随州古银杏遗传多样性的RAPD及ISSR分析
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摘要
银杏(Ginkgo biloba L. )属银杏科银杏属,为中生代第四纪冰川期的孓遗植物,有“活化石”之称。我国是银杏的原产地,许多地方有规模不等的银杏古树,湖北随州就是我国主要的银杏古树集中分布区之一。本研究采集随州121株古银杏的叶片样品和30株古银杏的种子样品为研究样本,利用RAPD和ISSR分子标记技术,从群体和个体2个层次,对随州古银杏的遗传多样性进行了分析研究。主要结果如下:
(1) 用9条RAPD引物和9条ISSR引物分别对随州大洪山区7个群体共121株古银杏的叶片DNA进行扩增,分别得到104条和107条清晰的扩增谱带,其中多态性带分别为103条和105条,多态性条带百分率(PPB)分别为99.04%和98.13%。RAPD所揭示的古银杏遗传多样性无论是物种水平(H_E=0.3241,H_O=0.4887)还是群体水平(H_E=0.2302,H_O=0.3440)均与ISSR分析所得的结果(物种水平:H_E=0.2516,H_O=0.3913;群体水平:H_E=0.1775,H_O=0.2697)相近。由RAPD所得的遗传一致度和由ISSR所得的遗传一致度之间的相关系数r=0.6932>r_(0.01)(P=99.65%),两者的相关性达到极显著水平。
(2) 依据RAPD和ISSR实验数据,对古银杏群体进行Nei's遗传多样性分析和AMOVA分析,揭示出随州古银杏群体间遗传分化水平。RAPD结果揭示出大洪山区7个群体的G_(ST)=0.2896,φ_(ST)=24.22%;ISSR结果揭示出这7个群体的G_(ST)=0.2944,φ_(ST)=23.12%。将桐柏山区的草店群体考虑在内,则遗传分化值更高,G_(ST)=0.3380,φ_(ST)=32.05%。
(3) 分别依据古银杏叶片样品的RAPD和ISSR标记数据,进行了群体和单株聚类分析。8个群体共129个单株的UPGMA聚类分析显示,来自同一群体的个体基本上都能各自聚在一起,表明各银杏群体间已有相当程度的遗传分化,特别是草店群体,与其它7个群体之间的分化程度最高,表现出明显的地区性差异,而群体聚类结果并没有表现出地域差异。群体间一定程度的遗传分化可能是人类生产活动和基因流障碍引起的。
(4) 从选出的9条ISSR引物中,再次挑选6条扩增谱带较多的ISSR引物,对30株古银杏的150粒种子的胚乳DNA进行ISSR分析,以分析随州古银杏在个体水平上的遗传多样性。共扩增出91条ISSR谱带,从单倍体水平和二倍体水平统计的多态性条带分别为90条和64条,多态性条带百分率分别为98.90%和70.33%。检测出的平均杂合位点有39个,平均杂合率为43.53%,杂合率最高的古银杏个体达到54.95%,最低的也有34.07%,显示出该地银杏古树的杂合程度都比较高。古银杏在个体水平的平均遗传多样性为H_E=0.1617、H_O=0.2393。古银杏单株间的遗
Ginkgo biloba L., a "living fossil" belonging to Ginkgoaceae and Ginkgo, is one of the oldest remained tree species. China is the native land of Ginkgo biloba and there are lots of ancient Ginkgo trees in many places in the country. Suizhou of Hubei Province is one of the main concentrated distribution regions of ancient Ginkgo trees. In this paper, we collected leaf samples from 121 ancient Ginkgo trees and seed samples from 30 ancient Ginkgo trees in Suizhou as research samples, analyzed the genetic diversity of ancient Ginkgo trees in Suizhou at population level and individual level by applying RAPD and ISSR molecular markers. Major results are as follows:(1) A total of 104 bands (including 103 polymorphic bands) were amplified using 9 RAPD primers and a total of 107 bands (including 105 polymorphic bands) were amplified using 9 ISSR primers, which were based on the DNA of those 121 Ginkgo individuals of 7 populations. The percentage of polymorphic bands (PPB) were 99.04% for RAPD and 98.13% for ISSR respectively. The genetic diversity of Ginkgo at species level (H_E = 0.3241, H_O= 0.4887) and population level (H_E = 0.2302, H_O = 0.3440) revealed by RAPD were both similar to those by ISSR (species level: H_E=0.2516, H_O= 0.3913; population level: H_E=0.1775, H_O=0.2697). Correlation coefficient between the genetic identity based on RAPD and that based on ISSR was 0.6932 (P=99.65%) and was significant.(2) Genetic differentiation among the ancient Ginkgo populations in Suizhou was revealed by Nei's genetic diversity analysis and AMOVA (Analysis of Molecular Variance) based on RAPD and ISSR experiment data. The RAPD results revealed that for the 7 polulations of ancient Ginkgo trees in the Dahong Mountain region, G_ST=0.2896,φ_ST= 24.22%; while ISSR results revealed that G_ST=0.2944, φ_ST = 23.12%. The genetic differentiation value was even higher when the Caodian population was concerned: G_ST=0.3380, φ_ST=32.05%.(3) According to the data of RAPD and ISSR molecular markers of the leaf samples, UPGMA cluster was made at population level and individual level. The clustering analysis of the 129 ancient Ginkgo individuals of 8 populations at individual level showed that most individuals from a same population could be clustered together in the
