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毛白杨多态SSR引物库和种质资源指纹图谱库构建

韩志强 任勇谕 夏宇飞 耿喜宁 杜康 康向阳

韩志强, 任勇谕, 夏宇飞, 耿喜宁, 杜康, 康向阳. 毛白杨多态SSR引物库和种质资源指纹图谱库构建[J]. 北京林业大学学报, 2019, 41(7): 10-18. doi: 10.13332/j.1000-1522.20190040
引用本文: 韩志强, 任勇谕, 夏宇飞, 耿喜宁, 杜康, 康向阳. 毛白杨多态SSR引物库和种质资源指纹图谱库构建[J]. 北京林业大学学报, 2019, 41(7): 10-18. doi: 10.13332/j.1000-1522.20190040
Han Zhiqiang, Ren Yongyu, Xia Yufei, Geng Xining, Du Kang, Kang Xiangyang. Construction of polymorphic SSR primer library and germplasm resource fingerprint database of Populus tomentosa[J]. Journal of Beijing Forestry University, 2019, 41(7): 10-18. doi: 10.13332/j.1000-1522.20190040
Citation: Han Zhiqiang, Ren Yongyu, Xia Yufei, Geng Xining, Du Kang, Kang Xiangyang. Construction of polymorphic SSR primer library and germplasm resource fingerprint database of Populus tomentosa[J]. Journal of Beijing Forestry University, 2019, 41(7): 10-18. doi: 10.13332/j.1000-1522.20190040

毛白杨多态SSR引物库和种质资源指纹图谱库构建

doi: 10.13332/j.1000-1522.20190040
基金项目: “十三五”国家重点研发计划课题(2016YFD0600403),北京市共建项目专项
详细信息
    作者简介:

    韩志强,博士。研究方向:细胞遗传与细胞工程。Email:zhiqiangybfq@163.com 地址:410004湖南省长沙市韶山南路498号

    责任作者:

    康向阳,教授,博士生导师。主要研究方向:林木细胞遗传与细胞工程育种。Email:kangxy@bjfu.edu.cn 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

Construction of polymorphic SSR primer library and germplasm resource fingerprint database of Populus tomentosa

  • 摘要: 目的针对毛白杨优树种质资源形态学差异小、不易区分等问题,利用多态SSR引物构建优树种质资源指纹图谱,为毛白杨种质资源管理、新品种选育、知识产权保护等提供参考。方法本研究以山东冠县毛白杨种质资源库469个优树无性系为对象,从毛白杨SSR多态引物筛选入手,通过荧光SSR引物PCR扩增和毛细管电泳仪检测筛选SSR引物,并构建指纹图谱。结果在2 317对SSR引物中,共计得到清晰、特异、多态、稳定扩增的SSR引物406对,这些SSR引物在19条染色体上的分布数量介于3 ~ 39对之间,利用BLAST比对分析可将其中389对SSR引物定位到毛果杨基因组,构建了毛白杨优树种质资源多态SSR引物库。应用多态性较高的SSR核心引物,以及特异的SSR辅助引物相结合的技术策略,筛选出25对多态性较高的SSR核心引物,以及13对特异的SSR辅助引物,构建了毛白杨种质资源库内469个优良无性系指纹图谱库,并完成了指纹图谱的QR编码。结论本研究成功构建了毛白杨种质资源多态SSR引物库和优树无性系指纹图谱库,对与毛白杨类似的林木种质资源遗传变异研究以及品种鉴定、系谱分析等具有重要意义。

     

  • 图  1  毛白杨优株无性系指纹图谱QR编码

    仅列举了18个毛白杨优株无性系材料的二维指纹图谱QR编码。The two-dimensional fingerprinting QR codes of only 18 superior clone materials of Populus tomentosa are listed.

