作物杂志,2019, 第3期: 66–72 doi: 10.16035/j.issn.1001-7283.2019.03.011

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

工业大麻雄性相关RAPD和SCAR标记的筛选与鉴定

姜颖1,2,冯乃杰2,王晓楠1,韩喜财1,韩承伟1,赵越1,曹焜1,孙宇峰1,李振伟1   

  1. 1 黑龙江省科学院大庆分院,163319,黑龙江大庆
    2 黑龙江八一农垦大学农学院,163319,黑龙江大庆
  • 收稿日期:2018-11-07 修回日期:2019-02-25 出版日期:2019-06-15 发布日期:2019-06-12
  • 通讯作者: 孙宇峰,李振伟
  • 作者简介:姜颖,助理研究员,研究方向为大麻遗传育种;
  • 基金资助:
    黑龙江省科学院院长基金(YZ2018DQ02);黑龙江省青年科学基金(QC2016037)

Screening and Identification of Male-Specific RAPD and SCAR Markers in Cannabis sativa L. (Industrial Hemp)

Jiang Ying1,2,Feng Naijie2,Wang Xiaonan1,Han Xicai1,Han Chengwei1,Zhao Yue1,Cao Kun1,Sun Yufeng1,Li Zhenwei1   

  1. 1 Daqing Branch of Heilongjiang Academy of Sciences, Daqing 163319, Heilongjiang, China
    2 College of Agronomy, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
  • Received:2018-11-07 Revised:2019-02-25 Online:2019-06-15 Published:2019-06-12
  • Contact: Yufeng Sun,Zhenwei Li

摘要:

利用42条RAPD(Random Amplified Polymorphic DNA)随机扩增引物分析工业大麻品种“火麻一号”组成的雄性或雌性DNA池(DNA pools),结果显示,引物OPV-08扩增得到一条大小为869bp与工业大麻雄性相关的特异条带。根据测序结果,合成了两条SCAR(Sequence Characterized Amplified Region)标记引物,该SCAR标记不仅可以对工业大麻雌雄异株材料花期已知性别的雌雄植株进行准确鉴定,还可以对幼苗期未知性别的大麻雌雄植株进行鉴定;也可对雌雄同株材料可能出现的雄化进行早期鉴定。这不仅为工业大麻早期性别鉴定提供基础,且为减少雌雄同株材料的雄化提供支撑。

关键词: 工业大麻, 性别鉴定, 雄性, RAPD, SCAR

Abstract:

The male DNA pools or female DNA pools of "Huoma No.1" were analyzed respectively by 42 RAPD random amplified primers. The results showed that a male-specific DNA fragment with a length of 869bp was amplified by OPV-08. Two SCAR primers were constructed according to the sequencing results. The primers of SCAR marker could identify the different genders of hemp not only in the known sex plants at flowering stage but also in the unknown sex seedlings at seedling stage. That could also be used for early identification of the appearance of male plant in monoecious industrial hemp. This provides a basis for early sex identification of industrial hemp, also provides support for reducing the appearance of male plant in monoecious industrial hemp.

Key words: Cannabis sativa L. (industrial hemp), Sex identification, Male, RAPD (random amplified polymorphic DNA), SCAR (sequence characterized amplified region)

表1

RAPD随机引物及核苷酸序列(5′-3′)"

引物Primer 序列Sequence 引物Primer 序列Sequence 引物Primer 序列Sequence
OPH-18 GAATCGGCCA OPP-08 ACATCGCCCA OPZ-06 GTGCCGTTCA
OPI-07 CAGCGACAAG OPP-11 AACGCGTCGG OPZ-13 GACTAAGCCC
OPO-10 TCAGAGCGCC OPP-12 AAGGGCGAGT OPU-05 TTGGCGGCCT
OPO-12 CAGTGCTGTG OPP-15 GGAAGCCAAC OPU-12 TCACCAGCCA
OPO-15 TGGCGTCCTT OPP-16 CCAAGCTGCC OPU-15 ACGGGCCAGT
OPY-05 GGCTGCGACA OPP-17 TGACCCGCCT OPU-16 CTGCGCTGGA
OPY-11 AGACGATGGG OPS-11 AGTCGGGTGG OPR-08 CCCGTTGCCT
OPY-15 AGTCGCCCTT OPS-17 TGGGGACCAC OPR-12 ACAGGTGCGT
OPY-16 GGGCCAATGT OPS-19 GAGTCAGCAG OPR-13 GGACGACAAG
OPT-07 GGCAGGCTGT OPX-12 TCGCCAGCCA OPV-06 ACGCCCAGGT
OPT-08 AACGGCGACA OPX-13 ACGGGAGCAA OPV-07 GAAGCCAGCC
OPT-13 AGGACTGCCA OPX-17 GACACGGACC OPV-08 GGACGGCGTT
OPQ-05 CCGCGTCTTG OPW-09 GTGACCGAGT OPV-10 GGACCTGCGT
OPQ-12 AGTAGGGCAC OPW-16 CAGCCTACCA OPV-17 ACCGGCTTGT

图1

DNA提取 M,分子量标准1kb plus;1~2,HM1的雌株;3~4,HM1的雄株;5,SH2的雌株;6,SH2的雄株;7,WD2的雌株;8,WD2的雄株;9,LJ的雌株;10,LJ的雄株;11,BQ的雌株;12,BQ的雄株;13,LB的雌株;14,LB的雄株;15~16,苗期QM1的叶片;17~18,苗期JM1的叶片;19,雌雄同株材料USO-14"

图2

引物OPV-08对工业大麻HM1雌雄株DNA池的PCR扩增结果 箭头表示在>750bp处有雄性特异片段;分子量标准D2000 DNA ladder"

图3

雄性特异DNA片段回收结果"

图4

雄性特异片段核苷酸序列"