Abstract
Male sterility is important to prevent self-pollination and loss of the onion hybrid genotype. Classic methods require 4-8 years of progeny testing before the cytoplasm type can be determined. An accurate and time-saving method was needed. Various types of markers were tested for application to South African onion parental lines of hybrid cultivars, and which could determine male sterility and maintainer genotypes accurately and easily with large numbers of samples. Five cytoplasmic (5’cob, orfA501, orf725, IGS and cob-type 2) and four nuclear markers (jnurf13, isotig34671_610, isotig30856_1351 and isotig29186_1830) were sourced. Genomic DNA was isolated from onion seedlings and young leaves growing from bulbs in the Agricultural Research Council (ARC) research field. PCR marker amplification products were separated by agarose or denaturing polyacrylamide gel electrophoresis (PAGE) gels. Real-time polymerase chain reaction (PCR) was performed with custom TaqMan® SNP genotyping assays containing primer/probe pairs designed to detect single nucleotide polymorphisms (SNPs) linked to the nuclear Ms locus. OrfA501 proved useful as a presence/absence marker for cytoplasmic male sterility, while TaqMan® SNP genotyping assays were superior to the jnurf13 nuclear marker in terms of rapid throughput. PCR molecular markers and custom TaqMan® SNP genotyping assays were efficient in screening the onion lines rapidly and accurately for their cytoplasmic and nuclear male sterility genotype. These methods reduced the time to identify the correct genotype of male sterile and maintainer lines, gave accurate genotypic information and proved to be useful on a larger scale. These molecular marker methods will facilitate the production of the correct seed for commercialization of onion lines worldwide.
Highlights
Cytoplasmic male sterility (CMS) is widely used in plant breeding schemes to produce F1 hybrid cultivars
Two CMS systems have been discovered in onion, CMS-S and CMS-T, but only the CMS-S type is widely used in breeding schemes, since it has only a single nuclear locus that can restore male sterility caused by the CMS-S cytoplasm, and has a simple inheritance pattern
The gene region, organelle genome origin, literature reference, polymerase chain reaction (PCR) primer sequences, primer annealing temperature (Tm) and expected amplification product expected for each genotype are indicated in Appendix A
Summary
Cytoplasmic male sterility (CMS) is widely used in plant breeding schemes to produce F1 hybrid cultivars. Two CMS systems have been discovered in onion, CMS-S and CMS-T, but only the CMS-S type is widely used in breeding schemes, since it has only a single nuclear locus that can restore male sterility caused by the CMS-S cytoplasm (restorer-of-fertility locus Ms), and has a simple inheritance pattern. The CMS-T system is influenced by three independently segregating restorer loci, with dominant alleles restoring fertility in specific combinations (Havey, 2000). This makes selection and breeding for onion male sterility using the CMS-T system more complicated, it is not as widely used compared to CMS-S
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