dendrogram of genetic relationship. It showed that there was great genetic differentiation among these populations. In particular, the genetic differentiation between Caodian population and other populations was the highest and the regional difference was distinct. But this difference was not so obvious in the dendrogram at population level. The genetic differentiation among populations might be caused by human production activities and barriers to gene flow.(4) Six ISSR primers which could amplify more bands were screened out secondly from those 9 polymorphic ISSR primers. These effective primers were used to analyze the genetic diversity of 150 seeds samples of 30 ancient Ginkgo trees at individual level. As a result, 91 bands were generated, of which polymorphic bands was 90 based on haploid data and 64 based on diploid data, the percentage of polymorphism bands were 98.90% and 70.33% respectively. Heterozygosity test showed that the average number of heterozygous loci was 39 and the average heterozygosity was 43.53%. The highest heterozygosity was 54.95%, and the lowest was 34.07%. These results indicated that high heterozygosity existed in the ancient Ginkgo trees in Suizhou. Genetic diversity at individual level was: He=0.1617, #0=0.2393. GSt and ?&St among the 30 Ginkgo individuals were 0.4111 and 25.65% respectively. Among the 3 seed nucleus types, Gst was 0.0674, and ?&st was 4.82%.(5) According to the amplification results with the 6 ISSR primers to the 30 ancient Ginkgo individuals, similar but not exactly the same dendrograms were generated through molecular genetic clustering at haploid level and diploid level. The former based on haploid level was more accurate than the latter based on diploid level, so the former was more credible than the latter. The result of genetic dendrogram was different from conventional classification based on seed nucleus morphological characteristics. As the genetic relationship among the Ginkgo individuals based on DNA was more accurate, it is necessary to use both molecular markers and morphological markers to classify or identify the Ginkgo types or varieties.
(1) 用9条RAPD引物和9条ISSR引物分别对随州大洪山区7个群体共121株古银杏的叶片DNA进行扩增,分别得到104条和107条清晰的扩增谱带,其中多态性带分别为103条和105条,多态性条带百分率(PPB)分别为99.04%和98.13%。RAPD所揭示的古银杏遗传多样性无论是物种水平(H_E=0.3241,H_O=0.4887)还是群体水平(H_E=0.2302,H_O=0.3440)均与ISSR分析所得的结果(物种水平:H_E=0.2516,H_O=0.3913;群体水平:H_E=0.1775,H_O=0.2697)相近。由RAPD所得的遗传一致度和由ISSR所得的遗传一致度之间的相关系数r=0.6932>r_(0.01)(P=99.65%),两者的相关性达到极显著水平。
(2) 依据RAPD和ISSR实验数据,对古银杏群体进行Nei's遗传多样性分析和AMOVA分析,揭示出随州古银杏群体间遗传分化水平。RAPD结果揭示出大洪山区7个群体的G_(ST)=0.2896,φ_(ST)=24.22%;ISSR结果揭示出这7个群体的G_(ST)=0.2944,φ_(ST)=23.12%。将桐柏山区的草店群体考虑在内,则遗传分化值更高,G_(ST)=0.3380,φ_(ST)=32.05%。
(3) 分别依据古银杏叶片样品的RAPD和ISSR标记数据,进行了群体和单株聚类分析。8个群体共129个单株的UPGMA聚类分析显示,来自同一群体的个体基本上都能各自聚在一起,表明各银杏群体间已有相当程度的遗传分化,特别是草店群体,与其它7个群体之间的分化程度最高,表现出明显的地区性差异,而群体聚类结果并没有表现出地域差异。群体间一定程度的遗传分化可能是人类生产活动和基因流障碍引起的。
(4) 从选出的9条ISSR引物中,再次挑选6条扩增谱带较多的ISSR引物,对30株古银杏的150粒种子的胚乳DNA进行ISSR分析,以分析随州古银杏在个体水平上的遗传多样性。共扩增出91条ISSR谱带,从单倍体水平和二倍体水平统计的多态性条带分别为90条和64条,多态性条带百分率分别为98.90%和70.33%。检测出的平均杂合位点有39个,平均杂合率为43.53%,杂合率最高的古银杏个体达到54.95%,最低的也有34.07%,显示出该地银杏古树的杂合程度都比较高。古银杏在个体水平的平均遗传多样性为H_E=0.1617、H_O=0.2393。古银杏单株间的遗
Ginkgo biloba L., a "living fossil" belonging to Ginkgoaceae and Ginkgo, is one of the oldest remained tree species. China is the native land of Ginkgo biloba and there are lots of ancient Ginkgo trees in many places in the country. Suizhou of Hubei Province is one of the main concentrated distribution regions of ancient Ginkgo trees. In this paper, we collected leaf samples from 121 ancient Ginkgo trees and seed samples from 30 ancient Ginkgo trees in Suizhou as research samples, analyzed the genetic diversity of ancient Ginkgo trees in Suizhou at population level and individual level by applying RAPD and ISSR molecular markers. Major results are as follows:(1) A total of 104 bands (including 103 polymorphic bands) were