@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 microPHAZIR TM 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 [r 2 (SE p 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 [r 2 (SE p ) = 0.93 (1.58), 0.87 (0.22), and 0.88 (0.18), respectively). Fit statistics for anabasine NIR models were intermediate with r 2 (SE p 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 r 2 (SE p 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{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={Inverse 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{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_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={Large-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}, 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}, 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}, 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}, 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{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={Pyridine 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}, 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{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={Regulations 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{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={Crop 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={Genetic 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{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}, 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}, 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={Abstract Investigation 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{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{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{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{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{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={Burley 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}, 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{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}, 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{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={Nicotine 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{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}, 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={Black 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={Methods 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{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{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{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{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={Multiple races of Phytophthora nicotianae (van Breda de Haan) cause the black shank disease of tobacco (Nicotiana tabacum L.). Identifi cation 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 identifi ed 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 fi eld data, the study authenticates the role of these two regions on black shank resistance. Correlations of line performance under fi eld 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 fi eld evaluations for mapping QTL affecting black shank resistance. Identifi ed 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{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_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={ABSTRACT Haploid 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 (TMV R ) 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 TMV R 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_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{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}, number={8}, journal={THEORETICAL AND APPLIED GENETICS}, author={Lewis, Ramsey S. and Kernodle, S. P.}, year={2009}, month={May}, pages={1499–1508} }
 @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={Little 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. fl ue-cured tobacco. Cluster analysis using various distance coeffi cients generated seven consistent groups. Patterns of geographic-specifi c 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-specifi c subpopulations. There was also a signifi cant level of population subdivision between country-specifi c 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={Technologies 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}, 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_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_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}, 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_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 BC 1 F 1 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}, 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}, 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, BC 1 F 1 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} }