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The genomic and cDNA sequences of BnSUT1C were isolated from B. napus. Combination of cDNA and genomic DNA sequences revealed that the BnSUT1C gene contained three exons and two introns. The cDNA encodes a protein of 513 amino acids with a calculated molecular mass of 54.7 kDa and an isoelectric point of 9.12. It exhibits typical features of sucrose transporter with 12 trans-membranes spanning domains. BnSUT1C showed highly homologous with AtSUC1 and AtSUC5. A histidine residue, which is conserved across all functional sucrose transporter proteins in higher plants, is located at position 66 of the BnSUT1C. Two putative pollen-specific cis-elements, AGAAA and GTGA motifs, are located in 5'-upstream of BnSUT1C. The spatial and temporal expression patterns carried out by semi-quantitative RT-PCR and Real-Time PCR, which indicated that BnSUT1C predominantly expressed in later developmental stages of anther, as tapetal cells began to shrink and collapse. BnSUT1C could mediate the uptake of sucrose in the pollen and retrieval of tapetal degenerated products during pollen maturation.

Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with different content of C18:3 in amphidiploid B. napus.

Yellow-seeded Brassica napus was for the first time developed from interspecific crosses using yellow-seeded B. juncea (AABB), yellow-seeded B. oleracea (CC), and black-seeded artificial B. napus (AACC). Three different mating approaches were undertaken to eliminate B-genome chromosomes after trigenomic hexaploids (AABBCC) were generated. Hybrids (AABCC, ABCC) from crosses AABBCC × AACC, AABBCC × CC and ABCC × AACC were advanced by continuous selfing in approach 1, 2 and 3, respectively. To provide more insight into Brassica genome evolution and the cytological basis for B. napus resynthesis in each approach, B-genome chromosome pairing and segregation were intensively analyzed in AABCC and ABCC plants using genomic in situ hybridization methods. The frequencies at which B-genome chromosomes underwent autosyndesis and allosyndesis were generally higher in ABCC than in AABCC plants. The difference was statistically significant for allosyndesis but not autosyndesis. Abnormal distributions of B-genome chromosomes were encountered at anaphase I, including chromosome lagging and precocious sister centromere separation of univalents. These abnormalities were observed at a significantly higher frequency in AABCC than in ABCC plants, which resulted in more rapid B-genome chromosome elimination in the AABCC derivatives. Yellow or yellow-brown seeds were obtained in all approaches, although true-breeding yellow-seeded B. napus was developed only in approaches 2 and 3. The efficiency of the B. napus construction approaches was in the order 1 > 3 > 2 whereas this order was 3 > 2 > 1 with respect to the construction of yellow-seeded B. napus. The results are discussed in relation to Brassica genome evolution and the development and utilization of the yellow-seeded B. napus obtained here.

A recessive epistatic genic male sterile two-type line, 7365AB (Bnms3ms3ms4msRrfRf/BnMs3ms3ms4ms4RfRf), combined with the fertile interim-maintainer 7365C (Bnms3ms3ms4ms4rfrf) is an effective pollination control system in hybrid rapeseed production. We report an effective strategy used to fine map BnMs4 and BnRf. The two genes were both defined to a common microsyntenic region with Arabidopsis chromosome 3 using intron polymorphism (IP) markers developed according to Arabidopsis genome information and published genome organization of the A genome. The near-isogenic lines 7365AC (Bnms3ms3ms4ms4Rfrf/Bnms3ms3ms4ms4rfrf) of BnRf and 736512AB (Bnms3ms3Ms4ms4RfRf/Bnms3ms3ms4ms4RfRf) of BnMs4 were constructed to screen developed markers and create genetic linkage maps. Nine polymorphic IP markers (P1-P9) were identified. Of these, P2, P3, P4, and P6 were linked to both BnMs4 and BnRf with genetic distances <0.6 cM. Three simple sequence repeat markers, SR2, SR3, and SR5, were also identified by using public information. Subsequently, all markers linked to the two genes were used to compare the micro-collinearity of the regions flanking the two genes with Brassica rapa and Arabidopsis. The flanking regions showed rearrangements and inversion with fragments of different Arabidopsis chromosomes, but a high collinearity with B. rapa. This collinearity provided extremely valuable reference for map-based cloning in polyploid Brassica species. These IP markers could be exploited for comparative genomic studies within and between Brassica species, providing an economically feasible approach for molecular marker-assisted selection breeding, accelerating the process of gene cloning, and providing more direct evidence for the presence of multiple alleles between BnMs4 and BnRf.

