DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance 1 first-author 1 1 1 1 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:renxuel@126.com †E-mail:renxuel@126.com renxuel@126.com Guizhou Institute of Tobacco Science, Guiyang 550081, China Guizhou Institute of Tobacco Science, Guiyang 550081, China Department of Agronomy, Zhejiang University, Hangzhou 310029, China Department of Agronomy, Zhejiang University, Hangzhou 310029, China DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum , using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits. Methylation-sensitive amplified polymorphism Epigenetic modification Tobacco mosaic virus (TMV) resistance Flue-cured tobacco Introduction 1 Plants usually adapt to complex and changeable environmental stresses through reversible epigenetic modifications to avoid unnecessary excessive genetic rearrangements and population diversification. The spectra of external and internal influences experienced during the life span may lead to specific changes in gene expression that can be epigenetically fixed and passed to progeny to form epigenetic memories (Boyko and Kovalchuk, 2008 2008 ). These changes are important for plants with regard to polyploidization and ecological adaptation (Lee and Chen, 2001 2001 ; Liu and Wendel, 2003 2003 ). DNA methylation is one of the most important epigenetic modification strategies, which is performed by DNA methyltransferases that catalyze the transfer of a methyl group from S -adenosyl-l-methionine to cytosine bases in DNA, and is especially limited to symmetrical CG and CHG sites (where H is A, C, or T) in plants (Bird, 2002 2002 ). It can inhibit gene expression and plays a crucial role in defending against foreign or mobile DNA elements and in maintaining specific patterns of gene expression during plant growth, development, and reproduction (Rassoulzadegan et al., 2006 2006 ). In maize, a significant decrease in DNA methylation is observed in roots upon cold treatment (Steward et al., 2002 2002 ) and in the P1-wr gene under a different pigment phenotype of kernel pericarp (Sekhon et al., 2007 2007 ). In tobacco, a glycerophosphodiesterase-like protein encoding gene ( NtGPDL ) and a pathogen-responsive gene ( NtAlix1 ) are demethylated and expressed in response to aluminum stress (Choi and Sano, 2007 2007 ) and tobacco mosaic virus (TMV) infection (Wada et al., 2004 2004 ), respectively. Tobacco is one of the most widely-grown non-food crop plants in the world and is a major economic force in almost 100 countries. Most cultivars belong to the species Nicotiana tabacum L. in the family Solanaceae. Flue-cured tobacco is a natural allotetraploid that presents wide environmental adaptability as a result of many years of extensive artificial cross-breeding which would increase its complexity through epigenetic modification and regulation. There have been no previous reports on the pattern of DNA methylation in N. tabacum cultivars or on its relationship with the genotype or ecotype. In this study we analyzed the DNA methylation pattern by a methylation-sensitive amplified polymorphism (MSAP) technique in 48 tobacco accessions that were selected to represent different geographic origins and assessed phenotype-related markers. The results should provide important insights for further studies on tobacco epigenetic polymorphism. Materials and methods 1 Plant materials and DNA extraction 2 A total of 61 accessions of flue-cured tobacco in genus N. tabacum L. were used for an MSAP analysis in this study. They included cultivars from America, Brazil, Zimbabwe, and China, and 24 accessions were used for a simultaneous analysis of TMV resistance (Table 1 1 ). Fresh tobacco seeds of all cultivars were selected and incubated in a Petri dish with wet gauze at 25 °C. Genomic DNA