用AFLP技术分析四川核桃资源的遗传多样性

doi:10.3773/j.issn.1005-264x.2008.06.017

摘要/Abstract

摘要：

利用AFLP分子标记技术, 运用EcoRⅠ/MseⅠ双酶切组合, 选用多态性高、分辨力强的4对选择性扩增引物组合E32/M48、E33/M61、E35/M61、E33/M62分别对四川省3个野生核桃(Juglans regia)种群和1个野生铁核桃(J. sigillata)种群共46个样品进行遗传多样性分析、居群遗传结构分析及种属关系探讨。结果表明: 1)共扩增出244个遗传位点, 其中146个多态位点, 多态率为59.84%; 核桃群体组和铁核桃群体的多态性百分率分别为55.33%和52.05%, 两个物种遗传多态性水平相当; 核桃群体组所检出的位点平均有效等位基因数Ae、Nei’s基因多样度H、平均Shannon信息指数I分别为1.322 9、0.190 8和0.286 3, 而铁核桃群体分别为1.339 9、0.196 1和0.289 8, 铁核桃群体遗传多样性水平略高于核桃群体。2)群体间特异带及群体间共有带占总扩增带数的15.16%, 其中铁核桃群体特异谱带最多, 群体特异谱带揭示了群体间的遗传差异及相似性。3) Shannon信息指数(I)、Nei’s基因多样性指数(H)和分子方差分析(AMOVA)表明核桃遗传多样性在群体间和群体内的分布分别为14.36%和85.64%、12.6%和87.4%、11.07%和88.93%, 表明群体内的遗传多样性大于群体间的遗传多样性; 核桃群体组与铁核桃群体的变异主要存在于群体组内, 组间的遗传变异仅占总变异的9.35%, 两者间的遗传分化系数G_st为0.093 5, 与AMOVA分析结果一致。4) 4个群体的Nei’s遗传距离在0.038 2~0.069 2之间, 遗传一致度在0.933 2~0.962 5之间, 表现出较高的遗传相似性; 运用Nei’s遗传一致度对供试种群进行了UPGMA聚类, 结果表明核桃的3个群体优先聚类, 大渡河流域群体与甘南地区群体聚类最近。AFLP所检测出的结果既是核桃与铁核桃生物学特性的反映, 又是其各自生态学特性的反映, 该研究结果对核桃种质资源的保护和育种提供一定的理论依据。

关键词: 核桃, 铁核桃, AFLP, 遗传多样性, 种群, 遗传结构

Abstract:

Aims Juglans regia and J. sigillata are important economic nut trees and are widespread in Sichuan Province. Accurate evaluation of genetic diversity and relationships between species is essential for effective preservation of germplasm resources and breeding. Traditional methods for assessment of genetic diversity in walnut, based on morphological, physiological and biochemical studies such as isozyme analysis or RAPD makers, are sensitive to environment so results are not reliable. AFLP has been applied extensively and effectively in population molecular ecology research and population genetic studies. Our aims were to identify genetic structure among populations and to examine genetic relationships between the two species.

Methods We compared three wild J. regiapopulations occurring at Qingba Mountain, Daduhe Valley, and southern Ganzhi District in Sichuan Province, and one wild J. sigillatapopulation at Panzhihua District in southeastern Sichuan Province. We selected 46 samples to analyze by AFLP molecular maker technology using 4 pairs of primer combinations screened.

Important findings We obtained 244 bands including 146 polymorphic bands. The percentage of polymorphic bands (P) was 59.84%. For the J. sigillatapopulation, percentage of polymorphic bands (P) was 52.05%, effective number of alleles per locus (A_e) was 1.339 9, and Nei’s gene diversity index (H) was 0.196 1, Shannon’s information index (I) was 0.289 8. For J. regiapopulations at species level, estimates were P=55.33%, A_e=1.322 9, H=0.190 8, andI=0.286 3. Although these findings showed that genetic diversity of J. sigillatawas slightly higher than the other species, genetic diversity level was generally equivalent. Shannon information index, Nei’s genetic diversity coefficient and analysis of molecular variance showed that 85.64%, 87.4%, 88.93% genetic diversities, respectively, distributed within populations at the species level. Most variation (80.65%) consistently originated from the interior of groups. The population ofJ. sigillatapossessed the greatest amount of unique bands, accounting for 4.5% of the total amplified bands, which indicated genetic variation between two species. The genetic differentiation coefficient (G_st=0.093 5) between two species is very low. Juglans regiashowed high genetic affinity to J. sigillata.Nei’s Genetic distances between populations varied from 0.038 2 to 0.069 2 and genetic similarities ranged from 0.933 2 to 0.962 5, which indicated there were high similarities among populations. UPGMA analysis revealed that threeJ. regiapopulations clustered first, and genetic distance was closest between the Daduhe Valley and southern Ganzhi District populations.

