基于高密度SNP标记的苹果属15种植物资源的亲缘关系与遗传结构分析

doi:10.3864/j.issn.0578-1752.2020.16.011

摘要/Abstract

摘要：

【目的】基于高通量简化基因组测序技术在全基因组开发的SNP标记,对中国原产苹果属植物种内和种间的亲缘关系和群体遗传结构解析,为苹果属植物的起源演化以及系统分类提供理论依据。【方法】对427份15种中国原产苹果属植物种质资源进行高通量简化基因组测序（SLAF-seq）,基于获得的SLAF标签,利用BWA软件将其通过比对定位到参考基因组上并获得多态性SLAF标签;利用GATK和SAMtools两种方法在多态性SLAF中开发多态性单核苷酸（SNP）,筛选两种方法共同得到的SNP作为开发的SNP标记数据集。根据完整度>0.94、次要等位基因频率（MAF）>0.05过滤筛选获得多态性的SNP。基于筛选多态性SNP,使用MEGA7的NJ（neighbor-joining）算法,构建苹果属不同种的系统进化树。利用Admixture软件进行群体遗传结构分析,假设样品的分群数（K）为1—15进行聚类,根据交叉验证错误率确定最佳K值,解析苹果属不同种间和种内的遗传结构。【结果】通过SLAF-seq技术对427份苹果属植物种质进行测序,最终获得586 454个SLAF标签,其中多态性SLAF标签463 612个。经过序列比对分析和筛选得到46 460个SNP位点,基于这些SNP位点构建苹果属植物不同种的系统发生树并分析群体结构。系统发育分析将15种苹果属植物分成4个类群,群体遗传结构在K=5和K=14为两个分群关键点。综合两种分析方法的结果,15种苹果属植物可分为4个基本的类群,分别为山荆子类群,新疆野苹果和少数中国苹果类群,变叶海棠、花叶海棠、陇东海棠、山楂海棠、滇池海棠和沧江海棠类群,4个苹果属植物栽培种中国苹果、八棱海棠、花红和楸子类群。中国苹果的部分种质中有新疆野苹果和山荆子的基因背景,但其中还有一部分种质可以独立代表类群基因库,其基因库中并没有新疆野苹果的参与,而与山荆子、花红、楸子和八棱海棠密切相关。【结论】利用SLAF技术快速发掘覆盖全基因组的46 460个多态性SNP标记可有效地对中国原产苹果属植物种内和种间的亲缘关系及遗传结构进行研究,为苹果属植物种质资源的鉴定评价、遗传多样性、系统分类和起源演化提供参考。15种苹果属植物分为4个基本类群,苹果属植物野生种和栽培种分类群明显,中国苹果与其他栽培种的亲缘关系密切。

关键词: 苹果属, SNP, 亲缘关系, 遗传结构

Abstract:

【Objective】 Based on SNP markers developed by high-throughput, the simplified genomic sequencing technology in the whole genome, the genetic relationship and population genetic structure of native germplasms of Malus Mill. in China were analyzed, which provided a theoretical basis for the origin, evolution and systematic classification of Malus Mill.【Method】Based on obtained SLAF tags, BWA software was used to locate them in the reference genome and to obtain the polymorphic SLAF tags. GATK and SAMtool were employed to develop SNPs in polymorphic SLAF tags and screened SNPs obtained by two methods as SNP markers data set. According to the integrity > 0.94 and the minor allele frequency (MAF) > 0.05, SNPs with polymorphism were screened. Based on the screening of polymorphic SNPs, the NJ (neighbor joining) algorithm of MEGA 7 was used to construct phylogenetic trees of different species of Malus Mill. The genetic structure was analyzed by using the software of admixture. Assuming that the number of clusters (k) was 1-15. The best K value was determined according to cross validation error rate, and the genetic structure among and within species of Malus Mill. was analyzed.【Result】427 accessions of Malus Mill. were sequenced by SLAF-seq, and 586 454 SLAF tags were obtained, including 463 612 polymorphic SLAF tags. 46 460 polymorphic single nucleotide (SNP) loci were obtained through sequence alignment analysis and screening. Phylogenetic tree of different species of Malus Mill. was constructed based on these SNP loci, and population structure was analyzed. In phylogenetic analysis, 15 species of Malus Mill. were divided into 4 groups, and K = 5 and K = 14 were key points in the genetic structure analysis. According to the results of two methods, 15 species of Malus Mill. could be divided into 4 basic groups, which were the group of Malus baccata, the group of Malus sieversii and a few accessions of Malus domestica subsp.chinensis, the group of Malus toringoides, Malus transitoria, Malus kansuensis, Malus komarovii, Malus yunnanensis and Malus ombrophila, and the group of Malus domestica subsp. chinensis, Malus robusta, Malus asiatica and Malus prunifolia. There were gene backgrounds of Malus sieversii and Malus baccata in some germplasms of Malus domestica subsp. chinensis, but there were also some germplasms of Malus domestica subsp. Chinensis, which could independently represent the group gene pool, and the gene pool was not involved in Malus sieversii, but closely related to Malus baccata, Malus asiatica and Malus prunifolia.【Conclusion】The rapid discovery of 46 460 polymorphic SNP markers covering the whole genome by SLAF technology could effectively study on the genetic relationship and structure within and among species of Malus Mill. in China, and provide references for identification and evaluation, genetic diversity, systematic classification, origin and evolution of germplasm resources of Malus Mill. 15 species of Malus Mill. could be divided into 4 basic groups, and the classification of wild and cultivated species of Malus Mill. was obvious. The genetic relationship between Malus domestica subsp. chinensis and other cultivated species was close.

