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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (11): 1649-1655.doi: 10.3724/SP.J.1006.2019.93009

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2019-11-12 Published:2019-06-03
  • Contact: Fa-Zhan QIU E-mail:qiufazhan@mail.hzau.edu.cn
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2018YFD0100202-3)

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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

Table 1

Primers used for Silky1"

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

Fig. 1

Tassel and ear phenotype of msm-6 mutant A: Tassel of WT (up) and msm-6 (down); B: Ear of WT (left) and msm-6 (right); C: Male flower of WT (left) and msm-6 (right); D: Female flower of WT (left) and msm-6 (right). Bar = 5 cm in Fig. A; bar = 3 cm in Fig. B; bar = 3 mm in Fig. C; bar = 3 cm in Fig. D."

Table 2

Chi-square test of the F2 population"

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

Fig. 2

Linkage markers analysis with msm-6 mutation loci via BSA"

Table 3

Information and exchange frequency between linkage markers and mutation sites"

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

Table 4

Genotype and phenotype of the recombinant plant"

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

Fig. 3

Allelism test of msm-6 and silky1 A: The wild-type plant from msm-6/msm-6×+/silky1-mum3; B: The mutant plant from msm-6/msm-6×+/silky1-mum3."

Fig. 4

Identification of msm-6 mutation sites. A: The sequence analysis of WT and msm-6; The arrow indicates the mutation site. B: Gene structure of Silky1 and mutation site of two mutants."

Fig. 5

Loss of exon 6 of Silky1 transcript in msm-6 mutant A: PCR product of Silky1 transcript between WT and msm-6; B: The sequence analysis of Silky1 transcript between WT and msm-6."

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