471-2148"]}, DOI={10.1186/1471-2148-14-149}, abstractNote={The relationship between allopolyploidy and plant virus resistance is poorly understood. To determine the relationship of plant evolutionary history and basal virus resistance, a panel of Nicotiana species from diverse geographic regions and ploidy levels was assessed for resistance to non-coevolved viruses from the genus Nepovirus, family Secoviridae. The heritability of resistance was tested in a panel of synthetic allopolyploids. Leaves of different positions on each inoculated plant were tested for virus presence and a subset of plants was re-inoculated and assessed for systemic recovery. Depending on the host-virus combination, plants displayed immunity, susceptibility or intermediate levels of resistance. Synthetic allopolyploids showed an incompletely dominant resistance phenotype and manifested systemic recovery. Plant ploidy was weakly negatively correlated with virus resistance in Nicotiana species, but this trend did not hold when synthetic allopolyploids were taken into account. Furthermore, a relationship between resistance and geographical origin was observed. The gradients of resistance and virulence corresponded to a modified matching allele model of resistance. Intermediate resistance responses of allopolyploids corresponded with a model of multi-allelic additive resistance. The variable virus resistance of extant allopolyploids suggested that selection-based mechanisms surpass ploidy with respect to evolution of basal resistance to viruses.}, journal={BMC EVOLUTIONARY BIOLOGY}, author={Gottula, John and Lewis, Ramsey and Saito, Seiya and Fuchs, Marc}, year={2014}, month={Jul} }
 @article{drake_moore_bertrand_fortnum_peterson_lewis_2015, title={Black Shank Resistance and Agronomic Performance of Flue-Cured Tobacco Lines and Hybrids Carrying the Introgressed Nicotiana rustica Region, Wz}, volume={55}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2014.02.0164}, abstractNote={ABSTRACTBlack shank, caused by Phytophthora nicotianae, is one of the most important diseases affecting tobacco (Nicotiana tabacum L.) production in the United States. Genetic mechanisms are needed that provide resistance to current races and that can be combined into cultivars that provide high yields of cured leaf with acceptable quality. Previous research identified DNA markers associated with an introgressed N. rustica genomic region (designated as Wz) found to contribute to resistance to race 0 and race 1 isolates. Objectives of the current research were to use DNA markers to transfer Wz into the elite genetic background of flue‐cured tobacco cultivar K 326 and to develop nearly isogenic lines and hybrids with and without the race 0 immunity gene Php. These materials were evaluated in multiple environments for black shank resistance, yield, and quality characteristics. Wz was observed to positively affect resistance in the seven diverse disease environments tested. Genotypes in which Wz was combined with Php exhibited the greatest levels of resistance. No evidence of a negative relationship between Wz and yield and/or quality was observed. Data suggest commercial value for Wz in flue‐cured tobacco‐breeding programs with the goal of developing high‐yielding tobacco cultivars with resistance to race 0 and race 1. Further studies are necessary to determine the durability of Wz‐mediated resistance, however.}, number={1}, journal={CROP SCIENCE}, author={Drake, Katherine E. and Moore, J. Michael and Bertrand, Paul and Fortnum, Bruce and Peterson, Paul and Lewis, Ramsey S.}, year={2015}, pages={79–86} }
 @article{eickholt_lewis_2014, title={Effect of an Introgressed Nicotiana tomentosa Leaf Number QTL on Yield and Quality Characteristics in Flue-Cured Tobacco}, volume={54}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2013.07.0464}, abstractNote={ABSTRACTIncreased cured leaf yield is an important breeding target for flue‐cured tobacco (Nicotiana tabacum L.), but genetic improvement for this trait has slowed in the last thirty years. Yield is positively correlated with days to flowering and leaf number in this species. Use of novel allelic diversity influencing these traits might contribute to further yield improvement. In this study, an introgressed quantitative trait locus (QTL) affecting flowering time and leaf number, designated as Many Leaves (Ml), was backcrossed into three genetic backgrounds of flue‐cured tobacco. Field evaluation of nearly isogenic MlMl lines and Mlml hybrids indicated that the QTL acted in an additive to partially dominant fashion to delay flowering and increase leaf number. This resulted in significant increases in yield and cash return per hectare. As the zygosity of Ml increased, corresponding decreases in percentage total alkaloids and increases in percentage reducing sugars were observed, however. Incorporation of Ml into flue‐cured tobacco breeding programs may be useful for increasing cured leaf yields in certain environments. Concurrent modification of the genetic potential to accumulate alkaloids would be also important under current industry requirements, however.}, number={2}, journal={CROP SCIENCE}, author={Eickholt, David P. and Lewis, Ramsey S.}, year={2014}, pages={586–594} }
 @article{cai_jack_lewis_dewey_bush_2013, title={(R)-nicotine biosynthesis, metabolism and translocation in tobacco as determined by nicotine demethylase mutants}, volume={95}, ISSN={["0031-9422"]}, DOI={10.1016/j.phytochem.2013.06.012}, abstractNote={Nicotine is a chiral compound and consequently exists as two enantiomers. Since (R)-nicotine consists of less than 0.5% of total nicotine pool in tobacco, few investigations relating to (R)-nicotine have been reported. However, previous studies of nicotine demethylases suggested there was substantial amount of (R)-nicotine at synthesis in the tobacco plant. In this study, the accumulation and translocation of (R)-nicotine in tobacco was analyzed. The accumulation of nicotine and its demethylation product the nornicotine enantiomers, were investigated in different tobacco plant parts and at different growth and post-harvest stages. Scion/rootstock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf tissue. The results indicate that 4% of nicotine is in the (R) form at synthesis in the root. After the majority of (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5v2 and CYP82E10 in the root, nicotine and nornicotine are translocated to leaf, where more nicotine becomes demethylated. Depending on the CYP82E4 activity in senescing leaf, constant low (R)-nicotine remains in the tobacco leaf and variable nornicotine composition is produced. These results confirmed the enantioselectivity of three nicotine demethylases in planta, could be used to predict the changes of nicotine and nornicotine composition, and may facilitate demethylase discovery in the future.}, journal={PHYTOCHEMISTRY}, author={Cai, Bin and Jack, Anne M. and Lewis, Ramsey S. and Dewey, Ralph E. and Bush, Lowell P.}, year={2013}, month={Nov}, pages={188–196} }
