用EST-SSR标记分析中国北部和中部地区天蓝苜蓿的遗传多样性和遗传结构

doi:10.3724/SP.J.1003.2008.07362

摘要/Abstract

摘要：

天蓝苜蓿(Medicago lupulina)隶属于苜蓿属, 是一年生或越年生、广布的草本植物。通常认为它是自交种, 但也有些研究报道它具有异交或者混合交配的繁育系统。为了了解它的居群遗传变异、基因流、繁育方式及其遗传背景, 我们用9个EST-SSR标记分析了中国新疆、内蒙古、甘肃、北京、山西、陕西、湖北7个省区的17个天蓝苜蓿野生居群。结果表明: (1) EST-SSR的多态位点百分率(PPL)为71.9%; 每个SSR位点的等位基因数(A)为4-11(平均为7.333); 遗传多样性(H_E)最高的居群是新疆那拉提(0.388), 最低的为陕西西安(0.042)。自交率达93.8%。(2)居群间的遗传分化水平高(F_ST= 0.528;R_ST = 0.499), AMOVA分析结果显示遗传变异主要存在于居群间, 占总变异的59.02%。(3) Mantel检验发现遗传距离和地理距离有显著的相关性(r = 0.4141, P ≤ 0.0003)。根据Nei’s遗传距离( D_a)得出的Neighbor-joining树显示, 地理距离近的居群聚在一起, 这进一步验证了Mantel检验的结果。由此推测, 天蓝苜蓿中等水平的遗传多样性和高度的居群间遗传分化主要受它的自交特性和分布方式影响。上述结果有助于初步了解天蓝苜蓿的种群动态和遗传结构, 同时对苜蓿属种质资源的保护和遗传育种有重要意义。

关键词: Medicago lupulina, EST-SSR, 遗传多样性, 遗传结构, 自交

Abstract

Abstract: Medicago lupulina is an annual or short-perennial and widespread herbaceous plant. It is generally considered a selfing species with a strong self-compatibility system, but a shift in mating system towards outcrossing or mix-mating has been reported. To estimate genetic variation, gene flow, mating system and genetic background of M. lupulina, we characterized 354 individuals from 17 wild populations from northern and central China using nine EST-SSRs markers. We found that: (1) EST-SSRs markers had a high percentage of polymorphic loci (PPL = 71.9%) and 4-11 alleles per loci. The Xinjiang-Nalati population had the highest level of genetic diversity (H_E= 0.388), whereas the Shaanxi-Xi’an population had the lowest (H_E= 0.042). Selfing rate was 93.8 %. (2) Genetic differentiation was high among populations (F_ST= 0.528; R_ST= 0.499). Of the total genetic variation, 59.02% was found among populations. (3) Mantel test revealed that genetic distance and geographic distance were positively correlated (r = 0.4141, P≤ 0.0003). Neighbor- joining trees based on Nei’s genetic distance (D_a) showed that neighboring populations clustered into the same clade, which was consistent with results of Mantel test. Our results indicated that the genetic structure of M. lupulina populations have been strongly affected by dispersal patterns and a strategy of inbreeding.

Key words: Medicago lupulina, EST-SSR, genetic diversity, genetic structure, selfing

闫娟, 楚海家, 王恒昌, 李建强 (2008) 用EST-SSR标记分析中国北部和中部地区天蓝苜蓿的遗传多样性和遗传结构. 生物多样性, 16, 263-270. DOI: 10.3724/SP.J.1003.2008.07362.

Juan Yan, Haijia Chu, Hengchang Wang, Jianqiang Li (2008) Genetic structure and diversity of Medicago lupulina populations in northern and central China based on EST-SSRs markers. Biodiversity Science, 16, 263-270. DOI: 10.3724/SP.J.1003.2008.07362.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2008.07362

