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.

Identification of Rfd1, a novel restorer-of-fertility locus for cytoplasmic male-sterility caused by DCGMS cytoplasm and development of simple PCR markers linked to the Rfd1 locus in radish (Raphanus sativus L.)

“…However, the gene responsible for CMS in DCGMS may differ from the orf138 gene, since orf138 was detected as a sublimon in the DCGMS cytotype (Lee et al 2008). Furthermore, the phylogenetic relationship based on chloroplast sequence polymorphisms among the four cytotypes revealed that the DCGMS cytotype was very close to the male-fertile DBRMF2 cytotypes rather than the Ogura cytotype (Kim et al 2009a). In addition, the two CMS lines had different male-sterility phenotypes and pollen development patterns, and many restorer lines for Ogura CMS failed to restore fertility to DCGMS male-sterility (Lee et al 2008).…”

“…One interesting feature of the male-fertile parental line (R171) is that this accession was introduced from Russia and contains DCGMS cytoplasm. The earliest identified male-sterile accession containing DCGMS cytoplasm was introduced from Uzbekistan ( Lee et al 2008), and a previous survey of four radish cytotype distribution showed that DCGMS cytotypes were predominantly identified from germplasm introduced from East European countries (Kim et al 2009a). Therefore, we assume that multiple restorer-of-fertility genes might exist in radish germplasm from European countries.…”

“…Previously, we classified radish germplasm into four cytotypes (DBRMF1, DBRMF2, Ogura and DCGMS) based on polymorphisms of the mitochondrial genome organizations and non-coding chloroplast sequences (Kim et al 2007(Kim et al , 2009aLee et al Fig. 5 Linkage analysis of the Rfo and Rfd1 loci in the backcross population (MS19 9 (MS19 9 R121)).…”

Construction of a high-resolution linkage map of Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility conferred by DCGMS cytoplasm in radish (Raphanus sativus L.) using synteny between radish and Arabidopsis genomes

“…The Brassicaceae family is estimated to consist of 338 genera and 3,700 species (Bailey et al 2006). Previous phylogenetic studies of the Brassicaceae family showed that radish was a relative of both Arabidopsis and Brassica species (Yang et al 1999;Kim et al 2009). In particular, radish was reported to be more closely related to Brassica rapa than other Brassica species when variations within the chloroplast genomes were used to construct phylogenetic trees (Warwick and Black 1997;Yang et al 2002;Kim et al 2009).…”

“…Previous phylogenetic studies of the Brassicaceae family showed that radish was a relative of both Arabidopsis and Brassica species (Yang et al 1999;Kim et al 2009). In particular, radish was reported to be more closely related to Brassica rapa than other Brassica species when variations within the chloroplast genomes were used to construct phylogenetic trees (Warwick and Black 1997;Yang et al 2002;Kim et al 2009). However, when the phylogeny were analyzed using RFLP and random amplified polymorphic DNA (RAPD) techniques (Thormann et al 1994) or nuclear internal transcribed spacer (ITS) sequences (Yang et al 1999), radish was shown to be more closely related to B. nigra than B. rapa.…”