    Figure  1.  Fingerprinting QR codes of Populus tomentosa superior clones

    表  1  毛白杨优良无性系种质特异SSR引物库信息

    Table  1.   Specific SSR primer information of Populus tomentosa superior clonal germplasm

    引物
    Primer
    上游引物(5′−3′)
    Forward primer sequence (5′−3′)
    下游引物(5′−3′)
    Reverse primer sequence (5′−3′)
    染色体
    Chromosome
    位置a
    Locationa
    等位标记配置类型
    Allele configuration type
    GCPM_1053-1 AAAAATGATAACCAGGAAAAA TCGAGTTATCTCAGCCTCAT Chr01 14.84 188/196, 188/215, 186/188, 188/227, 188/188
    GCPM_1153-1 TTCCTTTCACACAATGACAA TTTAAAAACTGGGTCCGTAA Chr11 11.14 176/179, 179/182, 189/189, 171/174, 180/189
    GCPM_1056-1 CCACATTTATGTCCCTCTGT AAGCTGAAGCTGCTCTCTTA Chr06 11.08 134/134, 132/132, 144/144, 124/124
    GCPM_1063 AGTTAATTGCGCATGTTCTT AAACAAACTCCAGCAAACAT Chr05 6.00 166/187, 166/185, 166/191, 167/174
    GCPM_1065 TGCAATCATATATTCCTCCC ATAAAATTACTGCGTGCCAT Chr06 24.97 178/178, 184/184, 172/172, 163/163
    GCPM_1005-1 CTAAGTAGTGAAAGAGGCGG CTCCTGTAAATGTATCCCCA Chr18 0.26 123/123, 128/128, 121/121, 126/130
    GCPM_108-1 CTGCAAGATGTAAGTGATCG CAGCATTAGAGCCAAGCTA Chr18 15.09 121/121, 123/123, 126/126, 117/117
    GCPM_1120-1 TGCAAATCCAAACACAGATA ACGAATCATCTTACGCACTT Chr10 6.05 236/257, 245/245, 236/265, 236/260
    GCPM_1157-1 CCTCCTACCACATATTCCAA AGTGGTTAAAATGCGAGTGT Chr14 17.49 170/170, 173/175, 172/172, 170/175
    GCPM_122-2 GAAACCAGAAAGCAACAATC TGTTTCAACATCACCATGTC Chr02 1.05 201/206, 203/203, 206/206, 201/206
    GCPM_1223-1 CCTGAAGATGAAGATTTTGC TTGAAGTTTGAAGGGAGAAA Chr12 1.04 117/123, 114/117, 117/117, 117/122
    GCPM_1224-2 TGTCTTCTCTCTACAGCAAGG CACCGAGAAGAAACTTTCAG Chr09 11.04 153/153, 109/153, 112/153, 142/142
    GCPM_1033-1 TGAATTAAAGGTGGTGGTTC GGGTGAAATTGCATTTGTAT Chr16 9.83 223/230, 228/228, 230/230
    GCPM_1036-1 AAGTGGAATATTCGCCAAC GCTGGGATGGATCTAGAAA Chr01 35.79 163/163, 170/170, 172/175
    GCPM_1002-1 AAGAACCAGCACTTCCTGTA ATGACTAATCACTTGTGGGC Chr01 11.45 149/149, 159/168, 159/159
    GCPM_1054-1 AGGTCTGTGCAAGGAATAAA GTCTGTAATCAAGCCAAAGC Chr07 0.86 159/164, 159/166, 159/172
