Modification On The Genic Mode For A Genic Male Sterile Line9012AB And Physical Mapping Of The BnRf Locus In Brassica Napus L.

The utilization of heterosis is an important approach to improve yield, resistance for biotic stress and abiotic stress, and alleviate yield and quality contradictions in rapeseed. As an effective pollination-control system, recessive genic male sterile (RGMS) lines are always regarded to possess several advantages, such as stable and thorough male sterility, almost no restriction of restorers and diverse sources of cytoplasplasm. However, a major drawback of most RGMS lines lies in the fact that the male-sterility can not be fully maintained. This shortcoming would inevitably cause a high labour cost for removal of the50%fertile from the mother line and a great risk of seed purity in commercial production of hybrids. This trouble has been basically solved with the introduction of a novel RGMS line9012AB, which has the capability of generating a complete male-sterile population by crossing9012A plants with the so-called temporary maintainers. In this study, we modified the hereditary mode on the inheritance of male sterility in9012AB, as a result of classical genetic analyses and molecular marker assay. Based on this result, we finely mapped the BnRf locus through map integration and comparative genomics, and finally delimited it into a13.8-kb DNA fragment of a BAC clone. The main results were as follows:1. Modification on the hereditary mode of male sterility in9012AB. By genetic analyses of the crosses between9012AB and its temporary maintainer line T45with some inbred lines and open-pollination (OP) cultivars, we found that the fertility segregation data cannot be united under the previous widely-accepted tri-genic hereditary mode on sterility inheritance. Instead, we proposed a new mode to interpret the fertility control in9012AB that it is conditioned by two individual loci, i.e. BnMs3and BnRf. At the BnMs3locus, the dominant allele can restore the male sterility caused by the recessive allele. The putative BnMs4locus is actually one allele of BnRf (BnRfa, the restoration allele), and the other two alleles are respectively BnRfb (the sterility allele from9012A) and BnRf (the wild type allele from TAM), with the dominance relationship of BnRfa>BnRfb>BnRf. According to this new mode, the genotypes of9012A,9012B and its temporary maintainer line T45are individually Bnms3ms3RfbRfb, BnMs3ms3Rfb Rfband Bnms3ms3RfcRfc. Similarly, the genotype of restoration lines is BnMs3Ms3__or__RfaRf 2. Molecular marker evidence on the multiple-allele BnRf locus. From the F2BC1families9012A×(9012A×DH206A)F2in which a fertility segregation ratio of1:1is observed, the fertile plants with the genotype of Bnms3ms3RfaRfb were identified. By successively backcrossing these plants with9012A, we generated a ferlity segregation population RG206AB (RG206A, genotyped as Bnms3ms3RfhbRfb;RG206B, genotyped as Bnms3ms3RfaRfb). Molecular marker screen among this population enabled us to yield13AFLP markers and5SSR markers associated with BnRf. Marker integration showed that all these5SSR markers are located on the A7linkage group of B. napus and linked to the allele of BnRfb. Similarly, three of the7AFLP-converted SCAR markers are also genetically close to BnRf. These results together drove us to finely map the BnRfa allele by using the previously identified markers from the high-resolution genetic map of BnRfb. As expected, both of the BnRf and BnRfb alleles were finally delimited to a region flanked by marker AT3G24240and AT3G23900, corresponding to a physical region of50kb in Arabidopsis chromosome3. This investigation on molecular markers around BnRfa and BnRf greatly supports the allelism of them at the BnRflocus.3. Physical mapping of the BnRf locus. We amplified the specific fragment from the genomic DNA of9012A by using the cosegregated marker AT3G23870. This PCR product as a hybridization probe fished16positive BAC clones from a B. napus BAC clone library, and. PCR analysis with the nearest flanking codominant markers (AT3G23900and AT3G24240) showed that BAC clones JBnB089D05and JBnB134D11are most likely to contain the candidate locus of BnRf. Subsequent mapping of the BAC end derived markers BES18and BES19on respective side of BnRf, further indicated that JBnB089D05is the target BAC clone which spans the BnRf locus. We successfully recovered the13.8-kb fragment between the closest flanking markers AT3G23870and AT3G23910-1from JBnB089D05. Gene prediction of the13.8-kb sequence showed that there are3complete ORFs and partial genomic sequences of2ORFs.