The recessive genic male sterility (RGMS) line 9012AB has been used successfully for rapeseed hybrid production in China. This male sterility was previously thought to be controlled by three independent genes (Bnms3, Bnms4, and BnRf). Here, we initially attempted to locate the BnMs4 locus and develop feasible molecular markers for application in practical rapeseed breeding. However, we found that three sequence characterized amplified region markers and five simple sequence repeat markers identified as linked to BnMs4 were also genetically associated with BnRf, suggesting the possible co-localization of these two loci. Moreover, we proved that four intron-based polymorphism markers tightly linked or co-segregated with BnRf could also be mapped to BnMs4 with a genetic distance ranging from 0.054 to 0.594 cM. Finally, integration of genetic maps around BnRf and BnMs4 allows for the physical restriction of both loci to a DNA fragment of about 50 kb. Systematic genetic tests also provided evidence that the candidate BnMs4 locus was allelic to the BnRf locus. These results confirmed a major modification of the sterility inheritance model in 9012A: specifically, that this male sterility was essentially controlled by two loci (BnMs3 and BnRf), whereas the previously designated BnMs4 locus (hereafter designated as BnRf (a) ) was just one allele of BnRf in addition to BnRf (b) (the allele from 9012A) and BnRf (c) (the allele from temporary maintainer), with a dominance relationship of BnRf (a) > BnRf (b) > BnRf (c) . This inheritance model will simplify the breeding process involved with this RGMS line, especially with the BnRf allele-specific molecular markers identified here.

The Polima (pol) system of cytoplasmic male sterility (CMS) in rapeseed is widely used in China for commercial hybrid seed production. Genetic studies have shown that its fertility restorer gene (Rfp) is monogenic dominant. For fine mapping of the Rfp gene, a near isogenic line comprising 3,662 individuals of BC14F1 generation segregating for the Rfp gene was created. Based on the sequences of two SCAR markers, SCAP0612ST and SCAP0612EM2, developed by Zhao et al. (Genes Genom 30(3):191–196, 2008) and the synteny region of Brassica napus and other Brassica species, 13 markers strongly linked with the Rfp gene were identified. By integrating three of these markers to the published linkage map, the Rfp gene was mapped on linkage group N9 of B. napus. Using these markers, the Rfp locus was narrowed down to a 29.2-kb genomic region of Brassica rapa. Seven open reading frames (ORFs) were predicted in the target region, of these, ORF2, encoding a PPR protein, was the most likely candidate gene of Rfp. These results lay a solid foundation for map-based cloning of the Rfp gene and will be helpful for marker-assisted selection of elite CMS restorer lines.

9012AB is a recessive epistatic genic male sterility(REGMS) two-type line of Brassica napus L.The sterility is controlled by two recessive duplicate genes ms3ms3ms4ms4 and one recessive epistatic inhibitor gene espesp.To effectively identify REGMS lines and their temporary maintainer lines,five AFLP(amplified fragment length polymorphism) markers(L1,L2,L3,L4,L5) in couple to esp and two AFLP markers(L6,L7) in repulsion to esp were identified through screening 1 024 primer combinations using 9012B and T45 segregation population.All the seven markers were located on one the same side of esp gene.Markers L6 and L7 co-segregated with Esp gene.Markers L1,L6 and L7 were successfully converted into SCAR(sequence characterized amplified region) markers as SCL1,SCL2 and WSCL3,respectively.The accuracy rates of SCL1,SCL2 and WSCL3 were 90%,100% and 100%,respectively.Result showed their potential in marker assisted selection of 9012AB.

Rapeseed yield is directly and indirectly influenced by the silique-traits, such as silique length (SL), seeds per silique (SS), seed weight (SW), because the silique is an organ which produced yield and a major photosynthesis organ as well. In this study, a linkage map comprising 150 simple sequence repeat and 195 amplified fragment length polymorphism markers covering 1,759.6 cM was constructed in a doubled haploid population from a cross between two genotypes of 'HZ396' and 'Y106'. In field experiments across three seasons and two locations in China 140 doubled haploid lines and their corresponding parents were evaluated for silique-traits. In total, 26 quantitative trait loci (QTL) were detected, of which 15 were clustered and integrated into 5 pleiotropic unique QTL by meta-analysis. These unique QTL, which in a certain sense reflected the significant positive correlation between SS and SL and the significant negative correlation between SW and SS by the genomic location and effects of QTL detected, were mapped on linkage groups N7, N8 and N13. A trait-by-trait meta-analysis revealed 5, 2 and 3 consensus QTL for SL, SS and SW, respectively. Epistatic effects varied according to the specific traits performed. All the epistatic interactions showed significant additive by additive effects while no significant epistasis by environment effect was identified. These findings provided a better understanding of the genetic factors controlling silique-traits and gained insights into the gene networks affecting silique-traits at QTL level in rapeseed.