https://www.biodiversity-science.net/CN/Y2008/V16/I3/263

图/表 8

参考文献 49

[1]	Bohonak AJ (2002) IBD (Isolation by Distance): a program for analyses of isolation by distance. Journal of Heredity, 93,153-154.
[2]	Bonnin I, Ronfort J, Wozniak F, Olivier I (2001) Spatial effects and rare outcrossing events in Medicago truncatula (Fabaceae) . Molecular Ecology, 10,1371-1383.
[3]	Bouck A, Vision T (2007) The molecular ecologist’s guide to expressed sequence tags. Molecular Ecology, 16,907-924.
[4]	Cao ZZ (曹致中), Feng YQ (冯毓琴), Ma HL (马晖玲), Liu XN (柳小妮), Zhou YL (周玉雷), Xu ZM (徐智明) (2003) Medicago lupulina—beautiful water-saving and easily-maintained turf legume . Pratacultural Science (草业科学), 20(4),58-60. (in Chinese with English abstract)
[5]	Chabane K, Ablett GA, Cordeiro GM, Valkoun J, Henry RJ (2005) EST versus genomic derived microsatellite markers for genotyping wild and cultivated barley. Genetic Resources and Crop Evolution, 52,903-909.
[6]	Cho YG, Ishii T, Temnykh S, Chen X, Lipovich L, McCouch SR, Park WD, Ayres N, Cartinhour S (2000) Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice ( Oryza sativa L.) . Theoretical and Applied Genetics, 100,713-722.
[7]	Clapham AR, Tutin TG, Warburg EF (1962) Flora of the British Isles. Cambridge University Press,Cambridge, UK.
[8]	Clauss MJ, Mitchell-Olds T (2006) Population genetic structure of Arabidopsis lyrata in Europe . Molecular Ecology, 15,2753-2766.
[9]	Crow JK, Kimura M (1970) An Introduction to Population Genetic Theory. Harper and Row, New York.
[10]	Diwan N, Bouton JH, Kochert G, Cregan PB (2000) Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa. Theoretical and Applied Genetics, 101,165-172.
[11]	Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19,11-15.
[12]	Ellis JR, Burke JM (2007) EST-SSRs as a resource for population genetic analyses. Heredity, 99,125-132.
[13]	Ennos RA (1994) Estimating the relative rates of pollen and seed migration among plant populations. Heredity, 72,250-259.
[14]	Eujayl I, Sledge MK, Wang L, May GD, Chekhovskiy K, Zwonitzer J, Mian MAR (2004) Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp. Theoretical and Applied Genetics, 108,414-422.
[15]	Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, 14,2611-2620.
[16]	Flajoulot S, Ronfort J, Baudouin P, Barre P, Huguet T, Huyghe C, Julier B (2005) Genetic diversity among alfalfa ( Medicago sativa) cultivars coming from a breeding program, using SSR markers . Theoretical and Applied Genetics, 111,1420-1429.
[17]	Fowler DP (1965) Effects of inbreeding in red pine, Pinus resinosa Ait. Ⅱ. Pollination studies . Silvae Genetica, 14,12-23.
[18]	Goudet J (2001) FSTAT, A Program to Estimate and Test Gene Diversities and Fixation Indices, version 2. 9.3. http://www.unil.ch/izea/softwares/fstat.html.
[19]	Govindaraju DR (1988) Relationship between dispersal ability and levels of gene flow in plants. Oikos, 52,31-35.
[20]	Gupta PK, Rustgi S, Sharma S, Singh R, Kumar N, Balyan HS (2003) Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat. Molecular Genetics and Genomics, 270,315-323. URL PMID
[21]	Hamrick JL, Godt MJW (1996) Effects of life-history traits on genetic diversity in plant species. Philosophical Transactions of the Royal Society of London Series B: Biological Sciences, 351,1291-1298.
[22]	Julier B, Flajoulot S, Barre P, Cardinet G, Santoni S, Huguet T, Huyghe C (2003) Construction of two genetic linkage maps in cultivated tetraploid alfalfa ( Medicago sativa) using microsatellite and AFLP markers . BMC Plant Biology, 3,9.
[23]	Kuroda Y, Kaga A, Tomooka N, Vaughan A (2006) Population genetic structure of Japanese wild soybean ( Glycine soja) based on microsatellite variation . Molecular Ecology, 15,959-974. URL PMID
[24]	Lammerink J (1968) Genetic variability in commencement of flowering in Medicago lupulina L. in the south island of New Zealand . New Zealand Journal of Botany, 6,33-42.
[25]	Langella O (2000) Populations 1.2: Population Genetic Software (Individuals or Population Distance, Phylogenetic Trees). http://bioinformatics.org/~tryphon/popula- tions/.
[26]	Loveless MD, Hamrick JL (1984) Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics, 15,65-95.
[27]	Mable BK, Adam A (2007) Patterns of genetic diversity in outcrossing and selfing populations of Arabidopsis lyrata. Molecular Ecology, 16,3565-3580. URL PMID
[28]	Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Research, 27,209-220.
[29]	Nei M, Tajima F, Tateno Y (1983) Accuracy of estimated phylogenetic trees from molecular data. Journal of Molecular Evolution, 19,153-170. DOI URL PMID
[30]	Moyle LC (2006) Correlates of genetic differentiation and isolation by distance in 17 congeneric Silene species . Molecular Ecology, 15,1067-1081.
[31]	Nybom H (2004) Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Molecular Ecology, 13,1143-1155. DOI URL PMID
[32]	Pashley CH, Ellis JR, McCauley DE, Burke JM (2006) EST databases as a source for molecular markers: lessons from Helianthus. Journal of Heredity, 97,381-388. URL PMID
[33]	Page RDM (1996) TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences, 12,357-358.
[34]	Pavone LV, Reader RJ (1985) Effect of microtopography on the survival and reproduction of Medicago lupulina . The Journal of Ecology, 73,685-694.
[35]	Peakall R, Gilmore S, Keys W, Morgante M, Rafalski A (1998) Cross species amplification of soybean ( Glycine max) simple sequence repeat (SSRs) within the genus and other legume genera: implication for transferability of SSRs in plants . Molecular Biology and Evolution, 15,1275-1287.
[36]	Peakall R, Smouse PE (2006) GenALEx6: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes, 6,288-295.
[37]	Pritchard JK, Stephans M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics, 155,945-959.
[38]	Pritchard JK, Wen XQ, Fslush D (2007) STRUCTURE: Documentation for Structure Software: version 2.2. http://pritch.bsd.uchicago.edu/software.
[39]	Rice WR (1989) Analyzing tables of statistical tests. Evolution, 43,223-225.
[40]	Rousset F (1997) Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance . Genetics, 145,1219-1228.
[41]	Sanguinetti CJ, Dias NE, Simpson G (1994) Rapid silver staining and recovery of PCR products separated on polyaerylamide gels. Biotechniques, 17,915-919.
[42]	Schneider S, Roessli D, Excoffier L (2000) ARLEQUIN ver. 2.000: A Software for Population Genetics Data Analysis. Genetics and Biochemistry Laboratory, University of Geneva, Switzerland.
[43]	Sidhu SS (1971) Some Aspects of the Ecology of Black Medick (Medicago lupulina L.). PhD dissertation, University of Western Ontario., .
[44]	Slatkin M, Barton NH (1989) A comparison of three indirect methods for estimating average levels of gene flow. Evolution, 43,1349-1368.
[45]	StenØien HK, Fenster CB, Tonteri A, Savolainen O (2005) Genetic variability in natural populations of Arabidopsis thaliana in northern Europe . Molecular Ecology, 14,137-148.
[46]	Turkington RA, Cavers PB (1979) The biology of Canadian weeds. 33. Medicago lupulina L. Canadian Journal of Plant Science, 59,99-110.
[47]	Wei Z (韦直), Huang YZ (黄以之) (1998) Leguminosae. In:Flora Reipublicae Popularis Sinicae (中国植物志), Tomus 42, pp.314-316. Science Press, Beijing.. (in Chinese)
[48]	Wright S (1965) The interpretations of population structure by F-statistics with special regards to systems of mating . Evolution, 19,395-420.
[49]	Zhu BC (朱邦长), He SJ (何胜江), Zhang CQ (张川黔), Song GX (宋高翔) (1996) Guizhou natural legume herbage—introduction and domestication of black medick (Medicago lupulina L.). Guizhou Agricultural Sciences (贵州农业科学), 24(4),36-40. (in Chinese with English abstract)

