Characterization of seedling proteomes and development of markers to distinguish the Brassica A and C genomes

The diploid species Brassica rapa(genome AA)and B.oleracea(genome CC)were compared by full-scale proteome analyses of seedling.A total of 28.2% of the proteins was common to both species,indicating the existence of a basal or ubiquitous proteome.However,a number of discriminating proteins(32.0%)and specific proteins(39.8%)of the Brassica A and C genomes,respectively,were identified,which could represent potentially species-specific functions.Based on these A or C genome-specific proteins,a number of PCR-bas...     

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.     

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.     

Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.     

Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica

The aim of this work was to find C genome specific repetitive DNA sequences able to differentiate the homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica, in order to assist in the physical identification of B. napus chromosomes. A repetitive sequence (pBo1.6) highly enriched in the C genome of Brassica was cloned from B. oleracea and its chromosomal organisation was investigated through fluorescent in situ hybridisation (FISH) in B. oleracea (2n = 18, CC), B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC) genomes. The sequence was 203 bp long with a GC content of 48.3%. It showed up to 89% sequence identity with telomere-like DNA from many plant species. This repeat was clearly underrepresented in the A genome and the in situ hybridisation showed its B. oleracea specificity at the chromosomal level. Sequence pBo1.6 was localised at interstitial and/or telomeric/subtelomeric regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus 18 to 24 chromosomes hybridised with pBo1.6. The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful for the determination of the chromosomal location of transgenic DNA in genetically modified oilseed rape.     

Studies on the Relationships Between Moricandia and Brassica Species

Moricandia is the only genus with C3-C4 species within the family of Cruciferae. To provide the basic information of transferring C3-C4 and other important characteristics from Moricandia to Brassica crops, the relationships between Moricandia and Brassica species were studied based on crossability and RFLP fingerprinting. The crossability was very low between the two genera in the experiment. There was no hybrid seed obtained between M. arvensis and B. rapa though 8 000 flowers were crossed. 2 989 cross-pollinated ovaries were cultured and also no hybrid embryo was developed. However, four intergeneric hybrid shoots were generated from 105 cultured ovaries in the combination of M. arvensis x B. napus. The nucleus DNA polymorphism of restriction loci was detected with 23 genic DNA clones of B. napus for the samples of B. napus, B. rapa and B. oleracea, M. arvensis and M. nit, ns. A high homology was found between Moricandia and Brassica species. The similarity between M. nitens and B. rapa was even greater than that between B. rapa and B. napus. The close relationships between Moricandia species and Brassica crops, especially European B. rapa, were also detected with 4 beta mitochondria probes. The intensive homology between Moricandia C3-C4 species and Brassica crops evaluated with the RFLP markers revealed the possibility of transferring some important genes from the C3-C4 species to the domesticated species by sexual hybridization or protoplast fusion followed by recombination of homoeologous chromosomes.     

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.     

芸苔属（Brassica）植物中microRNAs的生物信息学预测与分析

MicroRNA (miRNA) 是一类调控基因转录后表达的非编码的小分子RNA．它在生物的发育、细胞增殖、凋亡以及胁迫响应等生物过程中发挥着重要的调控作用．目前,分离和鉴定miRNA的方法主要包括实验方法（遗传筛选、直接克隆）和生物信息学方法．MiRNA存在表达丰度低,表达组织特异性,以及受特殊诱导等问题,用传统的实验法常难发现和鉴定miRNA．通过生物信息学方法在已有的各种基因库中寻找未知miRNA,大大提高了人们发现miRNA及其靶基因的效率．芸苔属（Brassica）的成员包括油菜、芜青、甘蓝等,是世界各国主要油料和食用作物．目前,油菜的miRNA的分离和鉴定工作已有文献报道,而其它的尚属空白．本文将拟南芥、水稻等植物已知的miRNA分别与芜青、甘蓝、野芥菜、黑芥菜、埃塞俄比亚芥的GSS和EST数据库进行比对搜索,采用一系列标准进行筛选,最后分别在芜青和甘蓝中预测到67个和95个miRNA．再把这些预测得到的miRNA分别与芜青和甘蓝的mRNA数据库进行比对搜索,分别找到120个和111个靶基因,除去未知功能及功能不详的,各有62个和48个靶基因．分析结果表明,上述大多数靶基因编码的产物为转录因子及重要代谢酶类,涉及植物的生长发育调控,信号转导及胁迫响应等方面．     

