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Identification of Ms2, a novel locus controlling male-fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.), and development of tightly linked molecular markers

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Abstract

Cytoplasmic male-sterility (CMS) has been exclusively used to produce F1 hybrid seeds of onion (Allium cepa L.). A single nuclear locus, Ms, is known to restore male-fertility of CMS in onions. Unstable male-sterile onions producing a small amount of pollen grains have been identified in a previous study. When such unstable male-sterile onions were crossed with stable male-sterile onions containing CMS-R cytoplasm, male-fertility was completely restored, although genotypes of the Ms locus were homozygous recessive. Inheritance patterns indicated that male-fertility restoration was controlled by a single locus designated as Ms2. A combined approach of bulked segregant analysis and RNA-seq was used to identify candidate genes for the Ms2 locus. High resolution melting markers were developed based on single nucleotide polymorphisms (SNPs) detected by RNA-Seq. Comparative mapping of the Ms2 locus showed that Ms2 was positioned at the end of chromosome 2 with a distance of approximately 70 cM away from the Ms locus. Although 38 contigs containing reliable SNPs were analyzed using recombinants selected from 1344 individuals, no contig showed perfect linkage to Ms2. Interestingly, transcription levels of orf725, a CMS-associated gene in onions, were significantly reduced in male-fertile individuals of segregating populations. However, no significant change in its transcription level was observed in individuals of a segregating population with male-fertility genotypes determined by the Ms locus, suggesting that male-fertility restoration mechanism of Ms2 might be different from that of the Ms locus.

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Acknowledgements

The authors thank Ji-hwa Heo, Jeong-An Yoo, and Su-jeong Kim for their dedicated technical assistance.

Funding

This study was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) via Golden Seed Project (Center for Horticultural Seed Development, No 213007-05-5-SBB10).

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  1. Department of Plant Biotechnology, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Republic of Korea

    Nari Yu & Sunggil Kim

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  1. Nari Yu
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NY performed experiments and drafted the manuscript. SK organized and coordinated this research project and edited the final manuscript.

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Correspondence to Sunggil Kim.

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10681_2021_2927_MOESM1_ESM.tif

Supplementary Fig. 1. Pedigree of populations used in this study. S1, OP1, OP2 populations were produced in a previous study (Kim et al. 2019a). MF and MS indicate male-fertile and male-sterile phenotypes, respectively. (TIF 60 KB)

10681_2021_2927_MOESM2_ESM.tif

Supplementary Fig. 2. Pollen morphologies of male-fertile onions of which male-fertility were restored by Ms and Ms2 loci, respectively. Male-fertile onions restored by the dominant Ms allele (A and B). Male-fertile onions restored by the dominant Ms2 allele (C and D). Scale bars in images of multiple and single pollen grains indicate 20 μm and 10 μm, respectively. (TIF 685 KB)

10681_2021_2927_MOESM3_ESM.tif

Supplementary Fig. 3. Genotypes of molecular markers showing linkage disequilibrium with the Ms locus in male-fertile and male-sterile individuals of the TUMS4 population. A, H, B: Homozygous dominant, heterozygous, and homozygous recessive Ms genotypes, respectively. Detail information about these molecular markers has been described by Kim et al. (2015a). (TIF 395 KB)

10681_2021_2927_MOESM4_ESM.tif

Supplementary Fig. 4. Correlation of transcription levels of all contigs between male-fertile and male-sterile bulked RNAs. A, B, C. First, second, and third RNA-seq analyses mapped to the reference transcriptome (Kim et al. 2015b), respectively. D, E, F. First, second, and third analyses mapped to de novo-assembled contigs, respectively. G, H. Second and third analyses mapped to reference transcripts (Sohn et al. 2016), respectively. (TIF 298 KB)

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Yu, N., Kim, S. Identification of Ms2, a novel locus controlling male-fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.), and development of tightly linked molecular markers. Euphytica 217, 191 (2021). https://doi.org/10.1007/s10681-021-02927-4

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