Identification of highly variable chloroplast sequences and development of cpDNA-based molecular markers that distinguish four cytoplasm types in radish (Raphanus sativus L.)

Abstract

Four types of cytoplasms (Ogura, DCGMS, DBRMF1, and DBRMF2) were identified in the previous studies using molecular markers based on mitochondrial genome variations in radish (Raphanus sativus L.). However, mtDNA markers have limitations in obtaining clear results due to complexity of radish mitochondrial genomes. To improve fidelity, molecular markers based on variation of chloroplast genome sequences were developed in this study. We searched for the sequence variations of chloroplast genome among the four cytoplasm types in 11 noncoding intergenic regions of ~8.7 kb. Highly variable intergenic regions between trnK and rps16 were identified, and a couple of 4-34 bp indels were used to develop a simple PCR-based marker that distinguished the four cytoplasm types based on the PCR product length polymorphism. Two additional cpDNA markers were developed by using a single nucleotide polymorphism and 17-bp insertion. Analysis of 90 accessions using both mtDNA and cpDNA markers showed the perfect match of results of both the markers, suggesting strict co-transmission of mitochondria and chloroplast in radish. Phylogenetic trees showed that two male-sterility inducing cytoplasms, Ogura and DCGMS, were closely related to DBRMF1 and DBRMF2, respectively. Analysis of 120 radish germplasms introduced from diverse countries showed that the frequency of male-sterility inducing mitotypes of Ogura and DCGMS was very low, and DCGMS was predominately detected in eastern European countries. Majority of accessions from Europe and Asia were shown to contain DBRMF2 and DBRMF1 mitotypes, respectively.