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基于RAD-seq技术的金银花SSR标记开发及鉴定

李慧 刘东超 徐瑞瑞 侯立娜 王天琪 刘忠华 付晓 李圣波

李慧, 刘东超, 徐瑞瑞, 侯立娜, 王天琪, 刘忠华, 付晓, 李圣波. 基于RAD-seq技术的金银花SSR标记开发及鉴定[J]. 北京林业大学学报, 2021, 43(6): 108-117. doi: 10.12171/j.1000-1522.20200337
引用本文: 李慧, 刘东超, 徐瑞瑞, 侯立娜, 王天琪, 刘忠华, 付晓, 李圣波. 基于RAD-seq技术的金银花SSR标记开发及鉴定[J]. 北京林业大学学报, 2021, 43(6): 108-117. doi: 10.12171/j.1000-1522.20200337
Li Hui, Liu Dongchao, Xu Ruirui, Hou Lina, Wang Tianqi, Liu Zhonghua, Fu Xiao, Li Shengbo. Development and identification of SSR markers based on RAD-seq of Lonicera japonica[J]. Journal of Beijing Forestry University, 2021, 43(6): 108-117. doi: 10.12171/j.1000-1522.20200337
Citation: Li Hui, Liu Dongchao, Xu Ruirui, Hou Lina, Wang Tianqi, Liu Zhonghua, Fu Xiao, Li Shengbo. Development and identification of SSR markers based on RAD-seq of Lonicera japonica[J]. Journal of Beijing Forestry University, 2021, 43(6): 108-117. doi: 10.12171/j.1000-1522.20200337

基于RAD-seq技术的金银花SSR标记开发及鉴定

doi: 10.12171/j.1000-1522.20200337
基金项目: 国家林业局业务委托项目(2019104011),北京林业大学一流学科建设项目(2019XKJS0308),北京市园林绿化局计划项目(2019-STBHXFC-02-022)
详细信息
    作者简介:

    李慧。主要研究方向:经济林品种鉴定。Email:hyacinth1107@163.com 地址:100083北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    刘忠华,博士,副教授。主要研究方向:植物生长发育与系统进化。Email:liuzh6@bjfu.edu.cn 地址:同上

  • 中图分类号: S793.9;S567.9

Development and identification of SSR markers based on RAD-seq of Lonicera japonica

  • 摘要:   目的  通过开发特异性高的多态性分子标记位点,以弥补金银花在简单重复序列(SSR)标记方面的匮乏,推动金银花在遗传资源管理及良种鉴定等方向的研究,为后续全基因组测序及组装策略奠定基础。  方法  运用RAD-seq技术对2份金银花样品进行简化基因组测序,利用MISA软件识别contig上的SSR序列并对各类型序列特征进行归纳分析,进而设计引物并利用生物信息学的方法进行引物筛选和有效性检测。  结果  对‘九丰一号’和‘亚特’金银花进行简化基因组测序,分别获得27.805 Mbp和42.560 Mbp干净读长。在组装的45 850个基因序列中共检测到46 999个SSR位点,其中单核苷酸重复单元比例最高(49.15%),六核苷酸重复单元最少(0.20%)。SSR位点的重复基序以(A/T)n为主,具有偏向性。除单碱基和二碱基重复类型外,SSR位点基序的重复次数集中在5 ~ 6次,且随重复次数增加,SSR位点重复类型出现的频率呈下降趋势。金银花基因组SSR序列长度介于10 ~ 310 bp之间,随重复次数增加,各SSR序列出现的频率逐渐下降。利用Primer3.0成功设计出38 507对SSR引物,设计成功率为81.93%。对挑选的35对SSR引物进行多态性分析,等位基因数、观测杂合度和多态性信息含量(PIC)的平均值分别为5.057、0.363和0.568,其中高多态信息位点26个,中度多态信息位点9个,均未偏离哈迪–温伯格平衡。  结论  利用RAD-seq技术可实现SSR标记的大量开发和多态性SSR引物的筛选,并为金银花在遗传多样性分析和种质鉴定等相关方面的研究提供数据支持。

     

