Crops ›› 2019, Vol. 35 ›› Issue (2): 84-89.doi: 10.16035/j.issn.1001-7283.2019.02.012

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Development and Application of Tobacco SSR Markers Based on Genome Re-Sequencing of Different Tobacco Types

Xinqi Geng1,Huijuan Yang1,Yanqing Qin2,Xingyou Yang2,Shimin Zhao3,Hongzhi Shi1   

  1. 1 Tobacco College/National Tobacco Cultivation & Physiology & Biochemistry Research Center, Henan Agricultural University, Zhengzhou 450002, Henan, China;
    2 Sichuan Provincial Tobacco Company, Chengdu 610000, Sichuan, China
    3 Luoyang Branch, Henan Provincial Tobacco Company, Luoyang 471026, Henan, China
  • Received:2018-09-29 Revised:2018-12-18 Online:2019-04-15 Published:2019-04-12
  • Contact: Hongzhi Shi

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Abstract:

The development of molecular markers based on genome re-sequencing is a new method and trend. In this research four tobacco varieties (lines) including two flue-cured tobacco types (LY1306 line, Qinyan 96), one sun-cured tobacco type (Wanmao 3) as well as one Maryland tobacco type (Wufeng 1) were re-sequenced on genomic nucleotide sequences. Combined with the genomic sequence of variety K326, TN90 which have been sequenced and released in NCBI, the InDel sites and SNP sites of these four re-sequenced tobacco varieties (lines) were analyzed. Seven SSR (simple sequence repeats) candidate alleles were identified from the genomic analysis which were supposed to be polymorphism among these varieties (lines). Five of them were confirmed to be applicable after amplifying the fragments from the SSR allele sites, respectively. Verification PCR tests were carried on ten tobacco varieties (lines) including different tobacco types revealed that they could be used to classify the tobacco varieties (lines) or tobacco types. Three of the SSR sites including NW-015889872.1, NW-015854676.1 and NW-015890969.1 were indicated that to be very effective on classification of the tobacco types among flue-cured tobacco, burley tobacco and Maryland tobacco as well as sun-cured tobacco.

Key words: Tobacco, Re-sequencing, Genome, SSR markers

Table 1

Sample quality control of genome sequences"

样本
Sample		 原始数据
Raw Reads data		 有效数据(×108)
Clean Reads data		 有效数据比例(%)
Clean Reads percent		 原始碱基量
Raw bases data		 有效碱基量
Clean bases data		 有效碱基量比例(%)
Clean base data percent		 G和C占比(%)
G and C percent
LY1306 974 594 470 8.87 91.01 146189170500
(146.189G)		 132816177340
(132.816G)		 90.85 38
QY96 985 001 064 9.04 91.79 147750159600
(147.75G)		 135428779630
(135.428G)		 91.66 38
WF1H 993 136 690 9.13 91.95 148970503500
(148.97G)		 136798225077
(136.798G)		 91.82 38
M3H 990 035 276 9.17 92.67 148505291400
(148.505G)		 137424500354
(137.424G)		 92.53 38

Table 2

Sample and reference genome quality control match result"

样本
Sample		 有效数据
Clean Reads data		 PCR重复比例(%)
PCR duplicate rate		 去除PCR重复后数据(×108)
Data after PCR duplication were removed		 对比参考基因组的数据(×108)
Compare reference genome data		 参考基因组匹配率(%)
Reference genome mapping rate
QY96 904 164 146 16.18 7.58 7.56 99.74
WM3H 917 495 832 15.53 7.75 7.74 99.91
LY1306 887 012 964 14.64 7.57 7.57 99.91
WF1H 913 227 010 15.97 7.67 7.66 99.88

Table 3

Sequencing species SNP variant results"

样本Sample WF1H WM3H LY1306 QY96 K326 BX
位于基因组上游的突变数The number of mutations in the upstream region 297 482 355 710 352 194 583 404 542 089 364 475
位于基因组下游的突变数The number of mutations in the downstream region 104 695 123 307 123 562 200 044 187 112 126 102
位于基因组区间的突变数The number of mutations in the intergenic region 3 152 030 3 528 347 3 463 461 5 285 151 4 736 983 3 386 957
位于5′端非翻译区域的突变数The number of mutations in the 5′ UTR region 6 916 9 009 8 867 12 127 10 357 8 526
位于3′端非翻译区域的突变数The number of mutations in the 3′ UTR region 9 323 12 474 12 396 17 041 14 327 11 539
位于剪切区域的突变数The number of mutations in the splice region 4 377 5 285 5 334 6 393 5 547 5 146
位于内含子区域的突变数The number of mutations in the intron region 111 300 132 075 133 106 191 130 172 108 134 178
位于外显子区域的突变数The number of mutations in the exon region 79 199 94 325 95 240 113 782 100 511 95 749
总突变数The total mutations 3 765 322 4 260 532 4 194 160 6 409 072 5 769 034 4 132 672