    GCPM_1224-1 AAAATGAATTGGCAGAAAGA GCTTCTTAGCTCAACCAGAA Chr09 11.04 196/216, 196/205, 196/207
    GCPM_1023-1 AGAAtAATTGTGGATGGACG TTTAATTGAGATTCGGGCTA Chr11 2.13 114/114, 110/110, 118/118
    GCPM_1175-1 TCATCAACCTGACCTCTACC CAAAGCAAAAACAAACACAA Chr14 13.38 190/209, 190/221, 193/196
    GCPM_1072-2 AGGAAAACAAAGGAGAGGAG ATGCTTAAAAGGGGATCTCT Chr06 4.20 141/146, 143/156, 152/157
    GCPM_11-1 TTGCTGTTAGCATCATTTTC TTGCTCTCTTTGTTGGAGAT Chr01 47.40 154/154, 151/151, 151/154
    GCPM_1116-1 GGGGCTAATATCAGTTTCCT TGAGAAAACCCTGGAAAATA Chr04 0.40 193/199, 199/199
    GCPM_1092-1 TTCTAAATATAAGAtAACC
    AATGAATG
    TATTCCCGCTTGTAATTCCT Chr01 4.82 217/217, 224/224
    GCPM_112-1 TTAGAGGAGAGAACTGCTGC TGGTCTGCAACACAAGATT Chr16 0.14 132/142, 128/132
    GCPM_1145-1 CTGAGAAATCCAACTCCTTG TACAGTAAGAAAGTGCTGCG Chr02 15.10 171/171, 191/191
    GCPM_1041-2 ATGACTTCATTGGGAAGAAA GACGATTTGGACTCCACTAA Chr13 14.59 167/167, 159/159
    GCPM_1079-1 CAAATTGGAGACTTCTCTGC TATTTCAATCTCCCATCGTC Chr05 20.82 222/222, 210/219
    GCPM_1184-1 TCTTGGCGAGAGAAGTAGAG GGATTTGGTGAAAATTGAAG Chr03 14.83 156/168, 154/164
    GCPM_1187-1 TGCTCTTGTATTGTTCCCTT TCTCAAGTAGGGTCCAGCTA Chr03 13.98 192/192, 189/189
    GCPM_1209-1 CGCTGTTGATGGATTACAA GAAAACATAGGAAAGTCGGA Chr12 12.35 132/137, 136/136
    GCPM_1220-1 ACTCTTGTTAAACCAAGCCA ACTACCTTTCTTCCCCTCAC Chr13 10.27 215/215, 204/215
    GCPM_1017-1 GTTTAATTCCCACGTCGTTA CGAATGAAGAAAAACCATTC Chr06 22.44 161/167, 169/173
    GCPM_1025-1 AAGGTGCTCCCACTTACTTT TGTGGCTAGGTAGTTTGGTT Chr03 10.31 135/230, 186/186
    GCPM_124 TTTGAGCACTTCAACTACCA TGTCTTCCCTTAGTCACCAC Chr01 6.33 205/208, 199/208
    GCPM_1242-1 TCGTGCAAATAAAAGCATTA ATGGATATTGTGCTCCTTTG Chr02 15.83 231/231, 229/229
    GCPM_1246 TCAAGTCAGAAACACTTCCA TTGATTCTATGGGTTTCACC Chr10 16.43 222/222, 222/236
    GCPM_1251-1 CTGCATTTGTTTCCTAATTTT AAAATGATAAGCTCGTTTGG Chr04 5.51 143/143, 130/130
    注:a表示SSR标记位点在染色体上的分布位置。Note: a SSR markers were distributed in chromosomes (size in Mb) of Populus.
    下载: 导出CSV

