Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Neither compatible nor self-incompatible pollinations of Brassica napus involve reorganization of the papillar cytoskeleton

Compatible pollination of Brassica napus necessitates pollen hydration, pollen germination and growth of the pollen tube through the loosened walls of stigmatic papillar cells, whereas self-incompatible (SI) pollinations fail at one of these stages. Analyses of the early stages of pollination show that at high (but not low) relative humidities, both compatible and SI pollen hydrates, but SI germination is reduced and the rare pollen tubes generally fail to penetrate the papillar walls, although there is some wall loosening. Inside the papillae, both compatible and SI interactions may induce the formation of callose, but there is no evidence for a major accumulation of cytoplasm or secretory vesicles in the vicinity of the pollen tubes and neither microtubule nor F-actin patterns re-arrange in this zone. These observations indicate that the source of the wall-loosening enzymes is probably the pollen tube or pollen coat, and that the common cellular responses of plants to attempted invasions have become suppressed in the papilla–pollen tube interaction.     

Exploiting comparative mapping among Brassica species to accelerate the physical delimitation of a genic male-sterile locus (BnRf) in Brassica napus

The recessive genic male sterility (RGMS) line 9012AB has been used as an important pollination control system for rapeseed hybrid production in China. Here, we report our study on physical mapping of one male-sterile locus (BnRf) in 9012AB by exploiting the comparative genomics among Brassica species. The genetic maps around BnRf from previous reports were integrated and enriched with markers from the Brassica A7 chromosome. Subsequent collinearity analysis of these markers contributed to the identification of a novel ancestral karyotype block F that possibly encompasses BnRf. Fourteen insertion/deletion markers were further developed from this conserved block and genotyped in three large backcross populations, leading to the construction of high-resolution local genetic maps where the BnRf locus was restricted to a less than 0.1-cM region. Moreover, it was observed that the target region in Brassica napus shares a high collinearity relationship with a region from the Brassica rapa A7 chromosome. A BnRf-cosegregated marker (AT3G23870) was then used to screen a B. napus bacterial artificial chromosome (BAC) library. From the resulting 16 positive BAC clones, one (JBnB089D05) was identified to most possibly contain the BnRf (c) allele. With the assistance of the genome sequence from the Brassica rapa homolog, the 13.8-kb DNA fragment covering both closest flanking markers from the BAC clone was isolated. Gene annotation based on the comparison of microcollinear regions among Brassica napus, B. rapa and Arabidopsis showed that five potential open reading frames reside in this fragment. These results provide a foundation for the characterization of the BnRf locus and allow a better understanding of the chromosome evolution around BnRf.     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

Sequence‐level comparative analysis of the Brassica napus genome around two stearoyl‐ACP desaturase loci

We conducted a sequence‐level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl‐ACP desaturase‐encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (～56%) is lower than has been reported previously between A. thaliana and Brassica (～66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 ± 3.9% and 85.8 ± 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 ± 3.1% between the B. napus A genome and B. rapa, and 93.1 ± 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法     

Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana

Over 1000 genetically linked RFLP loci in Brassica napus were mapped to homologous positions in the Arabidopsis genome on the basis of sequence similarity. Blocks of genetically linked loci in B. napus frequently corresponded to physically linked markers in Arabidopsis. This comparative analysis allowed the identification of a minimum of 21 conserved genomic units within the Arabidopsis genome, which can be duplicated and rearranged to generate the present-day B. napus genome. The conserved regions extended over lengths as great as 50 cM in the B. napus genetic map, equivalent to approximately 9 Mb of contiguous sequence in the Arabidopsis genome. There was also evidence for conservation of chromosome landmarks, particularly centromeric regions, between the two species. The observed segmental structure of the Brassica genome strongly suggests that the extant Brassica diploid species evolved from a hexaploid ancestor. The comparative map assists in exploiting the Arabidopsis genomic sequence for marker and candidate gene identification within the larger, intractable genomes of the Brassica polyploids.     

