AcSKP1, a multiplex PCR-based co-dominant marker in complete linkage disequilibrium with the male-fertility restoration (Ms) locus, and its application in open-pollinated populations of onion

Selection for maintainer lines using molecular markers that distinguish genotypes at the male restorer-of-fertility (Ms) locus would improve the efficiency of breeding F-1 hybrids. Here, we developed a multiplex PCR-based co-dominant marker (AcSKP1) in complete linkage disequilibrium with the Ms locus in onion through allele mining and demonstrated its robustness and utility in marker-assisted selection. F1890, the 5' flanking sequence of F1527, was obtained from male-fertility-restored line 12-12 by genome walking. S1887, homologous sequence of F1890, was acquired from male-sterile line 118. The coding sequences of F1890 and S1887 were predicted by FGENESH and BLASTP to encode putative S-phase kinase-associated protein 1 (SKP1). These were designated as AcSKP1 and have been deposited in GenBank (KM065384 and KM065385). On the basis of the alignment results for the AcSKP1 gene between F1890 and S1887, four compatible primers (FU898, FD898, SU628, and SD628) were selected to develop a multiplex PCR-based co-dominant marker, AcSKP1, which was established in 25 breeding lines, seven hybrid cultivars, one F-2 population, two BC1 populations, and four open-pollinated (OP) populations. Linkage disequilibrium among the Ms locus and AcSKP1 and jnurf05 markers was evaluated using the F-2 population. There was no recombination between the Ms locus and AcSKP1, and only 0.049 % recombination between AcSKP1 and jnurf05. Subsequently, the AcSKP1 marker was applied to directly select maintainer individuals from four OP populations with different genetic backgrounds. The results detected by the AcSKP1 marker perfectly predicted the genotypes at the Ms locus in the four OP populations according to the phenotypes of testcross progenies.