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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (8): 2077-2087.doi: 10.3724/SP.J.1006.2023.23062

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

Identification and gene localization of a novel maize nuclear male sterility mutant ms6

WANG Xing-Rong1(), ZHANG Yan-Jun1, TU Qi-Qi2, GONG Dian-Ming2,*(), QIU Fa-Zhan2,*()   

  1. 1 Crops Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
    2 Hubei Hongshan Laboratory, Wuhan 430070, Hubei, China
  • Received:2022-09-23 Accepted:2023-02-21 Online:2023-08-12 Published:2023-03-10
  • Contact: GONG Dian-Ming,QIU Fa-Zhan E-mail:wxr_0618@163.com;gongdianming@mail.hzau.edu.cn;qiufazhan@mail.hzau.edu.cn
  • Supported by:
    Wuhan Major Project of Key Technologies in Biological Breeding and New Variety Cultivation(2022021302024852);Research Condition Construction and Achievement Transformation Project of Gansu Academy of Agricultural Sciences(2021GAAS04);Key Talent Project of Gansu Province(Crop Germplasm Resources Protection and Utilization Talent Training). and National Natural Science Foundation of China(31960405)

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Abstract:

The localization, cloning, and functional mechanism research of male sterility genes can not only deepen our understanding of the molecular regulation mechanism of male flower growth and development, but also effectively promote the development of male sterility technology system and its application in maize breeding and seed production in maize. In this study, maize inbred line Mo17 was used as the wild-type background material, and a maize male sterile mutant, named male sterile 6 (ms6), was obtained by EMS mutagenesis. Phenotypic identification of ms6 mutant plants showed that they could perform normal tasseling, but the male flower glume could not dehiscence and powder could not disperse normally, and the pollen grains were dry, indicating pollen free sterility. Meanwhile, there were no significant differences between ms6 and Mo17 wild type (WT) in plant architecture, ear-related traits, kernel size, and other related traits, demonstrating that the ms6 gene only affected plant fertility, but did not affect other agronomic traits. Cytological observation revealed that the microspore of ms6 mutant was abnormal at the late stage of microspore development, which showed that tapetal cells were degenerated in advance, and the microspore could not undergo mitosis and gradually split. Scanning electron microscopy demonstrated that the anther epidermis of mutant ms6 were shrunk, and there was no intact pollen or ubisch on the inner surface of anther locule. Genetic analysis revealed that the ms6 phenotype was controlled by a pair of recessive nuclear genes. Combined with phenotypic and genotypic linkage analysis, ms6 was initially located between C6-19 and C6-30 on maize chromosome 6 by using the ms6 × B73 F2 genetic mapping population and about 200 pairs of polymorphic SSR markers in the whole genome, and 10 pairs of newly developed polymorphic markers in the interval were further used. Finally, ms6 was located within the interval of about 480 kb between M13-M14. Gene mapping located the mutation site into a 480 kb interval between M13 and M14. Zm00001d035201 was preliminarily identified as the key candidate gene of ms6 by RNA-Seq combined with qRT-PCR. Zm00001d035201 encoded an acid ribosomal protein. This study provides a solid foundation for further studies on the function of ms6 gene. Meanwhile, as a novel male sterile mutant, ms6 also provides the important material support for the production and application of new maize genetic sterile genes in the future.

Key words: maize, male sterile, ms6, gene mapping, identification of candidate genes

Fig. 1

Phenotypic comparison of wild type (WT) and ms6 mutant A: the tassel of wild-type WT and ms6 mutant during pollen dispersal period. B: the anthers of WT and ms6; bars: 2 mm. The pollen grains of WT (C) and ms6 (D) after I2- KI staining; bars: 0.1 mm."

Fig. 2

Investigation of agronomy traits between ms6 mutant and wild type Comparison of various agronomic traits between ms6 mutant and wild type. HKW: 100-grain weight; EW: ear weight; KRN: kernel row number; KL: kernel length; KW: kernel weight; PH: plant height; EH: ear height; SBN: the number of tassel branches. All data are shown as means ± SDs and significance is estimated by t-test, NS: not significant difference."

Fig. 3

SEM observation of the anther in WT and ms6 mutant A: the pollen grains of the wild-type anthers; B: the shriveled pollen grains of the ms6 mutant anthers; C-D are the wild-type and mutant anther outer walls, respectively; E-F are wild-type and mutant anther inner walls, respectively. Ub: Ubisch. Magnifications are labeled in Fig. 3."

Fig. 4

Paraffin slices analysis of anthers at different developmental stages in WT and ms6 A-F: cytological observation of wild type anthers; G-L: cytological observation of ms6 mutant anthers; A and G: the prosporangium stage; B and H: the dyad stage; C and I: the tetrad stage; D and G: the early stage of mononuclear microspore; E and K: the middle and late stages of mononuclear microspore; F and L: the early stage of trinuclear cell. E: epidermis; En: endothecium; ML: middle layer; T: tapetum; Dy: dyad cell; Tds: tetrads; Msp: microspore; Bp: biceullar pollen; Mp: mature pollen. Bars: 50 μm."