  • 图  1  简化基因组中SSR位点的基序重复次数分布

    Figure  1.  Distribution of repeat numbers of SSR motifs in simplified genome

    图  2  金银花基因组不同SSR序列长度的出现频率

    Figure  2.  Frequency of SSR with different lengths in L. japonica

    图  3  不同长度重复单元SSR序列的长度变异情况

    Figure  3.  Variation in sequence length of different types of SSR

    图  4  引物jap5在12个金银花样品中的毛细管电泳检测图

    Figure  4.  Capillary electrophoresis detection of 12 samples of L. japonica with jap5 primer

    表  1  RAD-seq基因组测序数据统计

    Table  1.   Data statistics of genomic sequencing generated by RAD-seq

    样本
    Sample
    过滤前 Before filter过滤后 After filter
    原始读长
    Raw read/Mbp
    原始碱基总数
    Total number of
    original bases/Gbp
    Q20/GbpQ30/Gbp干净读长
    Clean read/Mbp
    有效碱基总数
    Total number of
    bpeffective base/Gbp
    Q20/GbpQ30/Gbp平均长度
    Average
    length/bp
    ‘九丰一号’
    ‘Jiufengyihao’
    31.9294.7894.478
    (93.5%)
    4.147
    (86.6%)
    27.8054.0343.935
    (97.5%)
    3.758
    (93.2%)
    145.1
    ‘亚特’
    ‘Yate’
    45.9946.8996.652
    (96.4%)
    6.367
    (92.3%)
    42.5606.3046.211
    (98.5%)
    6.047
    (95.9%)
    148.1
    注:Q20. 质量得分不低于20的碱基数及比例;Q30. 质量得分不低于30的碱基数及比例。
    Notes:Q20, the number and proportion of bases with a quality score of not less than 20; Q30, the number and proportion of bases with a quality score of not less than 30.
    下载: 导出CSV

    表  2  金银花基因组中SSR位点信息

    Table  2.   Information of SSR loci in Lonicera japonica

    SSR类型
    SSR type
    数量
    Number
    重复次数
    Repeat time
    平均长度
    Average
    length/bp
    百分比
    Percentage/%
    重复单元类型 Motif type
    数量 Number主要基序 Main motif
    单核苷酸 Mono-nucleotide 23 101 10 ~ 82 11.83 49.15 2 A/T,C/G
    二核苷酸 Di-nucleotide 13 424 6 ~ 45 16.89 28.56 6 AT/TA,AG/CT,AC/GT
    三核苷酸 Tri-nucleotide 3 883 5 ~ 49 22.04 8.26 59 AAT/ATT,GAA/TTC,TAA/TAA,AAG/CTT,TCA/TGA
    四核苷酸 Tetra-nucleotide 540 5 ~ 14 23.03 1.15 86 AAAT/ATTT,TAAT/ATTA,TAAA/TTTA,CTTT/AAAG,TTTC/GAAA,ATAC/GTAT,TATC/GATA
    五核苷酸 Penta-nucleotide 147 5 ~ 18 27.99 0.31 92 TTTTC/GAAAA,TTTCT/AGAAA,AAAAT/ATTTT,GGGTT/AACCC,ATCCA/TGGAT
    六核苷酸 Hexa-nucleotide 94 5 ~ 13 34.40 0.20 74 GGCAAT/ATTGCC,GGAGAT/ATCTCC,GGTGGA/TCCACC,GATATA/TATATG,CAATCA/TGATTG,GCATTT/AAATGC
    复合型 Compound 5 810 70.01 12.36
    总计 Total 46 999 21.54 100.00 319
    下载: 导出CSV

