基于SSR的辽宁铁岭地区平榛遗传多样性与居群遗传结构分析

An assessment of the genetic diversity and population genetic structure concerning the Corylus heterophylla Fisch.,grown in the Tieling district of Liaoning province,using SSR markers

【目的】研究我国铁岭地区平榛的遗传多样性,指导平榛优良品种选育工作。【方法】采用SSR分子标记的方法,对铁岭市3个平榛主产区共124株平榛材料进行遗传多样性和居群遗传结构分析。【结果】利用12株表型差异大的平榛植株,从56对SSR引物中筛选出12对条带清晰、多态性强的引物。12对引物在124株平榛材料中共扩增出75个基因位点,平均等位基因数为6.25个,观测杂合度(Ho)平均值为0.535 6,预期杂合度(He)的平均值为0.615 7。Shannon’s信息指数(I=1.274 2)和Nei’s多样性指数(H=0.613 2)较高,遗传分化系数(Fst=0.061 6)较低,基因流(Nm=7.599 3)较高,居群间遗传距离(GD<0.214 5)较小,遗传一致度(GI>0.806 9)较大。【结论】研究结果表明我国铁岭地区平榛的遗传多样性较高,居群间的遗传分化水平较低,且在大部分位点均表现偏离Hardy-Weinberg平衡,杂合度不足,居群间有较高的基因流。

【Objective】The Corylus heterophylla Fisch. is widely found throughout the north of China,pri-marily within the three northeastern provinces. The total forest area of hazelnut is 1.67 million hm2 in ourcountry,and the proportion of the C. heterophylla is reaching 95% or more in our country. The Tielingwild hazelnut has a long history. To date,the area of the C. heterophylla in Tieling has reached up to80 000 hm2. Although the hazelnut has entered into the period of artificial cultivation,the higher qualityvarieties have still not been selected. Wang made a genetic diversity and genetic structure analysis of 14 C. heterophylla in our country using SSR molecular markers. This analysis provided a good foundation forthe application of SSR markers in the variety improvement of the C. heterophylla. However,her study hada large sampling range and low sampling quantity. The material she used did not involve the hazelnuts ofTieling,in which the C. heterophylla are widely distributed. In order to study the genetic diversity of the C. heterophylla in the Tieling area,and provide a guide for the breeding of hazelnuts,the genetic diversi-ty and population structure analysis of the C. heterophylla within three regions of Tieling were analyzed byusing SSR markers.【Methods】124 C. heterophylla from the three main-cultivation regions of Tieling were used as the materials for our study. These samples consist of 84 plants from Huangjingou,20 plantsfrom Xiaohongshi and 20 plants from Kaiyuan. SSR molecular markers were used for performing the genet-ic diversity and genetic structure analysis of the 124 C. heterophylla plants. The genome DNA was ob-tained using a new type of plant genomic DNA extraction kit. Then 1.0% agarose gel electrophoresis wasemployed for DNA integrity detection. We used an ultraviolet spectrophotometer to extract the purity andconcentration of the genomic DNA. The concentration of DNA was diluted to 10 mg · μL-1and it wasstored at-20 ℃ for later use. In order to screen the most suitable annealing temperature for different prim-ers,eight gradients were established with a range from 48 ℃ to 62 ℃. We established a PCR reaction sys-tem available for C. heterophylla. The 12 hazelnut plants were used for primer polymorphism-screening.Then the SSR primers with high polymorphism and clear bands were used for all the accesions and for per-forming the genetic diversity analysis. The PCR amplification products were detected through 8.0% of de-naturing polyacrylamide gel（PAGE）. After applying a silver stain,photos were taken to determine the sta-tistics of the bands. Popgene 3.2 software was used to calculate the genetic diversity index. The indexes in-cluded the number of alleles（Na）,effective number of alleles（Ne）,observed heterozygosity（Ho）,expect-ed heterozygosity（He）,Nei＇s genetic diversity index（H）,Shannon＇s information index（I） and the allelefrequency. The indexes for reflecting the population genetic structure included the coefficient of geneticdifferentiation（Fst）,the gene flow（Nm）,the degree of deviation from the Hardy-Weinberg equilibrium（Fis and Fit）,Nei＇s standard genetic distance（GD） and the genetic consistency（GI）. We employed theNTSYS-pc2.11 program for clustering analysis of the 124 C. heterophylla. The clustering figure was alsoestablished.【Results】The suitable annealing temperature for different primers was selected. The 12 pairs of SSR primers with clear and polymorphism bands were screened from the 56 pairs of SSR primers.The results of the genetic diversity analysis of the materials indicated that a total of 75 alleles were suc-cessfully detected with 12 pairs of SSR primers in the 124 C. heterophylla. The average number of alleles（Na） was 6.25,and the average number of effective alleles（Ne） was 3.441 0. The mean observed heterozy-gosity（Ho） and the mean expected heterozygosity（He） was 0.535 6 and 0.615 7 respectively. The Nei＇s ge-netic diversity（H） and Shannon＇s diversity（I） was 0.613 2 and 1.274 2 respectively,which showed thehigh genetic diversity in the C. heterophylla species level. The table of the allele frequency for the C. heterophylla indicates that the allele frequency for some of the locus was higher and some of the locus waslower. The overall distribution of the allele frequency was uneven. The genetic differentiation coefficient（Fst） was 0.061 6,which showed the most variation within the populations. The gene flow（Nm） was 7.599 3.The degree of deviation from the Hardy-Weinberg equilibrium within the populations was from-0.543 8to 0.767 5,and the total population of the degree of deviation from the Hardy-Weinberg equilibrium wasfrom-0.504 6 to 0.783 2. These results indicate that the 124 individuals deviated from the Hardy-Wein-berg equilibrium for most of the locus and lacked heterozygosity. To analyze the genetic relationshipsamong the C. heterophylla,the populations in Tieling,the genetic distance（GD） and genetic identity（GI）were calculated between the three populations. The results signified that the genetic distance betweenHuangjingou and Xiaohongshi was the lowest（0.107 4） and the highest genetic distance was between Xiao-hongshi and Kaiyuan（0.214 5）. The highest genetic identity was between Huangjingou ang Xiaohongshi（0.898 1） and the lowest was between Xiaohongshi and Kaiyuan（0.806 9）. Clustering analysis showed that124 C. heterophylla were divided into three groups. The first category included 50 individuals fromHuangjingou,18 individuals from Xiaohongshi and 5 individuals from Kaiyuan. The second category in-cluded 7 individuals from Huangjingou,5 individuals from Xiaohongshi and 15 individuals from Kaiyu-an. The third category was 27 individuals from Huangjingou.【Conclusion】The higher levels of gene diver-sity and Shannon ＇s diversity indicated a relatively high genetic diversity in the C. heterophylla popu-lations from Tieling,which can provide a foundation for varieties breeding and genetic improvement ofthe C. heterophylla The low genetic differentiation among populations was consistent with a low genetic dif-ferentiation coefficient,strong gene flow,low genetic distance and high genetic identity. And at most lo-cus,the 124 populations showed deviation from the Hardy-Weinberg Equilibrium and a low heterozygosi-ty. The gene flow was strong between the three populations. This might be related to the test materials,which were all from the Tieling area. Through cluster analysis,124 C. heterophylla plants can be dividedinto three groups. The results of genetic distance and genetic identity were consistent with the clusteringresults between the populations. The three categories all included the samples from Huangjingou,and theplants in the third group were all from Huangjinggou. These results indicated that the genetic distanceswere all close between Huangjingou and the other two populations.