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

doi:10.16035/j.issn.1001-7283.2019.02.012

摘要/Abstract

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。

关键词: 烟草, 重测序, 基因组, SSR标记

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

耿歆淇,杨惠娟,秦艳青,杨兴有,赵世民,史宏志. 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[J]. 作物杂志, 2019 (2): 84–89

Xinqi Geng,Huijuan Yang,Yanqing Qin,Xingyou Yang,Shimin Zhao,Hongzhi Shi. Development and Application of Tobacco SSR Markers Based on Genome Re-Sequencing of Different Tobacco Types[J]. Crops, 2019(2): 84–89

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参考文献 11

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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

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

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

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

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