 @article{drake_lewis_2013, title={An Introgressed Nicotiana rustica Genomic Region Confers Resistance to Phytophthora nicotianae in Cultivated Tobacco}, volume={53}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2012.10.0605}, abstractNote={ABSTRACTBlack shank, caused by the pathogen Phytophthora nicotianae, is an important disease affecting tobacco (Nicotiana tabacum L.) production in many parts of the world. Host resistance offers an efficient means of reducing economic loss due to this pathogen. Previous observations suggested that a genomic region introgressed from Nicotiana rustica L. may affect resistance to black shank. The effect of this genomic region, designated as Wz, on resistance to multiple races of P. nicotianae had not previously been investigated in a systematic way, however. We used growth chamber and field experiments to evaluate resistance of a doubled haploid (DH) mapping population segregating for the Wz region. We also genotyped the population with amplified fragment length polymorphism (AFLP) markers found to be polymorphic between the parental lines. In addition, we determined resistance and marker genotypes for individuals of a larger BC1F1 population segregating for Wz. A set of 29 AFLP markers determined to be of N. rustica origin were found to cosegregate with each other and were associated with resistance to both race 0 and race 1 of P. nicotianae in the DH population. A selected subset of these markers was also found to cosegregate with resistance in the BC1F1 population. Consistent with other introgressed alien genomic regions in N. tabacum, no recombination was observed between these selected markers. The N. rustica‐derived genetic variation and associated DNA markers will be of value for breeding for black shank resistance in tobacco.}, number={4}, journal={CROP SCIENCE}, author={Drake, Katherine and Lewis, Ramsey S.}, year={2013}, pages={1366–1374} }
 @article{eickholt_lewis_2013, title={Breeding Cycles Expedited by FT-mediated Reduction in Generation Time}, volume={53}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2013.03.0150}, abstractNote={ABSTRACTMethods for rapid inbreeding are desirable for reducing the time required to complete a breeding cycle and to increase genetic gain per year. Constitutive expression of FLOWERING LOCUS T (FT) derived from Arabidopsis has previously been shown to reduce generation time in tobacco (Nicotiana tabacum L.) and other crop plants. Here, we used tobacco as a model system to investigate the utility of a modified single‐seed descent (SSD) breeding method where transgenic expression of 35S:FT was used to reduce generation time during inbreeding. The transgene was maintained in hemizygous condition during inbreeding, and null segregants were isolated in the F4:5 generation to create nontransgenic F5:6 lines that were produced in nearly half the time that would have been required using conventional SSD. Opportunities for selection among 35S:FT plants during inbreeding were demonstrated by selecting for quantitative levels of resistance to the soil‐borne pathogen Phytophthora nicotianae. Populations of selected lines exhibited significantly higher levels of resistance relative to random lines, greater frequencies of highly resistant genotypes, and lower frequencies of highly susceptible genotypes. The system outlined here could lead to more rapid commercialization of improved cultivars in crop species amenable to manipulation of flowering time via transgenic expression of FT‐like genes. Strategies to enhance the frequency of favorable alleles in resulting populations can be incorporated into the method.}, number={6}, journal={CROP SCIENCE}, author={Eickholt, David P. and Lewis, Ramsey S.}, year={2013}, pages={2384–2391} }
 @article{bukan_sarcevic_buhinicek_palaversic_lewis_kozumplik_2013, title={STALK ROT RESISTANCE IN MAKSIMIR 3 SYNTHETIC MAIZE POPULATION AFTER FOUR CYCLES OF RECURRENT SELECTION}, volume={45}, ISSN={["1820-6069"]}, DOI={10.2298/gensr1303921b}, abstractNote={Fusarium stalk rot (FSR) and anthracnose stalk rot (ASR), caused by Fusarium
   spp. and Colletotrichum graminicola (Ces.) G.W. Wils. respectively, are the
   two most important stalk diseases in maize which increase the incidence of
   stalk lodging and reduce grain yield. The aim of the present study was to (1)
   evaluate the effect of four cycles of recurrent selection in the Maksimir 3
   Synthetic (M3S) maize population on ASR and FSR resistance and (2) to
   investigate the correlation among the different disease rating methods. The
   experiment included six M3S cycle populations per se and their test-crosses
   with a single cross hybrid. ASR resistance was estimated on artificially
   inoculated plant rows using three ratings (the number of infected internodes,
   the number of internodes rotten more than 75% and evaluation of outer stalk
   discoloration) whereas FSR resistance was estimated in artificially
   inoculated rows as well as in naturally inoculated rows by rating severity of
   disease symptoms on longitudinally cut stalks using the standard resistance
   scale. The results of the present study showed that four cycles of selection
   in the M3S maize population, conducted primarily for grain yield improvement,
   did not significantly affect its resistance to both ASR and FSR. Among the
   disease ratings a moderate positive correlation was found only between two
   ASR resistance ratings (the number of infected internodes and the number of
   internodes rotted more than 75%) in both population per se (r=0.49**) and
   population test-crosses (r=0.56**).}, number={3}, journal={GENETIKA-BELGRADE}, author={Bukan, Miroslav and Sarcevic, Hrvoje and Buhinicek, Ivica and Palaversic, Branko and Lewis, Ramsey S. and Kozumplik, Vinko}, year={2013}, pages={921–928} }
 @article{vontimitta_lewis_2012, title={Growth Chamber Evaluation of a Tobacco 'Beinhart 1000' x 'Hicks' Mapping Population for Quantitative Trait Loci Affecting Resistance to Multiple Races of Phytophthora nicotianae}, volume={52}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2011.06.0303}, abstractNote={ABSTRACTMultiple races of Phytophthora nicotianae (van Breda de Haan) cause the black shank disease of tobacco (Nicotiana tabacum L.). Identification of novel alleles affecting resistance and associated DNA markers might help increase the range and level of cultivar resistance. A doubled haploid mapping population generated from a highly resistant × susceptible cross (‘Beinhart 1000’ × ‘Hicks’) was evaluated for resistance after inoculation with either race 0 or race 1 in a growth chamber and also genotyped at 206 microsatellite marker loci. Multiple interval mapping identified two major genomic regions affecting resistance to both races. A quantitative trait locus (QTL) on linkage group 8 explained 54.7 and 45.5% of the observed variation for end percent survival after race 0 and race 1 inoculation, respectively. A QTL on linkage group 4 explained a larger proportion (16.8%) of phenotypic variation for race 1 resistance as compared to race 0 resistance (1.8%). In combination with previously reported field data, the study authenticates the role of these two regions on black shank resistance. Correlations of line performance under field and growth chamber environments were good, and agreement was excellent at extreme levels of resistance or susceptibility. With appropriate isolate selection, growth chamber inoculations may provide a superior alternative to field evaluations for mapping QTL affecting black shank resistance. Identified QTL and associated markers may be useful for increasing levels of resistance to P. nicotianae in tobacco cultivars.}, number={1}, journal={CROP SCIENCE}, author={Vontimitta, V. and Lewis, R. S.}, year={2012}, month={Jan}, pages={91–98} }