Polima cytoplasmic male sterility (pol CMS) has been widely used for exploiting heterosis in rapeseed breeding. The dominant restorer gene of pol CMS (Rfp) is found in the nucleus and is a key component of hybrid production by achieving F1 progeny with complete fertility restoration. To identify the molecular markers associated with the Rfp gene, a near isogenic line (NIL) population of 2,000 individuals segregating for the Rfp locus was generated by crossing and backcrossing for 12 times. This NIL population was used to screen Rfp markers by AFLP technique. Based on the sequence information of AFLP markers that have been identified in previous research, we identified a homologous region of Rfp locus in chromosome 1 of Arabidopsis. Then, six sequenced Brassica rapa BAC clones corresponding to this target region were chosen to design microsatellite (SSR) primer pairs. Twenty-two SSR markers were designed and one of them, KBrDP1, was verified in the 2, 000 NILs population and proved to be strongly linked to Rfp locus. The genetic distance between KBrDP1 and Rfp was 0.2 cM. KBrDP1 marker was found located on linkage group N9 of a published DH mapping population. This SSR marker was useful in marker assisted selection breeding of the elite pol CMS restorer lines in rapeseed.

The genetic model of a rapeseed recessive genic male sterility line 9012AB was re-evulated by traditional genetic analyses.Genetic analysis results showed that the male sterility of 9012A is controlled by two individual loci,i.e.Ms3 and Rf.There are two alleles at the Ms3 locus,the dominant wild type allele Ms3 is the fertile allele,while its recessive allele ms3 the sterile one.Three alleles are found at the Rf locus,individually Rf a(mutation restoration allele),Rf b(mutation sterile allele) and Rf c(wild type fertile allele),with the dominance relationship of Rf aRf bRf c.According to this new model,the genotype of 9012A,9012B and its temporary maintainer line T45 is Ms3ms3Rf bRf b,ms3ms3Rf bRf b and ms3ms3Rf cRf c,respectively.Similarly,the genotype of restoration line is Ms3 Ms3__or__Rf aRf a.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most severe diseases of rapeseed in China and other major growing regions. The objective of this study was to improve the S. sclerotiorum resistance of 'Hui5200', an elite 'Polima' CMS restorer line, by introgression and fixation of resistance alleles from the partially resistant cultivar 'NingRS-1' via phenotypic selection (PS), marker-assisted background selection (MAB) and microspore culture. A progeny designated as 'RSH' with greatly improved Sclerotinia resistance and a similar genetic background as 'Hui5200' was obtained by two backcrosses and one selfing. From a selected elite progeny line (named as 7-5) double haploid (DH) lines were developed. By three cycles of PS considering economic traits and genetic distance analysis, four resistant DH restorer lines with elite economic traits were finally selected. The obtained resistant restorer lines have been used to produce commercial F-1 hybrids. The results indicated that backcrossing plus PS and MAB is effective and suitable for improving resistance of rapeseed to S. sclerotiorum.

Seeds per silique (SS) is one of three important components of yield in rapeseed. Genetic control of this trait was primarily studied in 2006 in the cross of ‘HZ396’בY106’, in which ‘Y106’ is a higher-SS line and ‘HZ396’ is a lower-SS one. The frequency distribution of the F2 generation deviated from a normal distribution and appeared to have a multi-modal pattern, indicating the influence of major genes mixed with polygenes. To test this hypothesis and uncover the genetic basis of SS in diverse genotypes, we further used the joint segregation analysis (JSA) of six populations (P1, P2, F1, F2, B1 and B2) in the crosses of ‘HZ396’בY106’ and ‘HZ165’בY106’ and four populations (P1, P2, F1, F2) in the cross of ‘HZ168’בY106’ in 2008. The JSA revealed that SS was best described by the E-0 genetic model, a case of two additive-dominace-epistasis major genes as well as additive-dominace-epistasis polygenes. We observed considerable genetic diversity in all genotypes by evaluating the first- and second-order genetic parameters based on the JSA method. Variance analysis of genotype×year interaction revealed the steady genetic effects of the two major genes within different years. The additive effects of the two major genes ranged from −3.71 to −1.49 in the three crosses. Heritability of the major genes varied from 39% to 84% in diverse generations (B1, B2 and F2), which was much larger than that of the polygenes. These findings will facilitate breeding strategies for SS improvement as well as efforts to map the major genes.

