基于SSR标记的中亚生态区核桃(Juglans regia L.)遗传多样性与种群结构分析

Analysis of genetic diversity and population structure of walnut(Juglans regia L.)in Central Asia ecological region based on SSR markers

【目的】从分子水平探究中国新疆与吉尔吉斯斯坦和塔吉克斯坦2个中亚国家共6个区域核桃种质资源间的遗传多样性及亲缘关系。【方法】采用SSR分子标记方法对采自6个区域的137份核桃种质资源进行遗传多样性和居群遗传结构分析。【结果】利用14对引物共检测到130个等位变异位点,变异范围为6~13,平均每对SSR引物可检测到9.2857个等位位点;14个SSR位点的占比范围为0.6257~0.8651,平均值0.7634;平均Shannon’s多样性指数(I)为0.4598;种群总基因多样性(Ht)平均值为3.2481;种群内基因多样性(Hs)平均值为2.1089;种群间的遗传分化指数(Gst)范围为0.0578~0.4068,平均值为0.2595;基因流(Nm)平均值为3.0174;各个等位基因频率整体分布不均匀。UPGMA聚类结果显示,137份供试材料在遗传相似性系数为0.75时分成3个类群。其中,第一类群共42份,包括中国新疆阿克苏地区16份,中国新疆和田地区21份,吉尔吉斯斯坦5份;第二类群共44份,包括中国新疆伊犁地区12份、吉尔吉斯斯坦32份;第三类群共51份,包括中国新疆喀什地区19份、塔吉克斯坦13份和中国新疆伊犁地区19份。聚类结果在一定程度上表明了居群间亲缘关系的距离,总体分布与地理分布有关,不同区域之间存在一定的基因交换。聚类结果与居群间遗传距离和遗传一致度结果一致。【结论】SSR分子标记结果表明,中亚生态区不同地理分布的核桃种质资源遗传多样性存在差异,为揭示中亚生态区核桃种群结构及传播途径提供了依据。

【Objective】In the distribution areas of walnut(Juglans regia L.)in Central Asia,researchers in different countries have studied the genetic diversity of walnut in their own countries.However,there have been few studies on the germplasm of walnut in several regions,especially the related studies between Xinjiang and many other countries in Central Asia.In this study,genetic diversity and population genetic structure of 137 walnut germplasm resources collected from four regions of Ili,Kashgar,Hotan and Aksu and six regions of Kyrgyzstan and Tajikistan were analyzed by SSR(Simple Sequence Repeat)molecular markers.The genetic diversity and genetic relationships of walnut germplasm in six regions of Xinjiang and Central Asia were explored at the molecular level,in order to provide material references for further revealing the origin of walnut in the Central Asian ecology-region.【Methods】137 accessions of walnut germplasm resources were collected a in six areas with individual random sampling,including 19 of Kashgar region,15 of Aksu area,Hotan region in 21,31 of Ili region,37 of Kyrgyzstan,13 of Tajikistan.The fresh walnut leaves collected were wiped clean and immediately put into the self-sealed bag containing silica gel for preservation.The DNA of each material was extracted by Plant Genomic DNA Extraction Kit(Tiangen Company).After testing the quality and concentration of DNA,30 accessions of randomly selected walnut germplasms were used to construct the six group’s genomic DNA mixed gene pool,using published 64 SSR primers.The PCR products were detected by 1%agarose gel electrophoresis,and the bright and clear primers were screened and amplified.The 14 pairs of primers screened were used for PCR amplification of all the test materials.PCR products were separated with 6%non-denatured polyacrylamide gel electrophoresis.The bands were detected by silver staining.The amplification of each primer in each individual was counted.Genetic diversity indices including the number of alleles(Na),the number of effective alleles(Ne),Shannon's information index(I)and allele frequency were calculated by Popgene32 software,and the genetic structure indices were calculated.The results included genetic differentiation coefficient(Gst),gene flow between populations(Nm),Nei’s standard genetic distance(GD)and genetic consistency(GI).Clustering analysis of 137 walnut germplasm was carried out using NTSYS-PC2.10 program and the map was made.【Results】A total of 130 alleles were detected by 14 pairs of SSR primers,with the range of variation ranging from 6 to 13.An average of 9.2857 alleles could be detected by each pair of SSR primers.The percentage of 14 SSR loci ranged from 0.6257 to 0.8651,with an average of 0.7634.The average Shannon's diversity index(I)was 0.4598.The mean value of total genetic diversity(Ht)was 3.2481.The mean value of gene diversity(Hs)within the population was 2.1089;The genetic differentiation index(Gst)between populations ranged from 0.0578 to 0.4068,with an average of 0.2595.The mean value of gene flow(Nm)was 3.0174;All allele frequencies were not uniformly distributed.UPGMA clustering results showed that 137 samples were divided into three groups when the genetic similarity coefficient was 0.75.The first group consisted of 42 samples,including 16 samples from Aksu,21 samples from Hotan and 5 samples from Kyrgyzstan.The second group contained 44 accessions,including 12 samples from Ili and 32 samples from Kyrgyzstan.The third group contained 51 samples,including 19 samples from Kashgar,13 samples from Tajikistan and 19 samples from Ili.To a certain extent,the clustering results indicated the distance of the genetic relationship among the populations,and the overall distribution were related to the geographical distribution,and there was a certain gene exchange among the different regions.The analysis results of genetic distance and genetic consistency were consistent with the tree cluster diagram constructed by UPGMA method based on genetic distance frequency.【Conclusion】14 pairs of SSR primers with clear bands and strong polymorphism in walnut were screened,The genetic diversity of the six regions detected by SSR markers was high,among walnut germplasm resources from different geographical distribution in the Central Asian ecological region,and the level of genetic differentiation among populations was high.The gene exchange among walnut communities was high,which might be caused by the long-distance dispersal of walnut seeds by human or birds The walnut germplasm in Ili region would be likely the origin of walnut in Central Asia..