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鉴别紫芝菌株的PCR引物筛选及其序列比对验证

钟礼义 陈体强 刘新锐 应正河 杨驰

钟礼义,陈体强,刘新锐,等. 鉴别紫芝菌株的PCR引物筛选及其序列比对验证 [J]. 福建农业学报,2020,35(7):725−730 doi: 10.19303/j.issn.1008-0384.2020.07.005
引用本文: 钟礼义,陈体强,刘新锐,等. 鉴别紫芝菌株的PCR引物筛选及其序列比对验证 [J]. 福建农业学报,2020,35(7):725−730 doi: 10.19303/j.issn.1008-0384.2020.07.005
ZHONG L Y, CHEN T Q, LIU X R, et al. Selection and Sequence Alignment of PCR Primers for Identifying Zizhi Strain [J]. Fujian Journal of Agricultural Sciences,2020,35(7):725−730 doi: 10.19303/j.issn.1008-0384.2020.07.005
Citation: ZHONG L Y, CHEN T Q, LIU X R, et al. Selection and Sequence Alignment of PCR Primers for Identifying Zizhi Strain [J]. Fujian Journal of Agricultural Sciences,2020,35(7):725−730 doi: 10.19303/j.issn.1008-0384.2020.07.005

鉴别紫芝菌株的PCR引物筛选及其序列比对验证

doi: 10.19303/j.issn.1008-0384.2020.07.005
基金项目: 福建省财政专项-福建省农业科学院科技创新团队建设项目(STIT2017-2-8,STIT2017-1-6)
详细信息
    作者简介:

    钟礼义(1968−),男,推广研究员,研究方向:食药用菌技术推广应用(E-mail:fjzly99@163.com

    通讯作者:

    陈体强(1968−),男,研究员,研究方向:药用菌与闽产中药材(E-mail:chen_tiqiang@189.cn

  • 中图分类号: S 646.2

Selection and Sequence Alignment of PCR Primers for Identifying Zizhi Strain

  • 摘要:   目的  紫芝S2(品种名:武芝2号)是近年来在福建及周边省份推广应用的紫芝栽培新品种,为了避免与遗传背景不同的栽培菌株混杂而建立有效的分子标记。  方法  采用PCR扩增筛选条带清晰稳定、呈现多态性的引物,根据菌株间UPMGA聚类构建系统发生树,通过遗传距离确定主要栽培菌株间的亲缘关系,并与菌株间的拮抗反应结果相映证,进而与‘紫芝S2’基因组序列进行比对验证。  结果  筛选得到能清晰且稳定地扩增出特异性或多态性条带的2个RAPD-PCR引物和3个ISSR-PCR引物,比对发现这5个引物短序列与‘紫芝S2’基因组序列完全匹配上的位点与数目不同。  结论  基于紫芝S2基因组序列比对,确认其中3个引物(ISSR13、S1326和S1506)可以有效地用于紫芝栽培菌株鉴定。
  • 图  1  紫芝菌株间的拮抗现象

    注:A:紫芝S2-紫芝S2;B:紫芝S2-紫芝X5;C:紫芝S2-紫芝1;D:紫芝S2-紫芝2;E:紫芝S2-紫芝3;F:紫芝S2-紫芝4。

    Figure  1.  Antagonism between different Zizhi strains

    Note: A: Zizhi S2 vs. Zizhi S2; B: Zizhi S2 vs. Zizhi X5; C: Zizhi S2 vs. Zizhi 1; D: Zizhi S2 vs. Zizhi 2; E: Zizhi S2 vs. Zizhi 3; and, F: Zizhi S2 vs. Zizhi 4.

    图  2  RAPD-PCR扩增图谱

    注:泳道1~6:紫芝S2、紫芝X5、紫芝-1、紫芝-2、紫芝-3和紫芝-4, M为分子标记DL2000。

    Figure  2.  RAPD-PCR amplifications

    Note: Electrophoretic lines 1-6: Zizhi S2, Zizhi X5, Zizhi 1, Zizhi 2, Zizhi 3, and Zizhi 4; M: molecular marker DL2000.

    图  3  ISSR -PCR扩增图谱

    注:泳道1~6:紫芝S2、紫芝X5、紫芝-1、紫芝-2、紫芝-3和紫芝-4, M为分子标记物DL2000。

    Figure  3.  ISSR-PCR amplifications

    Note: Line 1-6: Electrophoretic results of Zizhi S2, Zizhi X5, Zizhi 1, Zizhi 2, Zizhi 3, Zizhi 4 and the M is the molecular marker DL2000.

    图  4  紫芝菌株的遗传聚类图

    Figure  4.  Clustering dendrogram of Zizhi strains

    表  1  供试紫芝菌株信息

    Table  1.   Strains of cultivated Zizhi used in study

    编号
    No.
    菌株名
    Strain name
    来源
    Source
    1 紫芝 S2 福建省农作物品种审定委员会认定品种(武芝2号)
    Zizhi S2 New varieties identified by Fujian Crop Variety Approval Committee(Wuzhi No.2)
    2 紫芝 X5 福建省武平县食用菌技术推广服务站
    Zizhi X5 Fujian Wuping County Edible Fungi Technology Extension Service Statio
    3 紫芝1 华中农业大学菌种实验中心
    Zizhi 1 Microbial Species Laboratory Center of Huazhong Agricultural Universit
    4 紫芝2 上海市农业科学院食用菌研究所
    Zizhi 2 Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences
    5 紫芝3 江苏省江都天达食用菌研究所
    Zizhi 3 Jiangdu Tianda Institute of Edible Fungi, Jiangsu Province
    6 紫芝4 辽宁省锦州市亚泰食用菌研究所
    Zizhi4 Yatai Institute of Edible Fungi, Jinzhou City, Liaoning Province
    下载: 导出CSV

    表  2  PCR引物与基因组比对结果

    Table  2.   Blast result of PCR primer sequences on genomes

    引物名称_碱基数
    Primer name _Base number
    比对上引物序列的 Scaffold 数目
    All scaffold number
    与引物匹配的位点数
    All primer number
    比对相似度
    Identity(%)
    不允许错配(空位为0)No mismatches allowed(Gap=0)
    ISSR13_1456100
    S1326_1048158100
    S1506_1047172100
    允许一个错配(空位为0)Allow one mismatch(Gap=0)
    ISSR2_17111493.75~94.12
    ISSR13_141831100
    S1326_1048158100
    S1506_1047172100
    注:引物ISSR13短序列出现在Scaffold_1上2个位点,引物S1326出现在Scaffold_1上15个位点;S1506引物出现在Scaffold_1上17个位点,出现在Scaffold_60上5个位点。
    Note: Short sequence of ISSR 13 appeared at two sites on Scaffold No. 1; S1326 at 15 sites on Scaffold No. 1; and S1506 at 17 sites on Scaffold No. 1 and 5 sites on Scaffold No. 60.
    下载: 导出CSV
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  • 收稿日期:  2020-01-16
  • 修回日期:  2020-06-03
  • 刊出日期:  2020-07-31

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