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Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq

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Abstract

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A combination of BSA and RNA-seq was performed to identify candidates for the restorer-of-fertility gene in onion. The AcPMS1 involved in DNA mismatch repair was identified as the best candidate.

Abstract

To identify candidate genes of the restorer-of-fertility gene (Ms) responsible for fertility restoration of onion cytoplasmic male-sterility, a combined approach of bulked segregant analysis and RNA-seq was employed. From 32,674 de novo assembled contigs, 430 perfectly homozygous SNPs between male-fertile (MF) and male-sterile (MS) bulks were identified in 141 contigs. After verifying the homozygosity of the SNPs by PCR amplification and sequencing, the SNPs on 139 of the contigs were genotypes for the two recombinants which contained crossover events between the Ms locus and two tightly linked molecular markers. As a result, 30 contigs showing perfect linkage with the Ms locus in the large-sized segregating population were identified. Among them, 14 showed perfect linkage disequilibrium (LD) with the Ms locus, as determined by genotyping 251 domestic breeding lines. Furthermore, molecular markers tagging the 14 contigs also showed almost perfect LD with each other in 124 exotic accessions introduced from 21 countries, except for one accession which contained a crossover event by which the 14 markers were divided into two groups. After sequencing of the full-length cDNA of the 14 contigs showing perfect LD, the deduced amino acids sequences of the MF and MS alleles were compared. Four genes were shown to harbor putative critical amino acid changes in the known domains. Among them, the gene encoding PMS1, involved in the DNA mismatch repair pathway, was assumed to be the best candidate gene responsible for fertility restoration of male-sterility in onion.

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Acknowledgments

This research was supported by the Agriculture Research Center program, Golden Seed Project (Center for Horticultural Seed Development, No 213003-04-2-SB910), and a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ011034). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.

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Authors and Affiliations

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

    Sunggil Kim

  2. National Institute of Horticultural & Herbal Science, RDA, Muan, 534-833, Korea

    Cheol-Woo Kim

  3. Department of Plant Science, Seoul National University, Seoul, Korea

    Minkyu Park & Doil Choi

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  1. Sunggil Kim
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  2. Cheol-Woo Kim
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122_2015_2584_MOESM1_ESM.tif

Supplementary material 1 Fig. 1. Physical map of rice chromosomes showing the positions of the genes orthologous to the onion genes linked to the Ms locus. The unit of distances in the map is kilobase pairs. The map was produced using MapChart 2.1 software (TIFF 301 kb)

122_2015_2584_MOESM2_ESM.tif

Supplementary material 2 Fig. 2. Phylogenetic relationship of onion PPR motif-containing proteins with PPR-containing Rf genes isolated from other plant species. Detailed information about the onion PPR genes is included in Supplementary Table 9. The GenBank accession numbers of Rf genes isolated from other species are shown in parenthesis. The tree was constructed by the neighbor-joining method using the deduced amino acid sequences. The numbers at the nodes indicate the bootstrap probability (%) with 1,000 replicates. (TIFF 108 kb)

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Kim, S., Kim, CW., Park, M. et al. Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq. Theor Appl Genet 128, 2289–2299 (2015). https://doi.org/10.1007/s00122-015-2584-z

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