    表  3  35个位点SSR引物多态性分析

    Table  3.   Polymorphism analysis of SSR primers at 35 loci

    引物
    Primer
    引物序列(5′—3′)
    Primer sequence (5′−3′)
    SSR类型
    Type of SSR
    重复基序
    Repeat motif
    NaNeHeHoHIPIC
    jap2 F:AGATGGATAGTGGGCCATTGT
    R:TAGAGTCACCGGGAGCTACC
    P3 (TGA)5 6.000 2.342 0.598 0.402 0.573 1.211 0.544
    jap5 F:GCACGTGCACTCAGAAACAG
    R:GCAACCGTTGAGCTCCTTTG
    P3 (GTA)7 8.000 6.000 0.870 0.130 0.833 1.932 0.814
    jap6 F:GCATAGACTCTGCTGGCCAA
    R:AGCAGTGGCAGATATGGCTG
    P3 (TCA)6 4.000 3.236 0.721 0.279 0.691 1.258 0.635
    jap15 F:TGAAAAGAAGGGAATAGGATTAGGGT
    R:CCTCTTGGTGAGACCGCTTT
    P4 (GAAT)5 5.000 2.220 0.576 0.424 0.550 1.097 0.512
    jap17 F:GAAGAAGAGCGGCTACTGCA
    R:AAGAGTGAAGCAGCAGCAGT
    P3 (CAC)5 5.000 3.723 0.766 0.234 0.731 1.417 0.685
    jap18 F:AGGCTACATGATGATTGAGACGA
    R:TTGGCCCCACCTGATCAAAA
    P3 (ACA)6 4.000 2.504 0.627 0.373 0.601 1.054 0.524
    jap21 F:ACCCTCACCTTCACCCTCAT
    R:TGGGTCATCGCATCGTCAAA
    P3 (CAT)5 4.000 1.419 0.308 0.692 0.295 0.624 0.281
    jap44 F:ATGGAGGGCTAGTGAATGCG
    R:TCCACACTCGGTACACCAAA
    P3 (GAT)5 4.000 1.959 0.511 0.489 0.490 0.882 0.431
    jap51 F:ACGTCGAATGTTAATGGCACG
    R:TGCAGCACATTTGGCATTCT
    P3 (ATT)6 4.000 2.575 0.641 0.359 0.612 1.121 0.555
    jap53 F:GTCTGGGGAATGAGTTGCCA
    R:AGCCTTTGATCGAGTCAGGT
    P3 (TTG)14 7.000 3.556 0.750 0.250 0.719 1.549 0.686
    jap73 F:TCAAACTCCTACCTGGCTGC
    R:CTGTATGTGCCGGACATGGT
    P3 (GCT)6 6.000 2.642 0.649 0.351 0.622 1.328 0.594
    yjap4 F:CAACTTCACCGGTGGATCCA
    R:TCCTTCCAATTCGAAACTAAACCT
    P3 (TTG)5 4.000 2.483 0.623 0.377 0.597 1.119 0.552
    yjap5 F:AAAGGCGTGGTGAAAGGTCT
    R:CACATATGGTTGCTGCAGCC
    P3 (GAA)5 10.000 6.698 0.888 0.112 0.851 2.084 0.835
    yjap6 F:TTCTAGGGCTTCGGCAACTG
    R:CCTTCCCCTCGAAAACTGCA
    P3 (CTG)5 4.000 1.714 0.435 0.565 0.417 0.837 0.393
    yjap9 F:TGGCAACTTTCCCCCAAATTC
    R:TGAGCGAGTCATATAGGCGAG
    P3 (TTC)6 4.000 2.361 0.601 0.399 0.576 0.980 0.485
    yjap10 F:AACGGGAAAGAGTGAGCGAG
    R:GTGTGTTTCCCTTTTCCGGC
    P3 (ATT)5 3.000 1.834 0.475 0.525 0.455 0.803 0.410
    yjap12 F:GCATAGACTCTGCTGGCCAA
    R:AGCAGTGGCAGATATGGCTG
    P3 (TCA)5 5.000 2.852 0.678 0.323 0.649 1.305 0.614
    yjap19 F:GGTGGATGAGCTTCTGAAGAGA
    R:CCAATGGCTTCTTCCCCTGT
    P3 (TTG)6 5.000 2.028 0.529 0.471 0.507 0.994 0.463
    yjap24 F:GCTTTTGCAGCTCCTACTTTGT
    R:TTTGCTGTTAAGACACGCGC
    P3 (CAC)5 4.000 3.429 0.739 0.261 0.708 1.309 0.659
