基于转录组序列的长柄双花木SSR标记开发
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中南林业科技大学林木遗传育种实验室,四川大学生命科学学院,中南林业科技大学林木遗传育种实验室

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国家“十三五”重点研发计划课题(2016YFC0503103),中南林业科技大学研究生科技创新基金(20183002)


Development of SSR markers based on transcriptome sequences of Disanthus cercidifolius var. Longipes
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The Laboratory of Forestry Genetics,Central South University of Forestry and Technology,College of life science,Sichuan University,The Laboratory of Forestry Genetics,Central South University of Forestry and Technology

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    摘要:

    双花木属(Disanthus)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)是日本特有植物双花木(D. cercidifolius)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。本研究结果为后续的长柄双花木种群遗传学及系统进化等方面研究奠定了基础,也为其他物种SSR标记开发提供了方法学参考。

    Abstract:

    Disanthus including one species and one variety, is a small eastern Asian genus of the family Hamamelidaceae. D. cercidifolius , which occurs in Japan and D. cercidifolius var. longipes , which only occurs in south China. D. cercidifolius var. longipes was listed in the Red List of Endangered Plant Species and Second Grade of the List of Wild Plants Under State Protection in China. This species is significant for the study of phylogeny and floristic geography in east Asia. However, the insufficient number of useful SSR molecular markers hinders current genetic research in this species. We performed transcriptome sequencing of samples D. cercidifolius var. Longipes using Illumina HiSeq4000. After assembling of transcriptome sequences, a total of 32325 uni-genes were obtained. There were 13779 SSR loci identified using MISA, and the occurrence frequency of SSR loci was 42.63%. The distribution frequency of SSR was 1/2.95kb. The di-nucleotide repeat motif was the most abundant among different repeat motifs, accounting for 44.10% of the total number of repeat motifs. Mono-nucleotide and tri-nucleotide repeat motifs accounted for 37.90% and 16.71%, respectively. 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. 46 pairs primers amplified with clear target bands, while 30 pairs were found to be polymorphic using TP-M13 -SSR technology. The amplifying rate and proportion of polymorphic loci were 76.67% and 65.22%, respectively. The results showed that the polymorphism of SSR loci developed based on the transcriptome sequences is higher. The large number of SSR markers in this study should be used for the study of population genetics and phylogeny of D. cercidifolius var. longipes, and provide the referential method of SSR developing for other species.

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孟艺宏,徐璕,徐刚标.基于转录组序列的长柄双花木SSR标记开发[J].植物遗传资源学报,2018,19(4):740-747.

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  • 收稿日期:2018-01-10
  • 最后修改日期:2018-05-12
  • 录用日期:2018-03-01
  • 在线发布日期: 2018-07-16
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