 @article{lewis_parker_danehower_andres_jack_whitley_bush_2012, title={Impact of Alleles at the Yellow Burley (Yb) Loci and Nitrogen Fertilization Rate on Nitrogen Utilization Efficiency and Tobacco-Specific Nitrosamine (TSNA) Formation in Air-Cured Tobacco}, volume={60}, ISSN={0021-8561 1520-5118}, url={http://dx.doi.org/10.1021/jf2053614}, DOI={10.1021/jf2053614}, abstractNote={Tobacco-specific nitrosamine (TSNA) formation in tobacco is influenced by alkaloid levels and the availability of nitrosating agents. Tobacco types differ in their potential for TSNA accumulation due to genetic, agronomic, and curing factors. Highest TSNA concentrations are typically measured in burley tobaccos. One of the main genetic differences between burley and all other tobacco types is that this tobacco type is homozygous for recessive mutant alleles at the Yellow Burley 1 (Yb(1)) and Yellow Burley 2 (Yb(2)) loci. In addition, burley tobacco is typically fertilized at higher nitrogen (N) rates than most other tobacco types. This study utilized nearly isogenic lines (NILs) differing for the presence of dominant or recessive alleles at the Yb(1) and Yb(2) loci to investigate the potential influence of genes at these loci on TSNA accumulation. Three pairs of NILs were evaluated at three different nitrogen fertilization rates for alkaloid levels, nitrogen physiology measures, and TSNA accumulation after air-curing. As previously observed by others, positive correlations were observed between N application rates and TSNA accumulation. Recessive alleles at Yb(1) and Yb(2) were associated with increased alkaloid levels, reduced nitrogen use efficiency, reduced nitrogen utilization efficiency, and increased leaf nitrate nitrogen (NO(3)-N). Acting together, these factors contributed to significantly greater TSNA levels in genotypes possessing the recessive alleles at these two loci relative to those carrying the dominant alleles. The chlorophyll-deficient phenotype conferred by the recessive yb(1) and yb(2) alleles probably contributes in a substantial way to increase available NO(3)-N during curing and, consequently, increased potential for TSNA formation.}, number={25}, journal={Journal of Agricultural and Food Chemistry}, publisher={American Chemical Society (ACS)}, author={Lewis, Ramsey S. and Parker, Robert G. and Danehower, David A. and Andres, Karen and Jack, Anne M. and Whitley, D. Scott and Bush, Lowell P.}, year={2012}, month={Jun}, pages={6454–6461} }
 @article{sarcevic_gunjaca_budimir_boic_bolaric_bukan_lewis_kozumplik_2013, title={Long-term Genetic Improvement and Genetic Diversity of Croatian Flue-cured Tobacco (Nicotiana tabacum L.) Cultivars}, volume={53}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2012.03.0173}, abstractNote={Use of breeding methodologies to develop improved flue‐cured tobacco (Nicotiana tabacum L.) cultivars in Croatia has been performed since the early 1960s. Since that time, >20 cultivars have been released. Between 1973 and 2007, only nine of these cultivars have been accepted by tobacco growers and the tobacco industry, however. To quantify the genetic improvement of agronomic traits and associated changes in morphological and chemical characters during this period of time, these nine cultivars were grown and studied in experiments conducted at three Croatian locations for 3 yr. Trait stability and genetic diversity of the same cultivars were also investigated. Regression analysis of agronomic traits over year of cultivar release showed a significant annual increase in yield of 17.39 kg ha−1 yr−1, price of $0.009 kg−1 yr−1, and value of $56.50 ha−1 yr−1. These increases can be attributed to genetic improvement of new cultivars. During the 1973–2007 time period, the average number of leaves increased significantly. Over the same period, nicotine content decreased slightly and reducing sugar content increased significantly. Newer cultivars were also found to have higher yield stability as compared to older cultivars. Genetic diversity among the studied cultivars as revealed by amplified fragment length polymorphism genetic markers was relatively low. Unweighted pair group method using arithmetic averages analysis clustered the cultivars in accordance with their pedigrees.}, number={1}, journal={CROP SCIENCE}, author={Sarcevic, H. and Gunjaca, J. and Budimir, A. and Boic, M. and Bolaric, S. and Bukan, M. and Lewis, R. S. and Kozumplik, V.}, year={2013}, month={Jan}, pages={112–120} }
 @article{li_lewis_jack_dewey_bowen_miller_2012, title={Development of CAPS and dCAPS markers for CYP82E4, CYP82E5v2 and CYP82E10 gene mutants reducing nicotine to nornicotine conversion in tobacco}, volume={29}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-011-9575-9}, number={3}, journal={MOLECULAR BREEDING}, author={Li, Dandan and Lewis, Ramsey S. and Jack, Anne M. and Dewey, Ralph E. and Bowen, Steve W. and Miller, Robert D.}, year={2012}, month={Mar}, pages={589–599} }
 @article{lewis_rose_2011, title={Identification of Tobacco Haploids on the Basis of Transgenic Overexpression of PAP1 from Arabidopsis thaliana}, volume={51}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2010.09.0546}, abstractNote={ABSTRACTHaploid plants have utility in plant breeding for several purposes. In tobacco (Nicotiana tabacum L.) gynogenic haploids can be produced from seed due to parthenogenesis. For practical use, a system is needed to identify infrequent haploid plants at the seed or seedling stage. Interspecific hybridization with N. africana is presently used to isolate gynogenic tobacco haploids because a suitable dominant seedling marker does not currently exist for N. tabacum. Here we investigated the utility of a purple seedling trait conferred by overexpression of the Arabidopsis gene, PAP1, to identify gynogenic haploids produced from seed. Two tobacco cultivars were crossed as females with a genetic stock homozygous at two 35S:PAP1 transgene loci. Gynogenic haploids were recognized as green seedlings among purple F1 hybrid seedlings. The average frequency of gynogenic haploidy using this system was f = 0.00027. For comparison, the same cultivars were also hybridized as females with N. africana. The average frequency of gynogenic haploidy using this system was approximately seven times higher than that observed for the 35S:PAP1 system. Having a dominant seedling marker for N. tabacum may permit development of genetic stocks that contribute to an increased predisposition for haploid formation via parthenogenesis. In addition, the 35S:PAP1 genetic marker may have utility for identifying androgenic haploids from seed for the purpose of rapidly generating alloplasmic lines of tobacco.}, number={4}, journal={CROP SCIENCE}, author={Lewis, Ramsey S. and Rose, Cara}, year={2011}, month={Jul}, pages={1491–1497} }
