Development of DNA Marker Enabling Selection of Male Sterile Citrus Seedlings Using PCR and Agarose Gel Electrophoresis

Abstract

Marker-assisted selection of male sterile seedlings improves breeding of seedless citrus varieties. We have identified a nuclear DNA region (MS-P1) related to male sterility caused by the Kishu cytoplasm, and classified the region into seven haplotypes (HT1–HT7) using SSR markers. The haplotype combination determines male sterility. This finding can be used in citrus breeding programs to select male sterile seedlings with the SSR markers. However, the high cost of SSR marker analysis prevents its widespread use in breeding programs at public research institutes in each prefecture. The aim of this study was to develop a simple and low-cost DNA marker that enables the selection of male sterile seedlings using PCR and agarose gel electrophoresis. Candidate markers were designed using InDel sequences near the MS-P1 region referred from Mikan Genome DB. The InDel marker (MS-P1 InDel) detected HT1 as the B allele, HT2 as the A allele, HT3, HT4, HT6, and HT7 as the C allele, and HT5 as the no-amplification allele. These alleles were discriminated by their amplified sizes using agarose gel electrophoresis. Correlation analysis with F₁ populations demonstrated that MS-P1 InDel could select male sterile seedlings with marked accuracy. HT4 was specifically detected by the CAPS marker (MS-P1 InDel-CAPS) using HT4-specific SNP in the sequence amplified by MS-P1 InDel. These haplotyping procedures were accelerated by a simple DNA extraction protocol. The results show that MS-P1 InDel and MS-P1 InDel-CAPS enable simple and low-cost selection of male sterile seedlings.