    表  2  毛白杨多态SSR引物库基本信息

    Table  2.   Basic information of polymorphic SSR primer library in Populus tomentosa

    项目 Item    所在染色体 Chromosomal localization未定位引物 Unlocated chromosome
    染色体 Chromosome 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
    引物对数 Number of primers 39 39 17 37 28 32 23 31 13 7 13 18 12 6 6 22 3 15 16 17
    下载: 导出CSV

    表  3  38对SSR核心引物信息表

    Table  3.   38 pairs of SSR core primer information

    编号
    No.
    引物
    Primer
    引物序列(5′−3′)
    Primer sequence (5′−3′)
    NaNeI杂合性
    Heterozygosity
    鉴定效率
    Identification efficiency
    PICHoHe区分种质数
    Distinguishing germplasm number
    区分率
    Distinguishing rate/%
    1 GCPM_1571-1 F: AAGAAGGGTGGTAAGCTTTT 14 1.694 0.958 0.392 0.054 0.410 81 17.27
    R: TCCGTAGATCTCTCTCCAAA
    2 GCPM_1063 F: AGTTAATTGCGCATGTTCTT 11 2.492 1.160 0.520 0.990 0.599 60 14.74
    R: AAACAAACTCCAGCAAACAT
    3 PMGC_2606 F: AATTTACATTTCTTTATCATCACC 10 2.756 1.225 0.569 0.990 0.638 50 12.29
    R: GCTGTCTAACATGCCATTGC
    4 GCPM_1411-1 F: TCAACGACTTTTTCATTGTG 10 1.476 0.778 0.312 0.206 0.323 48 11.79
    R: AGCATTCTTGCTGGTGTTAT
    5 PMGC_2571 F: TCTCGCAGATTCATGTAACCC 10 2.360 1.048 0.491 0.941 0.577 50 12.29
    R: GACTGTATGTTGACCATGCCC
    6 GCPM_1184-1 F: TCTTGGCGAGAGAAGTAGAG 9 1.696 0.913 0.389 0.435 0.411 39 9.58
    R: GGATTTGGTGAAAATTGAAG
    7 GCPM_1504-1 F: AGGTCTGTGCAAGGAATAAA 9 2.400 1.078 0.497 0.995 0.584 41 10.07
    R: GTCTGTAATCAAGCCAAAGC
    8 GCPM_1072-2 F: AGGAAAACAAAGGAGAGGAG 8 2.677 1.198 0.558 0.966 0.627 35 8.60
    R: ATGCTTAAAAGGGGATCTCT
    9 PMGC-2818 F: AAGCTTCATCGTCCTGCTTG 8 2.171 0.903 0.435 0.872 0.540 33 8.11
    R: CGTATCAATTCACGACTCTCG
    10 GCPM_112-1 F: TTAGAGGAGAGAACTGCTGC 7 2.647 1.168 0.548 0.990 0.623 25 6.14
    R: TGGTCTGCAACACAAGATT
    11 GCPM-2012 F: GGTGATGAAGATCTGGGATA 7 2.549 1.067 0.528 0.980 0.608 24 5.90
    R: ACCCAAATTACAGAACAACG
    12 GCPM_88-1 F: TAAACTGCTTTTAGTTGCCC 7 1.088 0.230 0.080 0.025 0.081 21 5.16
    R: TCCATGTTGTGTGATCTGAG
    13 PMGC_2088-2 F: TCACAAAAGGTTAACGACTTCG 7 1.381 0.561 0.254 0.017 0.276 24 5.89
    R: CAGTACTCAGCTGCAGGTCC
    14 GCPM_2627-1 F: TAAGTCCCACTACACCCAAC 6 2.325 0.950 0.477 0.995 0.571 20 4.91
    R: GAGTTCGAGAGAGGGAATCT
    15 LG_V-2 F: AAAGAAACCAGACCACACAC 6 2.312 0.967 0.473 0.995 0.568 19 4.67
    R: CGCTTGCCTTAATTAACAGT
    16 GCPM-2768 F: CAGCAAAATCATCACAAATC 6 2.136 0.852 0.422 0.988 0.532 19 4.67
    R: AGGGTTTGGTAGAGAAGACC
    17 GCPM_2570-1 F: AACCCACTTCCTCTCTCTGT 6 2.362 0.996 0.495 0.928 0.577 19 4.67
    R: TGAGACTTCCGACTCGTAG