 @article{nifong_nicholson_shew_lewis_2011, title={Variability for Resistance to Phytophthora nicotianae Within a Collection of Nicotiana rustica Accessions}, volume={95}, ISSN={["1943-7692"]}, DOI={10.1094/pdis-11-10-0862}, abstractNote={ Black shank, caused by Phytophthora nicotianae, is one of the most important diseases affecting tobacco (Nicotiana tabacum) production worldwide. Many current tobacco cultivars possess immunity to race 0 of this pathogen conferred by introgressed dominant genetic factors. Novel alleles conditioning resistance to alternative races are desired. The objective of this research was to evaluate variability for black shank resistance within a collection of N. rustica germplasm using both soilborne disease nurseries and controlled race-specific (race 0 and race 1) inoculations. Nearly all of the 86 accessions studied exhibited very high resistance to race 0, and many displayed levels of race 1 resistance greater than that exhibited by the resistant flue-cured tobacco check, ‘K 346’. Materials found to be highly resistant to race 0 and race 1 in growth-chamber experiments also had the best survivability in field disease nurseries. N. rustica accessions TR 6, TR 12, TR 16, TR 21, TR 20, TR 48, TR 54, TR 57, and TR 69 could be sources of novel alleles with large effects on black shank resistance, and could have value for burley and flue-cured tobacco breeding. }, number={11}, journal={PLANT DISEASE}, author={Nifong, J. M. and Nicholson, J. S. and Shew, H. D. and Lewis, R. S.}, year={2011}, month={Nov}, pages={1443–1447} }
 @article{lewis_rose_2010, title={Agronomic Performance of Tobacco Mosaic Virus-Resistant Tobacco Lines and Hybrids Possessing the Resistance Gene N Introgressed on Different Chromosomes}, volume={50}, ISSN={["0011-183X"]}, DOI={10.2135/cropsci2009.10.0615}, abstractNote={Resistance to tobacco mosaic virus (TMV) is conferred by the single dominant gene, N, in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) via interspecific hybridization and backcrossing. Current TMV‐resistant (TMVR) cultivars carry N introgressed on chromosome H of the N. tabacum genome. Undesirable linkage drag effects have caused associations with reduced yields and/or quality in flue‐cured tobacco, however. Other germplasm lines possess the gene transferred onto an alternative chromosome. The objective of this research was to compare the agronomic performance of nearly isogenic lines (NILs) and hybrids possessing N on different chromosomes and originating from four N donor lines. Regardless of the source of the gene, Nn heterozygotes were intermediate in value for yield, cash return, and cured leaf chemistry relative to nn and NN homozygotes. Lines and hybrids carrying N transferred from Xanthi nc produced the highest yields, whereas those possessing N introduced from TI 1473 exhibited the lowest yields. Overall, materials possessing N on chromosome H were not found to be significantly different for yield, grade index, value per hundred weight (US$ cwt−1), or cash return from those carrying the resistance gene on the alternative chromosome. Breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N are needed to develop TMVR flue‐cured tobacco cultivars that do not exhibit an accompanying yield penalty.}, number={4}, journal={CROP SCIENCE}, author={Lewis, Ramsey S. and Rose, Cara}, year={2010}, pages={1339–1347} }
 @article{vontimitta_lewis_2012, title={Mapping of quantitative trait loci affecting resistance to Phytophthora nicotianae in tobacco (Nicotiana tabacum L.) line Beinhart-1000}, volume={29}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-010-9528-8}, number={1}, journal={MOLECULAR BREEDING}, author={Vontimitta, Vijay and Lewis, Ramsey S.}, year={2012}, month={Jan}, pages={89–98} }
 @article{lewis_bowen_keogh_dewey_2010, title={Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L Functional characterization of the CYP82E10 gene}, volume={71}, ISSN={["1873-3700"]}, DOI={10.1016/j.phytochem.2010.09.011}, abstractNote={In most tobacco (Nicotiana tabacum L.) plants, nornicotine is a relatively minor alkaloid, comprising about 2-5% of the total pyridine alkaloid pool in the mature leaf. Changes in gene expression at an unstable locus, however, can give rise to plants that produce high levels of nornicotine, specifically during leaf senescence and curing. Minimizing the nornicotine content in tobacco is highly desirable, because this compound serves as the direct precursor in the synthesis of N'-nitrosonornicotine, a potent carcinogen in laboratory animals. Nornicotine is likely produced almost entirely via the N-demethylation of nicotine, in a process called nicotine conversion that is catalyzed by the enzyme nicotine N-demethylase (NND). Previous studies have identified CYP82E4 as the specific NND gene responsible for the unstable conversion phenomenon, and CYP82E5v2 as a putative minor NND gene. Here, by discovery and characterization of CYP82E10, a tobacco NND gene, is reported. PCR amplification studies showed that CYP82E10 originated from the N. sylvestris ancestral parent of modern tobacco. Using a chemical mutagenesis strategy, knockout mutations were induced and identified in all three tobacco NND genes. By generating a series of mutant NND genotypes, the relative contribution of each NND gene toward the nornicotine content of the plant was assessed. Plants possessing knockout mutations in all three genes displayed nornicotine phenotypes that were much lower (∼0.5% of total alkaloid content) than that found in conventional tobacco cultivars. The introduction of these mutations into commercial breeding lines promises to be a viable strategy for reducing the levels of one of the best characterized animal carcinogens found in tobacco products.}, number={17-18}, journal={PHYTOCHEMISTRY}, author={Lewis, Ramsey S. and Bowen, Steven W. and Keogh, Matthew R. and Dewey, Ralph E.}, year={2010}, month={Dec}, pages={1988–1998} }
 @article{lewis_kernodle_2009, title={A method for accelerated trait conversion in plant breeding}, volume={118}, ISSN={["1432-2242"]}, DOI={10.1007/s00122-009-0998-1}, abstractNote={Backcrossing is often used in cultivar development to transfer one or a few genes to desired genetic backgrounds. The duration necessary to complete such 'trait conversions' is largely dependent upon generation times. Constitutive overexpression of the Arabidopsis thaliana gene FT (FLOWERING LOCUS T) induces early-flowering in many plants. Here, we used tobacco (Nicotiana tabacum L.) as a model system to propose and examine aspects of a modified backcross procedure where transgenic FT overexpression is used to reduce generation time and accelerate gene transfer. In this method, the breeder would select for an FT transgene insertion and the trait(s) of interest at each backcross generation except the last. In the final generation, selection would be conducted for the trait(s) of interest, but against FT, to generate the backcross-derived trait conversion. We demonstrate here that constitutive FT overexpression functions to dramatically reduce days-to-flower similarly in diverse tobacco genetic backgrounds. FT-containing plants flowered in an average of 39 days, in comparison with 87-138 days for non-FT plants. Two FT transgene insertions were found to segregate independently of several disease resistance genes often the focus of backcrossing in tobacco. In addition, no undesirable epigenetic effects on flowering time were observed once FT was segregated away. The proposed system would reduce the time required to complete a trait conversion in tobacco by nearly one-half. These features suggest the possible value of this modified backcrossing system for tobacco or other crop species where long generation times or photoperiod sensitivity may impede timely trait conversion.}, number={8}, journal={THEORETICAL AND APPLIED GENETICS}, author={Lewis, Ramsey S. and Kernodle, S. P.}, year={2009}, month={May}, pages={1499–1508} }
