基于ISSR、SRAP和SSR标记的梅种质资源遗传多样性研究
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摘要
梅(Prunus mume Sieb. et Zucc.)是原产我国的十大名花之一,集色、香、姿、韵于一身。在7000多年的应用和3000多年的的栽培历史中,形成了众多的梅品种。为合理地对梅种质资源进行分类,形态学、孢粉学、染色体、同工酶、DNA水平上的特征相继应用于梅的系统分类研究,但得出的结论并不一致。本研究以中国梅花研究中心资源圃的135份梅种质为材料,采用ISSR, SRAP和SSR分子标记,对梅种质间的亲缘关系、系统演化及遗传多样性进行了分析比较。主要结果如下：
1、基于ISSR标记的梅种质遗传多样性分析
从38条ISSR引物中筛选得到13条扩增效果好的引物,共扩增得到144条谱带,其中11个为种质特异标记,平均多态性条带比率91.0%,平均多态性信息含量为0.6712。聚类分析表明：真梅品种、杏梅与野梅、美人梅明显聚为3个类群；真梅品种内朱砂品种群、垂枝品种群聚为独立的亚组,而宫粉品种群被分为两个亚组,一个亚组与单瓣品种群聚为一类,一个亚组与绿萼、玉蝶、黄香、跳枝混杂聚在一起。主坐标分析不完全支持聚类分析结果。
2、基于SRAP标记的梅种质遗传多样性分析
从170个引物组合中筛选得到17个引物组合,共扩增得到124条谱带,其中20个为种质特异标记,平均多态性条带比率87.5%,平均多态性信息含量为0.5120。聚类分析表明：真梅种质、杏梅与美人梅明显聚为2个类群；真梅种质内野梅、朱砂和垂枝品种群聚为独立的亚组,而宫粉品种群被分为两个亚组,一个亚组与单瓣品种群聚为一类,一个亚组与绿萼、玉蝶、黄香、跳枝混杂聚在一起。主坐标分析不完全支持聚类分析结果。
3.基于SSR标记的梅种质遗传多样性分析
利用FIASCO方法从‘雪梅’gDNA中开发SSR引物11对,其中扩增AG基序SSR引物10对,(GT)24(AG)16基序SSR引物1对。从43对SSR引物中筛选得到14对引物,共扩增得到177条谱带,其中20个为种质特异标记,平均多态性条带比率94.4%,平均多态性信息含量为0.6450。聚类分析结果表明：真梅品种、杏梅与野梅、美人梅明显聚为3个类群；真梅品种内朱砂品种群、垂枝品种群聚为独立的亚组,而宫粉品种群被分为两个亚组,一个亚组与单瓣品种群聚为一类,一个亚组与绿萼、玉蝶、黄香、跳枝混杂聚在一起。主坐标分析不完全支持聚类分析结果。
4.基于ISSR、SRAP及SSR整合数据的梅种质遗传多样性分析
SSR与ISSR相似系数矩阵的相关系数为0.8237,匹配良好,而SRAP与SSR、ISSR相似系数矩阵的相关系数低,分别为0.4852和0.5531,匹配性差。三者结合能更好解释梅种质亲缘关系；ISSR聚类分组结果与SSR分析结果基本相同,SSR多态性条带比率和多态性信息含量高于ISSR。
Mei flower (Prunus mume Sieb. et Zucc.), combined with rich flower color, fragrance, shape and special charm, was one of the ten famous flowers in China. A large number of cultivars developed during application history of over 7000 years and cultivation history of more than 3000 years. Morphological charaters, the number and morphology of chromosome, pollen morphology, isozyme and DNA characters were used to classification of P. mume, but the results were not uniform.135 accessions of P. mume in Center of Mei Flower Research of China were collected as experimental materials in this study. ISSR, SRAP and SSR were used to the studies on genetic diversity of Mei flower germplasm. The results were as follows:
1. Genetic diversity analysis of Mei flower based on ISSR data
A total of 144 bands generated by 13 informative ISSR primers screened from 38 ISSR primers,11 of which were unique to specific accessions in 135 P. mume samples. The mean polymorphic information content of ISSR markers was 0.6712 and the rate of polymorphic bands was 91.0%. The results of clustering analysis using UPGMA showed that the 135 genotypes could be divided into 3 groups. The first group included the wild Mei and Apricot Mei germplasm, while the second consisted of the Eumume cultivars, Meiren Group formed the third group. In group of Eummume cultivars, the cultivars in Cinnabar Group and Pendulous Group formed two relatively independent subgroups, while the cultivars in Pink Double Group were divided into two subgroups, the cultivars in one subgroup clustering with the ones in Single Flowered Group, and the cultivars in another subgroup clustering with the ones in Green Calyx Group, Flavescens Group, Albo-plena Group and Versicolor Group. The principal coordinate analysis partly supported the results of clustering analysis.