Key words: Malus Mill., SNP, genetic relationship, genetic structure

高源,王大江,王昆,丛佩华,李连文,朴继成. 基于高密度SNP标记的苹果属15种植物资源的亲缘关系与遗传结构分析[J]. 中国农业科学, 2020, 53(16): 3333-3343.

GAO Yuan,WANG DaJiang,WANG Kun,CONG PeiHua,LI LianWen,PIAO JiCheng. Genetic Relationship and Structure Analysis of 15 Species of Malus Mill. Based on SNP Markers[J]. Scientia Agricultura Sinica, 2020, 53(16): 3333-3343.

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序号 Code	供试种 Species	来源地 Origin	数量 Number	序号 Code	供试种 Species	来源地 Origin	数量 Number
1	新疆野苹果 Malus sieversii	新疆 Xinjiang	161	5	花红 Malus asiatica	黑龙江 Heilongjiang	7
2	中国苹果 Malus domestica subsp.chinensis	新疆 Xinjiang	2			甘肃 Gansu	1
		黑龙江 Heilongjiang	2			河北 Hebei	9
		甘肃 Gansu	4			云南 Yunnan	1
		河北 Hebei	14	6	八棱海棠 Malus robusta	河北 Hebei	32
		山西 Shanxi	10			山西 Shanxi	1
		山东 Shandong	1			吉林 Jilin	1
3	山荆子 Malus baccata	黑龙江 Heilongjiang	47	7	陇东海棠 Malus kansuensis	甘肃 Gansu	4
		甘肃 Gansu	3	8	垂丝海棠 Malus halliana	甘肃 Gansu	9
		河北 Hebei	10	9	山楂海棠 Malus komarovii	吉林 Jilin	1
		山西 Shanxi	14	10	变叶海棠 Malus toringoides	四川 Sichuan	2
		内蒙古 Inner Mongolia	41	10	变叶海棠 Malus toringoides	云南 Yunnan	1
		吉林 Jilin	19	11	花叶海棠 Malus transitoria	四川 Sichuan	1
4	楸子 Malus prunifolia	黑龙江 Heilongjiang	5	12	丽江山荆子 Malus rockii	云南 Yunnan	1
		甘肃 Gansu	4	13	滇池海棠 Malus yunnanensis	云南 Yunnan	1
		河北 Hebei	2	14	湖北海棠 Malus hupehensis	云南 Yunnan	1
		山西 Shanxi	8	15	沧江海棠 Malus ombrophila	云南 Yunnan	1
		内蒙古 Inner Mongolia	1
		吉林 Jilin	5

	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
1	0.000
2	0.030	0.000
3	0.970	1.000	0.000
4	0.600	0.606	0.394	0.000
5	0.030	0.000	1.000	0.606	0.000
6	0.030	0.000	1.000	0.606	0.000	0.000
7	0.030	0.000	1.000	0.606	0.000	0.000	0.000
8	0.187	0.167	0.833	0.571	0.167	0.167	0.167	0.000
9	0.030	0.000	1.000	0.606	0.000	0.000	0.000	0.167	0.000
10	0.953	0.981	0.019	0.398	0.981	0.981	0.981	0.821	0.981	0.000
11	0.143	0.120	0.880	0.581	0.120	0.120	0.120	0.247	0.120	0.866	0.000
12	0.030	0.000	1.000	0.606	0.000	0.000	0.000	0.167	0.000	0.981	0.120	0.000
13	0.134	0.111	0.889	0.582	0.111	0.111	0.111	0.241	0.111	0.874	0.204	0.111	0.000
14	0.279	0.265	0.735	0.550	0.265	0.265	0.265	0.343	0.265	0.727	0.321	0.265	0.317	0.000
15	0.030	0.000	1.000	0.606	0.000	0.000	0.000	0.167	0.000	0.981	0.120	0.000	0.111	0.265	0.000