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基于山茶转录组的SSR标记开发及亲缘关系分析

潘丽芹 李纪元 李绍翠 范正琪 殷恒福 何丽波

潘丽芹, 李纪元, 李绍翠, 范正琪, 殷恒福, 何丽波. 基于山茶转录组的SSR标记开发及亲缘关系分析[J]. 北京林业大学学报, 2019, 41(7): 111-120. doi: 10.13332/j.1000-1522.20190101
引用本文: 潘丽芹, 李纪元, 李绍翠, 范正琪, 殷恒福, 何丽波. 基于山茶转录组的SSR标记开发及亲缘关系分析[J]. 北京林业大学学报, 2019, 41(7): 111-120. doi: 10.13332/j.1000-1522.20190101
Pan Liqin, Li Jiyuan, Li Shaocui, Fan Zhengqi, Yin Hengfu, He Libo. Development of SSR markers based on transcriptome of Camellia japonica and analysis of genetic relationship[J]. Journal of Beijing Forestry University, 2019, 41(7): 111-120. doi: 10.13332/j.1000-1522.20190101
Citation: Pan Liqin, Li Jiyuan, Li Shaocui, Fan Zhengqi, Yin Hengfu, He Libo. Development of SSR markers based on transcriptome of Camellia japonica and analysis of genetic relationship[J]. Journal of Beijing Forestry University, 2019, 41(7): 111-120. doi: 10.13332/j.1000-1522.20190101

基于山茶转录组的SSR标记开发及亲缘关系分析

doi: 10.13332/j.1000-1522.20190101
基金项目: 浙江省农业(林木)新品种选育重大科技专项(2016C02056-12),政府间国际科技创新合作重点专项(2016YFE0126100)
详细信息
    作者简介:

    潘丽芹,副教授。主要研究方向:园林植物分子育种。Email:826673874@qq.com 地址:318020浙江省台州市黄岩区嘉木路288号台州科技职业学院

    责任作者:

    李纪元,研究员,博士生导师。主要研究方向:园林植物分子育种。Email:jiyuan_li@126.com 地址:311400浙江省杭州市富阳区大桥路73号中国林业科学研究院亚热带林业研究所

    何丽波,副教授。主要研究方向:风景园林。Email:helibo@hunau.edu.cn 地址:410128湖南省长沙市芙蓉区农大路1号湖南农业大学园艺园林学院

  • 中图分类号: S667.5; S794.9

Development of SSR markers based on transcriptome of Camellia japonica and analysis of genetic relationship

  • 摘要: 【目的】开发基于山茶转录组的EST-SSR分子标记,并应用该标记进行山茶种质的亲缘关系分析。【方法】本研究以山茶叶片转录组测序所获得的50 518条Unigenes为背景数据,利用MISA软件搜索SSR标记,设计并筛选SSR多态性引物,对8份山茶种质进行了UPGMA聚类。【结果】共检索到13 197个SSR位点,SSR的发生频率和分布频率分别为19.52%、26.12%,平均分布距离为4.33 kb;在6种SSR重复类型中,以单、二、三核苷酸这3种重复类型为主,分别占48.420%、34.917%和15.473%;出现频率高的基序类型为A/T、AG/CT和AT/TA,占总SSR位点的79.61%;三核苷酸中以AAG/CTT、ACC/GGT、AAT/ATT类型居多;在设计出的10 974对引物中,随机选用其中90对引物进行多态筛选,73对引物扩增产物与预期大小一致,有效扩增率为81.11%,29对引物存在扩增多态性,占39.73%。扩增得到72个等位基因,有效等位基因数平均达到3.264;观察杂合度和期望杂合度的平均值分别为0.208和0.638,引物多态信息含量平均为0.496;8份山茶种质在相似系数为0.58时,可以分为4类:山茶原种为Ⅰ类,‘黑蛋石’、‘红叶黑魔法’和‘黑骑士’为Ⅱ类,‘黑魔法’和‘金华美女’分别为Ⅲ类和Ⅳ类。【结论】山茶转录组测序的Unigenes信息可作为SSR标记开发的有效来源,为山茶的遗传多样性研究、亲缘关系鉴定以及分子标记辅助育种等奠定了一定的理论基础。

     

  • 图  1  山茶‘红叶黑魔法’转录组中SSR重复次数分布

    Figure  1.  Distribution of SSR repeats frequency in C. ‘Red Leaved Black Magic’ transcriptome

    图  2  山茶转录组SSR的基序类型分布

    A、B、C分别表示单核苷酸重复、二核苷酸重复、三核苷酸重复的基元比例。A, B, C, reperesent the ratio of mono-nucleotide repeats, di-nucleotide repeats and tri-nucleotide repeats in Camellia.