居群 Population	经纬度 Locality	采样数目 Sample size
新疆 Xinjiang
1 喀什 Kashi	39.44° N, 75.988° E	23
2 伊犁 Yili	43.88° N, 81.305° E	20
3 那拉提 Nalati	43.45° N, 83.283° E	20
4 库尔勒 Korla	41.75° N, 86.128° E	17
5 奇台 Qitai	43.70° N, 89.604° E	23
6 巴里坤 Balikun	43.58° N, 93.008° E	20
内蒙古 Inner Mongolia
7 海拉尔 Hailar	48.90° N, 119.837° E	18
8 白音敖包 Baiyinaobao	43.53° N, 117.236° E	21
9 兴和 Xinghe	40.89° N, 113.885° E	21
北京 Beijing
10 东灵山 Dongling Mountain	39.96° N, 115.439° E	12
山西 Shanxi
11 偏关 Pianguan	39.44° N, 111.461° E	22
陕西 Shaanxi
12 鱼河 Yuhe	37.98° N, 109.853° E	23
13 南泥湾 Nanniwan	36.32° N, 109.651° E	20
14 西安 Xi’an	34.28° N, 108.958° E	27
甘肃 Gansu
15 朱岔 Zhucha	36.94° N, 102.589° E	21
16 兴隆山 Xinglong Mountain 湖北 Hubei	35.78° N, 104.048° E	20
17 武汉 Wuhan	30.55° N, 114.415° E	26
总计 Total		354