 @article{vontimitta_danehower_steede_moon_lewis_2010, title={Analysis of a Nicotiana tabacum L. Genomic Region Controlling Two Leaf Surface Chemistry Traits}, volume={58}, ISSN={["1520-5118"]}, DOI={10.1021/jf903256h}, abstractNote={cis-Abienol and sucrose esters are Nicotiana tabacum leaf surface components that likely influence plant resistance to pests. Their breakdown products also contribute to flavor and aroma characteristics of certain tobacco types. Mapping of genes involved in the biosynthesis of these compounds could permit development of molecular-based tools for generating tobacco types with novel cured leaf chemistry profiles. A doubled haploid mapping population segregating for major genes (Abl and BMVSE) affecting the ability to accumulate cis-abienol and sucrose esters was generated and genotyped with a large set of microsatellite markers. The two genes were found to reside on chromosome A of the N. tabacum genome with a distance of 8.2 cM (centimorgans) between them. Seventeen microsatellite markers were also placed on this linkage group, several of which exhibited complete cosegregation with Abl and BMVSE. Results should aid breeding efforts focused on modification of this aspect of tobacco cured leaf chemistry.}, number={1}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Vontimitta, Vijay and Danehower, David A. and Steede, Tyler and Moon, Hyunsook S. and Lewis, Ramsey S.}, year={2010}, month={Jan}, pages={294–300} }
 @article{moon_nicholson_heineman_lion_hoeven_hayes_lewis_2009, title={Changes in Genetic Diversity of US Flue-Cured Tobacco Germplasm over Seven Decades of Cultivar Development}, volume={49}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2008.05.0253}, abstractNote={Plant breeding methodologies have been applied to flue‐cured tobacco (Nicotiana tabacum L.) for approximately seven decades. As has been observed in several other crops, stringent quality requirements have resulted in use of conservative breeding strategies in the development of new cultivars. The impact of breeding practices on genetic diversity within U.S. flue‐cured tobacco germplasm has not been investigated. In this study, we genotyped 117 tobacco cultivars from eight sequential time periods with 71 microsatellite primer pairs. A total of 294 alleles were scored. Only a fraction (48%) of alleles present in the initial germplasm pool was represented in cultivars released during the 1990s and 2000s. Only 13 and 18 alleles were detected in the 1990s and 2000s, respectively, which were undetected in the initial gene pool. The overall trend was one of gradual reduction in allelic counts at microsatellite loci, indicating a reduction in diversity over time at the gene level. Average genetic similarity was highest among cultivars of the 1990s and 2000s, reflecting a reduction in genetic diversity at the population level. This observed narrowing of the U.S. flue‐cured tobacco germplasm base in combination with low rates of genetic gain for yield in the last 20 years may point to a need for diversification of parental materials used in future breeding crosses. Reported genetic relationships among the group of genotyped cultivars may be valuable for future strategic germplasm choices.}, number={2}, journal={CROP SCIENCE}, author={Moon, H. S. and Nicholson, J. S. and Heineman, A. and Lion, K. and Hoeven, P. and Hayes, A. J. and Lewis, R. S.}, year={2009}, pages={498–508} }
 @article{moon_nifong_nicholson_heineman_lion_hoeven_hayes_lewis_2009, title={Microsatellite-based Analysis of Tobacco (Nicotiana tabacum L.) Genetic Resources}, volume={49}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2009.01.0024}, abstractNote={ABSTRACTLittle is currently known about the genetic variation within diverse gene pools of cultivated tobacco (Nicotiana tabacum L.). Knowledge in this area could aid in future germplasm maintenance activities, provide additional information in the area of N. tabacum population genetics, and assist in selection of materials for breeding or genetic investigations. To this end, 702 N. tabacum accessions from the U.S. Nicotiana Germplasm Collection were genotyped with 70 microsatellite markers. A total of 1031 alleles were detected, with an average of 14.7 alleles per marker. Average gene diversity was quite large (0.7362) and was three times the value for a diverse population of U.S. flue‐cured tobacco. Cluster analysis using various distance coefficients generated seven consistent groups. Patterns of geographic‐specific clustering were frequent but not universal. An analysis of molecular variance for tobacco collected from Central and South America revealed that 92% of the total genetic variation among samples could be attributed to country‐specific subpopulations. There was also a significant level of population subdivision between country‐specific subpopulations, however. Tobacco samples collected from Peru had both the largest average gene diversity and the largest mean genetic distance, although N. tabacum collected from Mexico had the greatest allelic richness. Tobacco samples collected from Argentina were the most divergent subpopulation.}, number={6}, journal={CROP SCIENCE}, author={Moon, H. S. and Nifong, J. M. and Nicholson, J. S. and Heineman, A. and Lion, K. and Hoeven, R. and Hayes, A. J. and Lewis, R. S.}, year={2009}, pages={2149–2159} }
 @article{elliott_lewis_shew_gutierrez_nicholson_2008, title={Evaluation of tobacco germplasm for seedling resistance to stem rot and target spot caused by Thanatephorus cucumeris}, volume={92}, ISSN={["0191-2917"]}, DOI={10.1094/PDIS-92-3-0425}, abstractNote={ Stem rot and target spot of tobacco, caused by Rhizoctonia solani and its teleomorph Thanatephorus cucumeris, respectively, can cause serious problems in production of tobacco (Nicotiana tabacum) seedlings. Previous screens for genetic resistance in tobacco have been limited. The objective of this study was to evaluate 97 genotypes composing several classes of tobacco and related Nicotiana spp. for seedling resistance to stem rot and target spot. Significant differences in disease incidence initially were observed among the genotypes for both stem rot and target spot; however, resistance to target spot was not observed when disease pressure was high. Partial resistance to stem rot was observed in several genotypes in repeated tests. These accessions may be useful as a source of resistance to R. solani in future breeding efforts. }, number={3}, journal={PLANT DISEASE}, author={Elliott, P. E. and Lewis, R. S. and Shew, H. D. and Gutierrez, W. A. and Nicholson, J. S.}, year={2008}, month={Mar}, pages={425–430} }