    18 LG_VIII-3 F: ATCCGACTTCGATATCTTCA 6 1.544 0.698 0.328 0.032 0.353 20 4.91
    R: CTACCTGAAACACAGGAAGC
    19 GCPM_1153-1 F: TTCCTTTCACACAATGACAA 6 1.381 0.633 0.265 0.273 0.276 18 4.42
    R: TTTAAAAACTGGGTCCGTAA
    20 GCPM_3652-1 F: TAAACTGCTTTTAGTTGCCC 6 1.069 0.180 0.065 0.015 0.065 18 4.42
    R: TCCATGTTGTGTGATCTGAG
    21 PMGC_223 F: CGATGAGGTTGAAGAAGTCG 5 2.309 0.939 0.472 0.831 0.568 14 3.44
    R: ATATATGTACCGGCACGCCAC
    22 GCPM_3474-1 F: GATCCGAAAACAACAACAAT 5 2.212 0.901 0.445 0.985 0.549 13 3.19
    R: ACCCCTTTCTCTTCTCAATC
    23 GCPM-124 F: TTTGAGCACTTCAACTACCA 5 2.045 0.757 0.391 0.993 0.512 11 2.70
    R: TGTCTTCCCTTAGTCACCAC
    24 LG_XVI-9 F: CTCGCAGCTCTTCTCATAGT 5 2.326 0.953 0.476 0.946 0.571 13 3.19
    R: CCTACCCATTTATGACCAAA
    25 PMGC_2140 F: GCTGTCAGAATCAAACACTTC 5 1.405 0.617 0.274 0.197 0.289 11 2.70
    R: AAGCAGATAACTAAGACATGCC
    26 GCPM_1056-1 F: CCACATTTATGTCCCTCTGT 5 1.485 0.589 0.288 0.017 0.327 13 3.19
    R: AAGCTGAAGCTGCTCTCTTA
    27 ORPM-221 F: TGGAGGCTGTCTGTTTTGTG 5 1.035 0.106 0.034 0.0074 0.034 10 2.26
    R: AGATTTGAGCGACTCCGAAA
    28 GCPM_1832-1 F: TTACTTGCTAGCTGCCAATC 5 1.222 0.428 0.176 0.039 0.182 10 2.46
    R: CCTAAAAGTTTGTCTATGCGA
    29 LG_XVI-7 F: ACAAATCAAAGTCACAGCCT 4 2.201 0.861 0.441 0.985 0.546 8 1.97
    R: ATAGTGTTCAATCGGACCTG
    30 GCPM_1572-2 F: TGTTGTGGATTGTGATTAGG 4 2.074 0.781 0.402 0.995 0.519 9 2.21
    R: ACACTTCCAAACAGAACACC
    31 LG_VIII-4 F: ATCTTTCAAGAAGCTTGACG 4 1.125 0.240 0.106 0.017 0.112 7 1.72
    R: AGCATTCTTGCTGGTGTTAT
    32 GCPM_3367-1 F: CAGGACATTTAACTCCTTCG 4 1.110 0.236 0.096 0.015 0.099 8 1.97
    R: TTAAGTCCTCAACAGAGGGA
    33 GCPM_XIX-11 F: CAACATGAAATGAGCTGCTA 3 2.043 0.746 0.391 0.872 0.511 10 2.46
    R: TCCACATGATGTCTGATTTG
    34 GCPM_1524-1 F: TTCAATGGAAAGGGATAATG 3 2.359 0.933 0.485 0.961 0.577 9 2.21
    R: TCATTTGTAAAACATCACGC
    35 LG_XVI-6 F: ATAGCGATCATCAAAGGAAA 3 2.295 0.906 0.470 0.985 0.565 8 1.97
    R: AAATATTCATGTGGAGGCAC
    36 LG_IX-1 F: CCAACTTCAATGCTAGGAAC 3 2.260 0.894 0.463 0.948 0.558 5 1.06
    R: TGGTAAGCCTGAGGATACAC
    37 ORPM_197 F: GTCAGTTTGCCCTCTTCGTC 2 1.380 0.447 0.237 0.138 0.275 3 0.74
    R: TGAGGGCGTCTCCTCTTTTA
    38 LG_III-2 F: ATTGATTATATTTGCCGCAT 2 1.244 0.347 0.177 0.202 0.197 2 0.49
    R: TGGACATCTCACTACCTTCC
    均值 Average value 6.017 1.912 0.770 0.378 0.601 0.427
    注:Na. 等位标记数量;Ne. 有效等位标记数量;I. Shannon’s信息指数;Ho. 观察杂合度;He. 期望杂合度;PIC. 多态性信息含量。Notes: Na, number of alleles per locus; Ne, effective number of alleles; I, Shannon’s information index; Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphism information content.
    下载: 导出CSV