was extracted from seeds germinated for 7 d using a DNA extraction kit (TaKaRa, Japan). The entire genomic DNA was diluted to 100 ng/μl and stored at −20 °C. Care was taken to collect samples at the same developmental stage to reflect genotype- or ecotype-specific variation in methylation patterns. S: TMV-susceptible; R: TMV-resistant ID Accession Geographic origin TMV resistance test 1 Qiandongnan retention Guizhou, China 2 Pianpianhuang Guizhou, China 3 Aizihuang Guizhou, China 4 Zimei Guizhou, China 5 Baiyanshi Guizhou, China 6 Nanjiang 3 Guizhou, China 7 Jiucaiping 2 Guizhou, China 8 Wuchun Guizhou, China 9 Qianxi 1 Guizhou, China 10 Jinyan 1 Guizhou, China 11 Dafang retention Guizhou, China 12 Bina Guizhou, China 13 Caohai 1 Guizhou, China 14 Guanghuang 21 Guangdong, China 15 Guidan 1 Guangxi, China 16 Honghuadajinyuan Yunnan, China 17 Renminliudui Yunnan, China 18 Yunyan 87 Yunnan, China 19 Liaoyan 7910 Liaoning, China 20 Mudan 78-7 Jilin, China 21 Qinyuanhuang Henan, China 22 Qianjinhuang Henan, China 23 Jingyehuang Henan, China 24 Xujin 1 Henan, China 25 Jintai 7618 Shanxi, China 26 Xiaohuangjin 1025 Shandong, China 27 Zhongyan 14 Shandong, China 28 Zhongyan 90 Shandong, China 29 T.T.6 Taiwan, China 30 Kutsaga E1 Zimbabwe 31 PVH06 Brazil 32 T.I.245 South America 33 Meck America 34 Nc37NF America 35 Nc82 America 36 Oxford 1 America 37 Oxford 2007 America 38 ReamsM-1 America 39 RG 11 America 40 RG 8 America 41 Special 400 America 42 V2 America 43 Va444 America 44 Vesta 64 America 45 Dixie Bright 101 America 46 Hicks (Broad leaf) America 47 K326 America 48 Coker 176 America R 49 Coker 86 America R 50 GY-22 Guizhou, China R 51 GY-41 Guizhou, China R 52 GY-89 Guizhou, China R 53 GY-107 Guizhou, China R 54 GY-151 Guizhou, China R 55 GY-162 Guizhou, China R 56 GY-204 Guizhou, China R 57 GY-A10 Guizhou, China R 58 K394 America S 59 NC89 America S 60 GY-1 Guizhou, China S 61 GY-3 Guizhou, China S 62 GY-7 Guizhou, China S 63 GY-12 Guizhou, China S 64 GY-32 Guizhou, China S 65 GY-49 Guizhou, China S 66 GY-50 Guizhou, China S 67 GY-52 Guizhou, China S 68 GY-97 Guizhou, China S 69 GY-A1 Guizhou, China S 70 GY-A8 Guizhou, China S 71 GY-76 Guizhou, China S MSAP analysis 2 About 400 ng of genomic DNA was digested by Hap II- Eco RI or Msp I- Eco RI (TaKaRa, Japan) in a total volume of 10 μl at 37 °C overnight. The reaction was stopped by heating the mixture at 80 °C for 10 min, and then adaptor ligation was carried out. Adaptors were prepared by mixing oligonucleotide pairs at equal concentrations of 100 μmol/L. The mixtures were heated at 95 °C for 5 min and allowed to anneal by cooling slowly. Next, 1.5 U of T4 DNA ligase (TaKaRa, Japan), 50 pmol of each adaptor, and 1× T4 ligase buffer were incubated at 16 °C overnight with the digestion product, with a total reaction volume of 20 μl. Pre-amplification was conducted in a total volume of 25 μl with 2 μl ligation product, 1× polymerase chain reaction (PCR) buffer, 200 μmol/L dNTPs, 0.5 μmol/L each of the pre-selective primers, and 1 U DNA polymerase. The reaction procedure was 94 °C for 5 min, followed by 21 cycles of 94 °C for 30 s, 56 °C for 1 min, and 72 °C for 1 min, then 72 °C for 10 min and holding at 12 °C. Selective amplification was carried out in a system similar to that described above with diluted DNA substrate of pre-amplification product and selective primers. The PCR parameters included: an initial hold at 94 °C for 5 min; 13 touchdown cycles (94 °C for 30 s, 65 °C for 30 s, and 72 °C for 1 min) during which the annealing temperature was decremented 0.7 °C each cycle; 23 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min; then 72 °C for 10 min; and a final hold at 12 °C. All primers and adaptors used are listed in Table 2 2 . Adaptor/primer Sequence (5′-3′) 2 Adaptor   Hap II/ Msp I adaptor-F