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作物学报 ›› 2020, Vol. 46 ›› Issue (7): 987-996.doi: 10.3724/SP.J.1006.2020.94158

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于黄麻转录组序列SNP位点的CAPS标记开发与验证

陶爱芬1,2,游梓翊2,徐建堂2,林荔辉2,张立武2,祁建民2,*(),方平平2,*()   

  1. 1 福建农林大学金山学院, 福建福州 350002
    2 福建农林大学教育部作物遗传育种与综合利用重点实验室 / 福建省分子设计育种实验室, 福建福州 350002
  • 收稿日期:2019-10-23 接受日期:2020-03-24 出版日期:2020-07-12 发布日期:2020-04-16
  • 通讯作者: 祁建民,方平平 E-mail:qijm863@163.com;fangpp1990@qq.com
  • 作者简介:E-mail: 281770126@qq.com, Tel: 0591-83789483
  • 基金资助:
    国家现代农业产业技术体系建设专项(CARS-19-E06)

Development and verification of CAPS markers based on SNPs from transcriptome of jute (Corchorus L.)

TAO Ai-Fen1,2,YOU Zi-Yi2,XU Jian-Tang2,LIN Li-Hui2,ZHANG Li-Wu2,QI Jian-Min2,*(),FANG Ping-Ping2,*()   

  1. 1 Jinshan College, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2 Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops / Key Laboratory of Crops by Design, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2019-10-23 Accepted:2020-03-24 Online:2020-07-12 Published:2020-04-16
  • Contact: Jian-Min QI,Ping-Ping FANG E-mail:qijm863@163.com;fangpp1990@qq.com
  • Supported by:
    China Agriculture Research System(CARS-19-E06)

摘要:

黄麻CAPS分子标记的开发, 可以为黄麻遗传多样性分析、种质资源鉴定和分子标记辅助选择等研究提供有效方法。本试验以黄麻179和爱店野生种为材料, 采用Illumina HiSeq 4000测序技术进行转录组测序, 对其SNP位点进行分析, 设计了与木质素合成基因4CL、COMT及转录因子MYB相关的SNP引物, 在此基础上, 应用dCAPS Finder2.0软件开发了CAPS标记, 并对其有效性进行了验证。结果表明: (1)组装后的黄麻unigene序列共72,674条, 序列总长度为29,705,997 bp, 检测到的SNP位点总数为67,567个, 平均每440 bp有1个SNP。(2)获得了39对分别与4CL、COMT和MYB相关的SNP引物, 从中筛选获得26对CAPS标记引物, 开发成功率为66.7%, 其中11对CAPS标记具有多态性, 多态性比例为43.2%。(3)开发的CAPS标记能较好地将12份不同类型的黄麻种质区分开来, 表明CAPS是适用于黄麻研究的较理想的分子标记方法, 为黄麻遗传基础研究提供了可靠工具。

关键词: 黄麻, 转录组, SNP位点, CAPS标记, 开发

Abstract:

Developing CAPS markers can provide a new and effective tool for genetic diversity analysis, germplasm identification and marker-assistant selection of jute. The transcriptional sequence analysis was carried out by Illumina HiSeq 4000 with jute 179 and the wild species in Aidian. Furthermore, the SNP loci were screened and primers related to lignin synthesis genes 4CL, COMT and transcription factor MYB were developed. CAPS markers were screened based on above SNP primers using dCAPS Finder2.0 and their polymorphism was verified. A total of 72,674 unigene sequences were got in jute transcriptome by assembly, with a length of 29,705,997 bp, a number of SNP loci of 67,567, and an average of 1 SNP per 440 bp. Thirty-nine pairs of SNP primers related to 4CL, COMT, and MYB were designed by Oligo8, of which 26 pairs of CAPS primers were screened, with a developing rate of 66.7%. Among the 26 pairs of CAPS markers, 11 pairs were polymorphic, with a polymorphic ratio of 43.2%. The screened CAPS markers could effectively distinguish 12 different accessions of jute germplasm, indicating CAPS is an ideal molecular marker which can lie a reliable tool used in genetic research of jute.