居群 Population	经纬度 Locality	采样数目 Sample size
新疆 Xinjiang
1 喀什 Kashi	39.44° N, 75.988° E	23
2 伊犁 Yili	43.88° N, 81.305° E	20
3 那拉提 Nalati	43.45° N, 83.283° E	20
4 库尔勒 Korla	41.75° N, 86.128° E	17
5 奇台 Qitai	43.70° N, 89.604° E	23
6 巴里坤 Balikun	43.58° N, 93.008° E	20
内蒙古 Inner Mongolia
7 海拉尔 Hailar	48.90° N, 119.837° E	18
8 白音敖包 Baiyinaobao	43.53° N, 117.236° E	21
9 兴和 Xinghe	40.89° N, 113.885° E	21
北京 Beijing
10 东灵山 Dongling Mountain	39.96° N, 115.439° E	12
山西 Shanxi
11 偏关 Pianguan	39.44° N, 111.461° E	22
陕西 Shaanxi
12 鱼河 Yuhe	37.98° N, 109.853° E	23
13 南泥湾 Nanniwan	36.32° N, 109.651° E	20
14 西安 Xi’an	34.28° N, 108.958° E	27
甘肃 Gansu
15 朱岔 Zhucha	36.94° N, 102.589° E	21
16 兴隆山 Xinglong Mountain 湖北 Hubei	35.78° N, 104.048° E	20
17 武汉 Wuhan	30.55° N, 114.415° E	26
总计 Total		354

位点 Locus	等位基因数 A	预期杂合度 H_E	观察杂合度 H_O	遗传分化系数 F_ST	基因流 N_m	遗传分化系数R_ST
MTIC189	10	0.256	0.017	0.555	0.201	0.478
MTIC432	5	0.177	0.034	0.643	0.139	0.58
MTIC339	4	0.039	0.000	0.058	4.038	-0.004
MTIC345	8	0.320	0.085	0.463	0.290	0.447
MTIC210	10	0.267	0.005	0.564	0.193	0.636
MTIC14	6	0.180	0.000	0.604	0.164	0.703
MTIC188	11	0.247	0.014	0.612	0.159	0.619
MAA660870	5	0.157	0.003	0.605	0.163	0.508
MtSSRNFAL05	7	0.266	0.017	0.649	0.135	0.528
平均 Mean	7.333	0.212	0.020	0.528	0.224	0.499

位点 Locus	等位基因数 A	预期杂合度 H_E	观察杂合度 H_O	遗传分化系数 F_ST	基因流 N_m	遗传分化系数R_ST
MTIC189	10	0.256	0.017	0.555	0.201	0.478
MTIC432	5	0.177	0.034	0.643	0.139	0.58
MTIC339	4	0.039	0.000	0.058	4.038	-0.004
MTIC345	8	0.320	0.085	0.463	0.290	0.447
MTIC210	10	0.267	0.005	0.564	0.193	0.636
MTIC14	6	0.180	0.000	0.604	0.164	0.703
MTIC188	11	0.247	0.014	0.612	0.159	0.619
MAA660870	5	0.157	0.003	0.605	0.163	0.508
MtSSRNFAL05	7	0.266	0.017	0.649	0.135	0.528
平均 Mean	7.333	0.212	0.020	0.528	0.224	0.499

居群 Population	平均等位基因数n_a	有效等位基因数n_e	观察杂合度 H_o	预期杂合度 H_E	近交系数 F	自交率 s	多态率 PPL (%)	私有基因数 Private alleles
1 喀什 Kashi	2.444	1.746	0.019	0.346	0.947	0.973	66.7	0
2 伊犁 Yili	3.556	1.717	0.056	0.342	0.845	0.916	100	5
3 那拉提 Nalati	2.667	1.868	0.028	0.388	0.932	0.965	88.9	1
4 库尔勒 Korla	2.222	1.779	0.007	0.348	0.982	0.991	77.8	1
5 奇台 Qitai	2.111	1.380	0.000	0.214	1.000	1.000	77.8	0
6 巴里坤 Balikun	1.667	1.196	0.034	0.118	0.725	0.841	33.3	0
7 海拉尔 Hailar	2.111	1.137	0.025	0.107	0.781	0.877	66.7	1
8 白音敖包 Baiyinaobao	1.778	1.196	0.011	0.145	0.93	0.964	66.7	0
9 兴和 Xinghe	1.889	1.223	0.011	0.153	0.931	0.964	77.8	0
10 东灵山 Dongling Mountain	2.111	1.467	0.009	0.270	0.968	0.984	77.8	0
11 偏关 Pianguan	2.000	1.183	0.035	0.137	0.752	0.858	88.9	0
12 鱼河 Yuhe	2.333	1.294	0.034	0.188	0.824	0.904	66.7	1
13 南泥湾 Nanniwan	1.667	1.246	0.000	0.152	1.000	1.000	55.6	1
14 西安 Xi’an	1.444	1.049	0.012	0.042	0.716	0.835	33.3	0
15 朱岔 Zhucha	3.556	1.448	0.042	0.252	0.840	0.913	77.8	8
16 兴隆山 Xinglongshan	2.333	1.576	0.000	0.302	1.000	1.000	66.7	0
17 武汉 Wuhan	2.333	1.122	0.009	0.104	0.921	0.959	100	3
平均 Mean	2.248	1.390	0.020	0.212	0.888	0.938	71.9	1