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作物学报 ›› 2021, Vol. 47 ›› Issue (11): 2163-2172.doi: 10.3724/SP.J.1006.2021.04236

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜桔红花显性基因候选区域的NGS定位及InDel标记开发

郭青青1,2**(), 周蓉1,2**(), 陈雪1,2, 陈蕾1,2, 李加纳1,2, 王瑞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:2020-10-31 接受日期:2021-01-13 出版日期:2021-11-12 网络出版日期:2021-02-18
  • 通讯作者: 王瑞
  • 作者简介:郭青青, E-mail: 1833266719@qq.com
    周蓉, E-mail: 1822701415@qq.com第一联系人：**同等贡献
  • 基金资助:
    西南大学校创项目(X202010635478);国家重点研发计划项目“七大作物育种”(2016YFD0101300)

Location and InDel markers for candidate interval of the orange petal gene in Brassica napus L. by next generation sequencing

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

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
  • Received:2020-10-31 Accepted:2021-01-13 Published:2021-11-12 Published online:2021-02-18
  • Contact: WANG Rui
  • About author:First author contact:** Contributed equally to this work
  • Supported by:
    Innovation Project of Southwest University Students(X202010635478);National Key Research and Development Program of China(2016YFD0101300)

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摘要：

甘蓝型油菜花色的选育和改良已成为种质资源鉴定和材料创制的重要研究方向。目前为止, 甘蓝型油菜桔红花显性基因定位的研究还未见报道。本研究以甘蓝型油菜双单倍体(doubled haploid, DH)纯系黄花Y05和桔红花R08杂交, 分析桔红花性状遗传模式。在F2群体中选取30株极端桔红花和30株极端纯黄花构建叶片DNA子代池和花瓣RNA子代池, 对亲本和DNA子代池进行30×重测序, 对RNA子代池进行6G测序。以法国甘蓝型油菜Darmor-bzh为参考序列, QTL-seq流程和MMAPPR流程相互结合鉴定桔红花基因候选区间。利用IGV软件可视化分析候选区间内插入缺失(InDel)变异位点, 根据候选区间信息设计InDel引物。结果表明, 桔红花性状受1对显性主效基因控制, 全基因组重测序定位桔红花性状基因候选区间结果与转录组测序定位结果高度一致, 均位于法国甘蓝型油菜Darmor-bzh A07染色体18~19 Mb。聚丙烯酰胺凝胶电泳筛选到3个与桔红花性状紧密连锁共分离的InDel标记。这些研究为精细定位桔红花显性候选基因以及分子标记辅助选育甘蓝型油菜桔红花新材料提供新思路。

关键词: 甘蓝型油菜, 桔红花性状, 基因定位, 测序, 分子标记

Abstract:

The petal color has been one of the major goals of breeding and genetic research in Brassica napus L. To date, there have been no reports about interval location of dominant orange petal gene trait in B. napus L. In this study, we constructed an F2 mapping population with 458 individuals from the cross between DH Y05 (yellow petal) and DH R08 (orange petal). Whole-genome re-sequencing of DNAs and transcriptome sequencing of RNAs were from two populations each composed of 30 individuals showing extreme opposite trait for a given phenotype in a segregating progeny. Then we performed 30× and 6G of sequencing. Darmor-bzh as the reference genome was aligned to sequence data from the two bulks and parents. QTL-seq and Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) workflow were applied to identify the candidate region of the orange petal gene. 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. The results indicated that the orange petal trait was controlled by a dominant major gene. A major candidate region was identified on chromosome A07 (18-19 Mb) of Darmor-bzh. Three InDel markers linked to the orange petal gene were screened by Polyacrylamide gel electrophoresis (PAGE). This study may provide a novel idea for fine mapping dominant orange petal gene as well as marker assisted selection.

Key words: Brassica napus L., orange petal, gene mapping, sequencing, molecular marker

图1

甘蓝型油菜黄花Y05和桔红花R08花瓣形态比较 A1与A2: 黄花Y05; B1与B2: 桔红花R08。"

表1

黄花Y05与桔红花R08杂交F2群体花色分离比例"

群体
Population		 总株数
Total plants		 桔红花
Orange petal		 黄花
Yellow petal		 期望比
Expected ratio		 χ2 P
2019 149 108 41 3:1 0.37 > 0.05
2020 458 333 125 3:1 1.16 > 0.05

图2

甘蓝型油菜19条染色体上重测序平均深度和覆盖度 黑线: 利用滑动窗口计算得到的平均测序深度曲线。"

图3

甘蓝型油菜19条染色体上delta (SNP-index)分布 蓝点: delta (SNP-index)位点; 红线: 利用滑动窗口数计算的delta (SNP-index)的变化趋势; 绿线: 显著性为95%的阈值; 橘线: 显著性为99%的阈值。"

图4

甘蓝型油菜19条染色体上ED4 (Loess fit)和A07染色体上ED4 (Loess fit)"

图5

IGV可视化候选区间InDel变异"

表2

基于候选区间设计的InDel引物序列"

引物名称
Primer name		 InDel位点
InDel site		 InDel 正向引物序列
Positive Primer sequence (5′-3′)		 反向引物序列
Reverse primer sequence (5′-3′)
InDel-65 18,968,634-18,968,641 D7 GGGTCGAGATACGGTTACGG CGTCGGTAAGTCTTCTTCACC
InDel-69 18,949,999-18,950,007 D8 TACCGAGCTCCAGGTCTCTC TGCGCAACTGTAACCATTCT
InDel-56 18,789,060-18,789,066 D6 ACAAGCAAGGCCTATATTTTGC TCGGGTGAAGCAAATGTGGA

图6

黄花和桔红花亲本及F2代单株电泳 M: 20 bp ladder; A: InDel-65; B: InDel-69; C: InDel-56; 1: 黄花亲本; 2: 桔红花亲本; 3~13: F2群体11个黄花单株; 14~24: F2群体11个桔红花单株。"

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