Key words: walnut, Juglans regia, Juglans sigillata, AFLP, genetic diversity, population, genetic structure

陈良华, 胡庭兴, 张帆, 李国和. 用AFLP技术分析四川核桃资源的遗传多样性. 植物生态学报, 2008, 32(6): 1362-1372. DOI: 10.3773/j.issn.1005-264x.2008.06.017

CHEN Liang-Hua, HU Ting-Xing, ZHANG Fan, LI Guo-He. GENETIC DIVERSITIES OF FOUR JUGLANSPOPULATIONS REVEALED BY AFLP IN SICHUAN PROVINCE, CHINA. Chinese Journal of Plant Ecology, 2008, 32(6): 1362-1372. DOI: 10.3773/j.issn.1005-264x.2008.06.017

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https://www.plant-ecology.com/CN/Y2008/V32/I6/1362

图/表 9

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群体名称及编号 Name of the population and the code	秦巴山与龙门山区 (核桃群体) Qingba Moutain (POP1)	邛崃山系及大渡河流域 (核桃群体) Daduhe vally (POP2)	甘南地区 (核桃群体) Gannan district (POP3)	攀枝花及大小凉山地区 (铁核桃群体) Panzhihua district (POP4)
经纬度范围 The range of longitude and latitude	108°24′~102°25′ E 32°59′~29°92′ N	102°25′~103°89′ E 29°04′~31°67′ N	99°00′~101°53′ E 28°71′~30°00′ N	101°52′~102°68′ E 26°30′~28°72′ N
海拔高度范围 The range of altitude (m)	507~970	1 440~2 463	2 100~2 869	976~1 944
采样地及编号 Site of the material and the code	朝天区(1, 2), 市中区(3, 4), 青川县(5, 6) 平武县(7, 8), 南江县(45, 46), 通江县(47, 48)	汶川县(21, 22), 茂县(23, 24), 泸定(25, 26), 小金县(27, 28), 石棉县(29, 30, 汉源县(31, 32), 宝兴县(33, 34)	巴塘县(36, 38)、乡城县(39, 40)、九龙县(41, 42)、得荣县(43, 44)	盐边县(9, 10), 米易县(11, 12), 仁和区(13, 14), 冕宁县(15, 16), 德昌县(17, 18), 盐源县(19, 20)
年平均气温 Annual average temperature (℃)	15.7	14.4	11.5	15.9
1月平均气温 The average temperature in January (℃)	4.4	4.8	2.9	8.1
7月平均气温 The average temperature in July (℃)	25.4	23.1	18.6	22.03
无霜期 The frost-free period (d)	254	268.4	201.2	260.5
大于10 ℃积温 Accumulative temperature over 10 ℃ degree (℃)	4 949.1	4 471.3	3 230.9	5 137.03
年降水量 Annual precipitation (mm)	1 086.9	683.9	559.4	1 015.3
日照时数 Annual sunshine time (h)	1 399.8	1 445.4	2 073.6	2 271.6
日照百分率 Annual sunshine percentage (%)	31.7	32.9	46.8	51.5
年平均相对湿度 Average annual relative humidity (%)	73.2	65.1	51.8	64.3

群体名称及编号 Name of the population and the code	秦巴山与龙门山区 (核桃群体) Qingba Moutain (POP1)	邛崃山系及大渡河流域 (核桃群体) Daduhe vally (POP2)	甘南地区 (核桃群体) Gannan district (POP3)	攀枝花及大小凉山地区 (铁核桃群体) Panzhihua district (POP4)
经纬度范围 The range of longitude and latitude	108°24′~102°25′ E 32°59′~29°92′ N	102°25′~103°89′ E 29°04′~31°67′ N	99°00′~101°53′ E 28°71′~30°00′ N	101°52′~102°68′ E 26°30′~28°72′ N
海拔高度范围 The range of altitude (m)	507~970	1 440~2 463	2 100~2 869	976~1 944
采样地及编号 Site of the material and the code	朝天区(1, 2), 市中区(3, 4), 青川县(5, 6) 平武县(7, 8), 南江县(45, 46), 通江县(47, 48)	汶川县(21, 22), 茂县(23, 24), 泸定(25, 26), 小金县(27, 28), 石棉县(29, 30, 汉源县(31, 32), 宝兴县(33, 34)	巴塘县(36, 38)、乡城县(39, 40)、九龙县(41, 42)、得荣县(43, 44)	盐边县(9, 10), 米易县(11, 12), 仁和区(13, 14), 冕宁县(15, 16), 德昌县(17, 18), 盐源县(19, 20)
年平均气温 Annual average temperature (℃)	15.7	14.4	11.5	15.9
1月平均气温 The average temperature in January (℃)	4.4	4.8	2.9	8.1
7月平均气温 The average temperature in July (℃)	25.4	23.1	18.6	22.03
无霜期 The frost-free period (d)	254	268.4	201.2	260.5
大于10 ℃积温 Accumulative temperature over 10 ℃ degree (℃)	4 949.1	4 471.3	3 230.9	5 137.03
年降水量 Annual precipitation (mm)	1 086.9	683.9	559.4	1 015.3
日照时数 Annual sunshine time (h)	1 399.8	1 445.4	2 073.6	2 271.6
日照百分率 Annual sunshine percentage (%)	31.7	32.9	46.8	51.5
年平均相对湿度 Average annual relative humidity (%)	73.2	65.1	51.8	64.3

	POP1	POP2	POP3	POP4
POP1	*
POP2	0.087 4	*
POP3	0.081 7	0.0472	*
POP4	0.154 6	0.139 1	0.117 8	*

	POP1	POP2	POP3	POP4
POP1	*
POP2	0.087 4	*
POP3	0.081 7	0.0472	*
POP4	0.154 6	0.139 1	0.117 8	*

	POP1	POP2	POP3	POP4
POP1	*	0.956 7	0.952 0	0.933 2
POP2	0.044 3	*	0.962 5	0.933 7
POP3	0.049 1	0.038 2	*	0.934 1
POP4	0.069 2	0.068 6	0.068 2	*