Here, we describe the characteristics of a Brassica napus male sterile mutant 7365A with loss of the BnMs3 gene, which exhibits abnormal enlargement of the tapetal cells during meiosis. Later in development, the absence of the BnMs3 gene in the mutant results in a loss of the secretory function of the tapetum, as suggested by abortive callose dissolution and retarded tapetal degradation. The BnaC.Tic40 gene (equivalent to BnMs3) was isolated by a map-based cloning approach and was confirmed by genetic complementation. Sequence analyses suggested that BnaC.Tic40 originated from BolC.Tic40 on the Brassica oleracea linkage group C9, whereas its allele Bnms3 was derived from BraA.Tic40 on the Brassica rapa linkage group A10. The BnaC.Tic40 gene is highly expressed in the tapetum and encodes a putative plastid inner envelope membrane translocon, Tic40, which is localized into the chloroplast. Transmission electron microscopy (TEM) and lipid staining analyses suggested that BnaC.Tic40 is a key factor in controlling lipid accumulation in the tapetal plastids. These data indicate that BnaC.Tic40 participates in specific protein translocation across the inner envelope membrane in the tapetal plastid, which is required for tapetal development and function., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The GRAS proteins are a family of transcription regulators found in plants and play diverse roles in plant growth and development. To study the biological roles of GRAS family genes in Brassica napus, an Arabidopsis LAS homologous gene, BnLAS and its two homologs were cloned from B. napus and its two progenitor species, Brassica rapa and Brassica oleracea. Relatively high levels of BnLAS were observed in roots, shoot tips, lateral meristems and flower organs based on the analysis of the transcripts by quantitative RT-PCR and promoter-reporter assays. Constitutive overexpression of BnLAS in Arabidopsis resulted in inhibition of growth, and delays in leaf senescence and flowering time. A large portion of transgenic lines had darker leaf color and higher chlorophyll content than in wild type plants. Interestingly, water lose rates in transgenic leaves were reduced, and transgenic plants exhibited enhanced drought tolerance and increased recovery after exposed to dehydration treatment. The stomatal density on leaves of the transgenic plants increased significantly due to the smaller cell size. However, the stomatal aperture on the leaves of the transgenic plants reduced significantly compared with wild type plants. More epidermal wax deposition on transgenic leaves was observed. Furthermore, several genes involved in wax synthesis and regulation, including CER1, CER2, KCS1 and KCS2, were upregulated in the transgenic plants. Our results indicate a potential to utilize BnLAS in the improvement of drought tolerance in plants.

In the present study, we have obtained one dwarf mutant (bnaC.dwf) from the Brassica napus inbred line T6 through chemical mutagen ethyl methanesulfonate (EMS). We have determined the phenotypic effects and genetic characteristics of dwarf mutant (bnaC.dwf). The dwarf mutant was insensitive to exogenous GA(3) for plant height, suggesting that it is significantly playing a crucial role in the gibberellins response pathway. Genetic analysis revealed that one recessive gene is responsible for controlling the phenotypic expression of dwarf mutant. Amplified Fragment Length Polymorphism (AFLP) technique was applied for selecting markers linked to the BnaC.DWF gene which assisted in screening of dwarf and normal individuals in the BC(4) population. We have screened 1,024 primer combinations and then identified nine AFLP markers linked to the BnaC.DWF gene. Identification and linkage of the markers were carried out by analysing 2,000 individuals from a larger population of the BC(4). Two markers EA10MC09 and EA12MC02 were located on the flanking region of the BnaC.DWF gene at a distance of 0.2 and 0.05 cM, respectively. Four AFLP markers EA09MG05, EA02MC07, EA01MC01 and EC04MC07 were successfully converted into Sequence Characterised Amplified Region markers namely SCA9G5, SCA2C7, SCA1C1 and SCC4C7. We further integrated BnaC.DWF linked Simple Sequence Repeat markers into two populations (Piquemal et al. Theor Appl Genet 111:1514-1523, 2005; Cheng et al. Theor Appl Genet 118:1121-1131, 2009). BnaC.DWF was mapped to the linkage region N18. The molecular markers developed from these investigations will greatly accelerate the selection process for developing dwarf varieties in B. napus by Marker Assisted Selection and genetic engineering.