GAT CAT GAG TCC TGC T   Hap II/ Msp I adaptor-R CGA GCA GGA CTC ATG A   Eco RI adaptor-F CTC GTA GAC TGC GTA CC   Eco RI adaptor-R AAT TGG TAC GCA GTC TAC 2 Preselective primer   Eco RI GTA GAC TGC GTA CCA ATT CA   Hap II/ Msp I ATC ATG AGT CCT GCT CGG T 2 Selective primer  H/M+TAA Hap II/ Msp I+AA  H/M+TCC Hap II/ Msp I+CC  H/M+TTC Hap II/ Msp I+TC  E+ATC Eco RI+TC  E+AGA Eco RI+GA  E+ACG Eco RI+CG  E+ATG Eco RI+TG  E+AGC Eco RI+GC  E+ACC Eco RI+CC  E+ACA Eco RI+CA  E+ACT Eco RI+CT  E+AGG Eco RI+GG  E+AGT Eco RI+GT The selective PCR product was mixed with a one-fifth volume of 6× loading buffer and heated at 95 °C for 10 min, and then quickly transferred to ice. A final 5 μl denatured mixture was electrophoresed on 6% denaturing polyacrylamide gel and silver-stained. The gel was then analyzed on a gel documentation system with Quantity One software (Bio-Rad, USA). The repeatability of the MSAP pattern was tested by analyzing the same DNA in triplicate. TMV-resistance investigation 2 Twenty four tobacco lines were selected for a TMV-resistance analysis (accession IDs 48 to 71), and all of the accessions were originated from self-bred tobacco lines, except that IDs 48, 49, 58, and 59 were introduced form America. The TMV strain was obtained by macerating leaf tissue of infected tobacco seedlings and diluting the sap in 20 mmol/L sodium phosphate buffer (pH 7.0) and carborundum. Viral inoculations were then carried out by rubbing the TMV suspension onto 4- to 5-week-old tobacco leaves with a sponge. Mock inoculations were performed as a control by rubbing tobacco leaves with phosphate buffer and carborundum alone. Plants were scored for the development of a resistance or susceptible response to TMV from 3 to 20 d after infection. Data analysis 2 Msp I and Hap II show different sensitivities in the outer cytosine methylation of the CCGG recognition sequence. When bands for one accession were present in one of the isoschizomer lanes, it was considered “methylated”, “hemimethylated” for Hap II and “fully methylated” for Msp I, while “not methylated” referred to bands present in both lanes, and “unknown” referred to bands absent from both lanes. A site was considered to be “methylation polymorphic”, if it was methylated for some accessions and not methylated for others accessions (Keyte et al., 2006 2006 ), as shown in Fig. 1 1 . The tobacco accession IDs and the endonucleases used are shown at the top (H and M refer to digestion with Hap II- Eco RI and Msp I- Eco RI, respectively; marker is the DNA ladder of 100–500 bp), and the types of methylation for accession ID 26 are labeled with arrows (A: hemimethylated site; B: fully methylated site; C: not methylated site; D: polymorphic allelic site) The statistical software NTSYSpc, version 2.10p (Applied Biostatistics, USA), was used for an unweighted pair grouping method of arithmetic averages (UPGMA) cluster analysis based on Jaccard’s genetic similarity matrix and a principal component analysis (PCA) of the correlation matrix of the methylation polymorphic sites among all accessions. All of the MSAP bands from the TMV-resistance test accessions (IDs 48 to 71) were used for a correlation analysis with TMV resistance. To calculate the association of MSAP bands with TMV resistance, a general linear regression model was used for association analyses, as implemented in TASSEL software [ http://www.maizegenetics.net/bioinformatics http://www.maizegenetics.net/bioinformatics ] (Bradbury et al., 2007 2007 ). Results 1 Methylation polymorphism 2 All 30 combinations of selective primers were tested on five tobacco accessions (IDs 48, 49, 58, 59, and 71) to identify the ideal conditions for the experiment, in which 16 combinations were selected with distinct and scorable amplified bands in the gel, while the others, which