 @article{lewis_jack_morris_robert_gavilano_siminszky_bush_hayes_dewey_2008, title={RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves}, volume={6}, ISSN={["1467-7644"]}, DOI={10.1111/j.1467-7652.2008.00324.x}, abstractNote={SummaryTechnologies for reducing the levels of tobacco product constituents that may contribute to unwanted health effects are desired. Target compounds include tobacco‐specific nitrosamines (TSNAs), a class of compounds generated through the nitrosation of pyridine alkaloids during the curing and processing of tobacco. Studies have reported the TSNA N′‐nitrosonornicotine (NNN) to be carcinogenic in laboratory animals. NNN is formed via the nitrosation of nornicotine, a secondary alkaloid produced through enzymatic N‐demethylation of nicotine. Strategies to lower nornicotine levels in tobacco (Nicotiana tabacum L.) could lead to a corresponding decrease in NNN accumulation in cured leaves. The major nicotine demethylase gene of tobacco has recently been isolated. In this study, a large‐scale field trial was conducted to evaluate transgenic lines of burley tobacco carrying an RNA interference (RNAi) construct designed to inhibit the expression of this gene. Selected transgenic lines exhibited a six‐fold decrease in nornicotine content relative to untransformed controls. Analysis of cured leaves revealed a commensurate decrease in NNN and total TSNAs. The inhibition of nicotine demethylase activity is an effective means of decreasing significantly the level of a key defined animal carcinogen present in tobacco products.}, number={4}, journal={PLANT BIOTECHNOLOGY JOURNAL}, author={Lewis, Ramsey S. and Jack, Anne M. and Morris, Jerry W. and Robert, Vincent J. M. and Gavilano, Lily B. and Siminszky, Balazs and Bush, Lowell P. and Hayes, Alec J. and Dewey, Ralph E.}, year={2008}, month={May}, pages={346–354} }
 @article{moon_nicholson_lewis_2008, title={Use of transferable Nicotiana tabacum L. microsatellite markers for investigating genetic diversity in the genus Nicotiana}, volume={51}, ISSN={["0831-2796"]}, DOI={10.1139/G08-039}, abstractNote={The recent development of microsatellite markers for tobacco, Nicotiana tabacum L., may be valuable for genetic studies within the genus Nicotiana . The first objective was to evaluate transferability of 100 N. tabacum microsatellite primer combinations to 5 diploid species closely related to tobacco. The number of primer combinations that amplified scorable bands in these species ranged from 42 to 56. Additional objectives were to assess levels of genetic diversity amongst available accessions of diploid relatives closely related to tobacco (species of sections Sylvestres and Tomentosae), and to evaluate the efficacy of microsatellite markers for establishing species relationships in comparison with existing phylogenetic reconstructions. A subset of 46 primer combinations was therefore used to genotype 3 synthetic tobaccos and an expanded collection of 51 Nicotiana accessions representing 15 species. The average genetic similarity for 7 diverse accessions of tobacco was greater than the average similarity for N. otophora accessions, but lower than the average genetic similarities for N. sylvestris , N. tomentosa , N. kawakamii , and N. tomentosiformis accessions. A microsatellite-based phylogenetic tree was largely congruent with taxonomic representations based on morphological, cytological, and molecular observations. Results will be useful for selection of parents for creation of diploid mapping populations and for germplasm introgression activities.}, number={8}, journal={GENOME}, author={Moon, H. S. and Nicholson, J. S. and Lewis, R. S.}, year={2008}, month={Aug}, pages={547–559} }
 @article{lewis_milla_kernodle_2007, title={Analysis of an introgressed Nicotiana tomentosa genomic region affecting leaf number and correlated traits in Nicotiana tabacum}, volume={114}, ISSN={["1432-2242"]}, DOI={10.1007/s00122-006-0482-0}, abstractNote={Germplasm from closely related diploid relatives of tobacco (Nicotiana tabacum L.) could be of value for continued genetic modification of this species and for mapping quantitative trait loci (QTLs). We examined near isogenic tobacco lines and hybrids differing for an introgressed genomic region from N. tomentosa Ruiz and Pavon designated as Many Leaves that exhibits a large influence on leaf number and correlated traits. Within a 'Red Russian' genetic background, the region acted in an additive to partially dominant fashion to delay flowering time, and increase leaf number, plant height, and green leaf yield. Evidence of epistasis was observed as the region affected these traits to varying degrees in diverse near isogenic hybrids. Fifteen amplified fragment length polymorphism (AFLP) markers of N. tomentosa origin were mapped within a single linkage group of 34.5 cM using a population of 207 BC(1)F(1) individuals segregating for Many Leaves. Composite interval mapping produced 2-LOD confidence intervals for likely QTL positions influencing leaf number (3.1 cM region), plant height (2.9 cM region), and days to flowering (3.3 cM region). These intervals were overlapping. Results demonstrate that genomic regions with large genetic effects can be transferred to tobacco from closely related diploid relatives, and that sufficient recombination within these regions may permit mapping of genes controlling quantitative traits. Materials and results described here may be useful in future research to gain insight on the genetic control of the transition from vegetative to reproductive development in Nicotiana.}, number={5}, journal={THEORETICAL AND APPLIED GENETICS}, publisher={Springer Science \mathplus Business Media}, author={Lewis, R. S. and Milla, S. R. and Kernodle, S. P.}, year={2007}, month={Mar}, pages={841–854} }
 @article{lewis_2007, title={Evaluation of Nicotiana tabacum genotypes possessing Nicotiana africana-derived genetic tolerance to potato virus Y}, volume={47}, ISSN={["0011-183X"]}, DOI={10.2135/cropsci2007.01.0001}, abstractNote={New alleles influencing resistance to potato virus Y (PVY) would be valuable for developing resistant tobacco (Nicotiana tabacum L.) cultivars. The first objective of this research was to evaluate materials possessing an introgressed genomic region (Nafr) from N. africana Merx. & Buttler for their resistance against an array of nine PVY isolates. Seven near‐isogenic genotypes of flue‐cured tobacco cultivar K326 were produced that possessed Nafr and the recessive resistance gene, va, in different combinations and zygosities. Nafr provided little protection against mild non‐necrotic isolates, but imparted significant increased resistance against the necrotic effects of two severe isolates. Data indicated that Nafr and va can be combined to increase the range and level of resistance or tolerance to PVY. A second objective was to evaluate five backcross‐derived K326 Nafr/Nafr isolines and corresponding hemizygous F1 hybrids for yield and quality characteristics in field experiments in the absence of PVY infection. The K326 Nafr/— genotypes were not significantly different from K326 for yield or cash return, and produced cured leaf with improved quality. The Nafr/Nafr genotypes were inferior to Nafr/— genotypes for yield, cash return, and cured leaf quality. Genotypes of the Nafr/— va/va combination may have the greatest practical value when yield, quality, and reaction to PVY infection are collectively taken into consideration.}, number={5}, journal={CROP SCIENCE}, author={Lewis, R. S.}, year={2007}, pages={1975–1984} }
