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基于InDel标记的斑节对虾早期性别鉴定方法的建立

黄智康 江世贵 周发林 黄建华 杨其彬 姜松 李运东 杨丽诗

黄智康, 江世贵, 周发林, 黄建华, 杨其彬, 姜松, 李运东, 杨丽诗. 基于InDel标记的斑节对虾早期性别鉴定方法的建立[J]. 南方水产科学, 2020, 16(3): 113-118. doi: 10.12131/20190222
引用本文: 黄智康, 江世贵, 周发林, 黄建华, 杨其彬, 姜松, 李运东, 杨丽诗. 基于InDel标记的斑节对虾早期性别鉴定方法的建立[J]. 南方水产科学, 2020, 16(3): 113-118. doi: 10.12131/20190222
Zhikang HUANG, Shigui JIANG, Falin ZHOU, Jianhua HUANG, Qibin YANG, Song JIANG, Yundong LI, Lishi YANG. Establishment of a sex identification method for black tiger shrimp (Penaeus monodon) in embryonic and early postembryonic development based on an InDel molecular marker[J]. South China Fisheries Science, 2020, 16(3): 113-118. doi: 10.12131/20190222
Citation: Zhikang HUANG, Shigui JIANG, Falin ZHOU, Jianhua HUANG, Qibin YANG, Song JIANG, Yundong LI, Lishi YANG. Establishment of a sex identification method for black tiger shrimp (Penaeus monodon) in embryonic and early postembryonic development based on an InDel molecular marker[J]. South China Fisheries Science, 2020, 16(3): 113-118. doi: 10.12131/20190222

基于InDel标记的斑节对虾早期性别鉴定方法的建立

doi: 10.12131/20190222
基金项目: 广东省现代农业产业技术体系创新团队建设专项 (2019KJ149);现代农业 (虾) 产业技术体系建设专项资金 (CARS-48);中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助 (2019TS09)
详细信息
    作者简介:

    黄智康 (1993—),男,硕士研究生,研究方向为水产动物遗传育种与繁育。E-mail: hzhzk1993@163.com

    通讯作者:

    杨丽诗 (1981—),女,博士,副研究员,从事水产遗传育种与生物技术研究。E-mail: yangls2016@163.com

  • 中图分类号: S 917.4

Establishment of a sex identification method for black tiger shrimp (Penaeus monodon) in embryonic and early postembryonic development based on an InDel molecular marker

  • 摘要:

    为分辨斑节对虾 (Penaeus monodon) 发育早期的性别,利用改良的插入/缺失 (InDel) 标记追踪了从受精卵、无节幼体、溞状幼体、糠虾幼体、仔虾后1 d (PL1)、仔虾后30 d (PL30)及亚成虾个体的性别,建立了测序和荧光定量PCR熔解曲线的检测方法。结果表明,InDel标记可扩增获得雌雄特异性序列,用测序法判定亚成虾性别的结果与外部观察结果一致,准确率达100%,对受精卵、无节幼体、溞状幼体及糠虾幼体均可得到雌雄特异性序列;进而建立了基于SYBR Green实时荧光定量PCR (Quantitative real-time polymerase chain reaction,qRT-PCR) 熔解曲线的快速鉴定斑节对虾性别的方法,其中雄性特异序列的熔解温度 (Melting temperature, Tm)为 (79.10±0.10) ℃,雌性为 (78.45±0.20) ℃,通过特异的熔解曲线可准确区分PL30、亚成虾和无节幼体Ⅵ期个体,准确率可达96.3%以上。

  • 图  1  斑节对虾性别特异基因组序列

    阴影表示雌雄特异的单个碱基和雌虾特有的6 bp插入序列

    Figure  1.  Sex-specific genome sequence of P. monodon

    The insertion in female shrimp and specific bases in both sexes are shaded.

    图  2  斑节对虾亚成虾雌雄个体测序结果

    a. 雌虾测序结果;b. 雄虾测序结果;红框表示雌雄特异的单个碱基和雌虾特有的6 bp插入序列

    Figure  2.  Sequencing results of female and male P. monodon subadult

    a. Sequencing results of female shrimp; b. Sequencing results of male shrimp. Insertion in female shrimp and specific bases in both sexes are in red frames.

    图  3  斑节对虾雌、雄亚成虾实时荧光定量熔解曲线及熔解峰

    a. 雌虾熔解曲线及熔解峰;b. 雄虾熔解曲线及熔解峰;c. 雌、雄虾熔解曲线及熔解峰对比

    Figure  3.  Melting curve and melting peak of quantitative real-time PCR of female and male P. monodon subadults

    a. Melting curve and melting peak of female shrimps; b. Melting curve and melting peak of male shrimps; c. Comparison of melting curve and melting peak between female and male shrimps

    表  1  InDel标记引物

    Table  1.   Primers of InDel marker

    引物
    Primer
    序列 (5'–3')
    Sequence
    Sex-InDel-F TAAAGATCCTATTTTCAAATGC
    Sex-InDel-R CGAGGGAATGTCACTTTG
    下载: 导出CSV

    表  2  斑节对虾性别鉴定方法的比较

    Table  2.   Comparison of different methods for sex identification of P. monodon %

    鉴定方法
    Identification method
    受精卵
    Zygote
    无节幼体Ⅰ−Ⅴ期
    Nauplii I−V
    无节幼
    体Ⅵ期
    Nauplii
    VI
    溞状
    幼体
    Zoea
    larvae
    糠虾
    幼体
    Mysis
    larvae
    仔虾后
    1 d
    PL1
    仔虾后
    30 d
    PL30
    雌性亚
    成虾
    Female subadult
    雄性亚
    成虾
    Male subadult
    外部观察
    External observation
    吻合率 100 100
    InDel标记测序法
    InDel marker sequencing
    成功率 100 100 100 100 100 100 100 100 100
    InDel标记实时荧光定量熔解曲线分析
    Melting curve of InDel marker by quantitative real-time PCR
    扩增
    成功率
    0 86 100 90 80 90 100 96.7 90
    吻合率 0 63 100 89 63 56 100 96.6 96.3
    注:−. 不能鉴别个体性别 Note: −. The sex can not be identified.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-11-02
  • 修回日期:  2020-02-11
  • 录用日期:  2020-02-25
  • 网络出版日期:  2020-09-28
  • 刊出日期:  2020-06-05

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