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基于石榴全基因组序列的SSR标记开发及鉴定

洪文娟 郝兆祥 刘康佳 罗华 毕润霞 苑兆和 宗世祥 王君

洪文娟, 郝兆祥, 刘康佳, 罗华, 毕润霞, 苑兆和, 宗世祥, 王君. 基于石榴全基因组序列的SSR标记开发及鉴定[J]. 北京林业大学学报, 2019, 41(8): 38-47. doi: 10.13332/j.1000-1522.20190167
引用本文: 洪文娟, 郝兆祥, 刘康佳, 罗华, 毕润霞, 苑兆和, 宗世祥, 王君. 基于石榴全基因组序列的SSR标记开发及鉴定[J]. 北京林业大学学报, 2019, 41(8): 38-47. doi: 10.13332/j.1000-1522.20190167
Hong Wenjuan, Hao Zhaoxiang, Liu Kangjia, Luo Hua, Bi Runxia, Yuan Zhaohe, Zong Shixiang, Wang Jun. Development and identification of SSR molecular markers based on whole genomic sequences of Punica granatum[J]. Journal of Beijing Forestry University, 2019, 41(8): 38-47. doi: 10.13332/j.1000-1522.20190167
Citation: Hong Wenjuan, Hao Zhaoxiang, Liu Kangjia, Luo Hua, Bi Runxia, Yuan Zhaohe, Zong Shixiang, Wang Jun. Development and identification of SSR molecular markers based on whole genomic sequences of Punica granatum[J]. Journal of Beijing Forestry University, 2019, 41(8): 38-47. doi: 10.13332/j.1000-1522.20190167

基于石榴全基因组序列的SSR标记开发及鉴定

doi: 10.13332/j.1000-1522.20190167
基金项目: 国家林业局业务委托项目(20180001),2019校专项−科技创新服务能力建设−科研基地建设−林果业生态环境功能提升协同创新中心(2011协同创新中心)(PXM2019_014207_000099)
详细信息
    作者简介:

    洪文娟。主要研究方向:经济林品种鉴定。 Email:1368030722@qq.com 地址:100083 北京市海淀区清华东路35号北京林业大学118信箱

    责任作者:

    王君,教授,博士生导师。主要研究方向:林木细胞遗传学与染色体工程育种。Email: wangjun@bjfu.edu.cn 地址:同上

  • 中图分类号: S665.4; Q943.2

Development and identification of SSR molecular markers based on whole genomic sequences of Punica granatum

  • 摘要: 目的利用cDNA文库筛选的石榴SSR多态性标记数量有限,为进一步推动石榴遗传多样性分析、遗传图谱构建、品种鉴定等研究,有必要系统开发高效稳定的分子标记位点。方法本研究根据已发表的石榴基因组测序数据利用MISA软件对1 ~ 6核苷酸重复的SSR位点进行了查找,分析了不同类型SSR位点的序列特征,进而设计引物并检测了引物的有效性和多态性。结果(1)石榴基因组中共检测到146 445个SSR位点,其中以单核苷酸重复型SSR最多(占51.95%),六核苷酸重复型最少(仅占0.38%);SSR序列以A/T碱基占主导,具有偏向性。(2)石榴基因组SSR序列长度变化范围为10 ~ 252 bp,平均长度15.48 bp。不同长度重复单元类型的SSR序列长度存在丰富变异,呈现随着重复次数增多,SSR序列丰度减少的趋势。(3)根据不同类型SSR位点设计并合成引物140对,其中119对在12份石榴种质中可扩增出有效条带,41对可产生多态性条带,PIC值在0.007 ~ 0.566之间;从中筛选出多态性较高、稳定性好的引物15对,共检测到等位基因44个,平均每个SSR位点检测到2.933 3个等位基因。结论利用石榴全基因组序列可实现SSR标记的大规模开发,并可鉴定出大量适用于石榴遗传多样性分析、遗传图谱构建、品种鉴定等研究的SSR引物。相关研究为石榴的遗传育种研究提供了丰富的SSR序列信息和标记资源。