yielded very few bands or resulted in smearing, were not used in further tests. A total of 9 741 Hap II- Eco RI/ Msp I- Eco RI (H/M) sites were amplified for 48 accessions with these 16 pairs of selective primers, of which 49.3% were methylated, including 2 484 hemimethylated sites and 2 317 fully methylated sites. Among these methylated sites, 402 were allelic sites, of which 281 (69.9%) were polymorphic. The pairs Eco RI (E)+AGT and H/M+TCC had a maximum polymorphic ratio of 92.6%, which was more than twice that of the pairs E+ATC and H/M+TTC, with a minimum polymorphic ratio of 40.9%. Overall, hemimethylated H/M sites were slightly more than fully methylated sites (Table 3 3 ). The methylation ratio indicates the percentage of methylated H/M sites among the total H/M sites, where methylation includes hemimethylation and full methylation; H and M indicate digestion with Hap II- Eco RI and Msp I- Eco RI, respectively No. Selective primer Methylation ratio (%) Hemimethylated H/M sites Fully methylated H/M sites Methylated allelic sites Polymorphic allelic sites Polymorphism ratio (%) 1 E+AGA-H/M+TCC 63.3 190 216 32 14 43.8 2 E+ACG-H/M+TAA 42.5 75 140 24 17 70.8 3 E+ACG-H/M+TCC 44.7 129 135 27 23 85.2 4 E+ATG-H/M+TAA 42.5 153 111 22 18 81.8 5 E+ATG-H/M+TCC 44.3 86 140 27 17 63.0 6 E+AGC-H/M+TCC 51.2 131 80 19 12 63.2 7 E+AGC-H/M+TTC 57.6 201 29 22 9 40.9 8 E+ACC-H/M+TAA 50.7 130 148 21 18 85.7 9 E+ACA-H/M+TAA 39.4 111 113 27 18 66.7 10 E+ACA-H/M+TCC 75.0 202 206 25 17 68.0 11 E+ACT-H/M+TCC 51.5 172 178 25 22 88.0 12 E+AGG-H/M+TCC 60.2 160 173 28 17 60.7 13 E+AGG-H/M+TTC 53.4 180 109 25 15 60.0 14 E+AGT-H/M+TAA 44.9 163 152 27 25 92.6 15 E+AGT-H/M+TCC 63.4 219 262 28 24 85.7 16 E+AGT-H/M+TTC 51.7 182 125 23 15 65.2 Total 49.3 2 484 2 317 402 281 69.9 Cluster analysis 2 Jaccard’s genetic similarity values based on pairwise comparisons among the 48 Nicotiana accessions were used for a cluster analysis. The dendrogram had coefficients ranging from 0.61 to 0.77, and the 48 accessions were clustered into three large groups and a single accession (ID 10; Jinyan 1) at a similarity level of 65% (Fig. 2a 2a ). The single accession Jinyan 1 collected from the Jinsha region of Guizhou Province was closely related to Group I, which consisted of three American cultivars (V2, Va444, and Vesta 64) and 12 accessions collected from other regions in Guizhou, China. Group III included 15 cultivars (accession IDs 14 to 28) from other provinces in China. The remaining accessions in Group IV included “T.T.6” from Taiwan and cultivars from foreign countries (accession IDs 30 to 48). Some accessions with distinct genetic relationships were not clustered closely together in the MSAP phylogenetic analysis. For example, “Qianjinhuang” is a selected seedling from “Jingyehuang”; “Zhongyan 90” is a filial generation of “Jingyehuang”; “Renminliudui” and “Nanjiang 3” were both selected seedlings from “Honghuadajinyuan”; “Oxford 2007”, “RG 11”, and “RG 8” have the same parent “K399”; and “Yunyan 87” and “RG 8” have the same parent “K326”. In addition, many Chinese flue-cured tobacco cultivars were bred or selected from foreign tobacco races, whereas members of these families were clustered into different groups or subgroups. We then performed a PCA analysis to further reveal geographic or genetic relationships. The results showed the same geographic relationship as the dendrogram (Fig. 2b 2b ). Thus, the cytosine methylation pattern of tobacco shows a strong ecotype profile through epigenetic memory. (a) Phylogenetic tree; (b) Principal component analysis (PCA). The first 2 principal components account for 9.6% and 9.3% of the total eigenvalues, respectively. Tobacco accession IDs for three main geographical regions (1–13 from Guizhou Province of China; 14–29 from other Chinese