基于GBS-seq的青藏扁蓿豆SNP标记开发

doi:10.13560/j.cnki.biotech.bull.1985.2021-0906

摘要/Abstract

摘要：

开发青藏扁蓿豆（Medicago archiducis-nicolai）单核苷酸多态性标记（single nucleotide polymorphism,SNP）,并开展遗传多样性初步分析,为青藏扁蓿豆种质资源的评价与利用提供理论支持。以5个青藏扁蓿豆群体共80份材料进行GBS（genotyping-by-sequencing）测序,采用GATK软件开发SNP标记,并以此进行遗传结构和遗传多样性的初步分析。测序产生的序列数据量为60.79 Gb,过滤后共获得12 796个SNP位点,且SNP突变类型存在明显的转换型偏差现象。不同地理来源青藏扁蓿豆群体观测杂合度（H_O）为0.187 68-0.304 36,期望杂合度（H_E）为0.201 97-0.364 34,遗传多样性指数（Pi）为0.178 32-0.241 34;祁连（QLCD）群体具有相对最高的遗传多样性水平。遗传结构分析表明5个群体分为2组,且群体遗传距离与地理距离之间存在极显著的正相关性。SNP标记适用于青藏扁蓿豆遗传多样性分析。

关键词: 青藏扁蓿豆, GBS, SNP, 遗传结构, 遗传多样性

Abstract:

In order to provide theoretical support for evaluating and exploiting the genetic resources in Medicago archiducis-nicolai,the SNPs were developed and the preliminary analysis of genetic diversity was carried out. A total of 80 individuals of M. archiducis-nicolai from 5 populations were selected to conduct GBS-seq and SNP markers were developed by using GATK software,and then preliminary analysis of genetic structure and genetic diversity was conducted. A total of 60.79 Gb sequence data was obtained,and 12 796 SNP sites were retained after screening,and SNP mutation types demonstrated obvious transition bias. The observed heterozygosity（H_o）for different geographic sources of M. archiducis-nicolai population was 0.187 68-0.304 36. The expected heterozygosity（H_e）was 0.201 97-0.364 34. The nucleotide diversity index（Pi）was 0.178 32-0.241 34,the population in Qilian of Qinhai province presented a relatively highest genetic diversity. The results of the genetic structure indicated that 5 populations could be divided into 2 groups,and there was highly significant positive correlation between geographical distance and genetic distance among populations of M. archiducis-nicolai. The SNP markers are suitable for analyzing the genetic diversity of M. archiducis-nicolai.

Key words: Medicago archiducis-nicolai, GBS, SNP, genetic structure, genetic diversity

周晓楠, 徐金青, 雷雨晴, 王海庆. 基于GBS-seq的青藏扁蓿豆SNP标记开发[J]. 生物技术通报, 2022, 38(4): 303-310.

ZHOU Xiao-nan, XU Jin-qing, LEI Yu-qing, WANG Hai-qing. Development of SNP Markers in Medicago archiducis-nicolai Based on GBS-seq[J]. Biotechnology Bulletin, 2022, 38(4): 303-310.

图/表 7

表1 青藏扁蓿豆材料来源

Table 1 Sources of M. archiducis-nicolai materials

采样地Location	经度Latitude（E）	纬度Longitude（N）	海拔Altitude/m	样本量Sample size
青海日月山RYS	101°7'11.7″	36°26'50.6″	3 311	16
青海祁连QLCD	100°28'3″	38°03'40″	3 008	16
青海大通DTXR	101°24'24″	37°1'1.7″	2 954	16
青海湟源HY	101°13'9″	36°34'30.3″	2 841	16
青海西宁西山XNXS	101°44'17.5″	36°37'17.5″	2 462	16