Seed weight is an important component of grain yield in oilseed rape (Brassica napus L.), but the genetic basis for the important quantitative trait is still not clear. In order to identify the genes for seed weight in oilseed rape, QTL mapping for thousand seed weight (TSW) was conducted with a doubled haploid (DH) population and an F(2) population. A complete linkage map of the DH population was constructed using 297 simple sequence repeat (SSR) markers. Among nine TSW QTLs detected, two major QTLs, TSWA7a and TSWA7b, were stably identified across years and collectively explained 27.6-37.9% of the trait variation in the DH population. No significant epistatic interactions for TSW detected in the DH population indicate that the seed weight variation may be primarily attributed to additive effects. The stability and significance of TSWA7a and TSWA7b were further validated in the F(2) population with different genetic backgrounds. By cloning BnMINI3a and BnTTG2a, two B. napus homologous genes to Arabidopsis thaliana, allele-specific markers were developed for TSWA5b and TSWA5c, two TSW QTLs on A5, respectively. The importance of the major and minor QTLs identified was further demonstrated by analysis of the allelic effects on TSW in the DH population.

S45A, a double recessive mutant at both the BnMs1 and BnMs2 loci in Brassica napus, produces no pollen in mature anthers and no seeds by self-fertilization. The BnMs1 and BnMs2 genes, which have redundant functions in the control of male fertility, are positioned on linkage groups N7 and N16, respectively, and are located at the same locus on Arabidopsis chromosome 1 based on collinearity between Arabidopsis and Brassica. Complementation tests indicated that one candidate gene, BnCYP704B1, a member of the cytochrome P450 family, can rescue male sterility. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the developing anther showed that pollen-wall formation in the mutant was severely compromised, with a lack of sporopollenin or exine. The phenotype was first evident at the tetrad stage (stage 7) of anther development, coinciding with the maximum BnCYP704B1 mRNA accumulation observed in tapetal cells at stages 7-8 (haploid stage). TEM also suggested that development of the tapetum was seriously defective due to the disturbed lipid metabolism in the S45A mutant. A TUNEL assay indicated that the pattern of programmed cell death in the tapetum of the S45A mutant was defective. Lipid analysis showed that the total fatty acid content was reduced in the S45A mutant, indicating that BnCYP704B1 is involved in lipid metabolism. These data suggest that BnCYP704B1 participates in a vital tapetum-specific metabolic pathway that is not only involved in exine formation but is also required for basic tapetal cell development and function., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Accumulated evidence has shown that each of the three basic Brassica genomes (A, B and C) has undergone profound changes in different species, and has led to the concept of the "subgenome". Significant intersubgenomic heterosis was observed in hybrids between traditional Brassica napus and first generation lines of new type B. napus. The latter were produced by the partial introgression of subgenomic components from different species into B. napus. To increase the proportion of exotic subgenomic components and thus achieve stronger heterosis, lines of first generation new type B. napus were intercrossed with each other, and subjected to intensive marker-assisted selection to develop the second generation of new type B. napus. The second generation showed better agronomic traits and a higher proportion of introgression of subgenomic components than did the first generation. Compared with the commercial hybrid and the hybrids produced with the first generation new type B. napus, the novel hybrids showed stronger heterosis for seed yield during the 2 years of field trials. The extent of heterosis showed a significant positive correlation with the introgressed subgenomic components in the parental new type B. napus. To increase the content of the exotic subgenomic components further and to allow sustainable breeding of novel lines of new type B. napus, we initiated the development of a gene pool for new type B. napus that contained a substantial amount of genetic variation in the A(r) and C(c) genome. We discuss new approaches to broaden the avenue of intersubgenomic heterosis in oilseed Brassica.

Male sterility is an important contributor to heterosis in Brassica napus L. The B. napus line 7-7365ABC is a recessive epistatic genic male sterile (REGMS) three-line system. The 7-7365A line with the genotype Bnms3ms3ms4ms4RfRf is male-sterile, while the 7-7365B line with the genotype BnMs3ms3ms4ms4RfRf is male-fertile, and 7-7365C with homozygous recessive genotypes at the three loci shows male fertility because the loss function of Bnrf gene causes the inhibition of the genetic trait of the double mutant Bnms3 Bnms4. Histological studies addressing male sterility, transcriptional regulation pathways and the role of abscisic acid (ABA) in the anther development of REGMS plants are reported here. In the male-sterile line 7-7365A, tapetum cell and microspore mother cell separation were affected, and this led to failure of microspore release. The activity of polygalacturonase and the expression of the pectin methylesterase gene (AT3g06830) were significantly downregulated. Nine genes were downregulated in 7-7365A compared to 7-7365B and 7-7365C, including genes specifically expressed in tapetum (A3, A9, MS1) and the ABA-responsive gene KIN1. ABA concentration in 7-7365B was significantly higher than in 7-7365A and 7-7365C in young flower buds. Furthermore, temperature treatment made some sterile 7-7365A flowers become fertile. The stamens in these flowers produced viable pollen, and filament elongation was restored to its level in 7-7365C. We propose that ABA might control the expression of genes involved in cell separation during early anther development. The REGMS phenotype could be controlled by a primary pathway of male sterile metabolism positively regulated by the BnMs3 gene and a supplementary pathway negatively regulated by the BnRf gene.