amplified using 9 RAPD primers and a total of 107 bands (including 105 polymorphic bands) were amplified using 9 ISSR primers, which were based on the DNA of those 121 Ginkgo individuals of 7 populations. The percentage of polymorphic bands (PPB) were 99.04% for RAPD and 98.13% for ISSR respectively. The genetic diversity of Ginkgo at species level (H_E = 0.3241, H_O= 0.4887) and population level (H_E = 0.2302, H_O = 0.3440) revealed by RAPD were both similar to those by ISSR (species level: H_E=0.2516, H_O= 0.3913; population level: H_E=0.1775, H_O=0.2697). Correlation coefficient between the genetic identity based on RAPD and that based on ISSR was 0.6932 (P=99.65%) and was significant.(2) Genetic differentiation among the ancient Ginkgo populations in Suizhou was revealed by Nei's genetic diversity analysis and AMOVA (Analysis of Molecular Variance) based on RAPD and ISSR experiment data. The RAPD results revealed that for the 7 polulations of ancient Ginkgo trees in the Dahong Mountain region, G_ST=0.2896,φ_ST= 24.22%; while ISSR results revealed that G_ST=0.2944, φ_ST = 23.12%. The genetic differentiation value was even higher when the Caodian population was concerned: G_ST=0.3380, φ_ST=32.05%.(3) According to the data of RAPD and ISSR molecular markers of the leaf samples, UPGMA cluster was made at population level and individual level. The clustering analysis of the 129 ancient Ginkgo individuals of 8 populations at individual level showed that most individuals from a same population could be clustered together in the
dendrogram of genetic relationship. It showed that there was great genetic differentiation among these populations. In particular, the genetic differentiation between Caodian population and other populations was the highest and the regional difference was distinct. But this difference was not so obvious in the dendrogram at population level. The genetic differentiation among populations might be caused by human production activities and barriers to gene flow.(4) Six ISSR primers which could amplify more bands were screened out secondly from those 9 polymorphic ISSR primers. These effective primers were used to analyze the genetic diversity of 150 seeds samples of 30 ancient Ginkgo trees at individual level. As a result, 91 bands were generated, of which polymorphic bands was 90 based on haploid data and 64 based on diploid data, the percentage of polymorphism bands were 98.90% and 70.33% respectively. Heterozygosity test showed that the average number of heterozygous loci was 39 and the average heterozygosity was 43.53%. The highest heterozygosity was 54.95%, and the lowest was 34.07%. These results indicated that high heterozygosity existed in the ancient Ginkgo trees in Suizhou. Genetic diversity at individual level was: He=0.1617, #0=0.2393. GSt and ?&St among the 30 Ginkgo individuals were 0.4111 and 25.65% respectively. Among the 3 seed nucleus types, Gst was 0.0674, and ?&st was 4.82%.(5) According to the amplification results with the 6 ISSR primers to the 30 ancient Ginkgo individuals, similar but not exactly the same dendrograms were generated through molecular genetic clustering at haploid level and diploid level. The former based on haploid level was more accurate than the latter based on diploid level, so the former was more credible than the latter. The result of genetic dendrogram was different from conventional classification based on seed nucleus morphological characteristics. As the genetic relationship among the Ginkgo individuals based on DNA was more accurate, it is necessary to use both molecular markers and morphological markers to classify or identify the Ginkgo types or varieties.
引文
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