Table 1

Genetic analysis of the ms6 locus"

群体
Population		 总株数
Total number		 可育株
Fertile plants		 不育株
Sterile plants		 可育/不育(3:1)
Fertility/sterile (3:1)		 χ (3:1) χ20.05
ms6×B73 396 300 96 3.13 0.12 3.84
ms6×Mo17 159 116 43 2.70 0.35

Table 2

Primers used in this study"

引物
Primer name		 序列
Primer sequence (5°-3°)		 引物
Primer name		 序列
Primer sequence (5°-3°)
IN34-F AGCACGGAGAAATGGAGTTG M13-F GAGGCCAAGCACAATGTATG
IN34-R AACAGAAAGCTTGGCGTCAT M13-R TTTCAGGTGGAACCAGCAAT
MY8860-F AATGGCAGGAGCACTACACC C6-11A-F CAACCAGGAGAAGCTGGCTA
MY8860-R GGTCTGCCTGATGCTCTTGT C6-11A-R GAAGAGCGCGTAGAAGATGC
M10-F GTGGAGCCCAGACACATTTT M14-F ACTGCAGCGTGTGAAAACAA
M10-R TTTGTTTATGCTTGGCCGTA M14-R GCGGGAGACTCATACCTGAA
M11-F ATGACCACTCCTGGGTTTTG M17-F GAGTTCTAAATAAGGGGGATGGA
M11-R CTGTAGCCAAGAAGCCTTGC M17-R TTGGTTTCTGTGATGCATTGA
M12-F AATTGGAGGAACCAGCTCAA C6-20-F ATTCGATCTAGGGTTTGGGTTCAG
M12-R ATTTGGAGGGCGGTTTTATC C6-20-R GATGCAGTAGCATGCTGGATGTAG

Fig. 5

Fine mapping of ms6 The red oval represents the centromere."

Table 3

Functional annotation of candidate genes"

基因ID Gene ID 注释 Gene annotation
Zm00001d035199 WPP domain-associated protein
Zm00001d035200 Putative AMP-dependent synthetize and ligase superfamily protein
Zm00001d035201 60S acidic ribosomal protein P0
AC187088.3_FG002 Guanylate kinase/Guanosine monophosphate kinase
Zm00001d035203 Unknown
Zm00001d035204 Unknown
Zm00001d035206 ATP binding protein
Zm00001d035207 Putative uncharacterized protein
Zm00001d035208 BTB/POZ domain-containing protein
Zm00001d035211 Ca(2)-dependent phospholipid-binding protein (Copine) family

Table 4

Gene expression within the interval region in anther of ms6 and WT"

基因ID
Gene ID		 表达差异Expression difference
花药发育时期
Anther development stage		 ms6 野生型
WT		 log2FC P-value 显著性
Significant
Zm00001d035199 四分体时期The tetrad stage 1024 1157 0.18 0.43 NS
双核期The binucleate stage 1069 911 -0.31 0.28 NS
Zm00001d035200 四分体时期The tetrad stage 103 140 0.45 0.38 NS
双核期The binucleate stage 135 75 -0.84 0.26 NS
Zm00001d035201 四分体时期The tetrad stage 9652 8941 -0.11 0.69 NS
双核期The binucleate stage 6690 17205 1.36 0.00018 DOWN
AC187088.3_FG002 四分体时期The tetrad stage NS NS NS NS NS
双核期The binucleate stage NS NS NS NS NS
Zm00001d035203 四分体时期The tetrad stage NS NS NS NS NS
双核期The binucleate stage NS NS NS NS NS
Zm00001d035204 四分体时期The tetrad stage NS NS NS NS NS
双核期The binucleate stage NS NS NS NS NS
Zm00001d035206 四分体时期The tetrad stage 600 943 0.65 0.0035 NS
双核期The binucleate stage 1196 1924 0.69 0.088 NS
Zm00001d035207 四分体时期The tetrad stage 736 819 0.15 0.56 NS
双核期The binucleate stage 763 729 -0.06 0.82 NS
Zm00001d035208 四分体时期The tetrad stage 827 1022 0.31 0.45 NS
双核期The binucleate stage 1184 877 -4.41 0.41 NS
Zm00001d035211 四分体时期The tetrad stage 167 134 -0.31 0.59 NS
双核期The binucleate stage 95 40 -1.06 0.16 DOWN

Fig. 6

Relative expression level of candidate genes by qRT-PCR Ubiquitin was used as an internal control gene and error bars show the SD (n = 3). Anthers of wild-type and mutant at binucleate stage were used for the relative expression level. WT: wild type; ms6: mutant. All data are shown as means ± SDs (n = 3). **: P < 0.01, NS: not significant difference."

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