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作物学报 ›› 2019, Vol. 45 ›› Issue (11): 1649-1655.doi: 10.3724/SP.J.1006.2019.93009

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一个新的玉米silky1基因等位突变体的遗传分析与分子鉴定

王晓娟1,潘振远2,刘敏2,刘忠祥1,周玉乾1,何海军1,邱法展2,*()   

  1. 1 甘肃省农业科学院作物研究所, 甘肃兰州730070
    2 华中农业大学作物遗传改良国家重点实验室, 湖北武汉430070
  • 收稿日期:2019-02-26 接受日期:2019-05-12 出版日期:2019-11-12 网络出版日期:2019-06-03
  • 通讯作者: 邱法展
  • 作者简介:王晓娟, E-mail: wangxj839@sina.com;|潘振远, E-mail: panzhenyuandawood@163.com
  • 基金资助:
    本研究由国家重点研发计划“七大农作物育种”专项(2018YFD0100202-3)

Genetic analysis and molecular characterization of a new allelic mutant of silky1 gene in maize

WANG Xiao-Juan1,PAN Zhen-Yuan2,LIU Min2,LIU Zhong-Xiang1,ZHOU Yu-Qian1,HE Hai-Jun1,QIU Fa-Zhan2,*()   

  1. 1 Crops Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
    2 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
  • Received:2019-02-26 Accepted:2019-05-12 Published:2019-11-12 Published online:2019-06-03
  • Contact: Fa-Zhan QIU
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2018YFD0100202-3)

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摘要：

前期试验发现一个玉米雄性不育突变体(male sterile mutant), 命名为msm-6。经过连续多代杂合单株自交发现该突变体性状能稳定遗传, 遗传模式分析表明该突变体为单个隐性 1核基因控制。以msm-6 与自交系B73 杂交构建F2 遗传定位群体, 利用BSA (Bulk Segregant Analysis)方法, 筛选到与msm-6位点连锁的4个SSR标记, 即C6-24、C6-30、C6-34和C6-40。进一步采用444个F2单株对这4个连锁标记验证, 将msm-6定位于标记C6-24与C6-34之间, 即玉米第6染色体68.5~98.1 Mb之间。通过基因组序列信息分析发现, 在此定位区间内存在一个已报道的Silky1基因。Silky1基因编码MADS-BOX蛋白, 是参与花器官建成ABCD模型的B功能基因, 该基因的突变会造成雄花不育、雌花花丝增多等表型。利用杂合体+/silky1-mum3与纯合突变体msm-6/msm-6杂交进行等位测验, 杂交后代中正常植株与突变植株分离比例符合1∶1。基因组序列以及转录本序列分析发现, msm-6突变体中第6个内含子5'端供体位点AG/GTAAG (外显子/内含子交接处)的序列+1位G突变为C, 导致第6外显子被错误剪切掉, 产生了第6个外显子缺失的Silky1异常转录本。以上实验证据表明, msm-6与所报道的由mutator转座子插入造成的Silky1突变体silky1-mum2、silky1-mum3和silky1-mum4的突变方式不同, 是一个新的Silky1基因等位突变体。msm-6的发现与鉴定为进一步深入解析玉米穗花器官决定的遗传机制提供丰富的实验材料, 同时也为RNA加工过程中剪接位点保守性提供重要证据。

关键词: 玉米, 雄性不育, 遗传分析, silky1, 基因定位, 等位突变

Abstract:

We identified a new maize male sterile mutant in early experiment, designated as msm-6. This mutant characters were steadily inherited and genetically regulated by a single recessive gene. An F2 population was developed by crossing B73 inbred line to msm-6, and four SSR markers (C6-24, C6-30, C6-34, and C6-40) closely linked to this locus were identified by BSA (Bulked Segregant Analysis) method. The 444 F2 individuals were used to map the target gene in an interval from 68.5 to 98.1 Mb between markers C6-24 and C6-34 on chromosome 6. Using genomic sequence database, we found that male sterile mutant gene Silky1 was located in this mapping region. Silky1 encodes a B function MADS box protein of ABCD model for floral organ establishment. The mutation of Silky1 led to sterile stamen and more silks of the ear. By crossing heterozygous +/silky1-mum3 plants to homozygous msm-6/msm-6 plants, we found a 1:1 segregation ratio for normal to male sterile plants. Genomic and cDNA sequences of msm-6 disclosed a single-nucleotide change from G to C at the first position of intron 6, which resulted in exon 6 skipping, producing aberrant mRNAs without exon 6. So msm-6 is a new allele mutant of Silky1, which is different from silky1-mum2, silky1-mum3, and silky1-mum4 caused by the insertion of mutator transposons. The identification of msm-6 provides not only abundant experimental materials for the study of the floral organ determination in maize, but also important evidence for the conservation of splicing sites in RNA processing.