    Figure  2.  Distribution of SSR types in Camellia

    图  3  引物32对8份山茶种质的PCR扩增信号图

    Figure  3.  PCR amplification signal figure of primer 32 in 8 Camellia germplasm

    图  4  8份山茶种质的UPGMA聚类图

    Figure  4.  UPGMA dendrogram for 8 Camellia samples

    表  1  山茶材料及采样地点

    Table  1.   List of Camellia collections and locations

    编号 No.名称 Genotype种源地 Origin编号 No.名称 Genotype种源地 Origin
    1 山茶
    C. japonica
    浙江杭州
    Zhejiang Hangzhou
    5 黑魔法
    C. japonica ‘Black Magic’
    浙江金华
    Zhejiang Jinhua
    2 黑蛋石
    C. ‘Black Opal’[11-13]
    浙江杭州
    Zhejiang Hangzhou
    6 黑魔法
    C. japonica ‘Black Magic’[11-13]
    浙江杭州
    Zhejiang Hangzhou
    3 红叶黑魔法
    C. ‘Red Leaved Black Magic’
    浙江杭州
    Zhejiang Hangzhou
    7 黑骑士
    C. ‘Night Rider’[11-13]
    浙江杭州
    Zhejiang Hangzhou
    4 金华美女
    C. japonica ‘Jinhua Meinǖ’[14]
    浙江杭州
    Zhejiang Hangzhou
    8 黑骑士
    C. ‘Night Rider’
    浙江金华
    Zhejiang Jinhua
    下载: 导出CSV

    表  2  山茶叶片转录组中SSR的分布情况

    Table  2.   Distribution of SSR loci in the transcriptome of Camellia leaves

    SSR类型 SSR type数量 Number比例 Percentage/%出现频率 Distribution frequency/%平均距离Average distance/kb
    单核苷酸 Mono-nucleotide 6 390 48.420 12.649 8.94
    二核苷酸 Di-nucleotide 4 608 34.917 9.121 12.40
    三核苷酸 Tri-nucleotide 2 042 15.473 4.042 27.97
    四核苷酸 Tetra-nucleotide 128 0.970 0.253 446.25
    五核苷酸 Penta-nucleotide 14 0.106 0.028 4 080.04
    六核苷酸 Hexa-nucleotide 15 0.114 0.029 3 808.04
    总计 Total 13 197 100 26.123 4.33
    下载: 导出CSV

    表  3  山茶转录组29对SSR引物信息及多态性信息

    Table  3.   29 pairs of SSR primers and polymorphic information of C. japonica transcriptome