     

  • 图  1  不同类型SSRs在石榴基因组最长的10条Scaffold上的数量变化

    Figure  1.  Variation in number of different SSR types in the longest 10 Scaffolds in Punica granatum genome

    图  2  石榴基因组不同长度微卫星的出现频率

    Figure  2.  Frequency of SSRs with different length in Punica granatum genome

    图  3  不同长度重复单元微卫星的序列长度变异情况

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

    图  4  有效SSR位点的PIC值与SSR长度相关分析

    Figure  4.  Correlation analyses between PIC value and SSR length in the valid loci

    图  5  引物PG_66_86在12个石榴样品中的毛细管电泳检测图

    Figure  5.  Analysis of capillary electrophoresis detection of 12 samples of pomegranate with PG_66_86 primer

    表  1  石榴测试品种编号

    Table  1.   Tested varieties of Punica granatum

    编号 No.品种 Variety来源 Source
    1 ‘秋艳’ P. ‘Qiuyan’ 山东枣庄市峄城 Yicheng, Zaozhuang City, Shandong Province
    2 ‘大叶满天红’ P. ‘Dayemantianhong’ 河北元氏县 Yuanshi County, Hebei Province
    3 ‘新疆和田酸’ P. ‘Xinjianghetiansuan’ 新疆和田 Hetian, Xinjiang Autonomous Region
    4 ‘开封四季红’ P. ‘Kaifengsijihong’ 河南开封市 Kaifeng City, Henan Province
    5 ‘怀远玉石籽’ P. ‘Huaiyuanyushizi’ 安徽怀远县 Huaiyuan County, Anhui Province
    6 ‘淮北丰产青皮’ P. ‘Huaibeifengchanqingpi’ 安徽淮北市 Huaibei City, Anhui Province
    7 ‘泰山三白甜’ P. ‘Taishansanbaitian’ 山东泰安市 Taian City, Shandong Province
    8 ‘峄城粉红牡丹’ P. ‘Yichengfenhongmudan’ 山东枣庄市峄城 Yicheng, Zaozhuang City, Shandong Province
    9 ‘墨石榴’ P. ‘Moshiliu’ 山东枣庄市峄城 Yicheng, Zaozhuang City, Shandong Province
    10 ‘会理青皮软籽’ P. ‘Huiliqingpiruanzi’ 四川会理县 Huili County, Sichuan Province
    11 ‘建水红玛瑙’ P. ‘Jianshuihongmanao’ 云南建水县 Jianshui County, Yunnan Province
    12 ‘突尼斯软籽’ P. ‘Tunisiruanzi’ 突尼斯 Tunisia
    下载: 导出CSV

    表  2  石榴基因组中部分SSR重复单元的分布特征

    Table  2.   Feature of parts of SSR motifs in Punica granatum genome

    重复单元 Repeat motif数目 Number重复次数 Repeat time平均长度 Average length/bp比例 Proportion/%
    A/T 72 892 10 ~ 56 11.42 49.77
    C/G 3 179 10 ~ 222 15.20 2.17
    AT/TA 34 270 6 ~ 58 19.07 23.40
    CG/GC 163 6 ~ 10 13.30 0.11
    AC/GT 3 470 6 ~ 41 16.52 2.37
    AG/CT 15 671 6 ~ 126 19.96 10.70
    AAC/GTT 326 5 ~ 26 18.51 0.22
    AAG/CTT 3 458 5 ~ 43 20.61 2.36
    AAT/ATT 5 339 5 ~ 24 21.76 3.65
    ACC/GGT 323 5 ~ 12 17.63 0.22
    ACG/CGT 127 5 ~ 15 17.55 0.09
    ACT/AGT 156 5 ~ 27 19.10 0.11
    AGC/CTG 599 5 ~ 19 17.69 0.41
    AGG/CCT 1 272 5 ~ 18 18.80 0.87
    ATC/ATG 983 5 ~ 16 18.17 0.67
    CCG/CGG 173 5 ~ 15 16.63 0.12
    下载: 导出CSV

