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作物学报 ›› 2019, Vol. 45 ›› Issue (5): 656-661.doi: 10.3724/SP.J.1006.2019.83058

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米穗发芽突变体vp-like8的遗传分析及突变基因鉴定

王瑞1,陈阳松1,孙明昊1,2,张秀艳3,杜依聪1,郑军1,*()   

  1. 1 中国农业科学院作物科学研究所, 北京 100081
    2 吉林农业大学农学院, 吉林长春 130118
    3 中国农业大学生物学院, 北京 100193
  • 收稿日期:2018-08-15 接受日期:2019-01-12 出版日期:2019-05-12 网络出版日期:2019-02-22
  • 通讯作者: 郑军
  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0101002);中国农业科学院创新工程专项经费资助

Genetic analysis and causal gene identification of maize viviparous mutant vp-like8

Rui WANG1,Yang-Song CHEN1,Ming-Hao SUN1,2,Xiu-Yan ZHANG3,Yi-Cong DU1,Jun ZHENG1,*()   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2 College of Agronomy, Jilin Agricultural University, Changchun 130118, Jilin, China
    3 School of Life Science, China Agricultural University, Beijing 100193, China
  • Received:2018-08-15 Accepted:2019-01-12 Published:2019-05-12 Published online:2019-02-22
  • Contact: Jun ZHENG
  • Supported by:
    This work was supported by the National Key Research and Development Program of China(2016YFD0101002);the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

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摘要：

玉米突变体vp-like8具有明显的穗发芽性状且能稳定遗传, 遗传分析表明该突变性状受隐性单基因控制。用vp-like8与自交系郑58杂交构建F2遗传定位群体, 利用BSR-Seq方法, 将基因初定在玉米第3染色体160.4 Mb~165.6 Mb区间内。参考玉米基因组信息, 发现已报道的穗发芽基因Vp1位于此定位区间内。分别利用vp1、vp-like8的杂合突变体进行等位测验, 发现杂交后代中正常与穗发芽籽粒符合3∶1遗传分离比。经序列分析, 发现vp-like8突变体中Vp1基因在第2内含子有343 bp碱基的缺失, 且第3内含子有222 bp重复序列的插入, 而已报道的vp1突变体只在第2个内含子有343 bp碱基的缺失。通过实时定量PCR检测发现, 与正常籽粒相比, vp-like8与Vp1突变籽粒中Vp1 基因的转录水平均明显降低。以上证据表明, vp-like8是一个新的Vp1基因等位突变体。

关键词: 玉米, 穗发芽, 突变体, Vp1, 基因定位

Abstract:

The maize mutant vp-like8 shows clear viviparous phenotype and stable inheritance, and genetic analysis showed that the mutant phenotype was controlled by a single recessive gene. Using an F2 segregation population derived from vp-like8 and inbred line Zheng 58, the causal gene was mapped to an interval from 160.4 Mb to 165.6 Mb on chromosome 3 by the BSR-Seq technology. According to the maize genomic database, a previously discovered viviparous gene Vp1 was identified to be in this mapping interval. The test crosses from vp1 and vp-like8 heterozygous plants showed a 3:1 segregation ratio between normal and viviparous kernels. The genomic sequence analysis revealed that vp-like8 mutant had a 343 bp deletion in the second intron and 222 bp insertion in the third intron of Vp1 gene, which is different from vp1 mutation of an only 343 bp deletion in the second intron of Vp1 gene. Further real time PCR analysis revealed that, compared with the normal kernels, the transcript level of Vp1 was significantly decreased both in vp-like8 and vp1 viviparous kernels. Taken together, these evidences suggest that vp-like8 is a new allele mutant of Vp1.

Key words: maize, viviparous, mutant, Vp1, gene mapping

表1

本研究所用的引物"

引物
Primer		 正向序列
Forward sequence (5°-3°)		 反向序列
Reverse sequence (5°-3°)
VP1-G-F1/R1 GCGAGACCTGAAAACACACA CATGGCGTTCTCTAGCATCA
VP1-G-F2/R2 CGCACTCCCAAGAGAACC ATAGGGTAAGAGCCCGTGGA
VP1-G-F3/R3 GTGGTCGTGAACAGCCAAC GCTCTGCTTCAGCACCTTCT
VP1-G-F4/R4 CATCGCTGTCGAGCAATAGA CTGTACCGCATGTTCCACAC
VP1-G-F5/R5 TAAAATCGGCCATGGATAGG TCTCTGGCCCAGTGGTTAGT
VP1-G-F6/R6 GCTGCTGTTTTCCTCGAATC GCACCTAGCTGCCAAACACT
VP1-G-F7/R7 AGTCCTCCGGATCTCTCGTT AAACGGTTGCGTAGATTTGG
VP1-G-F8/R8 CCAGTGCAATGTCAGTGCTT AATGGCCGAGAGATCAGGTA
VP1-CDS-F1/R1 AGAAGGTGCTGAAGCAGAGC AACGAACAAATTCCCCTGTG
Zm-GAPDH-F/R CCCTTCATCACCACGGACTAC AACCTTCTTGGCACCACCCT

图1

vp-like8突变体穗发芽表型 A: 授粉30 d 后突变体vp-like8杂合果穗上的穗发芽籽粒和正常籽粒; B: 授粉后60 d后突变体vp-like8杂合果穗上穗发芽籽粒和正常籽粒; C: 成熟的正常籽粒(WT)和穗发芽籽粒(vp); 标尺=1 cm。"

表2

vp-like8杂合突变体自交授粉后正常籽粒和穗发芽籽粒的分离情况"

年度
Year		 地点
Location		 植株基因型
Plant genotype		 籽粒表型Kernel phenotype
正常籽粒
Normal		 穗发芽籽粒
Viviparous		 总数
Total		 χ2
(3:1)
2014 海南Hainan vp-like8/+ 150 45 195 0.289
361 118 479 0.017
2016 北京Beijing vp-like8/+ 132 46 178 0.030
121 38 159 0.052

图2

利用BSR-Seq方法对vp-like8突变体基因的初定位"

图3

利用杂合体对vp-like8和vp1进行等位测验 A: 以vp1/+杂合突变体混粉杂交vp-like8的果穗上出现穗发芽籽粒。B: 以vp-like8/+杂合突变体混粉杂交vp1的果穗上出现穗发芽籽粒。"

图4

Vp1基因结构示意图及突变体的突变位置"

表3

vp-like8与vp1突变体等位测验"

父母本基因型
Parental genotype		 籽粒表型 Kernel phenotype
正常籽粒
Normal		 穗发芽籽粒
Viviparous		 总数
Total		 χ2
(3:1)
vp-like8/+ × vp1 /+ 208 72 280 0.042
vp1 /+ × vp-like8/+ 158 48 206 0.233

图5

实时定量PCR检测Vp1基因分别在vp-like8、vp1突变体的正常(normal)和穗发芽(vp)籽粒中的表达量"

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