Male sterility in a near-isogenic line S45AB after 25 generations of subcrossing is controlled by two pairs of duplicate genes. The genotype of S45A is Bnms1Bnms1Bnms2Bnms2, and that of S45B is BnMs1Bnms1Bnms2Bnms2, respectively. Histological observations revealed that abnormal anther development appeared in the tapetum and pollen exine during the tetrad stage. This male sterility was characterized by hypertrophy of the tapetal cells at the tetrad stage and a complete lack of microspore exine after the release of microspores from the tetrads. To elucidate the mechanism of this recessive genic male sterility, the flower bud expression profiles of the S45A and S45B lines were analyzed using an Arabidopsis thaliana ATH1 oligonucleotide array. When compared with the S45B line, 69 genes were significantly downregulated, and 46 genes were significantly upregulated in the S45A line. Real-time polymerase chain reaction (PCR) was then used to verify the results of the microarray analysis, and the majority of the downregulated genes in the S45A line were abundantly and specifically expressed in the anther. The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of Bnms1 may either block the biosynthesis of sporopollenin or block sporopollenin from being deposited on the microspore surface, thus, preventing pollen exine formation. The role of Bnms1 in the regulatory network of exine formation is also discussed as well.

Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding.

A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system.

S45AB, a recessive genic male sterile (RGMS) line, originated as a spontaneous mutant in Brassica napus cv. Oro. The genotypes of sterile (S45A) and fertile plants (S45B) are Bnms1ms1ms2ms2 and BnMs1ms1ms2ms2, respectively. In our previous studies, Yi et al. (Theor Appl Genet 113:643-650, 2006) mapped the BnMs1 locus to a region of 0.4 cM, candidates of which have been identified and genetic transformation is in progress. We describe the fine mapping of BnMs2 exploiting amplified fragment length polymorphism (AFLP) and amplified consensus genetic marker (ACGM) methodologies, and the identification of a collinear region probably containing BnMs2 orthologue in Arabidopsis thaliana. A near isogenic line (NIL) population S4516AB which segregated for BnMs2 locus was generated by crossing, allelism testing and repeated full-sib mating. From the survey of 1,024 AFLP primer combinations, 12 tightly linked AFLP markers were obtained and five of them were successfully converted into co-dominant or dominant sequence characterized amplified region (SCAR) markers. A population of 2,650 sterile plants was screened using these markers and a high-resolution map surrounding BnMs2 was constructed. The closest AFLP markers flanking BnMs2 were 0.038 and 0.075 cM away, respectively. Subsequently, an ACGM marker was developed to delimit the BnMs2 locus at an interval of 0.075 cM. We extended marker sequences to perform BlastN searches against the Arabidopsis genome and identified a collinear region containing 68 Arabidopsis genes, in which the orthologue of BnMs2 might be included. We further integrated BnMs2 linked AFLP or SCAR markers to two doubled-haploid (DH) populations derived from the crosses Tapidor x Ningyou7 (Qiu et al., Theor Appl Genet 114:67-80, 2006) and Quantum x No.2127-17 (available in our laboratory), and BnMs2 was mapped on N16. Molecular markers developed from these investigations will facilitate the marker-assisted selection (MAS) of RGMS lines, and the fine map and syntenic region identified will greatly hasten the process of positional cloning of BnMs2 gene.

