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首页 > 过刊浏览>2018年第19卷第4期 >740-747. DOI:10.13430/j.cnki.jpgr.20180110002
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基于转录组序列的长柄双花木SSR标记开发
作者:
作者单位:

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

基金项目:

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
Author:
Affiliation:

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.

    参考文献
    [1]高浦新, 李美琼, 周赛霞, 刘洁, 牛艳丽, 杜娟, 丁剑敏. 濒危植物长柄双花木(Disanthus cercidifolius var. longipes)的资源分布及濒危现状. 植物科学学报,2013, 31(1), 34-41.
    [2]缪绅裕, 曾庆昌, 陈志明, 刘嘉俊, 李晓杰, 黄金玲. 南岭大东山长柄双花木群落物种组成与种群结构特征分析. 植物资源与环境学报, 2014, 23( 1): 51－57
    [3]傅立国. 中国珍稀濒危植物. 上海:上海科技出版社,1989. 179.
    [4]肖宜安, 何平, 邓洪平, 时明芝. 井冈山长柄双花木种群遗传多样性与遗传分化. 西南师范大学学报: 自然科学版, 2003, 28(3):444-449.
    [5]谢国文, 赵俊杰, 李荣华, 李海生, 郭培国, 陈雅丽, 黄爱平. 南岭国家濒危植物长柄双花木遗传多样性研究. 广州大学学报: 自然科学版, 2014(4):47-52.
    [6]Mcgregor C E, Lambert C A, Greyling M M, et al. A comparative assessment of dna fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm. Euphytica, 2000, 113(2), 135-144.
    [7]Kalia R K, Rai M K, Kalia S, et al. Microsatellite markers: an overview of the recent progress in plants. Euphytica, 2011, 177(3):309-334.
    [8]Agarwal M, Shrivastava N, Padh H. Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Reports, 2008, 27(4):617-631.
    [9]Gao P X, Yang A H, Yao X H, et al. Isolation and characterization of nine polymorphic microsatellite loci in the endangered shrub Disanthus cercidifolius var. longipes (Hamamelidaceae). Molecular Ecology Resources, 2009, 9(3):1047-9.
    [10]Zane L,Bargelloni L,Patarnello T. Strategies for microsatellite isolation: A review. Molecular Ecology,2002, 11( 1) : 1-16．
    [11]Wang Z, Gerstein M, Snyder M. RNA-Seq: are volutionary tool for transcriptomics. Nature Reviews Genetics, 2009, 10(1):57.
    [12]Davey J W,SHohenlohe P A,SEtter P D,SBoone J Q,SCatchen J M,SBlaxter M L. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nature Reviews Genetics,2011, 12(7) :499- 510．
    [13]Marioni J C, Mason C E, Mane S M, Stephens M, Gilad Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Research, 2008, 18(9):1509.
    [14]Mortazavi A, Williams B A, Mccue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods, 2008, 5(7):621.
    [15]李金璐, 王硕, 于婧, 王玲, 周世良. 一种改良的植物DNA提取方法. 植物学报, 2013, 48(1):72-78.
    [16]李炎林, 杨星星, 张家银, 黄三文, 熊兴耀. 南方红豆杉转录组SSR挖掘及分子标记的研究. 园艺学报, 2014, 41(4):735-745.
    [17]林开勤, 李岩, 赵德刚. 杜仲转录组SSR发掘及标记开发. 分子植物育种, 2016, 14(6):1548-1558.
    [18]樊洪泓, 李廷春, 李正鹏, 林毅, 蔡永萍. 银杏EST序列中微卫星的分布特征. 基因组学与应用生物学, 2009, 28(5):869-873.
    [19]Xu M, Sun Y, Li H. EST-SSRs development and paternity analysis for Liriodendron spp. New Forests, 2010, 40(3):361-382.
    [20]杨华, 陈琪, 韦朝领, 史成颖, 方从兵, 宛晓春. 茶树转录组中SSR位点的信息分析. 安徽农业大学学报, 2011, 38(6):882-886.
    [21]甘霖, 覃碧, 刘实忠, 王秀全, 范高俊. 巴西橡胶树转录组中SSR位点的信息分析. 广东农业科学, 2014, 41(16):142-146.
    [22]Varshney, Rajeev K, Graner, Andreas, Sorrells, Mark E. Genic microsatellite markers in plants: features and applications. Trends in Biotechnology, 2005, 23(1):48-55.
    [23]张振, 张含国, 莫迟, 张磊. 红松转录组SSR分析及EST-SSR标记开发. 林业科学, 2015, 51(8):114-120.
    [24]Li H, Ruan C J, Wang L, Ding J, Tian X J. Development of RNA-Seq SSR Markers and Application to Genetic Relationship Analysis among Sea Buckthorn Germplasm. Journal of the American Society for Horticultural Science American Society for Horticultural Science, 2017, 142(3):200-208.
    [25]鄢秀芹, 鲁敏, 安华明. 刺梨转录组SSR信息分析及其分子标记开发. 园艺学报, 2015, 42(2):341-349.
    [26]Kantety R V, Rota M L, Matthews D E, Sorrells M E. Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Molecular Biology, 2002, 48(5-6):501-510.
    [27]Morgante M, Hanafey M, Powell W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nature Genetics, 2002, 30：194–200.
    [28]时小东, 朱学慧, 盛玉珍, 庄国庆, 陈放. 基于转录组序列的楠木SSR分子标记开发. 林业科学, 2016, 52(11):71-78.
    [29]李响, 杨楠, 赵凯歌, 陈玉星, 唐锐君, 陈龙清. 蜡梅转录组EST-SSR标记开发与引物筛选. 北京林业大学学报, 2013, 35(S1): 25-32.
    [30]文亚峰, 韩文军, 周宏, 徐刚标. 杉木转录组SSR挖掘及EST-SSR标记规模化开发. 林业科学, 2015, 51(11): 40-49.
    [31]Kelkar Y D, Tyekucheva S, Chiaromonte F, Makova K D. The genome-wide determinants of human and chimpanzee microsatellite evolution. Genome Research, 2008, 18(1):30.
    [32]Dreisigacker S, Zhang P, Warburton M L, Van Ginkel M, Hoisington D, Bohn M, Melchinger A E. SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different mega environments. Crop Science, 2004, 44 (2): 381-388.
    [33]Schuelke M. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology, 2000, 18(2):233.
    [34]李莉, 杨剑波, Mackill D J, Colowit P M. 应用自动荧光测序系统分析不同水稻基因型SSLPs. 作物学报, 2000, 26(5): 565-569.
    [35]刘志斋, 王天宇, 黎裕. TP-M13-SSR技术及其在玉米遗传多样性研究中的应用. 玉米科学, 2007, (06): 10-15+31.
    [36]高源, 刘凤之, 王昆, 王大江, 龚欣, 刘立军. 基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立. 植物遗传资源学报, 2015, 16(6):1290-1297.
    [37]Cao Y, Tian L, Gao Y, Liu F. Genetic diversity of cultivated and wild Ussurian Pear ( Pyrus ussuriensis, Maxim.) in China evaluated with M13-tailed SSR markers. Genetic Resources Crop Evolution, 2012, 59(1): 9-17.
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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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