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当前位置: 首页 > 文章 > 基于转录组序列的长柄双花木SSR标记开发 植物遗传资源学报 2018 (4) 740-747
Position: Home > Articles > Development of SSR Markers Based on Transcriptome Sequences of Disanthus cercidifolius var. longipes Journal of Plant Genetic Resources 2018 (4) 740-747

基于转录组序列的长柄双花木SSR标记开发

作  者：
孟艺宏;徐璕;徐刚标
关键词：
长柄双花木;转录组;TP-M13-SSR;引物开发
摘  要：
双花木属(Disanthus Maxim.)是金缕梅科(Hamamelidaceae)最原始的单种属,为东亚地区特有属。长柄双花木(D.cercidifolius var.longipes H.T.Chang)是日本特有植物双花木(D.cercidifolius Maxim.)的变种,仅分布于我国南方地区,为国家Ⅱ级保护植物,被列入我国濒危植物种名录,在研究金缕梅科系统发育和东亚植物区系地理演化等方面具有重要的科学价值。由于可利用的SSR标记引物数量不足,阻碍了长柄双花木遗传学研究。本研究对长柄双花木转录组序列进行高通量测序,采用de novo组装方法,共获得32325条unigene。利用MISA软件,搜索到13779个SSR位点,SSR位点发生频率为42.63%,位点分布频率为1/2.95 kb。不同重复基序类型中,二核苷酸重复基序数量最多,占总数44.10%;单核苷酸重复基序次之,占总数37.90%;三核苷酸重复基序,占总数16.71%。二核苷酸重复基序中,AG/CT重复基序数量最多;三核苷酸重复基序中,AAG/CTT重复基序数目最多,其次是ATC/ATG、ACC/GGT和AGC/CTG。从不同种群中任取1株构成8株无亲缘关系的随机样本,对随机合成的60对SSR引物的有效性进行琼脂糖电泳检测的结果表明,46对引物扩增出目的条带,扩增率达76.67%。进一步利用TP-M13-SSR技术基因分型结果显示,30对引物扩增产物显示出多态性,多态位点百分比为65.22%。这表明,基于转录组序列开发的长柄双花木SSR位点多态性较高。本研究结果有助于后续的长柄双花木种群遗传学及系统进化等方面研究。
译  名：
Development of SSR Markers Based on Transcriptome Sequences of Disanthus cercidifolius var. longipes
作  者：
MENG Yi-hong;XU Xun;XU Gang-biao;The Laboratory of Forestry Genetics,Central South University of Forestry and Technology;College of life science,Sichuan University;
单  位：
MENG Yi-hong%XU Xun%XU Gang-biao%The Laboratory of Forestry Genetics,Central South University of Forestry and Technology%College of life science,Sichuan University
关键词：
Disanthus cercidifolius var.longipes H.T.Chang;;transcriptome;;TP-M13-SSR;;primer development
摘  要：
Disanthus Maxim. belongs to a small eastern Asian genus of the family Hamamelidaceae. D. cercidifolius Maxim. is found in Japan,and this species is the relative of D. cercidifolius var. longipes H. T. Chang that is particularly distributed in the south part of China. D. cercidifolius var. longipes H. T. Chang is listed in the Red List of Endangered Plant Species and Second Grade of the List of Wild Plants Under State Protection in China. D. cercidifolius var. longipes H. T. Chang is of significant interest in phylogeny study and floristic geography in East Asia. However,the limited number of available SSR molecular markers inhibited the current genetic research. In this study,by unlocking the transcriptome datatets by RNA-seq,we obtained a total of 32325 uni-genes. 13779 SSR loci were found using MISA,with the occurrence frequency of 42. 63%. That included 37. 90% of mono-nucleotide repeat motifs,44. 10% of di-nucleotide repeat motifs and 16. 71% of tri-nucleotide repeat motifs. The AG/CT was the most abundant di-nucleotide repeat motifs,while the AAG/CTT was the most abundant tri-nucleotide repeat motif,followed by ATC/ATG,ACC/GGT,and AGC/CTG. 60 pairs of random-selected primers were used to test its availability in the random sample of 8 unrelated individuals from different populations using agarose electrophoresis. Forty-six pairs ofprimers amplified the target fragments,including 30 pairs that were found to be polymorphic using TP-M13-SSR technology. The rate of PCR amplification and polymorphism were 76. 67% and 65. 22%,respectively. Thus,the large number of SSR markers developed in this study might become valuable in population genetics and phylogeny analysis of D. cercidifolius var. longipes H. T. Chang,and also this work might provide an example in development of SSR markers in other species.

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