Key words: jute, transcriptome, SNP loci, CAPS markers, development

表1

用于CAPS引物多态性验证的黄麻品种名称和类型"

编号
Number
名称
Name
品种类型
Type of accessions
1 梅峰4号 Meifeng 4 圆果种 Corchorus capsularis
2 越南圆果 Yuenanyuanguo 圆果种 Corchorus capsularis
3 广东独尾麻 Guangdongduweima 圆果种 Corchorus capsularis
4 中黄麻1号 Zhonghuangma 1 圆果种 Corchorus capsularis
5 日本大分青皮 Japanese Dafenqingpi 圆果种 Corchorus capsularis
6 福农1号 Funong 1 长果种 Corchorus olitoris
7 马里野生种 Maliyeshengzhong 长果种 Corchorus olitoris
8 广西长果 Guangxichangguo 长果种 Corchorus olitoris
9 日本5号 Japanese 5 长果种 Corchorus olitoris
10 翠绿 Cuilyu 长果种 Corchorus olitoris
11 龙溪长果 Longxichangguo 长果种 Corchorus olitoris
12 台湾加利麻 Taiwanjialima 圆果种 Corchorus capsularis

图1

黄麻unigene中SNP位点分布的趋势图 横坐标为SNP的密度, 即每kb基因序列上的SNP数目; 纵坐标为拥有对应密度的unigene数目。"

表2

黄麻unigene中SNP位点的分布密度表"

SNP位点的数目
Number of SNP loci
对应unigene序列的数目
Corresponding number of unigenes
所占比例
Ratio of SNP (%)
0-1 66,525 91.53
1-2 2092 2.88
2-3 1277 1.76
3-4 948 1.30
4-5 735 1.01
5-6 258 0.35
6-7 234 0.32
7-8 174 0.24
>8 431 0.59

表3

39对SNP引物的编号、名称及序列"

引物名称
Primer name
正向引物序列
Forward primer sequence (5′-3′)
反向引物序列
Reverse primer sequence (5′-3′)
4CL SNP1-1 GTGCTTTTCATCGGTGGTTT TGTCGTAAAACTGCTGCTGG
4CL SNP1-2 CGATTTTCATCTCGGCATTT GCCACCCATAGTTTTGGCTA
4CL SNP2-1 AGCTCCCATGAAAGCAAAGA AGCTCCCATGAAAGCAAAGA
4CL SNP2-2 AGCTCCCATGAAAGCAAAGA CGATTTTCATCTCGGCATTT
4CL SNP3-1 GAGCTTAGCGCGAGAAGTGT TGTCGTAAAACTGCTGCTGG
4CL SNP3-2 TCTCGGCATTTCTAACCACC GCCACCCATAGTTTTGGCTA
COMT SNP1F1 CACCGTTTTCCGTTGTTCTT GTTGGAAAGCTAGCCAAACG
COMT SNP1F2 ACACTCCATCTTGGTTTGGC GCTACCCATGGCGTTAAAAA
COMT SNP1F3 CCAGGTTTTCAACACTGCAA GCATCTCCATTGGGAACACT
COMT SNP1F4 TGAAGATGACAATAAAGCGAAAA GTGGACAATCAGTGTGTGGC
COMT SNP1F5 ATGCAAGAAAGCCGAGTTTG GCAGGATTCCAAGGTTTCAA
COMT SNP2F1 AAGGTGATAGAAACACGCCG GTTGGAAAGCTAGCCAAACG
COMT SNP2F2 ACTCCATCTTGGTTTGGCAC GCTACCCATGGCGTTAAAAA
引物名称
Primer name
正向引物序列
Forward primer sequence (5′-3′)
反向引物序列
Reverse primer sequence (5′-3′)
COMT SNP2F3 GCTTAACCGGGTTGTTGATG GCATCTCCATTGGGAACACT
COMT SNP2F4 TGAAGATGACAATAAAGCGAAAA GCATCTCCATTGGGAACACT
COMT SNP2F5 ATGCAAGAAAGCCGAGTTTG TTGATTGCATCATGTTGGCT
COMT SNP3F1 AATTTCACCGTTTTCCGTTG GTTGGAAAGCTAGCCAAACG
COMT SNP3F2 ACTCCATCTTGGTTTGGCAC AAAAACTGCGGTGGAGATTG
COMT SNP3F3 GCTTAACCGGGTTGTTGATG CCATTGTCTGGAATGGCTTT
COMT SNP3F4 TGAAGATGACAATAAAGCGAAAA TGGACAATCAGTGTGTGGCT
COMT SNP3F5 ATGCAAGAAAGCCGAGTTTG CATAATCCGGGTGGAAAAGA
MYB SNP F1 TTGCAATGGATTTGTGCAAT GATTCCCAGCAAATGGAAGA
MYB SNP F2 TGCCTTGTCCTCTCACTAAGC TTGAGGCCACATGTTCTAATTG
MYB SNP F3 CCTTGCTATCATTGCCCATT TTTATGGCCCTCAAAACTGG
MYB SNP F4 CAAAAGCTCATCCTCTTCCG CAAACTGCTTGGCTCATCAA
MYB SNP F5 AAGTGTTCAAAGAGAAGCAGCA TTATAGATGGCGATGGAGGC
MYB SNP F6 AAGCAGAAGCAAAATCCCAA AAGTAGCCATGGAGGTGTGG
MYB SNP F7 GGCCATGAGTTTCAACGACT GACGGGAAGAGAAAACCCTT
MYB SNP F8 CTGCGTTTGTAACCCCAGAT GCTTCCTCTTCTGCTTCTCG
MYB SNP F9 CGACTCTTTCGGGACTCAAG TCGTCGGCGTTTAAGAAGTT
MYB SNP F10 TTACACCACCGTAACCGACA AGATGTGGATCGGATCAAGG
MYB SNP F11 AAGGAAGCCATGGAAGGACT ATGCTTCTACCAATGCCAGG
MYB SNP F12 TCGATCATCAGCAACCAAAA GCATTGAATTTTCCGTGGTT
MYB SNP F13 CCTGATCTTAAGCGTGGCTC TTTGGTGACGGAGTTGATGA
MYB SNP F14 GGCCATTGGAAAACTCAAAA TGGATGAAGAGCCTTTCACA
MYB SNP F15 ATCTTTGCCCAAAAATGCTG GCAATTACCTGCTTCCCAAA
MYB SNP F16 GAGAAGGGAAATGGCATCAA CTTTGGTGGAACTGGAAGGA
MYB SNP F17 TTCTTCAGCTCTCCAACGCT GGCTTGTGGTATTGTGAGCA
MYB SNP F18 CCCTTTCTAGCATATGGGCA AACATCCCTCCATATTCATGTGT