    引物编号
    Primer No.
    来源
    Gene ID
    引物序列
    Primer sequence
    SSR基元
    SSR motif
    有效等位基因
    Effective alleles
    观察杂合度
    Observed heterozygosity
    期望杂合度
    Expected heterozygosity
    多态信息含量
    Polymorphism
    information content
    P1 c105034.graph_c0 F:CTTCTTCTCGATCCACAGCC (GAG)5 4.414 0.250 0.773 0.556
    R:CGATCTCCTCCGTAACAAGC
    P6 c123499.graph_c0 F:GGACAATCTTTTTGGGAGCA (ATT)5 1.556 0.143 0.357 0.375
    R:CCCTACACAACCAGGAAACC
    P10 c126182.graph_c0 F:CCTTAACAATCAGCAATGCC (AAC)5 3.122 0.375 0.680 0.455
    R:TGCCATGTACCACATACCCA
    P12 c129722.graph_c0 F:TCAAAAGAGACCTTGGGCTG (GTA)7 4.741 0.375 0.789 0.545
    R:GGGGACTTCCGATAACACAA
    P16 c133766.graph_c0 F:GTCCCGAAAAATCCCAAAAT (CT)6 4.235 0.500 0.764 0.778
    R:AATTTGTCTGCAATGGCTCC
    P17 c133791.graph_c0 F:TGAATCACAATCTTGGCTGG (GA)8 2.909 0.500 0.656 0.417
    R:GGTGGCCTAATACAAGCTGC
    P18 c133791.graph_c0 F:TGAATCACAATCTTGGCTGG (A)12 2.667 0.500 0.625 0.417
    R:GGTGGCCTAATACAAGCTGC
    P21 c134194.graph_c1 F:ATGGTGGCAAGGAATCAAAG (TGG)5 4.000 0.750 0.750 0.500
    R:TGTAAGCTCCTGTGCTGTGG
    P22 c135100.graph_c0 F:TTCCTCTTTCAAATGCCAATG (TA)6 2.844 0.125 0.648 0.333
    R:TTAACGGGGAGCCATATCAA
    P24 c135126.graph_c1 F:GTGAAACAAAGCCGGAGAGT (GA)7 5.565 0.625 0.820 0.615
    R:ACCTGGTTCAATCTATGGCG
    P25 c136711.graph_c1 F:CGTTTCAAGGGCAATATCGT (TTG)5 2.390 0.429 0.582 0.400
    R:GCTACCATGAAGCTCCAACC
    P26 c137110.graph_c0 F:AGATTTGCAAGGTTGGGTTG (GCA)6 2.579 0.286 0.612 0.375
    R:TCTACCACACTCCCACTCCC
    P28 c137365.graph_c0 F:TGGTTGCTGTTGTTGAGGAG (TGG)6 3.200 0.250 0.688 0.444
    R:CCGCCTAATCAGAACCCTTT
    P32 c138380.graph_c1 F:TCGTTAAGGCAGCTCTCGAT (CGT)7 2.723 0.750 0.633 0.286
    R:CCACCACTGTGTACGGTCAA
    P37 c141066.graph_c0 F:ACACACGAACCACTCCATCA (TAA)6 2.844 0.375 0.648 0.273
    R:TTTGGTTGTTGGCATTTTCA
    P40 c142086.graph_c0 F:AACAATACCCGACTCCTCCC (CAT)6 5.333 0.500 0.813 0.583
    R:CCTATGGCGAGACGTTCAAT
    P41 c142086.graph_c1 F:AATAGCACGGTAATCACGGC (CAT)7 4.129 0.125 0.758 0.556
    R:GAATTTTCTGGGCCATCTGA
    P42 c143100.graph_c0 F:TTCCACAATTCCCACCCTTA (ACC)5 3.200 0.250 0.688 0.500
    R:CCAGTCAAGCCCTGTAGCTC
    P44 c143532.graph_c1 F:CACCATCACCAAAGAAGGCT (CCA)6 4.455 0.429 0.776 0.600
    R:TGCAAGAATTTTAACCAAACG
    P45 c144082.graph_c1 F:AATACCTTGGCAAATGACGC (AGA)5 3.161 0.571 0.684 0.364
    R:CGCCAACCTATCTTCAAAGC
    P47 c144342.graph_c0 F:CGTCCAGCATTTCTCCATTT (TTC)6 3.122 0.125 0.680 0.556
    R:CAAGAAGGCTCTGGAGGATG
    P61 c147427.graph_c0 F:GGGGAGTAGAAGAAGGGACG (GTG)5 3.657 0.125 0.727 0.556
    R:TGATCTCTCACTCCGACACG
    P70 c148755.graph_c0 F:TGGTGGAGCTCAGAACAAGA (GGT)6 3.657 0.375 0.727 0.455
    R:TCCATTGAAGTATCCACCGC
    P71 c148777.graph_c3 F:CTCGCGTCTCAAAACTTTCC (AAG)5 2.977 0.125 0.664 0.556
    R:ATCACTGGCTCATCTCCGTC
    P75 c151957.graph_c0 F:GGGGGCAGGTTAACTTTGTT (TA)7 2.977 0.250 0.664 0.500
    R:CCCGTCCTGATCTACCTCCT
    P78 c151961.graph_c0 F:TCCCCATGTAGACTCTTCCG (CCG)5 5.818 0.750 0.828 0.500
    R:AAGACATGTTCGGTTCCGTC
    P84 c153951.graph_c1 F:CCCACATGTTTCCTCCACTT (TTA)5 3.556 0.625 0.719 0.385
    R:TAGGGCAGAATTTGGGTTTG
    P85 c154034.graph_c0 F:GCGTTGATCATGGTTTATCG (ATG)5 4.900 0.571 0.796 0.636
    R:CCGTTGATCCCTTCGACTTA
    P89 c76886.graph_c0 F:AGATCTATTGGCCACGGATG (TCT)5 4.414 0.375 0.773 0.456
    R:GTTGCGAAAAGACGAAGAGG
    平均值 Average 3.264 ± 0.158 0.208 ± 0.029 0.638 ± 0.020 0.496 ± 0.038
    下载: 导出CSV

    表  4  8份山茶种质的遗传相似系数

    Table  4.   Genetic similarity coefficients of 8 Camellia germplasms

    编号 No.12345678
    1 1.000 0
    2 0.260 9 1.000 0
    3 0.217 4 0.752 2 1.000 0
    4 0.449 0 0.693 9 0.489 8 1.000 0
    5 0.367 3 0.612 2 0.693 9 0.576 9 1.000 0
    6 0.347 8 0.521 7 0.565 2 0.445 0 0.671 4 1.000 0
    7 0.461 5 0.692 3 0.692 3 0.545 5 0.618 2 0.538 5 1.000 0
    8 0.375 0 0.666 7 0.625 0 0.549 0 0.627 5 0.500 0 0.814 8 1.000 0
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-03-26
  • 修回日期:  2019-05-26
  • 网络出版日期:  2019-07-06
  • 刊出日期:  2019-07-01

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