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.     

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.     

甘蓝型油菜Toc33基因编码区的分离及蛋白序列的比较

以甘蓝型油菜新鲜嫩叶为实验材料提取其总DNA,以其为模板,根据拟南芥Toc33基因编码区序列设计引物,PCR扩增甘蓝型油菜叶绿体外膜蛋白转运机器的构件蛋白基因Toc33,得到两条扩增带,测序结果显示克隆到的两个片段分别长1370bp、1490bp,将这两个片段分别命名为Bn Tpc33-1,Bn Toc33-2,序列比较发现它们之间的同源性为78％,其中外显子的同源性为96％,而内含子的同源性仅为60％。为研究Toc33与同一基因家族的Toc34基因功能间的关系,对拟南芥、油菜、诸葛菜等植物的Toc33、Toc34蛋白序列进行比较分析并构建了分子系统进化树。     

Comparison of Flowering Time Genes in Brassica Rapa, B. Napus and Arabidopsis Thaliana

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.     

与甘蓝型油菜重要经济性状有关的DNA克隆在拟南芥遗传图谱中的整合

拟南芥与油菜同属十字花科植物芸寡族,亲缘关系很近,基因组间的同源性很高,在用拟南芥EST克隆和油菜DNA克隆作探针定位了甘蓝型油菜一系列重要性状的基础上,对25个与油菜雄性不育恢复基因,硼高效利用基因,抗菌核病QTL及油菜种间杂种营养优势相关联的克隆进行了测序,在拟南芥基因组数据库中寻找到与这25个克隆高度同源的序列,根据这些高度同源序列在拟南芥染色体上的相位位置,将油菜DNA克隆整合到了拟南芥遗传图谱上,其中油菜硼高效基因BE1两侧的标记克隆整合在拟南芥第一染色体长臂一个较小的区段内,以该目标区段内的拟南芥EST克隆PA24为探针对甘蓝型油菜基因组比较作图,将该克隆定位在油菜连锁图BE1两侧标记之间,表明了利用基因组间的相互比较作图来精细定位芸薹属作物重要基因的可能性。     

Molecular mapping of Arabidopsis thaliana lipid-related orthologous genes in Brassica napus

Quantitative Trait Loci (QTL) for oil content has been previously analyzed in a SG-DH population from a cross between a Chinese cultivar and a European cultivar of Brassica napus. Eight QTL with additive and epistatic effects, and with environmental interactions were evaluated. Here we present an integrated linkage map of this population predominantly based on informative markers derived from Brassica sequences, including 249 orthologous A. thaliana genes, where nearly half (112) are acyl lipid metabolism related genes. Comparative genomic analysis between B. napus and A. thaliana revealed 33 colinearity regions. Each of the conserved A. thaliana segments is present two to six?times in the B. napus genome. Approximately half of the mapped lipid-related orthologous gene loci (76/137) were assigned in these conserved colinearity regions. QTL analysis for seed oil content was performed using the new map and phenotypic data from 11 different field trials. Nine significant QTL were identified on linkage groups A1, A5, A7, A9, C2, C3, C6 and C8, together explaining 57.79% of the total phenotypic variation. A total of 14 lipid related candidate gene loci were located in the confidence intervals of six of these QTL, of which ten were assigned in the conserved colinearity regions and felled in the most frequently overlapped QTL intervals. The information obtained from this study demonstrates the potential role of the suggested candidate genes in rapeseed kernel oil accumulation.     

Conserved structure and function of the Arabidopsis flowering time gene CONSTANS in Brassica napus

The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.     

Characterization and linkage mapping of R-gene analogous DNA sequences in pea (Pisum sativum L.)

Pea (Pisum sativum L.) sequences that are analogous to the conserved nucleotide binding site (NBS) domain found in a number of plant disease resistance genes (R-genes) were cloned. Using redundant oligonucleotide primers and the polymerase chain reaction (PCR), we amplified nine pea sequences and characterised their sequences. The pea R-gene analog (RGA)- deduced amino acid sequences demonstrated significant sequence similarity with known R-gene sequences lodged in public databases. The genomic locations of eight of the pea RGAs were determined by linkage mapping. The eight RGAs identified ten loci that mapped to six linkage groups. In addition, the genomic organization of the RGAs was inferred. Both single-copy and multicopy sequence families were present among the RGAs, and the multicopy families occurred most often as tightly linked clusters of related sequences. Intraspecific copy number variability was observed in three of the RGA sequence families, suggesting that these sequence families are evolving rapidly. The genomic locations of the pea RGAs were compared with the locations of known pea R-genes and sym genes involved in the pea-rhizobia symbiosis. Two pea RGAs mapped in the genomic region containing a pea R-gene, Fw, and four pea RGAs mapped in regions of the genome containing sym genes. Received: 4 August 1999 / Accepted: 11 November 1999     

