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作物学报 ›› 2022, Vol. 48 ›› Issue (3): 759-769.doi: 10.3724/SP.J.1006.2022.14025

• 研究简报 • 上一篇    下一篇

甘蓝型油菜白花基因InDel连锁标记开发

王瑞1,2(), 陈雪1,2, 郭青青1,2, 周蓉1,2, 陈蕾1,2, 李加纳1,2,*()   

  1. 1西南大学农学与生物科技学院, 重庆 400715
    2重庆市油菜工程技术研究中心, 重庆 400715
  • 收稿日期:2021-02-08 接受日期:2021-07-12 出版日期:2022-03-12 网络出版日期:2021-08-09
  • 通讯作者: 李加纳
  • 作者简介:E-mail: Ruiwang71@163.com
  • 基金资助:
    高等学校学科创新引智计划(111计划)项目(B12006)

Development of linkage InDel markers of the white petal gene based on whole-genome re-sequencing data in Brassica napus L.

WANG Rui1,2(), CHEN Xue1,2, GUO Qing-Qing1,2, ZHOU Rong1,2, CHEN Lei1,2, LI Jia-Na1,2,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
  • Received:2021-02-08 Accepted:2021-07-12 Published:2022-03-12 Published online:2021-08-09
  • Contact: LI Jia-Na
  • Supported by:
    Project of Intellectual Base for Discipline Innovation in Colleges and Universities (111 Program)(B12006)

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摘要：

碱基插入/缺失(InDel)是基因组上广泛分布的遗传变异形式。但甘蓝型油菜白花基因InDel连锁标记还未见有关研究报道。本研究以甘蓝型油菜双单倍体(doubled haploid, DH)纯系黄花Y05和甘蓝型油菜纯系白花W01杂交构建F2群体。在F2群体中选取30株极端纯白花和30株极端纯黄花构建叶片DNA子代池, 对亲本和DNA子代池进行30×重测序。以法国甘蓝型油菜Darmor-bzh为参考序列, QTL-seq流程和PoPoolation2流程相互结合鉴定白花基因候选区间, 2种方法均将白花基因定位于法国甘蓝型油菜Darmor-bzh C03染色体52~54 Mb区间。利用IGV软件可视化白花基因候选区间插入缺失(InDel)变异位点, 依据候选区间序列信息设计InDel引物, 聚丙烯酰胺凝胶电泳筛选到8个与白花基因连锁共分离的InDel标记。上述研究为甘蓝型油菜白花基因精细定位和分子标记辅助选育以及白花基因功能标记开发奠定了研究基础和工作思路。

关键词: 甘蓝型油菜, 重测序, 白花基因, InDel标记

Abstract:

InDel is widely distributed across the genome and occurs in a high density and large numbers in a genome. To date, the researches about linkage InDel markers of the white petal gene in B. napus L are very less yet. In this study, we constructed the F2 mapping population from the cross between DH Y05 (yellow petal) and DH W01 (white petal). Two bulks with 30 yellow petal lines and 30 white petal lines of F2 population were constructed by mixing an equal amount of DNA. Then two bulks and parents were performed 30× whole-genome re-sequencing. Darmor-bzh as the reference genome was aligned to sequence data from the two bulks and parents. QTL-seq and PoPoolation2 workflow were applied to identify the candidate region of the white petal gene. A major candidate region was identified on chromosome C03 (52-54 Mb) of Darmor-bzh. The insertion-deletion (InDel) sites can be visualized in candidate interval by Integrative Genomics Viewer (IGV). Based on these Indel variations, we used Vector and Blast to design InDel primers. Eight InDel markers closely linked to the white petal gene were screened by Polyacrylamide gel electrophoresis (PAGE). In summary, these results provide a basis for fine mapping white petal gene and InDel molecular marker located on functional genes as well as molecular marker assisted selection breeding.

Key words: Brassica napus L., re-sequencing, white petal genes, InDel markers

图1

染色体上∆(SNP-index)分布 蓝点: ∆(SNP-index)位点; 红线: 利用滑动窗口数计算∆(SNP-index)变化趋势; 绿线: 显著性为95%的阈值; 橘线: 显著性为99%的阈值。"

图2

白花基因定位于C03 (Chr13)染色体上"

图3

IGV可视化候选区间InDel变异"

表1

InDel引物序列"

引物名称
Primer name		 Chr. C03 InDel 正向引物序列
Forward sequence (5°-3°)		 反向引物序列
Reverse sequence (5°-3°)
InDel-2 52375493-52375505 D10 GCTAAGCGTGTATCTTATCCT CGGGAATGGCGATCTAAAAA
InDel-3 52396484 I14 GAAAAACCATTGGAAATGCC TGGGAAGTTTGTTAAAGGGT
InDel-4 52348627-52348640 D12 GACAAGTCTTGGACAGCTAA CGAGAGGAATCCAAAGATGT
InDel-5 52377696-52377718 D20 TAGAGAGAGCTTAGACCTCC CGGCTATGAGTGATGTTCTT
InDel-6 52374279 I12 GGTTCTCTCTCTCTTCAGGTT TAATACTAATCGAAATTGTTGTCCC
InDel-8 52358540-52358551 D11 ATGCAACATCCAAAACACCT TTTTGCTCTTTTCGCGTTTG
InDel-9 52358775-52358788 D11 TCTGCGATCACGAATACCT CAAATCGAAAGGAAAGGAACA
InDel-10 52373701-52373721 D19 ACGTTAAAACAGCTTCACTTTC TCCGTGTCTGCCAAGTATC

图4

黄花和白花亲本及F2代单株电泳 1: 黄花亲本; 2: 白花亲本; 3~13: F2群体11个黄花单株; 14~24: F2群体11个白花单株。M: 20 bp梯度。"

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