    yjap36 F:GACACACACGGTTGCTTGAC
    R:AGAGTTTCTGCTGTTGGTGGA
    P3 (GCA)6 4.000 2.969 0.692 0.308 0.663 1.200 0.600
    yjap39 F:TCACTACCTGTATCCGCTCT
    R:TGGAAGCAGAAAGGGCATCT
    P3 (ATC)7 4.000 2.743 0.663 0.337 0.635 1.140 0.566
    yjap47 F:GCTTCGCCTTTGGTTCGGTA
    R:CAACTCGCTCTCTCCCTTCC
    P3 (ATG)7 4.000 1.419 0.308 0.692 0.295 0.624 0.281
    yjap48 F:GGGCATTTTCTGGTGGAGGA
    R:AGCATCCAAACCTGATTTGTTT
    P3 (GTT)5 4.000 2.642 0.649 0.351 0.622 1.105 0.553
    yjap51 F:CGGAGAGAGATACTAGGCGC
    R:CGCGGCTGAATTTTAGAGCC
    P2 (TA)6 7.000 3.692 0.761 0.239 0.729 1.595 0.701
    yjap53 F:CATTCATTCCCCCACCAGGT
    R:GGGACCGGTCGAAACCAATA
    P2 (TC)10 5.000 2.034 0.533 0.468 0.508 1.056 0.483
    yjap59 F:GGATCCAGCATGTCTCGAACT
    R:TGCTTTGGCCCTTCCTCATT
    P2 (AT)8 4.000 2,215 0.573 0.428 0.549 1.013 0.500
    yjap89 F:GAGTAAAGGTGGAGAGCGTGT
    R:AGTTTCCGAGAAGTGCGTGT
    P4 (CTAT)5 4.000 3.310 0.728 0.272 0.698 1.283 0.645
    yjap105 F:TGTTTAGCAACAAGGGATGGT
    R:GCTGGCAGGGACAAAAAGTG
    P2 (GA)7 6.000 4.033 0.788 0.212 0.752 1.547 0.714
    yjap109 F:AACTTCTGTGACAGGGCAGG
    R:GCATATGCGGCAATGGGAAG
    P2 (GT)6 5.000 2.504 0.627 0.373 0.601 1.188 0.559
    yjap120 F:GCCAGAGCTCCGATAAGGTC
    R:TGCATCGCGTGTGTATGTGA
    P2 (AT)6 8.000 4.966 0.833 0.167 0.799 1.848 0.779
    yjap153 F:ACATGAGGGTTTGCCGATCC
    R:GGAGTATGGCCAGGTTCGTC
    P3 (GTG)6 5.000 2.852 0.678 0.323 0.649 1.253 0.596
    yjap169 F:AGTTCGAGGGATTGTGCGTT
    R:TGTGGAATTCGTCAATGGCG
    P3 (TTC)7 3.000 1.674 0.420 0.580 0.403 0.722 0.363
    yjap171 F:TCAGAGAGGGGGTAACAGCA
    R:TCGGCTTATACGACTGCGAG
    P3 (ACC)5 4.000 2.483 0.623 0.377 0.597 1.119 0.552
    yjap175 F:GTTTCAATGCGAGCTCGACG
    R:TCTCATTCTTCCCAAAATTCATCAGA
    P3 (TTA)6 9.000 3.388 0.736 0.265 0.705 1.647 0.681
    yjap183 F:GGGGCCTCATTTTCCCTCAA
    R:TTTTCCGGTGGTGGAGCAAT
    P3 (CAC)5 5.000 3.236 0.721 0.279 0.691 1.342 0.644
    均值 Mean 5.057 2.907 0.638 0.363 0.611 1.217 0.568
    注:Ho. 观测杂合度;Na. 观测等位基因数;Ne. 有效等位基因数;He. 期望杂合度;I. Shannon信息指数;H. Nei’s遗传多样性指数;PIC. 多态性信息含量。Notes: Ho, observation of heterozygosity; Na, observed number of alleles; Ne, effective number of alleles; He, expected heterozygosity; I, Shannon information index; H, Nei ’s genetic diversity index; PIC, polymorphism information content.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-11-09
  • 修回日期:  2020-12-16
  • 网络出版日期:  2021-06-03
  • 刊出日期:  2021-06-30

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