表4

CAPS引物名称、对应的unigene编号及内切酶名称"

序号
Code
引物名称
Primer name
unigene编号
unigene code
内切酶的名称
Endonuclease name
1 4CL CAPS1-1 unigene_17597 Ssp I
2 4CL CAPS2-1 unigene_17597 Ssp I
3 4CL CAPS3-1 unigene_17597 Ssp I
4 COMT CAPS1-1 unigene_04800 Cla I
5 COMT CAPS1-3 unigene_09002 Bsi YI
6 COMT CAPS1-5 unigene_08713 Ava III
7 COMT CAPS2-1 unigene_04800 Cla I
8 COMT CAPS2-3 unigene_09002 Bsi YI
9 COMT CAPS2-5 unigene_08713 Ava III
10 COMT CAPS3-1 unigene_04800 Cla I
11 COMT CAPS3-3 unigene_09002 Bsi YI
12 COMT CAPS3-5 unigene_08713 Ava III
13 MYB CAPS1 unigene_47261 Psi I
14 MYB CAPS2 unigene_17018 Mse I
序号
Code
引物名称
Primer name
unigene编号
unigene code
内切酶的名称
Endonuclease name
15 MYB CAPS3 unigene_53709 Mbo II
16 MYB CAPS5 unigene_64377 Mnl I
17 MYB CAPS6 unigene_10508 Mbo I
18 MYB CAPS7 unigene_21631 Mfe I
19 MYB CAPS8 unigene_31305 Sec I
20 MYB CAPS9 unigene_16840 Hpy99 I
21 MYB CAPS11 unigene_02399 Alu I
22 MYB CAPS12 unigene_02162 Asu I
23 MYB CAPS13 unigene_46122 Sec I
24 MYB CAPS14 unigene_10460 Tsp RI
25 MYB CAPS17 unigene_59879 Rsa I
26 MYB CAPS18 unigene_49512 Mae I

图2

COMT CAPS1-3引物扩增和酶切结果 M: marker; 1~12表示试验所用的12份黄麻种质资源的代号。"

图3

12份黄麻品种基于CAPS标记的聚类图"

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