Table 4

Sequencing species InDeL variant results"

样本Sample WF1H WM3H LY1306 QY96 K326 BX
位于基因组上游的突变数The number of mutations in the upstream region 32 016 44 902 41 378 50 940 85 775 69 880
位于基因组下游的突变数The number of mutations in the downstream region 9 836 13 438 12 432 15 352 25 873 20 177
位于基因组区间的突变数The number of mutations in the intergenic region 179 646 226 119 212 089 250 865 437 125 295 984
位于5′端非翻译区域的突变数The number of mutations in the 5′ UTR region 1 334 2 029 1 854 2 299 3 488 3 456
位于3′端非翻译区域的突变数The number of mutations in the 3′ UTR region 1 104 1 604 1 405 1 998 2 968 2 241
位于剪切区域的突变数The number of mutations in the splice region 662 812 749 874 1 081 931
位于内含子区域的突变数The number of mutations in the intron region 12 614 16 880 15 448 19 796 32 146 24 199
位于外显子区域的突变数The number of mutations in the exon region 2 059 2 641 2 450 3 045 4 706 3 468
总突变数The total mutations 239 271 308 425 287 805 345 169 593 162 420 336

Fig.1

The distribution of mutation sites of SNP and InDeL in the whole genome"

Fig.2

Phylogenetic map of the sequenced samples"

Table 5

SSR polymorphism based on genomic sequence analysis"

突变位点所在染色体编号
The chromosome number
of the mutation site		 突变位点所在染色体上位置
The position of mutation
site on the chromosome		 突变位点左端染色体编号
The left end chromosome
number of the mutation site		 突变位点右端染色体编号
The right end chromosome
number of the mutation site
NW015795276.1 14233 >NW015795276.1:14032-14232 >NW015795276.1:14233-14433
NW015854676.1 34338 >NW015854676.1:34137-34337 >NW015854676.1:34338-34538
NW015889872.1 10709 >NW015889872.1:10508-10708 >NW015889872.1:10709-10909
NW015890969.1 7031 >NW015890969.1:6830-7030 >NW015890969.1:7031-7231
NW015894675.1 7242 >NW015894675.1:7041-7241 >NW015894675.1:7242-7442
NW015920095.1 7693 >NW015920095.1:7492-7692 >NW015920095.1:7693-7893
NW015930318.1 14777 >NW015930318.1:14576-14776 >NW015930318.1:14777-14977

Fig.3

PCR pre-test electrophoresis"

Table 6

SSR polymorphic site primers used to distinguish different types of tobacco"

编号No. 序列名称Seq. name 正向引物Forward primer (5′→3′) 反向引物Reverse primer (5′→3′)
1 NW015795276.1 AAAGTGCGTGGAAAAAATC AGCAGCAGAAGAAAAAGTG
2 NW015854676.1 ATCAAATCCATGCTCCCAA GACCCCAAGATACCCCAAC
3 NW015889872.1 CAAAAGCAGCAAAAACACA GAGAAATGAACGGAAAGTC
4 NW015890969.1 CTTAACCACAAAACCCACA TTCGTAACAATTATCACCG
5 NW015930318.1 GTGTTCCTCTGACTTCTCT CCTTAATCTCAAATCTCTC

Table 7

SSR polymorphism loci analysis results between different tobacco types"