Bacterial wilt (BW) caused by Ralstonia solanacearum is an important constraint to peanut (Arachis hypogaea L.) production in several Asian and African countries, and planting BW-resistant cultivars is the most feasible method for controlling the disease. Although several BW-resistant peanut germplasm accessions have been identified, the genetic diversity among these has not been properly investigated, which has impeded efficient utilization. In this study, the genetic relationships of 31 peanut genotypes with various levels of resistance to BW were assessed based on SSR and AFLP analyses. Twenty-nine of 78 SSR primers and 32 of 126 AFLP primer combinations employed in this study were polymorphic amongst the peanut genotypes tested. The SSR primers amplified 91 polymorphic loci in total with an average of 3.14 alleles per primer, and the AFLP primers amplified 72 polymorphic loci in total with an average of 2.25 alleles per primer. Four SSR primers (14H06, 7G02, 3A8, 16C6) and one AFLP primer (P1M62) were found to be most efficient in detecting diversity. The genetic distance between pairs of genotypes ranged from 0.12 to 0.94 with an average of 0.53 in the SSR data and from 0.06 to 0.57 with an average of 0.25 in the AFLP data. The SSR-based estimates of the genetic distance were generally larger than that based on the AFLP data. The genotypes belonging to subsp. fastigiata possessed wider diversity than that of subsp. hypogaea. The clustering of genotypes based on the SSR and AFLP data were similar but the SSR clustering was more consistent with morphological classification of A. hypogaea. Optimum diverse genotypes of both subsp. hypogaea and subsp. fastigiata can be recommended based on this analysis for developing mapping populations and breeding for high yielding and resistant cultivars.

The Brassica napus oilseed rape line, 7-7365AB, is a recessive epistatic genic male sterile (RGMS) two-type line system. The sterility is controlled by two pairs of recessive duplicate genes (Bnms3 and Bnms4) and one pair of recessive epistatic inhibitor gene (Bnrf). Homozygosity at the Bnrf locus (Bnrfrf) inhibits the expression of the two recessive male sterility genes in homozygous Bnms3ms3ms4ms4 plants and produces a male fertile phenotype. This line has a good potential for heterosis utilization but it is difficult to breed heterotic hybrids without molecular markers. To develop markers linked to the BnMs3 gene, amplified fragment length polymorphism (AFLP) technology was applied to screen the bulks of sterile and fertile individuals selected randomly from a population of near-isogenic lines (NIL) consisting of 2,000 plants. From a survey of 1,024 primer combinations, we identified 17 AFLP markers linked to the BnMs3 gene. By integrating the previous markers linked to the BnMs3 gene into the genetic map of the NIL population, two markers, EA01MC12 and EA09P06, were located on either side of the BnMs3 gene at a distance of 0.1 and 0.3 cM, respectively. In order to use the markers for male sterile line breeding, five AFLP markers, P05MG05, P03MG04, P11MG02, P05MC11(250), and EA09P06, were successfully converted into sequence characterized amplified region (SCAR) markers. Two of these, P06MG04 and sR12384, were subsequently mapped on to linkage group N19 using two doubled-haploid mapping populations available at our laboratory derived from the crosses Tapidor x Ningyou7 and Quantum x No2127-17. The markers found in the present study should improve our knowledge of recessive genic male sterility (RGMS), and accelerate the development of male sterile line breeding and map-based cloning.

A recessive genic male sterility (RGMS) system, S45 AB, has been developed from spontaneous mutation in Brassica napus canola variety Oro, and is being used for hybrid cultivar development in China. The male sterility of S45 was controlled by two duplicated recessive genes, named as Bnms1 and Bnms2. In this study, a NIL (near-isogenic line) population from the sib-mating of S45 AB was developed and used for the fine mapping of the Bnms1 gene, in which the recessive allele was homozygous at the second locus. AFLP technology combined with BSA (bulked segregant analysis) was used. From a survey of 2,560 primer combinations (+3/+3 selective bases), seven AFLP markers linked closely to the target gene were identified, of which four were successfully converted to sequence characterized amplified region (SCAR) markers. For further analysis, a population of 1,974 individuals was used to map the Bnms1 gene. On the fine map, Bnms1 gene was flanked by two SCAR markers, SC1 and SC7, with genetic distance of 0.1 cM and 0.3 cM, respectively. SC1 was subsequently mapped on linkage group N7 using doubled-haploid mapping populations derived from the crosses Tapidor x Ningyou7 and DH 821 x DHBao 604, available at IMSORB, UK, and our laboratory, respectively. Linkage of an SSR marker, Na12A02, with the Bnms1 gene further confirmed its location on linkage group N7. Na12A02, 2.6 cM away from Bnms1, was a co-dominant marker. These molecular markers developed from this research will facilitate the marker-assisted selection of male sterile lines and the fine map lays a solid foundation for map-based cloning of the Bnms1 gene.