白菜和甘蓝基因组转座子表达及其对基因调控的潜在影响

转座子或转座元件是大多数真核生物基因组的主要组成成分。甘蓝(Brassica oleracea)基因组比白菜(B. rapa)大主要是转座子的扩增差异造成的。然而, 这两个芸薹属近缘物种转座子表达水平以及对基因的调控和功能的影响目前还不清楚。文章对白菜和甘蓝叶、根、茎3个器官的转录组数据进行了初步分析。结果显示, 转座子的表达量很低, 转录组reads中有1%来自转座子的转录本; 转座子的表达存在器官差异, 且不同类别和家族的转座子表达量相差很大, 相同类别和同一家族的转座子在白菜和甘蓝基因组中的表达活性也不相同。进一步鉴定到转录读出的LTR反转座子, 其与下游基因距离小于2 kb的有41个, 小于100 bp的有9个, 这些LTR的转录读出很可能通过正义或反义的转录本激活或干扰下游基因的表达。同时, 具有转录读出的intact LTR比solo LTR具有更强的读出活性。通过深入分析转座子的插入位点发现, 白菜基因组中转座子插入基因内部的频率比甘蓝基因组中的高; 与反转座子相比, DNA转座子更偏向于插入或保留在基因的内含子当中。这些结果为认识转座子对其他蛋白编码基因的影响提供了基础。     

Identification of the A and C genomes of amphidiploid Brassica napus (oilseed rape).

A genetic linkage map consisting of 399 RFLP-defined loci was generated from a cross between resynthesized Brassica napus (an interspecific B. rapa x B. oleracea hybrid) and "natural" oilseed rape. The majority of loci exhibited disomic inheritance of parental alleles demonstrating that B. rapa chromosomes were each pairing exclusively with recognisable A-genome homologues in B. napus and that B. oleracea chromosomes were pairing similarly with C-genome homologues. This behaviour identified the 10 A genome and 9 C genome linkage groups of B. napus and demonstrated that the nuclear genomes of B. napus, B. rapa, and B. oleracea have remained essentially unaltered since the formation of the amphidiploid species, B. napus. A range of unusual marker patterns, which could be explained by aneuploidy and nonreciprocal translocations, were observed in the mapping population. These chromosome abnormalities were probably caused by associations between homoeologous chromosomes at meiosis in the resynthesized parent and the F1 plant leading to nondisjunction and homoeologous recombination.     

Genetic investigation of the origination of allopolyploid with virtually synthesized lines: application to the C subgenome of Brassica napus

Although there are a number of different allopolyploids in the plant kingdom, the exact ancestral parents of some allopolyploids have not been well characterized. We propose a strategy in which virtual allopolyploid lines derived from different types of parental species are used to investigate the progenitors of an allopolyploid. The genotypes of the parental lines and the natural allopolyploid were established using a set of DNA molecular markers. The genotypes of the virtual lines were then derived from those of the parental lines, and compared extensively with that of the natural allopolyploid. We applied this strategy to investigate the progenitors of the C subgenome of Brassica napus (rapeseed, AACC). A total of 39 accessions from 10 wild and 7 cultivated types of the B. oleracea cytodeme (CC), and 4 accessions of B. rapa (AA) were used to construct 156 virtual rapeseed lines. Genetic structure was compared among natural rapeseed, virtual rapeseed lines, and their parental lines by principal component analysis and analysis of ancestry. Our data showed that the C subgenome of natural rapeseed was related closely to the genome of cultivated B. oleracea and its related wild types, such as B. incana, B. bourgeaui, B. montana, B. oleracea ssp. oleracea and B. cretica. This finding indicated that these types or their progeny might be ancestral donors of the C subgenome of rapeseed. The successful application of the strategy of virtual allopolyploidy in rapeseed demonstrates that it can possibly be used to identify the progenitors of an allopolyploid species.     

Numerous and rapid nonstochastic modifications of gene products in newly synthesized Brassica napus allotetraploids

Polyploidization is a widespread process that results in the merger of two or more genomes in a common nucleus. To investigate modifications of gene expression occurring during allopolyploid formation, the Brassica napus allotetraploid model was chosen. Large-scale analyses of the proteome were conducted on two organs, the stem and root, so that >1600 polypeptides were screened. Comparative proteomics of synthetic B. napus and its homozygous diploid progenitors B. rapa and B. oleracea showed that very few proteins disappeared or appeared in the amphiploids (<1%), but a strikingly high number (25-38%) of polypeptides displayed quantitative nonadditive pattern. Nonstochastic gene expression repatterning was found since 99% of the detected variations were reproducible in four independently created amphiploids. More than 60% of proteins displayed a nonadditive pattern closer to the paternal parent B. rapa. Interspecific hybridization triggered the majority of the deviations (89%), whereas very few variations (approximately 3%) were associated with genome doubling and more significant alterations arose from selfing (approximately 9%). Some nonadditive proteins behaved similarly in both organs, while others exhibited contrasted behavior, showing rapid organ-specific regulation. B. napus formation was therefore correlated with immediate and directed nonadditive changes in gene expression, suggesting that the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.     

