基于不同烟草类型基因组重测序的烟草SSR标记的开发和应用
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摘要
基于基因组重测序的分子标记研究是一种新的方法。本研究在普通烟草基因组序列已完全测定的基础上重测序了4个烟草品种(系),包括烤烟类型(LY1306,秦烟96)2个,晒烟类型(Wanmao 3)1个,马里兰烟草类型(Wufeng 1)1个。结合在NCBI中测序并发布的K326和TN90品种的基因组序列,分析了这4个重测序的烟草品种的InDel突变和SNP突变位点,鉴定出7个具有品种多态性的SSR候选位点,其中5个SSR位点可扩增出DNA片段。通过对包括不同烟草类型在内的10个烟草品种进行PCR检测,表明本研究选出的SSR位点可用于烟草品种或烟草类型的分类,其中NW-015889872.1、NW-015854676.1和NW-015890969.1等3个SSR位点可有效区分烤烟、白肋烟和马里兰烟以及晒烟烟草类型。
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 suncured 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.
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 suncured 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.
引文
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[11]Li H,Handsaker B,Wysoker A,et al. The sequence alignment/map(SAM)format and SAMtools. Transplantation Proceedings,2009,25(16):2078-2079.
[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,2004(6):11-15.
[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,2004(3):254-257.
[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,2005(6):68-71.
[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.
[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.
[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.
[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.
[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.