 @article{lewis_linger_wolff_wernsman_2007, title={The negative influence of N-mediated TMV resistance on yield in tobacco: linkage drag versus pleiotropy}, volume={115}, ISSN={["0040-5752"]}, DOI={10.1007/s00122-007-0552-y}, abstractNote={Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar 'K326') via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha(-1)) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha(-1) (P < 0.05) and generated $1,365 ha(-1) more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.}, number={2}, journal={THEORETICAL AND APPLIED GENETICS}, author={Lewis, R. S. and Linger, L. R. and Wolff, M. F. and Wernsman, E. A.}, year={2007}, month={Jul}, pages={169–178} }
 @article{lewis_nicholson_2007, title={Aspects of the evolution of Nicotiana tabacum L. and the status of the United States Nicotiana Germplasm Collection}, volume={54}, ISSN={["1573-5109"]}, DOI={10.1007/s10722-006-0024-2}, number={4}, journal={GENETIC RESOURCES AND CROP EVOLUTION}, author={Lewis, R. S. and Nicholson, J. S.}, year={2007}, month={Jun}, pages={727–740} }
 @article{lewis_2006, title={Identification of germplasm of possible value for confronting an unfavorable inverse genetic correlation in tobacco}, volume={46}, ISSN={["0011-183X"]}, DOI={10.2135/cropsci2005.12-0519}, abstractNote={To be commercially viable in the USA, a flue‐cured tobacco (Nicotiana tabacum L.) cultivar must be high‐yielding and also meet minimum requirements for percent total alkaloids (PTA). The negative correlation between yield and PTA complicates development of higher‐yielding cultivars with acceptable leaf chemistry, however. Identification and use of germplasm possessing alternative alleles positively affecting PTA could be an important component of strategies to develop commercially acceptable, higher‐yielding cultivars. Choice of donor germplasm should be done carefully, however, because yield modulates the phenotypic expression of PTA. Consequently, comparison of materials for genetic potential to accumulate alkaloids might best be done at common levels of yield. This investigation used manual control of leaf number to manipulate yield of fifteen diverse tobacco genotypes grown in a split‐plot design in two North Carolina environments. Within genotypes, the relationships between PTA and yield were found to be negative. Through statistical analyses, germplasm accessions TI 464 and TI 959 were found to exhibit the highest levels of PTA at given levels of yield in both environments. Data on N‐partitioning indicated that these genotypes may have increased genetic potential for utilizing accumulated N for alkaloid synthesis. Transfer of alleles from these genotypes to elite germplasm pools may facilitate development of higher‐yielding cultivars with acceptable PTA levels.}, number={4}, journal={CROP SCIENCE}, author={Lewis, R. S.}, year={2006}, pages={1764–1771} }
 @article{lewis_milla_levin_2005, title={Molecular and genetic characterization of N. glutinosa L. chromosome segments in tobacco mosaic virus (TMV)-resistant tobacco accessions}, volume={45}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2005.0121}, abstractNote={Tobacco mosaic virus (TMV)‐resistant flue‐cured tobacco (Nicotiana tabacum L.) cultivars have been developed using the N gene derived from N. glutinosa L. Their adoption has been low, however, because of unfavorable linkage drag effects. Strategies to overcome this problem might include pursuit of alternative introgression events and/or use of molecular markers for selection against deleterious alien chromatin. Previous workers demonstrated the presence of a TMV‐resistance mechanism on more than one chromosome of the tobacco genome. The objectives of this research were to determine the relative genomic positions of TMV resistance loci in a set of 12 TMV‐resistant tobacco accessions and to use amplified fragment length polymorphism (AFLP) markers for characterization of this material with respect to linked alien chromatin. Five accessions were found to carry a TMV resistance gene on chromosome H. Seven accessions were found to carry a resistance factor on an alternative chromosome. Polymerase chain reaction results indicated that the N gene from N. glutinosa is responsible for resistance in all 12 accessions. A set of 168 AFLP markers specific to the N. glutinosa donor chromosome was identified and used to reveal variability among the 12 accessions for the relative amounts of N. glutinosa chromatin linked to the N gene. The relative propensity for crossing over within the alien segment when in different genomic positions was evaluated in BC1F1 families derived from three different accessions. Lines possessing the N gene on chromosome H may be of greater practical value because of relatively smaller introgressed alien segments and increased potential for obtaining crossover events within the segments.}, number={6}, journal={Crop Science}, publisher={Crop Science Society of America}, author={Lewis, R.S. and Milla, S.R. and Levin, J.S.}, year={2005}, pages={2355–2362} }
 @article{milla_levin_lewis_rufty_2005, title={RAPD and SCAR markers linked to an introgressed gene conditioning resistance to Peronospora tabacina D.B. Adam in tobacco}, volume={45}, ISSN={["1435-0653"]}, DOI={10.2135/cropsci2004.0754}, abstractNote={Blue mold, caused by the fungal pathogen Peronospora tabacina D.B. Adam, is one of the most important foliar diseases of tobacco (Nicotiana tabacum L.). Identification of molecular markers linked to genetic factors controlling resistance would facilitate development of resistant cultivars. Bulked segregant analysis was used to screen 1216 random amplified polymorphic DNA (RAPD) primers for their ability to reveal polymorphism between DNA bulks from susceptible doubled haploid (DH) lines and resistant DH lines possessing resistance derived from cultivar Ovens 62. Fifteen RAPD markers were tentatively identified as being linked to a major gene conditioning resistance to blue mold. These 15 markers (12 in coupling phase linkage with resistance and three in repulsion phase) were found to lie within a single linkage group of 36.6 cM and were subsequently tested on 122 DH lines derived from crosses between resistant and susceptible parents. F tests revealed statistically significant associations between resistance and each of the 15 RAPD markers. Interval mapping was used to more accurately place the quantitative trait locus (QTL) controlling resistance on the linkage map. The RAPD markers were screened on a set of 45 resistant and susceptible cultivars or breeding lines and four Nicotiana species. At variance with previous reports, marker genotypes indicated that resistance in Ovens 62 and most other blue mold resistant lines likely originated from N. debneyi Domin. Two RAPD markers flanking the most likely QTL position were converted to sequence characterized amplified region (SCAR) markers. These markers should aid in development of blue mold‐resistant tobacco cultivars worldwide.}, number={6}, journal={Crop Science}, publisher={Crop Science Society of America}, author={Milla, S.R. and Levin, J.S. and Lewis, R.S. and Rufty, R.C.}, year={2005}, pages={2346–2354} }