Identification and analysis of expressed resistance gene sequences in wheat

Forty-eight resistance (R) genes conferring resistance to various types of pests have been cloned from 12 plant species. Irrespective of the host or the pest type, most R genes share a strong protein sequence similarity especially for domains and motifs. The objective of this study was to identify expressed R genes of wheat, the fraction of which is expected to be very low in the genome. Using modified RNA fingerprinting and data mining approaches we identified 220 expressed R-gene candidates. Of these, 125 sequences structurally resembled known R genes. In addition to 25-87% protein sequence similarity with the known R genes, the sequence, order, and distribution of the domains and motifs were also the same. Among the remaining 95, 17 were probable R-related, 21 were a new class of nucleotide-binding kinases, 21 were probable kinases, and 36 were p-loop-containing unknown sequences. About 76% were rare including 73 novel sequences. Three new R-gene specific motifs were also identified. Physical mapping of the 164 best R-gene candidates on 339 deletion lines localized 121 mappable R-gene candidates to 26 small chromosomal regions encompassing about 16% of the genome. About 90 of the 110 phenotypically characterized wheat R genes corresponding to 18 different pests also mapped in these regions.     

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.     

Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance

An SSR-based linkage map was constructed in Brassica rapa. It includes 113 SSR, 87 RFLP, and 62 RAPD markers. It consists of 10 linkage groups with a total distance of 1005.5 cM and an average distance of 3.7 cM. SSRs are distributed throughout the linkage groups at an average of 8.7 cM. Synteny between B. rapa and a model plant, Arabidopsis thaliana, was analyzed. A number of small genomic segments of A. thaliana were scattered throughout an entire B. rapa linkage map. This points out the complex genomic rearrangements during the course of evolution in Cruciferae. A 282.5-cM region in the B. rapa map was in synteny with A. thaliana. Of the three QTL (Crr1, Crr2, and Crr4) for clubroot resistance identified, synteny analysis revealed that two major QTL regions, Crr1 and Crr2, overlapped in a small region of Arabidopsis chromosome 4. This region belongs to one of the disease-resistance gene clusters (MRCs) in the A. thaliana genome. These results suggest that the resistance genes for clubroot originated from a member of the MRCs in a common ancestral genome and subsequently were distributed to the different regions they now inhabit in the process of evolution.     

Cloning of dehydrin coding sequences from Brassica juncea and Brassica napus and their low temperature-inducible expression in germinating seeds.

A novel subclass of dehydrin genes, homologous to the Raphanus sativus late embryogenesis-abundant (LEA) protein (RsLEA2) and the Arabidopsis thaliana dehydrin, was isolated from Brassica juncea and Brassica napus, here designated BjDHN1 and BnDHN1, respectively. The cDNA of BjDHN1 and BnDHN1 genes share 100% nucleotide identity. The encoded protein is predicted to consist of 183 amino acid residues (molecular mass of 19.2 kDa and pI of 7.0). It shares 85.3% and 65.4% amino acid sequence identity with the RsLEA2 and Arabidopsis dehydrin, respectively. This Brassica dehydrin also features a "Y(3)SK(2)" plant dehydrin structure. Expression analysis indicated that the Brassica dehydrin gene is expressed at the late stages of developing siliques, suggesting that the gene expression may be inducible by water-deficit. Analysis of gene expression also indicated that in germinating seeds the gene expression was inducible by low temperature. Seed germination under low temperature was compared between B. juncea and B. napus. The results showed that B. juncea seeds germinated faster than B. napus seeds. Expression of Brassica dehydrin gene was also examined as a function of seed germination under low temperature.     

Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.     

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.     

Birth, death and subfunctionalization in the Arabidopsis genome

Arabidopsis thaliana is now a model system, not just for plant biology but also for comparative genomics. The completion of the sequences of two closely related species, Arabidopsis lyrata and Brassica rapa, is complemented by genomic comparisons among A. thaliana accessions and mutation accumulation lines. Together these genomic data document the birth of new genes via gene duplication, transposon exaptation and de novo formation of new genes from noncoding sequence. Most novel loci exhibit low expression, and are undergoing pseudogenization or subfunctionalization. Comparatively, A. thaliana has lost large amounts of sequence through deletion, particularly of transposable elements. Intraspecific genomic variation indicates high rates of deletion mutations and deletion polymorphisms across accessions, shedding light on the history of Arabidopsis genome architecture.     

Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis

The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.     

Comparison of a Brassica oleracea genetic map with the genome of Arabidopsis thaliana

Brassica oleracea is closely related to the model plant, Arabidopsis thaliana. Despite this relationship, it has been difficult to both identify the most closely related segments between the genomes and determine the degree of genome replication within B. oleracea relative to A. thaliana. These difficulties have arisen in part because both species have replicated genomes, and the criteria used to identify orthologous regions between the genomes are often ambiguous. In this report, we compare the positions of sequenced Brassica loci with a known position on a B. oleracea genetic map to the positions of their putative orthologs within the A. thaliana genome. We use explicit criteria to distinguish orthologous from paralogous loci. In addition, we develop a conservative algorithm to identify collinear loci between the genomes and a permutation test to evaluate the significance of these regions. The algorithm identified 34 significant A. thaliana regions that are collinear with >28% of the B. oleracea genetic map. These regions have a mean of 3.3 markers spanning 2.1 Mbp of the A. thaliana genome and 2.5 cM of the B. oleracea genetic map. Our findings are consistent with the hypothesis that the B. oleracea genome has been highly rearranged since divergence from A. thaliana, likely as a result of polyploidization.