表2 青藏扁蓿豆群体测序数据统计

Table 2 Summary of sequencing data of M. archiducis-nicolai populations

群体名称 Population name	测序片段数 Raw reads	碱基数 Raw bases/Gb	过滤后测序片段数Clean reads	过滤后碱基数Clean bases/Gb	酶捕获率 Enzyme catch ratio/%	测序质量 Sequencing quality Q30/%	GC含量 GC content/%
RYS	4 815 069	0.72	3 856 582	0.58	99.39	93.80	53.23
XNXS	4 360 916	0.65	3 468 857	0.52	98.74	93.13	46.40
HY	6 638 025	0.99	5 456 407	0.82	98.85	93.39	49.90
QLCD	4 800 074	0.71	3 981 534	0.60	99.16	93.07	42.29
DTXR	4 884 748	0.73	4 044 807	0.61	99.46	93.33	51.73

图1 SNP位点碱基突变类型及其数量

Fig.1 Base mutation type and number of SNPs

图2 群体遗传结构分析 A：利用ADMIXTURE软件对80份青藏扁蓿豆材料进行群体结构分析,计算K为2-5时的CV error;B：对80份青藏扁蓿豆进行主成分分析;C：K=2时80份青藏扁蓿豆的群体结构

Fig.2 Population genetic structure of populations A：ADMIXTURE software was used to analyze the population structure of 80 M.archiducis-nicolai materials,and the cross-validation（CV）error was calculated when K was 2-5. B：Principal component analysis（PCA）of the 80 M.archiducis-nicolai materials. C：The population structure of 80 M. archiducis-nicolai at K=2

表3 群体间遗传分化指数（FST）

Table 3 Index of genetic differentiation（FST）between populations

群体 Population	HY	RYS	QLCD	XNXS	DTXR
HY	0.0000	0.0093**	0.0282**	0.0138**	0.0146**
RYS		0.0000	0.0369**	0.0213**	0.0171**
QLCD			0.0000	0.0338**	0.0258**
XNXS				0.0000	0.0215**
DTXR					0.0000

图3 群体遗传距离与地理距离的相关性注：**：群体间存在极显著（P<0.01）遗传分化

Fig.3 Correlation between population genetic distance and geographical distance Note：** indicate extremely significant differentiation among populations at 0.01 levels,respectively

表4 青藏扁蓿豆野生群体遗传参数统计

Table 4 Statistics of genetic parameters in M. archiducis-nicolai wild populations

群体 Population	等位基因数 Number of alleles（N_A）	观测杂合度 Observed heterozygosity（H_O）	期望杂合度 Expected heterozygosity（H_E）	遗传多样性指数 Nucleotide diversity index（Pi）
DTXR	2	0.224 02	0.222 41	0.202 46
QLCD	2	0.304 36	0.364 34	0.241 34
XNXS	2	0.243 44	0.234 05	0.213 86
HY	2	0.234 03	0.226 40	0.209 63
RYS	2	0.187 68	0.201 97	0.178 32