烟草类型
Tobacco type		 品种(系)
Variety
(Line)		 NW-015795276.1
(205-A 208-B 214-C 220-D)		 NW-015854676.1
(114-A 115-B 116-C 120-D 121-E)		 NW-015889872.1
(197-A 198-B 199-C 200-D 201-E 202-F)		 NW-015890969.1
(168-A 169-B 170-C 171-D 174-E)		 NW-015930318.1
(198-A 225-B 231-C 255-D)
长度Size (bp) 类型Classification 长度Size (bp) 类型Classification 长度Size (bp) 类型Classification 长度Size (bp) 类型Classification 长度Size (bp) 类型Classification
烤烟
Flue-cured tobacco		 K326 208 B 121 E 201 E 170 C 198
255		 AD
NC89
 205 A 121 E 201 E 170 C 198
225		 AB
中烟100
Zhongyan 100		 205 A 116
121		 CE 200 D 170 C 198
255		 AD
LY1306 214 C 121 E 198 B 170 C 198
255		 AD
QY96 205
208		 AB 121 E 201 E 170 C 231
255		 CD
晾烟（白肋烟）
Air-cured tobacco		 TN90
 205
 A
 116
120		 CD 202 F 174 E 198
255		 AD
(Burley tobacco)
 TN86 205 A 116
120		 CD 202 F 174 E 198
225		 AB
晾烟（马里兰烟）
Air-cured tobacco
(Maryland tobacco)		 WF1H 220 D 114
121		 AE 199 C 171 D 198
255		 AD
晒烟（雪茄烟）
Sun-cured tobacco
(Cigar filler)		 茄芯1
Jiaxin1		 205 A 116
120		 CD 201 E 169 B 198
255		 AD
WM3H 205 A 115
121		 BE 198 A 168 A 198
255		 AD
[1] 刘齐元, 刘飞虎, 黄海泉 , 等. 烟草胞质雄性不育系与保持系线粒体DNA的RAPD分析. 江西农业大学学报, 2017(6):826-830.
doi: 10.3969/j.issn.1000-2286.2005.06.006
[2] 朴红梅, 王玉民, 刘宪虎 , 等. 简单重复序列的研究与应用. 吉林农业科学, 2017(6):11-15.
doi: 10.3969/j.issn.1003-8701.2004.06.003
[3] 张志敏, 熊国梅, 王慧娟 , 等. 简单重复序列的筛选与应用. 生命的化学, 2017(3):254-257.
doi: 10.3969/j.issn.1000-1336.2004.03.028
[4] 罗玉娣, 李建国 . SSR标记及其在蔬菜育种中的应用. 热带农业科学, 2017(6):68-71.
doi: 10.3969/j.issn.1009-2196.2005.06.018
[5] 杨育峰, 史典义, 王雁楠 , 等. 基于转录组测序数据的甘薯SSR标记开发及群体聚类分析. 分子植物育种, 2018,16(11):3569-3579.
[6] 聂琼, 刘仁祥 . 23份烟草种质遗传多样性的SSR和ISSR标记分析. 西南农业学报, 2011,24(1):15-19.
doi: 10.3969/j.issn.1001-4829.2011.01.004
[7] 陈云, 陈磊, 乐超银 . 马里兰烟SSR分子标记分析. 湖北农业科学, 2014,53(6):1440-1441,1464.
doi: 10.3969/j.issn.0439-8114.2014.06.057
[8] Nicolas S, James N D B,Sonia O ,et al.The tobacco genome sequence and its comparison with those of tomato and potato. Nature Communications, 2014,5:1-9.
doi: 10.1038/ncomms4833 pmid: 4024737
[9] Dai M, Thompson R C, Maher C , et al. NGSQC: cross-platform quality analysis pipeline for deep sequencing data. BMC Genomics, 2010,1(S4):1-9.
doi: 10.1186/1471-2164-11-S4-S7 pmid: 21143816
[10] Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2010,25(5):1754-1760.
[11] Li H, Handsaker B, Wysoker A , et al. The sequence alignment/map (SAM) format and SAMtools. Transplantation Proceedings, 2009,25(16):2078-2079.
doi: 10.1046/j.1440-1665.1999.0178e.x
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[4] . [J]. Crops, 2002, 18(3): 15 -17 .
[5] Yaoyan Li,Yanyan Pei,Shanyan Huang,Yingyan Zhang,Songji Wei,Yangjiao Xie,Qiong Zhou. Pharmacognosy of Zhongliuteng (Pileostegia tomentella Hand. Mazz) of Yao Medicine[J]. Crops, 2018, 34(1): 61 -65 .
[6] . [J]. Crops, 2000, 16(5): 30 -31 .
[7] . [J]. Crops, 1997, 13(2): 21 -22 .
[8] . [J]. Crops, 1990, 6(1): 9 .
[9] . [J]. Crops, 1995, 11(3): 37 .
[10] . [J]. Crops, 1990, 6(2): 8 -9 .