Comparative Genome Mapping in Brassica

A Brassica nigra genetic linkage map was developed from a highly polymorphic cross analyzed with a set of low copy number Brassica RFLP probes. The Brassica genome is extensively duplicated with eight distinct sets of chromosomal segments, each present in three copies, covering virtually the whole genome. Thus, B. nigra could be descended from a hexaploid ancestor. A comparative analysis of B. nigra, B. oleracea and B. rapa genomes, based on maps developed using a common set of RFLP probes, was also performed. The three genomes have distinct chromosomal structures differentiated by a large number of rearrangements, but collinear regions involving virtually the whole of each the three genomes were identified. The genic contents of B. nigra, B. oleracea and B. rapa were basically equivalent and differences in chromosome number (8, 9 and 10, respectively) are probably the result of chromsome fusions and/or fissions. The strong conservation of overall genic content across the three Brassica genomes mirrors the conservation of genic content observed over a much longer evolutionary span in cereals. However, the rate of chromosomal rearrangement in crucifers is much higher than that observed in cereal genomes.     

芸薹属多倍体植物基因组进化的RAPD分析

多倍化是促进高等植物发生进化的重要力量。为了更清楚地了解多倍体在形成之后其基因组是如何进化的,利用38个随机引物对芸薹属Brassica L.禹氏三角（U’Triangle）中的多倍体物种及其祖先二倍体物种进行了研究。根据扩增出的273条带计算了遗传距离,并用UPGMA法进行了聚类分析。结果发现,二倍体物种B.campestris（AA）与B.oleracea（CC）的亲缘关系比与B.nigra（BB）的要近;异源多倍体B.napus（AACC）比起其二倍体祖先之一B.campestris（AA）与另一个     

Synthesis of intergeneric hybrids and establishment of genomic affinity between <Emphasis Type="Italic">Diplotaxis catholica</Emphasis> and crop <Emphasis Type="Italic">Brassica</Emphasis> species

Intergeneric hybrids of the wild crucifer Diplotaxis catholica (2n = 18, D(C)D(C)) as female with two crop Brassica species, namely Brassica rapa (2n = 20; AA) and Brassica juncea (2n = 36; AABB) as male, were developed, using ovary and sequential culture. Reciprocal crosses were not successful, suggesting unilateral cross incompatibility. Morphologically, the hybrid plants resembled the crop brassica parents, but were nearly male- as well as female-sterile. Induction of amphiploidy helped to improve pollen fertility for the D. catholica x B. rapa cross (73%), but less so for the D. catholica x B. juncea cross (35-40%). Female fertility was also higher in both the amphiploids. Cytological analysis of the F(1) hybrids revealed aberrant meiosis with predominant occurrence of the univalents. Partial genomic homoeology between the A genome of B. rapa and the D(C) genome of D. catholica was indicated by the presence of up to five bivalents in 14.7% of the PMCs in the D. catholica x B. rapa hybrid, and 1-2 trivalents or a quadrivalent in nearly 44% of the PMCs in the derived amphiploid. In the second cross, D. catholica x B. juncea, up to six bivalents and one trivalent were observed indicating homoeology between the A/B genomes of B. juncea and the D(C) genome of D. catholica. The possibility of introgression of desirable genes from D. catholica into crop Brassica species exists in view of significant affinity between the D(C) and A/B genomes.     

Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.     

Diversification and alteration of recognition specificity of the pollen ligand SP11/SCR in self-incompatibility of Brassica and Raphanus

The recognition specificity of the pollen ligand of self-incompatibility (SP11/SCR) was investigated using Brassica rapa transgenic plants expressing SP11 transgenes, and SP11 of Raphanus sativus S-21 was found to have the same recognition specificity as that of B. rapa S-9. In a set of three S haplotypes, whose sequence identities of SP11 and SRK are fairly high, R. sativus S-6 showed the same recognition specificity as Brassica oleracea S-18 and a slightly different specificity from B. rapa S-52. B. oleracea S-18, however, showed a different specificity from B. rapa S-52. Using these similar S haplotypes, chimeric SP11 proteins were produced by domain swapping. Bioassay using the chimeric SP11 proteins revealed that the incompatibility response induction activity was altered by the replacement of Region III and Region V. Pollen grains of Brassica transgenic plants expressing chimeric SP11 of the B. oleracea SP11-18 sequence with Region III and Region V from B. rapa SP11-52 (chimeric BoSP11-18[52]) were partially incompatible with the B. rapa S-52 stigmas, and those expressing the R. sativus SP11-6 sequence with Region III and Region V from B. rapa SP11-52 (chimeric RsSP11-6[52]) were completely incompatible with the stigmas having B. rapa S-52. However, the transgenic plant expressing chimeric RsSP11-6(52) also showed incompatibility with B. oleracea S-18 stigmas. These results suggest that Regions III and Region V of SP11 are important for determining the recognition specificity, but not the sole determinant. A possible process of the generation of a new S haplotype is herein discussed.