2. Genetic diversity analysis of Mei flower based on SRAP data
17 informative SRAP primer combinations screened from 170 SRAP primer pairs produced 124 bands,20 of which were unique to specific accessions and 87.5% were polymorphic in 135 accessions of P. mume. The polymorphic information content was 0.5120. The results of clustering analysis based on UPGMA showed that the 135 genotypes could be divided into 2 groups. The first group included the Eumume germplasm, while the second consisted of the cultivars in Apricot Mei and Meiren Group introgressing other related species genes. In Eummume germplasm, wild Mei and the cultivars in Cinnabar Group and Pendulous Group formed three relatively independent subgroups, respectively, while the cultivars in Pink Double Group were divided into two subgroups, the cultivars in one subgroup clustering with the ones in Single Flowered Group, and the cultivars in another subgroup clustering with the ones in Green Calyx Group, Flavescens Group, Albo-plena Group and Versicolor Group. The principal coordinate analysis partly supported the results of clustering analysis.
3. Genetic diversity analysis of Mei flower based on SSR data.
11 SSR primer pairs were developed from genome DNA of'Xue Mei'using FIASCO method, of which 10 primer pairs amplied the AG motif and 1 primer pairs amplified the (GT)24(AG)16 motif.14 informative SSR primer pairs screened from 43 SSR primer pairs produced 177 bands,20 of which were unique to specific accessions and 94.4% were polymorphic in 135 accessions of P. mume. The polymorphic information content was 0.6450. The results of clustering analysis based on UPGMA showed that the 135 genotypes could be divided into 3 groups. The first group included the wild Mei and Apricot Mei germplasm, while the second consisted of the Eumume cultivars, Meiren Group formed the third group. In group of Eummume cultivars, the cultivars in Cinnabar Group and Pendulous Group formed two relatively independent subgroups, while the cultivars in Pink Double Group were divided into two subgroups, the cultivars in one subgroup clustering with the ones in Single Flowered Group, and the cultivars in another subgroup clustering with the ones in Green Calyx Group, Flavescens Group, Albo-plena Group and Versicolor Group. The principal coordinate analysis partly supported the results of clustering analysis.
4. Genetic diversity analysis based on integrated data of ISSR and SSR.
The study on the phylogeny of 135 P. mume accessions was conducted based on the integrated ISSR, SRAP and SSR markers data. The correlation between the similarity coefficients matrix of SSR and ISSR was r=0.8237, which meant SSR and ISSR were identical to each other, while the correlation value between the similarity coefficients matrix of SRAP and ISSR, SRAP and SSR, was low (0.5531 and 0.4852, respectively). The genetic relationship among the 135 Mei flower accessions could be well defined based on the integrated ISSR, SRAP and ISSR markers data. The pedigrees revealed by SSR and ISSR were basically similar, but the average polymorphic rate and polymorphic information content amplified by SSR in the 135 Mei flower accessions were higher than ones by ISSR.
1、基于ISSR标记的梅种质遗传多样性分析
从38条ISSR引物中筛选得到13条扩增效果好的引物,共扩增得到144条谱带,其中11个为种质特异标记,平均多态性条带比率91.0%,平均多态性信息含量为0.6712。聚类分析表明：真梅品种、杏梅与野梅、美人梅明显聚为3个类群；真梅品种内朱砂品种群、垂枝品种群聚为独立的亚组,而宫粉品种群被分为两个亚组,一个亚组与单瓣品种群聚为一类,一个亚组与绿萼、玉蝶、黄香、跳枝混杂聚在一起。主坐标分析不完全支持聚类分析结果。
2、基于SRAP标记的梅种质遗传多样性分析
从170个引物组合中筛选得到17个引物组合,共扩增得到124条谱带,其中20个为种质特异标记,平均多态性条带比率87.5%,平均多态性信息含量为0.5120。聚类分析表明：真梅种质、杏梅与美人梅明显聚为2个类群；真梅种质内野梅、朱砂和垂枝品种群聚为独立的亚组,而宫粉品种群被分为两个亚组,一个亚组与单瓣品种群聚为一类,一个亚组与绿萼、玉蝶、黄香、跳枝混杂聚在一起。主坐标分析不完全支持聚类分析结果。
3.基于SSR标记的梅种质遗传多样性分析
利用FIASCO方法从‘雪梅’gDNA中开发SSR引物11对,其中扩增AG基序SSR引物10对,(GT)24(AG)16基序SSR引物1对。从43对SSR引物中筛选得到14对引物,共扩增得到177条谱带,其中20个为种质特异标记,平均多态性条带比率94.4%,平均多态性信息含量为0.6450。聚类分析结果表明：真梅品种、杏梅与野梅、美人梅明显聚为3个类群；真梅品种内朱砂品种群、垂枝品种群聚为独立的亚组,而宫粉品种群被分为两个亚组,一个亚组与单瓣品种群聚为一类,一个亚组与绿萼、玉蝶、黄香、跳枝混杂聚在一起。主坐标分析不完全支持聚类分析结果。
4.基于ISSR、SRAP及SSR整合数据的梅种质遗传多样性分析
SSR与ISSR相似系数矩阵的相关系数为0.8237,匹配良好,而SRAP与SSR、ISSR相似系数矩阵的相关系数低,分别为0.4852和0.5531,匹配性差。三者结合能更好解释梅种质亲缘关系；ISSR聚类分组结果与SSR分析结果基本相同,SSR多态性条带比率和多态性信息含量高于ISSR。
Mei flower (Prunus mume Sieb. et Zucc.), combined with rich flower color, fragrance, shape and special charm, was one of the ten famous flowers in China. A large number of cultivars developed during application history of over 7000 years and cultivation history of more than 3000 years. Morphological charaters, the number and morphology of chromosome, pollen morphology, isozyme and DNA characters were used to classification of P. mume, but the results were not uniform.135 accessions of P. mume in Center of Mei Flower Research of China were collected as experimental materials in this study. ISSR, SRAP and SSR were used to the studies on genetic diversity of Mei flower germplasm. The results were as follows:
