杨梅栽培品种的SSR标记指纹数据库和多重荧光标记鉴别技术
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摘要
杨梅(Myrica rubra)原产于我国东南沿海和云贵高原,属于亚热带常绿果树,具有悠久的栽培历史,丰富的品种资源,复杂的遗传多样性。近年来随着杨梅产业的发展,有关杨梅亲缘关系,品种鉴别等的研究越来越受到人们的关注。本研究利用微卫星标记(SSR)与荧光标记相结合建立了杨梅大多数栽培品种的指纹图谱,为进一步开展杨梅品种鉴定提供了理论基础。主要研究结果如下：
1杨梅品种SSR标记的指纹数据库以及信息分析：选择应用14对荧光SSR引物,分析123份杨梅材料的遗传多样性,每个SSR位点分别产生了3-16个等位基因数(A)；多态性信息含量(PIC)从0.07到0.83,平均每对引物的多态性信息含量为0.62；观察杂合度observed heterozygosity(Ho)从0.0080(MRU147)到0.9200(MYBSSR1)；期望杂合度expected heterozygosity (HE)从0.0701(MRU147)到0.8525(MYB-SSR1)；将14个荧光SSR标记在123份杨梅材料上的扩增结果进行UPGMA聚类分析,相似系数为0.78时处理美国杨梅之外的122个杨梅材料可以分成4组,其中第一组又可以分成6小类,第一小类是包括月粒盘和晚稻杨梅在内的41个材料,其中来自浙江得有36个,第二小类包括荸荠在内32份材料,第三小类包括大叶光、小乐江冲黑杨梅、条把梅、乌梅和太波梅这5个材料,第四小类只有松毛荔、湘红、葡萄荔这3个材料,第五小类包括黑瑞林和瑞光梅等24个材料,第六小类由来自温州的11个材料组成。利用这14对SSR标记可以区分实验中所有的杨梅材料,而建立的SSR指纹图谱数据库,也为杨梅品种的鉴定了基础。而且通过比对发现利用其中的10个标记就可以区分实验中的所有材料。在实际生产中的应用有两个方面,对于一个未知的杨梅材料我们利用SSR荧光标记得到这14个位点的片段图谱,与现有的数据库进行比对,就可以达到鉴别、鉴定品种的目的；对于新品种、系与现有数据库中的品种进行聚类分析也可以达到分析其亲缘关系的效果。
2多重荧光SSR标记组合鉴定杨梅果实品种来源：进一步选择了10对SSR引物(MRU155, MRU386, MYB-SSR1, my0043, my0186, my0427,my0792,my0793,my0889和my0972),根据退火温度和引物扩增片段长度这两个因素,经过试验建立了适合ABI3130遗传序列分析仪的4个多重PCR扩增组合,组合A包括my0186和my0427；组合B包括MRU386、MYB-SSR1和my0043；组合C含有my0793,my0889和my0972；组合D包含MRU155和my0792。所用的模板是从8个杨梅果实中提取的DNA,得到的SSR指纹图谱和已有的杨梅指纹图谱数据库比较,验证鉴定了水晶,粉红,月粒盘,丁岙梅和晚稻杨梅这5个品种；亲缘关系方面炭梅、早酸和野生品种分别和余杭大炭梅、黑炭儿、柳叶梅最近。以上结果也能说明这种方法可靠性高,省时省力,为快速、高通量的检测提供的捷径,有利于推动多重SSR技术在杨梅品种鉴别种广泛应用。
Chinese bayberry(Myrica rubra) is a subtropical evergreen fruit tree native to the southeast coastal and yunnan-guizhou plateau of China. It has a long history of cultivation with varoius local varieties and complex genetic diversity. As for the rapid development of Chinese bayberry industy, the genetic relationship and cultivar identification have been paid more and more attentions both by consumers and researchers. This research established the fingerprint of 123 commonly cultivated bayberry accessions based on fluorescent SSR markers, providing a basis for further cultival identification. The main results were summarized as follows:
Establishment of Chinese bayberry fingerprint on SSR marker:A total of 14 fluorescent SSR primers were used to analyse the genetic diversity of the 122 Chinese bayberry accessions and one wax myrtle. The number of alleles (A) produced on each locus varied from 3 to 16; Polymorphism information content (PIC) ranged from 0.07 to 0.83 with an average of 0.62. observed heterozygosity(Ho) and expected heterozygosity(HE) ranged from 0.0080(MRU147) to 0.9200(MYB-SSR1) and 0.0701(MRU147) to 0.8525(MYB-SSR1) respectively. An unweighted pair-group method of the arithmetic averages(UPGMA) was also used to analyze the genetic relationships. When the Dice's similarity coefficient was 0.78, the 122 Chinese bayberries were divided into four groups. The first group further divided into 6 subgroups. Subgroupl contained 41 samples of which 36 accessions were from Zhejiang, Subgroup2 included a total of 32 accessions, Subgroup3 included 5 accessions, Subgroup4 include 3 accessions, Subgroup5 included 