Map-Based Cloning Of A Male Sterility Locus Bnms3in Brassica Napus L. And Development Of Functional Marker

A novel recessive genic male sterility line,9012AB, has been characterized by generating a complete male-sterile population through pollinating9012A male-sterile plants with the temporary maintainers. Preliminary genetic analyses suggested that three loci (BnMs3, BnMs4and BnRf) are responsible for the male sterility in9012AB (Chen et al.,1998). Recent study, however, suggests that the BnMs4locus should be allelic to BnRf, turning the trigenic inheritance model to a digenic one (Dong et al.,2010). In this research, we constructed three new populations with different genetic compositions (Pop B, Pop C and Pop D, respectively) to isolate the BnMs3locus, on basis of the fine genetic mapping of BnMs3(He et al.,2008). With the integration of classic map-based cloning strategy and comparative genomics, we successfully cloned the gene, BnMs3. Main results are as follows:1. Genetyping of more than8,000individuals among the new populations showed that BnMs3, still cosegregated with SCR1, can be restricted by two previously BnMs-cosegregated markers9W21and MT7. each with one recombination event. Hereby, we designed new SSR primers according to the sequence between9W21and SCR1on BAC clone JBnB004A17. The development of a new marker (BSR73) on the same side as9W21further reduced the candidate region of BnMs3to a39-kb fragment on JBnB004A17.2. We used DM1, a BnMs3-linked marker which can effectively differentiate the A and C subgenome in B. napus, to identify the BAC clone JBnB004A17. It was revealed that the resulting fragment corresponds to the band in B.rapa, and is different from the polymorphic band associated with BnMs3in the mapping polulations, suggesting that JBnB004A17is actually originated from a BnMs3paralogous region in the A subgenome. The BnMs3-linked fragment amplified by DM1from Tapidor was therefore employed to screen the BAC library again. PCR and Southern blotting analysis found that6BAC clones from C subgenome possibly cover the BnMs3candidate region. One BAC clone from them, JBnB043L23, was then shotgun sequenced, harvesting an insertion fragment of118kb.3. High sequence similarity was observed on a large scale between JBnB043L23and JBnB004A17, while extensive variations including fragment insertion/deletion and massive SNPs were also revealed. According to the sequence differences between the two homologous BAC clones, SCAR primers specifically binding to JBnB043L23as well as SSR primers were designed from the61-kb region delimited by marker BSR73and MT7. Population analysis showed that massive primers located in this region could generate good polymorphism. Assay of recombinantion individuals with these markers led to our final delimitation of BnMs3to a9.3-kb physical region, restricted by closest flanking markers RCP145and RCP137.4. Gene prediction analysis of the9.3-kb fragment identified only one complete ORF (ORF2) and partial genomic sequences of its two flanking genes (ORF1and ORF3). Complementation test indicated that the ORF1, restoring the fertility of the male-sterile9012A plants, is the target gene of BnMs3. We designed BnMs3as BnaC9.TIC40.a, since ORF1was highly homologous with Arabidopsis gene At5g16620(Tic40), and the mutant of9012A was braC9.tic40.a-2. Consistently, the A10paralogue of BnMs3was referred to BnaA10.TIC40.a. The allele of BnaC9.TIC40.a from Tapidor includes14exons, and is predicted to encode a protein with455amino acids, which contains four conserved domains, i.e.. a Ser/Pro-rich domain, transmembrane domain. TPR domain and Stilp/H op/Hip domain. RT-PCR analysis showed that BnaC9. TIC40.a can be expressed in all the tissues tested in9012B, including stamens with diffenerent sizes, roots, stems, petals and leafs. Additionly, whether in9012A and9012B, bnaC9.1ic40.a-2and BnaA10. TIC40.a were found to be expressed in all the tissues tested.5. The genomic sequences corresponding to the9.3-kb BAC fragment were individually isolated from9012A,9012F and T45. Comparison of these fragments among different lines showed that9012A has an85.5%similarity with JBnB004A17, much higher than that with JBnB043L23(79.1%). In contrast,9012F has a93.3%similarity with JBnB043L23, while its similarity with JBnB004A17is only80.8%. In most cases,9012A was found to have an identical sequence with JBnB004A17in the sites polymorphic between JBnB004A17and JBnB043L23, whereas9012B is closely identical to JBnB043L23. However, the sequence similarity in the candidate region between T45and JBnB043L23was99.9%. Thus, we proposed that the Bnms3allele in9012A was originally caused by an occasional homeologous reciprocal recombination between chromosome A10and C9, whereas the Bnms3allele from T45was most likely to be derived from a spontaneous mutation. So, there are two Bnms3alleles with distinctive origins. 6. We designed an InDel marker according to the2-bp nucleotide variation between T45and Tapidor in BnaC9.TIC40.a. RCP170L/R can accurately differentiate the BnMs3allele from all the inbred lines (OP cultivars) from the Bnms3allele in T45, indicating it is a functional marker for the Bnms3locus in T45. We also developed another codominant marker RCP170L2-3/R2according to the differences between9012A and9012B of BnaC9.TIC40.a, which can successfully differentiate the Bnms3allele in9012A from all the other materials with a wild-type BnMs3allele as well as T45. In principle, utilization of these two markers can accurately trarget the genotype of the BnMs3locus in any segregation generation involved in RGMS breeding.