参考文献 33

[1]	Small E, Jomphe M. A synopsis of the genus Medicago(Leguminosae)[J]. Can J Bot, 1989, 67(11):3260-3294. doi: 10.1139/b89-405 URL
[2]	Jin DM, Ma JJ, Ma WH, et al. Legumes in Chinese natural grasslands:species, biomass, and distribution[J]. Rangel Ecol Manag, 2013, 66(6):648-656. doi: 10.2111/REM-D-12-00159.1 URL
[3]	Wu XP, Liu DM, Gulzar K, et al. Population genetic structure and demographic history of Medicago ruthenica(Fabaceae)on the Qinghai-Tibetan Plateau based on nuclear ITS and chloroplast markers[J]. Biochem Syst Ecol, 2016, 69:204-212. doi: 10.1016/j.bse.2016.10.005 URL
[4]	Robledo-Arnuncio JJ, Klein EK, Muller-Landau HC, et al. Space, time and complexity in plant dispersal ecology[J]. Mov Ecol, 2014, 2(1):16. doi: 10.1186/s40462-014-0016-3 pmid: 25709828
[5]	吴小培, 沈迎芳, 王海庆. 基于trnL-trnF序列的扁蓿豆和青藏扁蓿豆遗传多样性及其群体遗传结构分析[J]. 草业科学, 2016, 33(6):1136-1146.
	Wu XP, Shen YF, Wang HQ. Analysis of genetic diversity and population genetic structure of Medicago archiducis-nolai and Medicago ruthenica populations based on cpDNA trnL-trnF sequences[J]. Pratacultural Sci, 2016, 33(6):1136-1146.
[6]	王英芳, 张业猛, 刘德梅, 等. 基于转录组测序的青藏扁蓿豆EST-SSR标记开发与验证[J]. 草业科学, 2020, 37(4):718-727.
	Wang YF, Zhang YM, Liu DM, et al. Development and verification of EST-SSR markers in Medicago archiducis-nicolai by transcriptome sequencing[J]. Pratacultural Sci, 2020, 37(4):718-727.
[7]	Wang YF, Shen YF, Liu DM, et al. Insights from putatively neutral EST-SSR markers on the population genetic structure and genetic diversity of the Qinghai-Tibetan Plateau endemic Medicago archiducis-nicolai Sirjaev[J]. Genet Resour Crop Evol, 2021, 68(6):2537-2548. doi: 10.1007/s10722-021-01147-y URL
[8]	Barendse W, Harrison BE, Bunch RJ, et al. Genome wide signatures of positive selection:the comparison of independent samples and the identification of regions associated to traits[J]. BMC Genomics, 2009, 10:178. doi: 10.1186/1471-2164-10-178 pmid: 19393047
[9]	Singh A, Singh PK, Singh R, et al. SNP haplotypes of the BADH1 gene and their association with aroma in rice(Oryza sativa L.)[J]. Mol Breed, 2010, 26(2):325-338. doi: 10.1007/s11032-010-9425-1 URL
[10]	Stephens JC, Schneider JA, Tanguay DA, et al. Haplotype variation and linkage disequilibrium in 313 human genes[J]. Science, 2001, 293(5529):489-493. doi: 10.1126/science.1059431 pmid: 11452081
[11]	Hyten DL, Choi IY, Song QJ, et al. A high density integrated genetic linkage map of soybean and the development of a 1536 universal soy linkage panel for quantitative trait locus mapping[J]. Crop Sci, 2010, 50(3):960-968. doi: 10.2135/cropsci2009.06.0360 URL
[12]	申淑慧, 戴习林. 基于生长和抗逆功能基因SNP分子标记的凡纳滨对虾野生及选育群体遗传多样性分析[J]. 南方农业学报, 2020, 51(11):2836-2845.
	Shen SH, Dai XL. Genetic diversity analysis in wild and selected populations of Penaeus vannamei based on SNP markers in growth and stress resistance genes[J]. J South Agric, 2020, 51(11):2836-2845.
[13]	Glaubitz JC, Casstevens TM, Lu F, et al. TASSEL-GBS:a high capacity genotyping by sequencing analysis pipeline[J]. PLoS One, 2014, 9(2):e90346.
[14]	Doyle J. A rapid DNA isolation procedure for small quantities of fresh leaf tissue[J]. Phytochemical Bulletin, 1987, 19:11-15.
[15]	Elshire RJ, Glaubitz JC, Sun Q, et al. A robust, simple genotyping-by-sequencing(GBS)approach for high diversity species[J]. PLoS One, 2011, 6(5):e19379.