provinces; 30–48 from abroad. For details see Table 1 1 ) are shown Candidate MSAP markers for TMV resistance 2 All of the MSAP bands scored by the 7th pair of primers (Table 3 3 ) were used for a correlation analysis with TMV resistance; three polymorphism allelic sites showed a significant correlation ( P < 0.001) and explained the phenotypic variation with values of 0.3231, 0.258, and 0.2117, respectively. Therefore, to assess the sequence of marker sites, we isolated and sequenced selected MSAP bands. One band was similar to the Gossypium hirsutum MSAP sequence, and the other two sequences were similar to plant cDNA-AFLP segments (Table 4 4 ). This indicated that the sites related to TMV resistance were protein- or enzyme-coding sequences, and methylated modification could possibly regulate gene expression and the stress response of tobacco. 2 Band ID 2 Size (bp) 2 Correlation to resistance 2 Sequence similarity 2 Length similarity (bp) P- value Req_marker P7-19 134 0.000025041 0.3231 Rice cold stress cDNA-AFLP 32 P7-13 201 0.000228100 0.2580 Nicotiana benthamiana cDNA 26 P7-7 261 0.000999960 0.2117 Gossypium hirsutum MSAP sequence 24 Discussion 1 In this study, we analyzed the diversity of cytosine methylation at CCGG sites for allotetraploid flue-cured tobacco. The results showed that methylation polymorphism is widespread in N. tabacum . Among all of the H/M sites for 48 tobacco accessions, 49.3% were methylated and 69.9% of the allelic sites were polymorphic. High methylation polymorphism has also been observed in Gossypium arboreum L. (67%) (Keyte et al., 2006 2006 ) and Brassica oleracea (95%) (Salmon et al., 2008 2008 ), while low methylation polymorphism has been reported in navel orange (max. 22.2%) (Hong and Deng, 2005 2005 ), wild soybean (34.65%), and cultivated soybean (47.05%) (Zhong et al., 2009 2009 ). Even though these 48 flue-cured tobacco cultivars showed high methylation polymorphism, narrow genetic diversity was also revealed by a phylogenetic cluster analysis, in which the similarity coefficient ranged from 0.61 to 0.77. Moon et al. ( 2009 2009 ) reported that the genetic diversity of 702 N. tabacum accessions from the US (0.7362) was about three times the value for the US flue-cured tobacco (0.2489) as analyzed by simple sequence repeat (SSR) markers. This suggests that breeders should extend tobacco germplasm collection to increase genetic diversity. A cluster pattern analysis showed that, among the accessions studied, geographic origin was closely related to methylation polymorphism; however, the genetic relationship was obscure. This result was similar to other reports on methylation analysis in Arabidopsis (Cervera et al., 2002 2002 ) and rice (Ashikawa, 2001 2001 ). Similar results were also reported in tobacco amplified fragment length polymorphism (AFLP) (Denduangboripant et al., 2010 2010 ) and SSR analyses. For example, 46 tobacco cultivars from 18 different countries were grouped based on their geographic origins and manufacturing quality traits (Ren and Timko, 2001 2001 ). Fifty-one Virginia cultivars with desirable agronomic characteristics also formed groupings based on their geographic origins (Zhang et al., 2006 2006 ). In addition, 702 tobacco accessions collected from Central and South America were frequently clustered in a geography-specific manner using various distance coefficients (Moon et al., 2009 2009 ). 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Merr.) 10.1631/jzus.B1000417 Project supported by the Program for High-Quality Tobacco Development of China (No. [2010]221), the Foundation of Science and Technology of Guizhou Province (No. J[2010]2251), and the Program for Guizhou Tobacco Science of China (No. 200910) 2010-12-03 2011-10-10 2011-05-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 12