 @article{lewis_2005, title={Transfer of resistance to potato virus Y (PVY) from Nicotiana africana to Nicotiana tabacum: possible influence of tissue culture on the rate of introgression}, volume={110}, ISSN={["0040-5752"]}, DOI={10.1007/s00122-004-1893-4}, abstractNote={A disomic chromosome addition line of tobacco, Nicotiana tabacum L., was established previously that possesses a single chromosome pair from N. africana [Merxm. and Buttler]. This addition chromosome carries a gene that confers increased resistance to severe strains of potato virus Y (PVY). Methods to increase the probability of gene transfer from alien chromosomes to tobacco (2n=48) are desired. In the research described here, the PVY resistance gene was transferred to a tobacco chromosome from the N. africana addition chromosome in seven independent cases. One introgression event was obtained using conventional backcrossing of the disomic addition line to N. tabacum cv. Petite Havana, while the remaining six events were obtained using a scheme that involved exposure of explants of the addition line to tissue culture. Twenty-six derived 2n=48 individuals heterozygous for PVY resistance were found to exhibit 24 bivalents or 23 bivalents + 2 univalents at metaphase I. Ovular transmission rates for the PVY resistance factor ranged from 25% to 52%, while pollen transmission rates were much lower, ranging from 0 to 39%. Fifty-one random amplified polymorphic DNA (RAPD) markers specific for the intact addition chromosome were identified and used to characterize derived 2n=48/PVY-resistant genotypes. Variability was observed among these plants with respect to the total number of N. africana RAPD markers that were present, which is an indication that crossing over was occurring within each of the seven introgressed chromosome segments. A limited molecular marker-assisted backcrossing experiment allowed for selection of a 2n=48/PVY-resistant individual that possessed only 6 of the 51 original N. africana RAPD markers. In vitro culture is potentially a valuable system for increasing the rate of alien gene transfer in tobacco, and the successful transfer of PVY resistance from N. africana may allow for an increased level and range of resistance to this virus in tobacco.}, number={4}, journal={THEORETICAL AND APPLIED GENETICS}, author={Lewis, RS}, year={2005}, month={Feb}, pages={678–687} }
 @article{lewis_goodman_2003, title={Incorporation of tropical maize germplasm into inbred lines derived from temperate x temperate-adapted tropical line crosses: agronomic and molecular assessment}, volume={107}, ISSN={["1432-2242"]}, DOI={10.1007/s00122-003-1341-x}, abstractNote={Exotic maize ( Zea mays L.) germplasm may allow for increased flexibility and greater long-term progress from selection if it can be incorporated at high rates into U.S. breeding programs. Crosses were made between a temperate line, NC262A, and each of eight different lines consisting of 100% temperate-adapted tropical germplasm. Pedigree selection was used to generate a set of 148 F(5)S(2) lines that were evaluated in testcrosses with FR992/FR1064 in nine North Carolina environments. Several entries had grain yield, grain moisture content and standability that were comparable to three commercial checks. The best testcrosses outyielded the cross NC262A x FR992/FR1064 by 9.5 to 10.9%, suggesting that a significant amount of tropical germplasm was retained in these lines and that this germplasm combined well with the Stiff Stalk tester. Previous researchers had suggested that tropical alleles could be rapidly lost during inbreeding in populations derived from tropical x temperate bi-parental crosses, leading to the development of lines that possess significantly less than 50% tropical germplasm. F(5)S(5) sub-lines corresponding to the 14 best testcrosses were genotyped at 47 to 49 polymorphic simple sequence repeat (SSR) loci across all ten chromosomes to estimate the amount of tropical germplasm that was retained. The estimated genetic contribution from the tropical parent ranged from 32 to 70%, with the average being 49%. Only two of the 14 lines deviated significantly from a 50%-tropical/50%-temperate ratio, suggesting limited overall selection against germplasm from the tropical parents. These experiments collectively demonstrated that tropical maize germplasm can be incorporated at high rates into a temperate line via pedigree breeding methods in order to derive new inbred lines with acceptable agronomic performance.}, number={5}, journal={THEORETICAL AND APPLIED GENETICS}, author={Lewis, RS and Goodman, MM}, year={2003}, month={Sep}, pages={798–805} }
 @article{lewis_wernsman_2001, title={Efforts to initiate construction of a disease resistance package on a designer chromosome in tobacco}, volume={41}, ISSN={["0011-183X"]}, DOI={10.2135/cropsci2001.4151420x}, abstractNote={Gene cloning and transformation can be used to circumvent linkage drag effects that can plague conventional interspecific gene transfers. These techniques can also be used to create desirable genetic linkages. Use of Nicotiana glutinosa L. N‐gene mediated TMV (tobacco mosaic virus) resistance in flue‐cured tobacco, N tabacum L., has been limited due to linkage drag effects. Transformation was used to introduce the cloned N‐gene into NC152, a chromosome addition line possessing a chromosome pair from N africana. This chromosome has been proposed to be used as a “designer chromosome” into which numerous transgenes could be inserted to form a desirable linkage package. The system could be used to shuttle a large number of transgenes from genotype to genotype. One hundred thirty‐six primary transformants possessing the N transgene were produced and hybridized with TMV‐susceptible ‘Petite Havana.’ These may serve as valuable TMV‐resistant breeding materials. For each independent transformant, BC1F1 families which segregated for TMV resistance and the addition chromosome were generated. Data from cosegregation, transmission, and molecular analyses were used to conclude that one transformant possessed an insertion of the N‐gene in the addition chromosome. By inserting N in the chromosome, we initiated construction of a disease resistance package by linking the TMV resistance gene with a potyvirus resistance gene(s) native to the chromosome. Occasional loss of the transgene, however, may be evidence of previously undetected interchromosomal recombination, and may have implications for use of this system in cultivar development.}, number={5}, journal={CROP SCIENCE}, author={Lewis, RS and Wernsman, EA}, year={2001}, pages={1420–1427} }