24 samples and Subgroup6 included 11 accessions both from Wenzhou. All the samples in this study could be distinguished by the 14 SSR markers, and the fingerprint layed foundation for identification of Chinese bayberry. Ten selected SSR markers were able to distinguish most accessions. There are two applications in actual production:for an unknown material, get the fingerprints on the 14 loci by fluorescent SSR markers and compared with the existing fingerprint profiles, achieve the goal on discrimination or identification of varities; for new varieties, genetic relationships can be deduced by cluster analysis with fingerprint profiles existed.
Identify Chinese bayberry cultivars by multiplex PCR with fluorcent SSR markers:Ten SSR locis(MRU155, MRU386, MYB-SSR1, my0043, my0186, my0427, my0792, my0793, my0889 and my0972)were choosed, according to the factors of annealing temperature and the length of amplification, formed 4 multiplex PCR combination suitable for ABI3130 genetic sequences analyzer. Combination A includes my0186 and my0427; Combination B contains MRU386, MYB-SSR1 and my0043; Combination C includes my0889, my0972 and my0793; Combination D contains MRU155 and my0792. DNA used in the amplification were extracted from 8 Chinese bayberry fruits. By comparing with the established fingerprints above, we can identify 5 cultivars:'Shuijin','Fenhong', 'Yuelipan','Dingaomei'and'Wandaoyangmei';'Tanmei','Zaosuan'and the 'Y2010-75'showed a close relationship with'Yuhangdatanmei'and'Heitaner' 'Liuyemei'respectively. All these showed that this method is reliable, convienent and and high-throughput.
1杨梅品种SSR标记的指纹数据库以及信息分析：选择应用14对荧光SSR引物,分析123份杨梅材料的遗传多样性,每个SSR位点分别产生了3-16个等位基因数(A)；多态性信息含量(PIC)从0.07到0.83,平均每对引物的多态性信息含量为0.62；观察杂合度observed heterozygosity(Ho)从0.0080(MRU147)到0.9200(MYBSSR1)；期望杂合度expected heterozygosity (HE)从0.0701(MRU147)到0.8525(MYB-SSR1)；将14个荧光SSR标记在123份杨梅材料上的扩增结果进行UPGMA聚类分析,相似系数为0.78时处理美国杨梅之外的122个杨梅材料可以分成4组,其中第一组又可以分成6小类,第一小类是包括月粒盘和晚稻杨梅在内的41个材料,其中来自浙江得有36个,第二小类包括荸荠在内32份材料,第三小类包括大叶光、小乐江冲黑杨梅、条把梅、乌梅和太波梅这5个材料,第四小类只有松毛荔、湘红、葡萄荔这3个材料,第五小类包括黑瑞林和瑞光梅等24个材料,第六小类由来自温州的11个材料组成。利用这14对SSR标记可以区分实验中所有的杨梅材料,而建立的SSR指纹图谱数据库,也为杨梅品种的鉴定了基础。而且通过比对发现利用其中的10个标记就可以区分实验中的所有材料。在实际生产中的应用有两个方面,对于一个未知的杨梅材料我们利用SSR荧光标记得到这14个位点的片段图谱,与现有的数据库进行比对,就可以达到鉴别、鉴定品种的目的；对于新品种、系与现有数据库中的品种进行聚类分析也可以达到分析其亲缘关系的效果。
2多重荧光SSR标记组合鉴定杨梅果实品种来源：进一步选择了10对SSR引物(MRU155, MRU386, MYB-SSR1, my0043, my0186, my0427,my0792,my0793,my0889和my0972),根据退火温度和引物扩增片段长度这两个因素,经过试验建立了适合ABI3130遗传序列分析仪的4个多重PCR扩增组合,组合A包括my0186和my0427；组合B包括MRU386、MYB-SSR1和my0043；组合C含有my0793,my0889和my0972；组合D包含MRU155和my0792。所用的模板是从8个杨梅果实中提取的DNA,得到的SSR指纹图谱和已有的杨梅指纹图谱数据库比较,验证鉴定了水晶,粉红,月粒盘,丁岙梅和晚稻杨梅这5个品种；亲缘关系方面炭梅、早酸和野生品种分别和余杭大炭梅、黑炭儿、柳叶梅最近。以上结果也能说明这种方法可靠性高,省时省力,为快速、高通量的检测提供的捷径,有利于推动多重SSR技术在杨梅品种鉴别种广泛应用。