    表  4  毛白杨指纹图谱构建最佳引物组合筛选

    Table  4.   Optimum primer combination applied to construction of the fingerprint of Populus tomentosa

    引物组合
    Primer combination
    鉴定效率
    Identification efficiency
    区分种质数
    Distinguishing
    germplasm number
    区分率
    Distinguishing rate/%
    No.1—No.5 156 33.17
    No.1—No.10 262 55.77
    No.1—No.15 361 76.97
    No.1—No.20 448 95.52
    No.1—No.25 456 97.29
    No.1—No.30 463 98.77
    No.1—No.38 469 100.00
    下载: 导出CSV

    表  5  毛白杨优株无性系SSR指纹代码

    Table  5.   SSR fingerprint code of Populus tomentosa superior clones

    编号 No.SSR指纹代码 SSR fingerprint code
    2-3 1A391/396B236/246C165/184D206/206E191/191F178/178G388/388H237/237I133/143J237/257K387/387L157/164M242/242N196/207O180/189P170/173Q115/130R221/234S138/140T194/196U352/368V126/138W152/152X121/121Y145/145Z144/144AA121/133AB398/398AC178/178AD118/118AE151/157AF185/185AG153/163AH166/191AI146/146AJ106/118AK220/220AM205/209
    314 1A396/396B236/246C165/184D206/206E191/191F176/178G388/405H237/237I131/133J237/257K387/387L157/162M236/236N196/207O180/180P170/175Q115/130R218/221S138/140T194/204U352/364V129/138W156/156X121/121Y145/145Z144/144AA121/133AB398/405AC178/178AD118/123AE148/151AF185/185AG153/153AH166/185AI146/146AJ106/118AK220/220AM205/209
    3-3-4 2A396/396B236/246C165/184D206/208E191/191F176/178G388/405H237/237I131/133J237/257K387/387L157/162M220/236N196/207O180/180P170/173Q115/130R218/221S138/140T194/204U352/364V129/138W156/156X121/121Y145/145Z144/144AA121/133AB398/405AC178/178AD118/123AE148/151AF185/185AG153/153AH166/185AI146/146AJ106/118AK220/220AM205/209
    3000-4 1A396/396B236/246C165/184D206/206E191/191F176/178G388/405H237/237I131/133J237/257K387/387L157/162M236/236N196/207O180/180P170/173Q115/130R218/221S138/140T194/204U352/364V129/138W156/156X121/121Y145/145Z144/144AA121/133AB405/405AC178/178AD118/123AE185/185AF185/185AG153/153AH166/185AI146/146AJ106/118AK220/220AM205/209
    6319 2A396/396B236/246C165/184D206/206E191/191F176/178G388/405H237/237I131/133J237/257K387/387L157/162M236/236N196/207O180/180P170/170Q115/130R218/221S138/140T194/204U352/364V129/138W156/156X121/121Y145/145Z144/144AA121/133AB398/405AC178/178AD118/123AE148/151AF185/185AG153/153AH166/185AI146/146AJ106/118AK220/220AM205/209
    注:本表格只示例部分毛白杨优株无性系指纹代码。Note: only part of the clone fingerprint code of Populus tomentosa is showed in this table as example.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-01-15
  • 修回日期:  2019-05-12
  • 网络出版日期:  2019-07-04
  • 刊出日期:  2019-07-01

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