A male-sterile (MS) radish (Raphanus sativus L.) was found in an accession collected from Uzbekistan. Unlike Ogura MS radishes in which no pollen grain is typically visible during anthesis, a small number of pollen grains stuck together in the dehiscing anthers was observed in the newly identified MS radish. Fluorescein diacetate tests and scanning electron micrographs showed that pollen grains in the new MS radish were severely deformed and non-viable. Cytological examination of pollen development stages showed a clear difference in the defective stage from that seen in Ogura male-sterility. Reciprocal cross-pollination with diverse male-fertile lines indicated that pollen grains of the new MS radish were completely sterile, and the female organs were fully fertile. When the new MS radish and Ogura MS lines were cross-pollinated with a set of eight breeding lines, all F1 progeny originating from crosses with the new MS radish were male-sterile. In contrast, most of the F1 progeny resulting from crosses with Ogura MS lines were male-fertile. These results demonstrated that factors associated with induction of the newly identified male-sterility are different from those of Ogura male-sterility. The lack of restorer lines for the newly identified male-sterility led us to predict that it might be a complete cytoplasmic male-sterility without restorer-of-fertility genes in nuclear genomes. However, cross-pollination with more diverse radish germplasm identified one accession introduced from Russia that could completely restore fertility, proving the existence of restorer-of-fertility gene(s) for the new male-sterility. Meanwhile, the PCR amplification profile of molecular markers for the classification of radish mitochondrial genome types revealed that the new MS radish contained a novel mitotype.

Plant mitochondrial genomes have complex configurations resulting from the multipartite structures and highly rearranged substoichiometric molecules created by repetitive sequences. To expedite the reliable classification of the diverse radish (Raphanus sativus L.) cytoplasmic types, we have developed consistent molecular markers within their complex mitochondrial genomes. orf138, a gene responsible for Ogura male-sterility, was detected in normal cultivars in the form of low-copy-number substoichiometric molecules. In addition to the dominant orf138-atp8 Ogura mitochondrial DNA (mtDNA) organization, three novel substoichiometric organizations linked to the atp8 gene were identified in this study. PCR amplification profiles of seven atp8- and atp6-linked sequences were divided into three groups. Interestingly, the normal cytoplasm type, which had previously been considered a single group, showed two patterns by PCR amplification. The most prominent difference between the two normal mtDNAs was size variation within four short-repeat sequences linked to the atp6 gene. This variation appeared to be the result of a double crossover, mediated by these homologous, short-repeat sequences. Specific PCR amplification profiles reflecting the stoichiometry of different mtDNA fragments were conserved within cultivars and across generations. Therefore, the specific sequences detected in these profiles were used as molecular markers for the classification of diverse radish germplasm. Using this classification system, a total of 90 radish cultivars, or accessions, were successfully assigned to three different mitotypes.

Previously, novel cytoplasmic male-sterility (CMS) caused by DCGMS cytoplasm was discovered in radish (Raphanus sativus L.) introduced from Uzbekistan. We performed extensive progeny tests and identified two fertility restorer lines ('R171' and 'R121') for this new CMS. Two F 1 hybrid populations were self-pollinated and backcrossed to produce F 2 and BC populations. Inheritance patterns of male-sterility in segregating populations varied depending on paternal lines. Segregation of malesterility in F 2 populations originating from the cross between MS19 and R121 showed that a single locus was involved in fertility restoration. However, populations originating from the cross between MS15 and R171 showed the involvement of more than one restorer-of-fertility genes. The single fertility restorer locus identified in the cross between MS19 and R121 was designated Rfd1 locus. Bulked segregant analysis was performed using RAPD and AFLP, which identified one marker each. Both RAPD and AFLP markers were converted into simple PCR-based co-dominant markers after their isolated flanking sequences were analyzed. Indels 773-bp and 67-bp in length were identified between two Rfd1 allele-linked flanking sequences of the RAPD and AFLP fragments, respectively, then utilized to develop simple PCR markers. In addition, we prove that the newly identified Rfd1 locus is independent of the Rfo locus, another radish fertility restorer for CMS caused by Ogura cytoplasm.

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.

A novel cytoplasmic male-sterility (CMS) radish (Raphanus sativus L.) and its associated mitotype (DCGMS) were previously identified; however, no mtDNA fragments flanking the atp6 gene were found in the DCGMS mitotype. Unlike three other mitotypes in this study, a unique mtDNA organization, atp6-nad3-rps12, was found to be the major mtDNA structure associated with this mitotype. This organization may have arisen from short repeat sequence-mediated recombination events. The short repeat clusters involved in the mtDNA rearrangement around the atp6 gene also exist as repetitive sequences in the complete mitochondrial genomes of other members of the Brassicaceae family, including rapeseed and Arabidopsis. These sequences do not exist as repetitive elements in the mtDNA of tobacco, sugar beet, or rice. While studying the regions flanking atp6, we identified a truncated atp6 mtDNA fragment which consists of the 5' part of the atp6 gene linked to an unidentified sequence. This mtDNA structure was present in all mitotypes; however, a single nucleotide insertion mutation leading to a frame-shift was identified only in the DCGMS mitotype. Although this truncated atp6 organization was transcribed, there was no significantly different expression between male-sterile and fertile segregating individuals from the BC(1)F(1) population originating from a cross between male-sterile and restorer parents. Comprehensive survey of the single base-pair insertion showed that it was maternally inherited and unique to the DCGMS mitotype. Therefore, this single nucleotide polymorphism (SNP) in the coding sequence of the mtDNA will be a useful molecular marker for the detection of the DCGMS mitotype.

A detailed restriction map of squash chloroplast DNA (cpDNA) was constructed with five restriction endonucleases, SalI, PvuII, BglI, SacII, and PstI. The cleavage sites were mapped by sequential digestion of cpDNA using low-gelling temperature agarose. The restriction map shows that squash cpDNA is an approximately 153 kilobase (kb) circle with a large inverted repeat sequence of 23.3 kb, separated by a large (83.7 kb) and a small (22.7 kb) single copy region. Genes for a number of chloroplast polypeptides were localized on the map by hybridizing the cpDNA restriction fragments to heterologous gene-specific probes from tobacco, pea, tomato, maize, and spinach chloroplasts. The gene locations and organization of squash cpDNA are highly conserved and similar to chloroplast genomes of tomato, pepper, and Ginkgo.