Molecular Identification Of The Male-sterile Gene BnRf~b Of A Genic Male Sterility Line 9012A In Brassica Napus L.

Rapeseed is an important crop worldwide, sustainably meeting the demands of edible and industrial oil and protein-rich animal feed. Application of hybrids significantly contributed to the productivity increase of rapeseed. A genic male sterility(GMS) line 9012 A and its analogues have been commercially used as an efficient pollination control system for hybrid production in major rapeseed cultivation districts. In 9012 A, the male-sterile phenotype is controlled by two gene loci Bn Ms3 and Bn Rf. To dissect the molecular basis of male sterility and restoration in 9012 A, the restorer gene Bn Ms3 has been isolated which encodes a translocon of the inner envelope of chloroplasts 40(Tic40) homologue. Fine mapping of the Bn Rf locus has been reported previously, and the genomic sequence of the candidate region for a temporary maintainer allele(Bn Rf c) has been acquired from a bacterial artificial chromosome(BAC) clone. Here in this study, a new genomic BAC library carrying both the male sterility gene(BnRf~b) and the fertility restoration gene(Bn Rf a) was constructed. Screening and sequencing of the BAC clones respectively containing Bn Rf a and BnRf~b reveals complex chromosomal structure variations in the Bn Rf candidate regions compared with the Bn Rf c-containing region. Further fine mapping of Bn Rf and genetic transformation validated that the male steritliy gene BnRf~bcorresponds to a chimeric gene, Bna A7.mt HSP70-1-lik. The major results were as follows:1、To get the genomic sequences covering Bn Rf a and BnRf~b, we identified independent BAC clones respectively encompassing Bn Rf a(HBn B029B10) and BnRf~b(HBn B114M18) from a well designed B. napus BAC library. Sequence comparison found a large DNA fragment insertion/deletion(In Del) and a local inversion in Bn Rf a and BnRf~b candidate regions relative to Bn Rf c, which made the closest flanking markers delimit Bn Rf to a 58.1 Kb DNA fragment on BAC clone HBn B029B10.2、We developed eight new molecular markers polymorphic between Bn Rf a and BnRf~b and further mapped Bn Rf a to a 35.9 Kb fragment including seven predicted open reading frames(ORFs). The previously identified candidates(G1-G5) from Bn Rfc-containing BAC were all excluded from this region, suggesting Bn Rf c is indeed a deleted nonfunctional allele.3、Considering two In Dels(Insertion/Deletion) in the promoter regionof G14, we preferred to validate the possibility of G14 as the candidate. To this end, we prepared a construct carrying the complete genomic sequences of G14 from RG206A(AGS14-1) and introduced it into the wild-type Arabidopsis(WT, Col-0) plants which shows a Bnms3 genetic background. All the resulting 27 independent transgenic-positive T1 plants displayed a male-sterile phenotype similar to that of RG206 A, with shorter filaments and severely retrograded anthers without pollen storage inside. Detail cytological observations showed the aberrant anther development is highly identical between sterile Arabidopsis plants and RG206 A. Moreover, we showed the additional introduction of Bn Ms3 from B. napus could completely restore the sterility of transgenic Arabidopsis plants carrying AGS14-1, consistent with the restoration function of Bn Ms3 specific to BnRf~b in B. napus. These facts comprehensively confirmed AGS14-1contain the male sterility gene BnRf~b that confers the GMS phenotype in RG206 A.4、Because Bn Rf a is regarded to be fully dominant over BnRf~b, the transfer of Bn Rf a to Arabidopsis is expected to reverse the sterility as Bn Ms3 showed. Nevertheless, we found all plants harboring the allelic G14 genomic DNA fragment from the restorer RG206H(AGS14-2) also showed a similar male-sterile phenotype. Accordingly, the allelic G14 from RG206 H is indeed a different haplotype of BnRf~b, while the true Bn Rf a is probably not allelic to BnRf~b but corresponds to another tightly associated gene in the 35.9 Kb candidate regions.5、The CDS of G14 from RG206A and RG206 H are completely identical, containing 9 exons and encoding a protein of 1386 amino acids. G14 is a chimeric gene fused by three independent members. Due to the high similarity with mt HSP70-1, we designated G14 as Bna A7.mt HSP70-1-like.Bna A7.mt HSP70-1-like showed continuous expression throughout the whole bud developmental stages with relatively high concentration around meiosis stages in RG206 H, while displayed weak and specific accumulation only in the microsporocyte and meiosis stages in RG206 A.6、The ectopic expression of Bna A7.mt HSP70-1-like in Arabidopsis, though not significantly affecting genes responsible for tapetum differentiation, negatively regulates genes functioning in tapetum degeneration. The retardation of tapetum PCD clearly observed in RG206 A further supported this conclusion.7、The only homologous gene of Bna A7.mt HSP70-1-like in the public database is a gene from Brassica oleracea. Although Bna A7.mt HSP70-1-likeanditshomologous gene are absent on the reference genomic sequence of Brassica rapa and Brassica napus, Bna A7.mt HSP70-1-likewere identified from 52 Brassica napus and 20 Brassica rapa in our study.