    表  3  筛选的15对石榴基因组SSR位点引物及多态性信息

    Table  3.   SSR site primer and polymorphic information for selected 15 pairs of pomegranate genomic groups

    引物名称
    Primer ID
    SSR类型
    Type of SSR
    重复单元
    Repeat motif
    引物序列
    Primer sequence
    等位
    基因数
    Number of alleles (Na)
    有效等位
    基因数
    Effective allelic marker number (Ne)
    Shannon
    信息指数
    Shannon diversity index (I)
    多态
    信息含量
    Polymorphic information content (PIC)
    PG_25_548 p2 (AT)9 F: ATATGGCGGCATGAGAGTTC 4.000 0 2.742 9 1.139 5 0.566
    R: GTAATAGCATGCCCTTTGCC
    PG_98_83 p2 (TA)11 F: AGGGGCAAAACCCTTACATC 5.000 0 2.285 7 1.117 1 0.525
    R: TCGGCCCAATAAATGGAAT
    PG_44_17 p3 (AGA)6 F: GGGCGAAGAATTACAGGTGA 2.000 0 1.945 9 0.679 2 0.368
    R: ATAATGGCTCCTGTGGAACG
    PG_140_166 p4 (TATC)9 F: AAAGCATGCGAAAGGATGAT 3.000 0 2.341 5 0.922 2 0.479
    R: CAATGCCAATGTTACGGATG
    PG_4_1646 p4 (AAAT)5 F: CCAAAGGATGAGGAATCGAA 2.000 0 1.882 4 0.661 6 0.359
    R: TTTGACCCGATCTACCTCGT
    PG_105_152 p4 (GTCT)7 F: CGATGTGTACAGTTGTGGGC 3.000 0 2.104 3 0.896 1 0.466
    R: CCGTTTTAGCTCCAGTCTGC
    PG_128_96 p5 (ATAGG)7 F: GAGGAGTCAATTCGACCCAA 3.000 0 2.071 9 0.860 2 0.444
    R: CATCGAATTCTATTCCCTCCC
    PG_13_2441 p5 (AGTTG)5 F: CCGTTTTGGTTTGTTCTGGT 3.000 0 1.967 5 0.792 5 0.408
    R: CTCAACTCAACTCCACTTATCTTCA
    PG_32_463 p5 (TTGTT)5 F: CCCACGTAGGAAGGTGAAAA 2.000 0 1.945 9 0.679 2 0.368
    R: GGCTGGCATTTCAGTTCAGT
    PG_47_500 p5 (AATAT)5 F: TATGCCCGGTGTTAACCAAT 2.000 0 1.945 9 0.679 2 0.368
    R: TGATCACTCACACCAGCCTC
    PG_20_939 p6 (TATAGA)6 F: GGGTCCACAACAACTCCACT 4.000 0 2.594 6 1.109 7 0.557
    R: TTTTCCATTCCTTTTCCCCT
    PG_22_2120 p6 (AATCAA)5 F: CCCCGTTTGGATTCAAAGAT 3.000 0 1.811 3 0.778 1 0.397
    R: GAGGGGAAGCTGAGACAGAG
    PG_66_86 p6 (GAGGGA)6 F: GCTGGTAGGAGTGCTTGAGG 3.000 0 2.133 3 0.829 3 0.428
    R: TTCCCCTAATTTGGTGGGTT
    PG_28_765 p6 (CAAAAT)5 F: TTGTCGACGAACTCGAACAG 2.000 0 2.000 0 0.693 1 0.375
    R: TGCATGCAGACAGCTTTAGG
    PG_117_30 C (GCA)5(GCC)8 F: GGACGAGATACGGCAGAGAC 3.000 0 1.766 9 0.723 3 0.420
    R: TGCAGAGGATTGCTGAGATG
    平均值 Mean 2.933 3 2.102 7 0.837 4 0.437
    下载: 导出CSV
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  • 收稿日期:  2019-03-28
  • 修回日期:  2019-05-11
  • 网络出版日期:  2019-06-12
  • 刊出日期:  2019-08-01

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