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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (11): 2913-2922.doi: 10.3724/SP.J.1006.2023.33002

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

Genetic analysis and molecular identification of a multiple allele mutant of ZmMs7 gene in maize

CAO Xiao-Xiong1,2(), LIU Yi-Fan1,2, ZHOU Yu-Qiang2, WANG Jing2, WU Yu-Jin2, WANG Hong-Wu2, LI Kun2, LIU Xiao-Gang2, HUANG Chang-Ling2, LIU Zhi-Fang2, GUO Jin-Jie1,*(), HU Xiao-Jiao2,*()   

  1. 1College of Agronomy, Hebei Sub-center of National Maize Improvement Center of China / State Key Laboratory of North China Crop Improvement and Regulation / Hebei Agricultural University, Baoding 071001, Hebei, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / National Engineering Research Center of Crop Molecular Breeding, Beijing 100081, China
  • Received:2023-01-05 Accepted:2023-04-17 Online:2023-11-12 Published:2023-05-05
  • Supported by:
    National Key Research and Development Program of China(2022YFD1200802);Agricultural Science and Technology Innovation Program(CAAS-ZDRW202004);National Engineering Research Center of Crop Molecular Breeding, and the Science and Technology Innovation Team of Maize Modern Seed Industry in Hebei(21326319D)

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Abstract:

We identified a maize male sterile mutant in the natural population, designated as ms20s1. The mutant had complete male sterility and lacked pollen grains in the withered anthers. Cytological analysis showed that, compared with the wild type, the ms20s1 mutant exhibited shrinkage locule, swollen tapetum cells, and aborted microspore at S11 stage, indicating that the ms20s1 mutant had abnormal tapetal programmed cell death and complete pollen abortion. Genetic analysis revealed that the male sterility trait was controlled by a single recessive nuclear gene. To clone the target gene, we constructed the F2 populations by crossing ms20s1 with different inbred lines and analyzed the population genotype using genotyping by target sequencing (GBTS) technology. The gene was initially mapped to the 124.95-128.47 Mb region on chromosome 7, and the interval was narrowed down to 0.68 Mb after fine mapping. Bioinformatics analysis indicated that there was one known gene ZmMs7 in this region. The ZmMs7 gene encoded a PHD-finger transcription factor that played an important role in tapetum development and pollen wall formation. Allelism test demonstrated that ms20s1 was an allelic mutant of ZmMs7 gene. Gene sequencing results showed that the ms20s1 mutant had multiple sequence variants in the exon region, which were different from the reported mutants ms7-6007 and ms7gl, confirming ms20s1 was a new allelic mutant of ZmMs7. The discovery and identification of the ms20s1 mutant provide a new material for exploring the molecular mechanism and breeding application of maize genic male sterility.

Key words: maize, male sterility, ms20s1, gene mapping, allelic mutant

Table 1

PCR primers used in this study"

引物名称
Primer name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)		 用途
Usage
InD124 GCACTGAAGGCTTATTTCGTCG TTTTTGGTCGTTGCTGCTGAT 基因定位
Gene mapping
InD125 AGCAGGAACGAAAAGGCACT GCAAAACAGGACACGCATCA
InD127 CCCTGCTATGACGACTTTTTTTCT ACTCTTGGTTGTCCACCGTGC
InD128 CCCGCTCATTGCTCTGTTG CCAAGCGAGCAGGCACAT
InD130 GCTCTTGATTTGCGAGGTGGT GCTGTGAGTGTGATGCGTGTG
20q CTTGGACACCAAGCACTTCGTC CAGGGTGACCGTCTCGTACG 基因表达分析
Gene expression
Tubulin GTGTCCTGTCCACCCACTCTCT GGAACTCGTTCACATCAACGTTC
Ms7-P1 CAGAACAGAGCAGAGGAACCAT GGATAACCAAACGAAACACGAGCC 基因克隆
Gene cloning
Ms7-P2 GGAGAAACCGTCCAAAGGC AAACCTTCGTGGTAATCTTTGAC
Ms7-P3 GTCTTGGACACCAAGCACTTCGT TGCCATGGCGGCTATAGGAGTT 基因克隆/CAPS鉴定
Gene cloning/CAPS

Fig. 1

Comparison of phenotypes between wild type and ms20s1 mutant A: the comparison of plant morphology between wild type and ms20s1 mutant at anthesis stage, bar: 20 cm; B: the comparison on tassel traits between the WT and ms20s1 mutant at anthesis stage, bar: 20 cm; C, D: the comparison of the anther morphology between the WT (C) and ms20s1 mutant (D), bar: 1 mm; E, F: pollen grains of WT (E) and ms20s1 mutant (F) stained with I2-KI, bar: 200 μm."