Rs1046AB is a dominant genic male sterility (DGMS) line derived from a spontaneous mutant in Brassica napus. The sterility of this mutant was previously regarded as to be conditioned by the interaction of a dominant male sterility gene Ms and its non-allelic dominant restorer gene Mf (or Rf in previous reports). Recent genetic analyses, however, indicated that Ms and Mf may be allelic. In this research, we confirmed that a multiple allele model should be more appropriate to elucidate the heredity of DGMS line Rs1046AB. Based on this result, the present study emphasized on identifying DNA markers linked to Ms/Mf an F-2 population constructed by crossing Rs1046A with a double haploid (DH) restorer line (19514A). Twenty amplified fragment length polymorphism (AFLP) markers linked to the Ms/Mf locus were identified by combination of bulked segregant analysis (BSA) with AFLP technique. The target locus was detected to be co-segregating with the marker S05T05 and bracketed by two nearest markers, E14M01 and E01M02, with a genetic distance of 0.1 cM and 1.2 cM respectively. Furthermore, we successfully converted two AFLP markers into sequence characterized amplified region (SCAR) markers. These findings provided direct molecular marker proofs on the inheritance model of this DGMS line, and the high density molecular markers linkage map around the Ms/Mf locus will be more informative for both marker-assisted selection MAS) of elite male sterile lines and isolation of the Ms/Mf genes by positional cloning in future.

Rs1046AB is a dominant genic male sterility (DGMS) line in rapeseed, in which the sterility has always been thought to be conditioned by the interaction of a male sterility gene (Ms) and its non-allelic restorer gene (Rf). This system provides not only a tool for assisting in recurrent selection but also a promising system for hybrid production. Based on previous studies, two amplified fragment length polymorphism markers linked with the Ms gene were converted into a dominant and a co-dominant sequence characterized amplified region (SCAR) marker, respectively. The putative linear order relationship of three dominant SCAR markers with the same genetic distance from the Rf gene, was also determined by an examination of whether the homologues of these markers are present or not in different lines carrying Rf. A bigger fragment generated by the closest marker linked to the Rf gene was observed in all lines carrying the recessive allele rf, suggesting that this marker is a co-dominant marker, which was further confirmed by nucleotide sequence comparison of these fragments. SCAR markers specific for Ms and Rf will be especially valuable in marker-assisted DGMS three-line breeding.

RAPD,SSR and AFLP technology combined with bulked segregant analysis were performed to identify genetic markers for pol CMS restorer gene Rfp and to map the markers using an F2 population of a pol CMS line 1141A crossed to its restorer line Huayehui.One AFLP marker E7P16230 and one RAPD marker S1-500 were identified linked to Rfp locus.These two genetic markers flank Rfp gene on different sides,and closely linked to Rfp with a genetic distance of 4.3 cM for E7P16230 and 10.8 cM for S1-500,respectively.

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene ( Ms ) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene ( Rf ). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus . In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual ( MsMsrfrf ) and a fertile individual ( MsMsRfrf ) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.

In this study, AFLP (amplified fragment length polymorphism) was improved on the combination of restriction enzymes by using two average cutting frequency restriction enzymes (EcoRⅠ+PstⅠ) to take place the combination of an average cutting frequency and a higher cutting frequency restriction enzyme (EcoRI+MseI and PstI+MseI) in a single digestion. The results showed that the combination of EcoRⅠ+PstⅠ could generate fewer fragments with larger size (most of bands distribute among 150 bp to 800bp) as well as a higher percentage of polymorphism than EcoRⅠ+MseⅠ and PstⅠ+MseⅠ. The signal strength and polymorphism of PCR products were improved too. Furthermore, the results described here have great commercial potential because the average cutting frequency enzymes EcoRI and PstI are much cheaper than the high cutting frequency enzyme MseI. In this way, the cost of AFLP analysis can be reduced.

In the regional trial of Hubei province and middle and lower reaches of Yangtze River,Huayouza No9 increaced yield by 24.76% and 16.68% than Zhongyou 821 andZhongshuang 6.In nearly two years production,This cultivar appeared high and steady yield,good quality,steady yield,anti-lodging and broad adaptability.Huayouza No9 increased production remarkably in the districts of Yangtse river such as Hubei,Hunan,Shnghai,Zhejiang,Jiangsu,Anhui and the south of Huai river.

Huaza No.8 is a new3-linehybrid rapeseed with using invention patent donated by national oilseed improvement center of Huazhong Agriculture University. The variety could be applied in Yangtze River valley very well. It is identical for high and steady yield, improved quality, high oil content as well as strong disease resistance. So it is an ideal variety to realize rapeseed industrialization.