1. Genetic diversity analysis of Mei flower based on ISSR data
A total of 144 bands generated by 13 informative ISSR primers screened from 38 ISSR primers,11 of which were unique to specific accessions in 135 P. mume samples. The mean polymorphic information content of ISSR markers was 0.6712 and the rate of polymorphic bands was 91.0%. The results of clustering analysis using UPGMA showed that the 135 genotypes could be divided into 3 groups. The first group included the wild Mei and Apricot Mei germplasm, while the second consisted of the Eumume cultivars, Meiren Group formed the third group. In group of Eummume cultivars, the cultivars in Cinnabar Group and Pendulous Group formed two relatively independent subgroups, while the cultivars in Pink Double Group were divided into two subgroups, the cultivars in one subgroup clustering with the ones in Single Flowered Group, and the cultivars in another subgroup clustering with the ones in Green Calyx Group, Flavescens Group, Albo-plena Group and Versicolor Group. The principal coordinate analysis partly supported the results of clustering analysis.
2. Genetic diversity analysis of Mei flower based on SRAP data
17 informative SRAP primer combinations screened from 170 SRAP primer pairs produced 124 bands,20 of which were unique to specific accessions and 87.5% were polymorphic in 135 accessions of P. mume. The polymorphic information content was 0.5120. The results of clustering analysis based on UPGMA showed that the 135 genotypes could be divided into 2 groups. The first group included the Eumume germplasm, while the second consisted of the cultivars in Apricot Mei and Meiren Group introgressing other related species genes. In Eummume germplasm, wild Mei and the cultivars in Cinnabar Group and Pendulous Group formed three relatively independent subgroups, respectively, while the cultivars in Pink Double Group were divided into two subgroups, the cultivars in one subgroup clustering with the ones in Single Flowered Group, and the cultivars in another subgroup clustering with the ones in Green Calyx Group, Flavescens Group, Albo-plena Group and Versicolor Group. The principal coordinate analysis partly supported the results of clustering analysis.
3. Genetic diversity analysis of Mei flower based on SSR data.
11 SSR primer pairs were developed from genome DNA of'Xue Mei'using FIASCO method, of which 10 primer pairs amplied the AG motif and 1 primer pairs amplified the (GT)24(AG)16 motif.14 informative SSR primer pairs screened from 43 SSR primer pairs produced 177 bands,20 of which were unique to specific accessions and 94.4% were polymorphic in 135 accessions of P. mume. The polymorphic information content was 0.6450. The results of clustering analysis based on UPGMA showed that the 135 genotypes could be divided into 3 groups. The first group included the wild Mei and Apricot Mei germplasm, while the second consisted of the Eumume cultivars, Meiren Group formed the third group. In group of Eummume cultivars, the cultivars in Cinnabar Group and Pendulous Group formed two relatively independent subgroups, while the cultivars in Pink Double Group were divided into two subgroups, the cultivars in one subgroup clustering with the ones in Single Flowered Group, and the cultivars in another subgroup clustering with the ones in Green Calyx Group, Flavescens Group, Albo-plena Group and Versicolor Group. The principal coordinate analysis partly supported the results of clustering analysis.
4. Genetic diversity analysis based on integrated data of ISSR and SSR.
The study on the phylogeny of 135 P. mume accessions was conducted based on the integrated ISSR, SRAP and SSR markers data. The correlation between the similarity coefficients matrix of SSR and ISSR was r=0.8237, which meant SSR and ISSR were identical to each other, while the correlation value between the similarity coefficients matrix of SRAP and ISSR, SRAP and SSR, was low (0.5531 and 0.4852, respectively). The genetic relationship among the 135 Mei flower accessions could be well defined based on the integrated ISSR, SRAP and ISSR markers data. The pedigrees revealed by SSR and ISSR were basically similar, but the average polymorphic rate and polymorphic information content amplified by SSR in the 135 Mei flower accessions were higher than ones by ISSR.
引文
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