A multiplex PCR system for testing the genetic purity of hybrid rice (Oryza sativa L.)

Commercial exploitation of rice hybrid for sustainable production and productivity largely depends on genetic purity of hybrid seed used. To detect genetic impurity in the hybrid seeds developed through the three-line system using wild abortive (WA) cytoplasmic-based male sterility, a multiplex PCR assay was designed . A total of six primers, namely, DRRM-Rf3-10, Rf4-STS, RM6100, CMS-WA, osWA352 and RMS-3-WA352 were designed based on fertility restorer genes Rf3, Rf4 and Wa352. The primer combinations having RF4-STS and CMS-WA markers of Rf4 and WA352 genes, respectively showed clear and distinct PCR banding patterns among the WA-cytoplasm possessing 31A, fertile restorer PK117 lines and their cognate hybrid rice, Ajay. This multiplex PCR will be useful for assessment of genetic purity of hybrid rice seeds.

Identification of Suitable Restorers for WA-CMS Lines of Rice (Oryza sativa L.) through Conventional and Molecular Breeding Methods

The current study extensively evaluates 51 genotypes for their fertility restoration potential using test crosses with five WA (wild abortive) Cytoplasmic Male Sterile lines namely IR58025A, IR6897A, IR79156A, IR80559A and APMS6A. Also the genotypes were screened using SSR markers RM6100 and RM10313, tightly linked with the fertility restorer genes Rf4 and Rf3 respectively. The two way approach helped in identifying potential restorers for five WA-CMS lines and also detected the presence of dominant Rf genes in their genetic background. The R-lines identified can be safely presumed to be strong restorers for consecutive A-lines they were crossed with. The study also identified a potential maintainer CN1039-9 for the A-line IR58025A. The maintainer line identified can be later exploited for developing new CMS lines.

Homologous recombination changes the context of Cytochrome b transcription in the mitochondrial genome of Silene vulgaris KRA

BackgroundSilene vulgaris (bladder campion) is a gynodioecious species existing as two genders – male-sterile females and hermaphrodites. Cytoplasmic male sterility (CMS) is generally encoded by mitochondrial genes, which interact with nuclear fertility restorer genes. Mitochondrial genomes of this species vary in DNA sequence, gene order and gene content. Multiple CMS genes are expected to exist in S. vulgaris, but little is known about their molecular identity.ResultsWe assembled the complete mitochondrial genome from the haplotype KRA of S. vulgaris. It consists of five chromosomes, two of which recombine with each other. Two small non-recombining chromosomes exist in linear, supercoiled and relaxed circle forms. We compared the mitochondrial transcriptomes from females and hermaphrodites and confirmed the differentially expressed chimeric gene bobt as the strongest CMS candidate gene in S. vulgaris KRA. The chimeric gene bobt is co-transcribed with the Cytochrome b (cob) gene in some genomic configurations. The co-transcription of a CMS factor with an essential gene may constrain transcription inhibition as a mechanism for fertility restoration because of the need to maintain appropriate production of the necessary protein. Homologous recombination places the gene cob outside the control of bobt, which allows for the suppression the CMS gene by the fertility restorer genes. In addition, by analyzing RNA editing, we found the loss of three editing sites in the KRA mitochondrial genome and identified four sites with highly distinct editing rates between KRA and another S. vulgaris haplotypes (KOV). Three of these highly differentially edited sites were located in the transport membrane protein B (mttB) gene. They resulted in differences in MttB protein sequences between haplotypes despite completely identical gene sequences.ConclusionsFrequent homologous recombination events that are widespread in plant mitochondrial genomes may change chromosomal configurations and also the control of gene transcription including CMS gene expression. Posttranscriptional processes, e.g RNA editing shall be evaluated in evolutionary and co-evolutionary studies of mitochondrial genes, because they may change protein composition despite the sequence identity of the respective genes. The investigation of natural populations of wild species such as S. vulgaris are necessary to reveal important aspects of CMS missed in domesticated crops, the traditional focus of the CMS studies.