Chinese bayberry(Myrica rubra) is a subtropical evergreen fruit tree native to the southeast coastal and yunnan-guizhou plateau of China. It has a long history of cultivation with varoius local varieties and complex genetic diversity. As for the rapid development of Chinese bayberry industy, the genetic relationship and cultivar identification have been paid more and more attentions both by consumers and researchers. This research established the fingerprint of 123 commonly cultivated bayberry accessions based on fluorescent SSR markers, providing a basis for further cultival identification. The main results were summarized as follows:
Establishment of Chinese bayberry fingerprint on SSR marker:A total of 14 fluorescent SSR primers were used to analyse the genetic diversity of the 122 Chinese bayberry accessions and one wax myrtle. The number of alleles (A) produced on each locus varied from 3 to 16; Polymorphism information content (PIC) ranged from 0.07 to 0.83 with an average of 0.62. observed heterozygosity(Ho) and expected heterozygosity(HE) ranged from 0.0080(MRU147) to 0.9200(MYB-SSR1) and 0.0701(MRU147) to 0.8525(MYB-SSR1) respectively. An unweighted pair-group method of the arithmetic averages(UPGMA) was also used to analyze the genetic relationships. When the Dice's similarity coefficient was 0.78, the 122 Chinese bayberries were divided into four groups. The first group further divided into 6 subgroups. Subgroupl contained 41 samples of which 36 accessions were from Zhejiang, Subgroup2 included a total of 32 accessions, Subgroup3 included 5 accessions, Subgroup4 include 3 accessions, Subgroup5 included 24 samples and Subgroup6 included 11 accessions both from Wenzhou. All the samples in this study could be distinguished by the 14 SSR markers, and the fingerprint layed foundation for identification of Chinese bayberry. Ten selected SSR markers were able to distinguish most accessions. There are two applications in actual production:for an unknown material, get the fingerprints on the 14 loci by fluorescent SSR markers and compared with the existing fingerprint profiles, achieve the goal on discrimination or identification of varities; for new varieties, genetic relationships can be deduced by cluster analysis with fingerprint profiles existed.
Identify Chinese bayberry cultivars by multiplex PCR with fluorcent SSR markers:Ten SSR locis(MRU155, MRU386, MYB-SSR1, my0043, my0186, my0427, my0792, my0793, my0889 and my0972)were choosed, according to the factors of annealing temperature and the length of amplification, formed 4 multiplex PCR combination suitable for ABI3130 genetic sequences analyzer. Combination A includes my0186 and my0427; Combination B contains MRU386, MYB-SSR1 and my0043; Combination C includes my0889, my0972 and my0793; Combination D contains MRU155 and my0792. DNA used in the amplification were extracted from 8 Chinese bayberry fruits. By comparing with the established fingerprints above, we can identify 5 cultivars:'Shuijin','Fenhong', 'Yuelipan','Dingaomei'and'Wandaoyangmei';'Tanmei','Zaosuan'and the 'Y2010-75'showed a close relationship with'Yuhangdatanmei'and'Heitaner' 'Liuyemei'respectively. All these showed that this method is reliable, convienent and and high-throughput.
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