Key words: maize, male sterile, genetic analysis, silky1, gene mapping, allelic mutant

表1

Silky1基因相关的引物"

引物名称
Primer		 序列
Sequence (5°-3°)		 用途
Use
Silky1_F1 TCTGTCGCCGAGTATTGGTAT Silky1 gene
Silky1_R1 CGCGTTTCCAATCAATCC Silky1 gene
Silky1_F2 GAAGCCAAAACCGTGCTAAC Silky1 gene
Silky1_R2 CGGATCCATCCCTCGTAGTA Silky1 gene
Silky1_F3 TGGTCTGCGCACAGAGATTA Silky1 gene
Silky1_R3 AGCTCTCAAACTTGCCCAAT Silky1 gene
Silky1_F4 CAGCACCGTTGGATGTAGAG Silky1 gene
Silky1_R4 GATGAGCTGGATGCAACGTA Silky1 gene
Silky1_F5 GAGGTATGGAAATGGGACTCA Silky1 gene
Silky1_R5 TGTTTTTCGGGCAAAAATTG Silky1 gene
Silky1-cDNA_F TCCCAACTCCCAAGTCCCAT Silky1 cDNA
Silky1-cDNA_R GACGATGCATGAGCGAACTA Silky1 cDNA
silky1-mum3_F TTGTCCTTGCTTGCTTCCTT silky1-mum3
silky1-mum3_R CGGATCCATCCCTCGTAGTA silky1-mum3
TIR6 AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC silky1-mum3

图1

msm-6突变体雄穗和雌穗表型 A: 野生型(上)和msm-6突变体(下)的雄穗表型; B: 野生型(左)和msm-6突变体(右)的雌穗表型; C: 野生型(左)和msm-6突变体(右)的雄花表型; D: 野生型(左)和msm-6突变体(右)的雌花表型。A图, 标尺=5 cm; B图, 标尺=3 cm; C图, 标尺=3 mm; D图, 标尺=3 cm。"

表2

F2分离群体的卡方测验"

群体
Population		 观察值Observations 期望值Expectations 卡方检验
Chi-square
野生型WT 突变体Mutant 野生型WT 突变体Mutant
msm-6 × 黄早4 F2 140 48 141 47 0.87
msm-6 × B73 F2 335 109 333 111 0.83

图2

BSA筛选到的与msm-6连锁标记"

表3

连锁标记信息以及与突变位点的交换频率"

标记Primer 左引物序列
Left sequence (5°-3°)		 右引物序列
Right primer sequence (5°-3°)		 物理位置
Physical location		 交换频率
Exchange frequency
C6-24 ATTCGATCTAGGGTTTGGGTTCAG GATGCAGTAGCATGCTGGATGTAG 68,453,519 0.045
C6-30 GGAGGAGTACGGCTTCGAG AAGTGGTTGCTGTTGTGGTG 87,429,100 0
C6-34 CTGATCGTGGATATCCCCTCC GACTCGAAGCCAAAACCAACTC 98,078,061 0.011
C6-40 CGCTGAGGCTTAAGATGGTGTT AACGCCTTTACGAGCACGAAC 107,626,979 0.034

表4

重组单株基因型以及表型信息"

标记Marker 单株数
Plant
number		 表型
Phenotype
C6-24 C6-30 C6-34 C6-40
H B B B 12 突变体Mutant
B B H H 3 突变体Mutant
B B B H 8 突变体Mutant
H H H B 7 野生型WT
H H B B 2 野生型WT
B H H H 8 野生型WT

图3

msm-6与silky1突变体等位测验 A: 以杂合+/ silky1-mum3单株和mus-6突变体杂交后代的野生型; B: 以杂合+/ silky1-mum3单株和msm-6突变体杂交后代的突变体。"

图4

msm-6突变位点鉴定 A: 野生型以及msm-6突变体的序列分析; 箭头指示突变位点。B: Silky1基因结构示意图及2个突变体的突变位置。"

图5

msm-6突变体中Silky1转录本Exon6的缺失 A: Silky1转录本在野生型和msm-6突变体中的扩增; B: Silky1转录本在野生型和msm-6突变体中的序列分析。"

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