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Identification of a variant of CMS-T cytoplasm and development of high resolution melting markers for distinguishing cytoplasm types and genotyping a restorer-of-fertility locus in onion (Allium cepa L.)

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Abstract

Cytoplasmic male-sterility (CMS) has been used in F1 hybrid seed production in onion (Allium cepa L.). Two types of CMS (CMS-S and CMS-T) have been reported in onions. Almost complete mitochondrial genome sequences of normal, CMS-T, and CMS-S cytoplasm types have been obtained in previous studies. Unlike highly divergent CMS-S mitochondrial genome, there were only three single nucleotide polymorphisms (SNPs) between normal and CMS-T mitochondrial genomes except for orf725, the causal gene for CMS induction. Cleaved amplified polymorphic sequence (CAPS) and high resolution melting (HRM) markers were developed based on one of these SNPs. When these markers were tested for 243 diverse breeding lines, four of them containing CMS-T cytoplasm showed SNP genotypes of the normal cytoplasm. Although mitochondrial genome organizations of these four accessions were assumed to be similar to those of normal and CMS-T, the copy number of orf725 was less than half of that of general CMS-T, suggesting that male-sterility of this variant of CMS-T might be induced by an independent event of substoichiometric shifting of orf725. To distinguish normal and all types of CMS cytoplasms, an HRM marker was developed on the basis of cox1 and orf725. In addition, for genotyping the Ms locus, a nuclear restorer-of-fertility (Rf) gene, approximately 15 kb full-length genomic DNA sequences of AcPMS1, a candidate Rf gene, were obtained. Two reproducible HRM markers were developed among ten candidate HRM markers designed on the basis of more than 800 SNPs or InDels between AcPMS1 alleles.

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Acknowledgements

This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program (Vegetable Breeding Research Center) funded by the Ministry of Agriculture, Food and Rural Affairs (710011-03), Golden Seed Project (Center for Horticultural Seed Development, No. 213007-05-3-SBB10), and a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ013400). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.

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  1. Department of Horticulture, Biotechnology Research Institute, Chonnam National University, Gwangju, 61186, Republic of Korea

    Bongju Kim & Sunggil Kim

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  1. Bongju Kim
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BK performed most experiments and drafted the manuscript. SK organized and coordinated this research project and edited the final manuscript. All authors read and approved the final manuscript.

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Correspondence to Sunggil Kim.

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Supplementary Fig. 1

CAPS and HRM markers designed on the basis of a single SNP between normal and CMS-inducing cytoplasms. A. PCR products of the CAPS marker digested with a Dra I restriction enzyme. B. Normalized melting peak patterns of the HRM marker (TIFF 233 kb)

Supplementary Fig. 2

PCR patterns of three previously reported molecular markers developed for distinguishing onion cytoplasm types. N: normal; T: CMS-T; S: CMS-S. PCR amplifications were carried out according to respective references (TIFF 106 kb)

Supplementary Fig. 3

Normalized melting peak patterns of 10 HRM markers designed on the basis of AcPMS1 genomic DNA polymorphisms. Position of each marker is shown in Fig. 5A (TIFF 1521 kb)

Supplementary material 4 (XLSX 16 kb)

Supplementary material 5 (XLSX 12 kb)

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Kim, B., Kim, S. Identification of a variant of CMS-T cytoplasm and development of high resolution melting markers for distinguishing cytoplasm types and genotyping a restorer-of-fertility locus in onion (Allium cepa L.). Euphytica 215, 164 (2019). https://doi.org/10.1007/s10681-019-2492-4

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