Table 2

Comparison of important agronomic traits between WT and ms20s1"

农艺性状
Agronomic trait		 野生型
WT		 突变体
ms20s1
株高Plant height (cm) 188.13±3.88 178.83±3.53*
穗位高Ear height (cm) 77.20±6.04 76.50±4.89
雄穗主轴长Total tassel length (cm) 41.01±2.29 38.48±2.21*
雄穗分枝数Tassel branch number 16.00±1.73 19.00±2.00*
穗上1叶长Leaf length-1 (cm) 88.33±3.94 90.65±3.27
穗上2叶长Leaf length-2 (cm) 82.26±3.03 84.32±4.25
穗上3叶长Leaf length-3 (cm) 75.17±3.20 74.75±3.80
穗上1叶宽Leaf width-1 (cm) 8.03±0.41 7.70±0.43
穗上2叶宽 Leaf width-2 (cm) 7.61±0.43 7.43±0.35
穗上3叶宽 Leaf width-3 (cm) 7.22±0.41 6.93±0.47
穗上1叶夹角Leaf angle-1 (°) 30.74±1.54 30.18±1.37
穗上2叶夹角Leaf angle-2 (°) 31.19±1.47 31.27±1.52
穗上3叶夹角Leaf angle-3 (°) 32.13±2.63 32.98±3.43

Fig. 2

Transverse section analyses of anther (from stage 8a to stage 11) in WT and ms20s1 mutant E: epidermis; En: endothecium; ML: middle layer; Ta: tapetum; Msp: microspore; CMsp: collapsed microspore; Dy: dyad; Tds: tetrads. Bar: 50 μm."

Table 3

Chi-square test for phenotypic segregation in F2 population"

群体
Population		 总株数
Total number of plants		 可育植株
Number of fertile plants		 不育植株
Number of male sterile plants		 期望比
Expected rate		 χ2
F2 (ms20s1×B73) 379 285 94 3:1 0.0009
F2 (ms20s1×Mo17) 181 139 42 3:1 0.2228
F2 (ms20s1×C7-2) 990 769 221 3:1 3.6418

Fig. 3

Distribution of SNP-index on whole genome"

Fig. 4

Fine mapping of ms20s1 mutant and structure schematic diagram of ZmMs7 gene A: fine mapping of ms20s1 mutant. Marker: marker names; N: F2 recessive population used in mapping; recombinant: the number of recombinants; B: the structure schematic diagram of ZmMs7 gene. The grey box represents the 5° un-translated region. The black boxes represent the exons and the horizontal lines represent introns."

Fig. 5

Allelism test of ms7-6007 heterozygotes and ms20s1 mutants A: plants from left to right are the tassels of the ms20s1 mutant, the fertile hybrid plant, the sterile hybrid plant, and the ms7-6007 mutant, respectively, bar: 10 cm; B: plants from left to right are the anthers of the ms20s1 mutant, the fertile hybrid plant, the sterile hybrid plant, and the ms7-6007 mutant, respectively, bar: 1 mm; C: plants from left to right are the I2-KI staining of pollen grains of the ms20s1 mutant, the fertile hybrid plant, the sterile hybrid plant, and the ms7-6007 mutant, respectively, bar: 100 μm."

Table 4

Allelism test of ms20s1 with ms7-6007"

父母本基因型
Parental genotype		 表型统计Phenotypic statistics 总株数
Number of the total plants		 χ2(1:1)
可育植株Fertile plants 不育植株Sterile plants
ms20s1×+/ms7-6007 96 100 196 0.0459
ms7-6007×+/ms20s1 151 145 296 0.0844

Fig. 6

Genotype identification A: PCR products of the CAPS markers; B: SnaB I digestion product. AA, Aa, and aa represent homozygous wild type, heterozygous wild type, and homozygous mutant, respectively."

Fig. 7

Relative expression level of ZmMs7 genes in different tissues"

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