[16]	Catchen J, Hohenlohe PA, Bascham S, et al. Stacks:an analysis tool set for population genomics[J]. Mol Ecol, 2013, 22(11):3124-3140. doi: 10.1111/mec.12354 pmid: 23701397
[17]	Ginestet C. ggplot2:elegant graphics for data analysis[J]. J Royal Stat Soc:Ser A Stat Soc, 2011, 174(1):245-246.
[18]	Excoffier L, Lischer HE. Arlequin suite ver 3. 5:a new series of programs to perform population genetics analyses under Linux and Windows[J]. Mol Ecol Resour, 2010, 10(3):564-567. doi: 10.1111/j.1755-0998.2010.02847.x pmid: 21565059
[19]	Peakall R, Smouse PE. GenAlEx 6. 5:genetic analysis in Excel. Population genetic software for teaching and research——an update[J]. Bioinformatics, 2012, 28(19):2537-2539. pmid: 22820204
[20]	蔡露, 杨欢, 王勇, 等. 利用GBS技术开发烟草SNP标记及遗传多样性分析[J]. 中国烟草科学, 2018, 39(5):17-24.
	Cai L, Yang H, Wang Y, et al. Analysis of genetic diversity of tobacco germplasm resources based on SNP markers via genotyping-by-sequencing technology[J]. Chin Tob Sci, 2018, 39(5):17-24.
[21]	Yu LX, Zheng P, Zhang T, et al. Genotyping-by-sequencing-based genome-wide association studies on Verticillium wilt resistance in autotetraploid alfalfa(Medicago sativa L.)[J]. Mol Plant Pathol, 2017, 18(2):187-194. doi: 10.1111/mpp.12389 URL
[22]	Zhao H, Li QZ, Li J, et al. The study of neighboring nucleotide composition and transition/transversion bias[J]. Sci China Ser C:Life Sci, 2006, 49(4):395-402. doi: 10.1007/s11427-006-2002-5 URL
[23]	严佳文, 肖图舰, 袁启凤, 等. 基于转录组序列的火龙果SSR和SNP多态性分析[J]. 热带作物学报, 2018, 39(7):1338-1343.
	Yan JW, Xiao TJ, Yuan QF, et al. SSR and SNP polymorphism analysis based on Hylocereus polyrhizus transcriptome[J]. Chin J Trop Crops, 2018, 39(7):1338-1343.
[24]	Collins DW, Jukes TH. Rates of transition and transversion in coding sequences since the human-rodent divergence[J]. Genomics, 1994, 20(3):386-396. pmid: 8034311
[25]	Gojobori T, Li WH, Graur D. Patterns of nucleotide substitution in Pseudogenes and functional genes[J]. J Mol Evol, 1982, 18(5):360-369. pmid: 7120431
[26]	Li WH, Wu CI, Luo CC. Nonrandomness of point mutation as reflected in nucleotide substitutions in Pseudogenes and its evolutionary implications[J]. J Mol Evol, 1984, 21(1):58-71. pmid: 6442359
[27]	Zhao Z, Boerwinkle E. Neighboring-nucleotide effects on single nucleotide polymorphisms:a study of 2. 6 million polymorphisms across the human genome[J]. Genome Res, 2002, 12(11):1679-1686. doi: 10.1101/gr.287302 URL
[28]	Stojak J, Borowik T, Górny M, et al. Climatic influences on the genetic structure and distribution of the common vole and field vole in Europe[J]. Mammal Res, 2019, 64(1):19-29. doi: 10.1007/s13364-018-0395-8 URL
[29]	McCauley DE. What is the influence of the seed bank on the persistence and genetic structure of plant populations that experience a high level of disturbance?[J]. New Phytol, 2014, 202(3):734-735. doi: 10.1111/nph.12732 pmid: 24716514
[30]	Hamrick JL, Trapnell DW. Using population genetic analyses to understand seed dispersal patterns[J]. Acta Oecologica, 2011, 37(6):641-649. doi: 10.1016/j.actao.2011.05.008 URL
[31]	Rousset F. Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance[J]. Genetics, 1997, 145(4):1219-1228. doi: 10.1093/genetics/145.4.1219 pmid: 9093870
[32]	Wright S. Variability within and among natural populations[M]. Chicago: University of Chicago Press, 1978.
[33]	Favre A, Päckert M, Pauls SU, et al. The role of the uplift of the Qinghai-Tibetan Plateau for the evolution of Tibetan biotas[J]. Biol Rev, 2015, 90(1):236-253.