Determining the physical limits of the Brassica S locus by recombinational analysis

A genetic analysis was performed to study the frequency of recombination for intervals across the Brassica S locus region. No recombination was observed between the S locus glycoprotein gene and the S receptor kinase gene in the segregating populations that we analyzed. However, a number of recombination breakpoints in regions flanking these genes were identified, allowing the construction of an integrated genetic and physical map of the genomic region encompassing one S haplotype. We identified, based on the pollination phenotype of plants homozygous for recombinant S haplotypes, a 50-kb region that encompasses all specificity functions in the S haplotype that we analyzed. Mechanisms that might operate to preserve the tight linkage of self-incompatibility specificity genes within the S locus complex are discussed in light of the relatively uniform recombination frequencies that we observed across the S locus region and of the structural heteromorphisms that characterize different S haplotypes.     

Intrahaplotype polymorphism at the Brassica S locus

The S locus receptor kinase and the S locus glycoproteins are encoded by genes located at the S locus, which controls the self-incompatibility response in Brassica. In class II self-incompatibility haplotypes, S locus glycoproteins can be encoded by two different genes, SLGA and SLGB. In this study, we analyzed the sequences of these genes in several independently isolated plants, all of which carry the same S haplotype (S(2)). Two groups of S(2) haplotypes could be distinguished depending on whether SRK was associated with SLGA or SLGB. Surprisingly, SRK alleles from the two groups could be distinguished at the sequence level, suggesting that recombination rarely occurs between haplotypes of the two groups. An analysis of the distribution of polymorphisms along the S domain of SRK showed that hypervariable domains I and II tend to be conserved within haplotypes but to be highly variable between haplotypes. This is consistent with these domains playing a role in the determination of haplotype specificity.     

Detection of self-incompatible oilseed rape plants (Brassica napus L.) based on molecular markers for identification of the class I S haplotype

The selection of desirable genotypes with recessive characteristics, such as self-incompatible plants, is often difficult or even impossible and represents a crucial barrier in accelerating the breeding process. Molecular approaches and selection based on molecular markers can allow breeders to overcome this limitation. The use of self-incompatibility is an alternative in hybrid breeding of oilseed rape. Unfortunately, stable self-incompatibility is recessive and phenotype-based selection is very difficult and time-consuming. The development of reliable molecular markers for detecting desirable plants with functional self-incompatible genes is of great importance for breeders and allows selection at early stages of plant growth. Because most of these reliable molecular markers are based on discrimination of class I S-locus genes that are present in self-compatible plants, there is a need to use an internal control in order to detect possible PCR inhibition that gives false results during genotyping. In this study, 269 double haploid F₂ oilseed rape plants obtained by microspore embryogenesis were used to verify the applicability of an improved PCR assay based on the detection of the class I SLG gene along with an internal control. Comparative analysis of the PCR genotyping results vs. S phenotype analysis confirmed the applicability of this molecular approach in hybrid breeding programs. This approach allows accurate detection of self-incompatible plants via a different amplification profile.     

RFLP mapping of Brassica napus using doubled haploid lines

The combined use of doubled haploid lines and molecular markers can provide new genetic information for use in breeding programs. An F₁-derived doubled haploid (DH) population of Brassica napus obtained from a cross between an annual canola cultivar (Stellar) and a biennial rapeseed (Major) was used to construct a linkage map of 132 restriction fragment length polymorphism loci. The marker loci were arranged into 22 linkage groups and six pairs of linked loci covering 1016 cM. The DH map was compared to a partial map constructed with a common set of markers for an F₂ population derived from the same F₁ plant, and the overall maps were not significantly different. Comparisons of maps in Brassica species suggest that less recombination occurs in B. napus (n = 19) than expected from the combined map distances of the two hypothesized diploid progenitors, B. oleracea (n = 9) and B. rapa (n=10). A high percentage (32%) of segregating marker loci were duplicated in the DH map, and conserved linkage arrangements of some duplicated loci indicated possible intergenome homoeology in the amphidiploid or intragenome duplications from the diploid progenitors. Deviation from Mendelian segregation ratios (P < 0.05) was observed for 30% of the marker loci in the DH population and for 24% in the F₂ population. Deviation towards each parent occurred at equal frequencies in both populations and marker loci that showed deviation clustered in specific linkage groups. The DH lines and molecular marker map generated for this study can be used to map loci for agronomic traits segregating in this population. Present address Embrapa/Cenargen, C.P. 0.2372, CEP 70.770, Brasilia DF, Brazil     

Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars

Oil bodies were purified from mature seed of two Brassica napus crop cultivars, Reston and Westar. Purified oil body proteins were subjected to both 2-DE followed by LC-MS/MS and multidimensional protein identification technology. Besides previously known oil body proteins oleosin, putative embryo specific protein ATS1, (similar to caleosin), and 11-beta-hydroxysteroid dehydrogenase-like protein (steroleosin), several new proteins were identified in this study. One of the identified proteins, a short chain dehydrogenase/reductase, is similar to a triacylglycerol-associated factor from narrow-leafed lupin while the other, a protein annotated as a myrosinase associated protein, shows high similarity to the lipase/hydrolase family of enzymes with GDSL-motifs. These similarities suggest these two proteins could be involved in oil body degradation. Detailed analysis of the two other oil body components, polar lipids (lipid monolayer) and neutral lipids (triacylglycerol matrix) was also performed. Major differences were observed in the fatty acid composition of polar lipid fractions between the two B. napus cultivars. Neutral lipid composition confirmed erucic acid and oleic acid accumulation in Reston and Westar seed oil, respectively.     

Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, we compared the gene expression during seed development between two lines of Brassica napus with a 10% difference in oil content. We isolated the immature seeds 15 and 25 days after flowering at periods preceding and including the major accumulation of storage oils and proteins. The differentially expressed gene clones between the two rape lines were isolated by subtractive suppression hybridization (SSH). All SSH clones were arrayed and screened by dot blot hybridization, followed by RT-PCR analysis for selected clones. A total of 217 cDNA clones corresponding to 30 genes were found to have a high expression in seeds with high oil content. Six genes were highly expressed in seeds with low oil content. Northern blot and enzyme activity analysis demonstrated a change in expression pattern of several genes. The results provide information on gene-encoding factors responsible for the regulation of oil synthesis. The possible role of these genes in seeds is discussed. The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies of rape breeding.     

Characterization of the quantitative trait locus OilA1 for oil content in Brassica napus

Increasing seed oil content has become one of the most important breeding criteria in rapeseed (Brassica napus). However, oil content is a complex quantitative trait. QTL mapping in a double haploid population (SG population) emerging from a cross between a German (Sollux) and Chinese (Gaoyou) cultivars revealed one QTL for oil content on linkage group A1 (OilA1), which was mapped to a 17 cM genetic interval. To further validate and characterize the OilA1, we constructed a high-resolution map using B. rapa sequence resources and developed a set of near-isogenic lines (NILs) by employing a DH line SG-DH267 as donor and Chinese parent Gaoyou as recurrent background. The results showed highly conserved synteny order between B. rapa and B. napus within the linkage group A1 and revealed a possible centromere region between two markers ZAASA1-38 and NTP3 (2.5 cM). OilA1 was firstly validated by 250 BC₅F₂ plants and was confirmed in a 10.6 cM interval between the markers ZAASA1-47 and ZAASA1-77. Further substitution mapping was conducted by using two generations of QTL-NILs, 283 lines from eight BC₅F_3:4 families and 428 plants from six BC₅F₄ sub-NILs and thus narrowed the OilA1 interval to 6.9 cM and 4.3 cM (1.4 Mb), respectively. Field investigations with two replications using homozygous BC₅F_3:4 sister sub-NILs indicated that NILs, which carry a Sollux chromosome segment across the target region showed significant higher oil content (1.26 %, p < 0.001) than their sister NILs containing Gaoyou chromosome. The OilA1 locus is of particular interest for breeding purpose in China because 80 % of Chinese cultivars do not carry this desirable allele.     

Sequence and structural diversity of the S locus genes from different lines with the same self-recognition specificities in Brassica oleracea

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. In Brassica, it is controlled by a single multi-allelic locus, S, and it is believed that two highly polymorphic genes in the S locus, SLG and SRK, play central roles in self-recognition in stigmas. SRK is a putative receptor protein kinase, whose extracellular domain exhibits high similarity to SLG. We analyzed two pairs of lines showing cross-incompatibility (S(2) and S(2-b); S(13) and S(13-b)). In S(2) and S(2-b), SRKs were more highly conserved than SLGs. This was also the case with S(13) and S(13-b). This suggests that the SRKs of different lines must be conserved for the lines to have the same self-recognition specificity. In particular, SLG(2-b) showed only 88. 5% identity to SLG(2), which is comparable to that between the SLGs of different S haplotypes, while SRK(2-b) showed 97.3% identity to SRK(2) in the S domain. These findings suggest that the SLGs in these S haplotypes are not important for self-recognition in SI.