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.     

植物CO基因研究进展

CO(constans)是植物开花时间光周期调控途径中的一个重要基因.目前从拟南芥、水稻、油菜、马铃薯等多个物种中都已经克隆到CO同源基因.CO基因在不同物种中具有保守的锌指结构和核定位区域,但是不同植物中的作用机理并不完全相同.序列分析表明该基因在被子植物与裸子植物之间、双子叶植物与单子叶植物之间以及不同科、属的植物之间均有明显分化,说明CO基因可能在植物进化中起到了重要作用.本文综述了近年来有关植物CO基因的研究进展,并对其在物种中的进化进行分析,为CO基因进一步研究提供参考.     

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.     

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.     

Flowering time quantitative trait Loci analysis of oilseed brassica in multiple environments and genomewide alignment with Arabidopsis

Most agronomical traits exhibit quantitative variation, which is controlled by multiple genes and are environmentally dependent. To study the genetic variation of flowering time in Brassica napus, a DH population and its derived reconstructed F(2) population were planted in 11 field environments. The flowering time varied greatly with environments; 60% of the phenotypic variation was attributed to genetic effects. Five to 18 QTL at a statistically significant level (SL-QTL) were detected in each environment and, on average, two new SL-QTL were discovered with each added environment. Another type of QTL, micro-real QTL (MR-QTL), was detected repeatedly from at least 2 of the 11 environments; resulting in a total of 36 SL-QTL and 6 MR-QTL. Sixty-three interacting pairs of loci were found; 50% of them were involved in QTL. Hundreds of floral transition genes in Arabidopsis were aligned with the linkage map of B. napus by in silico mapping; 28% of them aligned with QTL regions and 9% were consistent with interacting loci. One locus, BnFLC10, in N10 and a QTL cluster in N16 were specific to spring- and winter-cropped environments respectively. The number of QTL, interacting loci, and aligned functional genes revealed a complex genetic network controlling flowering time in B. napus.     

Complexities of chromosome landing in a highly duplicated genome: toward map-based cloning of a gene controlling blackleg resistance in Brassica napus

The LmR1 locus, which controls seedling resistance to the blackleg fungus Leptosphaeria maculans in the Brassica napus cultivar Shiralee, was positioned on linkage group N7. Fine genetic mapping in a population of 2500 backcross lines identified three molecular markers that cosegregated with LmR1. Additional linkage mapping in a second population colocalized a seedling resistance gene, ClmR1, from the cultivar Cresor to the same genetic interval on N7 as LmR1. Both genes were located in a region that showed extensive inter- and intragenomic duplications as well as intrachromosomal tandem duplications. The tandem duplications seem to have occurred in the Brassica lineage before the divergence of B. rapa and B. oleracea but after the separation of Brassica and Arabidopsis from a common ancestor. Microsynteny was found between the region on N7 carrying the resistance gene and the end of Arabidopsis chromosome 1, interrupted by a single inversion close to the resistance locus. The collinear region in Arabidopsis was assayed for the presence of possible candidate genes for blackleg resistance. These data provided novel insights into the genomic structure and evolution of plant resistance loci and an evaluation of the candidate gene approach using comparative mapping with a model organism.     

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.     

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.     

Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris.

The spectrum of resistance to isolates of Leptosphaeria maculans and the map location of a new blackleg resistance gene found in the canola cultivar Brassica napus 'Surpass 400' are described. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris and introgressed in B. napus were identified previously. 'Surpass 400' also has blackleg resistance introgressed from B. rapa subsp. sylvestris. Using 31 diverse isolates of L. maculans, the disease reaction of 'Surpass 400' was compared with those of the resistant breeding lines AD9 (which contains LepR1), AD49 (which contains LepR2), and MC1-8 (which contains both LepR1 and LepR2). The disease reaction on 'Surpass 400' was different from those observed on AD9 and MC1-8, indicating that 'Surpass 400' carries neither LepR1 nor both LepR1 and LepR2 in combination. Disease reactions of 'Surpass 400' to most of the isolates tested were indistinguishable from those of AD49, which suggested 'Surpass 400' might contain LepR2 or a similar resistance gene. Classical genetic analysis of F1 and BC1 plants showed that a dominant allele conferred resistance to isolates of L. maculans in 'Surpass 400'. The resistance gene, which mapped to B. napus linkage group N10 in an interval of 2.9 cM flanked by microsatellite markers sR12281a and sN2428Rb and 11.7 cM below LepR2, was designated LepR3. A 9 cM region of the B. napus genome containing LepR3 was found to be syntenic with a segment of Arabidopsis chromosome 5.     

Arabidopsis thaliana: a source of candidate disease-resistance genes for Brassica napus.

Common structural and amino acid motifs among cloned plant disease-resistance genes (R genes), have made it possible to identify putative disease-resistance sequences based on DNA sequence identity. Mapping of such R-gene homologues will identify candidate disease-resistance loci to expedite map-based cloning strategies in complex crop genomes. Arabidopsis thaliana expressed sequence tags (ESTs) with homology to cloned plant R genes (R-ESTs), were mapped in both A. thaliana and Brassica napus to identify candidate R-gene loci and investigate intergenomic collinearity. Brassica R-gene homologous sequences were also mapped in B. napus. In total, 103 R-EST loci and 36 Brassica R-gene homologous loci were positioned on the N-fo-61-9 B. napus genetic map, and 48 R-EST loci positioned on the Columbia x Landsberg A. thaliana map. The mapped loci identified collinear regions between Arabidopsis and Brassica which had been observed in previous comparative mapping studies; the detection of syntenic genomic regions indicated that there was no apparent rapid divergence of the identified genomic regions housing the R-EST loci.     

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。     

Comparative mapping in Arabidopsis and Brassica, fine scale genome collinearity and congruence of genes controlling flowering time

The model dicotyledonous plant, Arabidopsis thaliana , is closely related to Brassica crop species. It is intended that information concerning the genetic control of basic biological processes in Arabidopsis will be transferable to other species. Genome collinearity and its potential to facilitate the identification of candidate genes in Arabidopsis homologous to genes controlling important agronomic traits in Brassica was investigated. Genetic mapping in B. nigra identified two loci influencing flowering time (FT), with loci on linkage groups 2 and 8 explaining 53% and 12% of the total variation in FT, respectively. The CO gene exerts an important control over FT in A. thaliana , and B. nigra homologues of CO probably also play an important role in regulating FT. B. nigra homologues of CO were identified on linkage groups 2 and 8, the homologue on group 2 was coincident with the major locus controlling FT while the homologue on group 8 was within the 90% confidence interval of the weaker FT gene. The CO homologue on group 2 exhibits abundant allelic variation suggesting that it naturally controls a wide range of flowering times. Fine-scale A. thaliana/B. nigra comparative mapping demonstrated short-range collinearity between the genomes of Arabidopsis and Brassica . Eleven DNA fragments spaced over a 1.5 Mb contig in A. thaliana were used as RFLP probes in B. nigra . Three collinear representations of the A. thaliana contig were identified in B. nigra , with one interrupted by a large chromosomal inversion. Collinearity over this range will allow the resources generated by the Arabidopsis genome project to facilitate map-based cloning in Brassica crops.     

Multiple flowering time QTLs within several Brassica species could be the result of duplicated copies of one ancestral gene.

Quantitative trait locus (QTL) analysis was used to study the evolution of genes controlling the timing of flowering in four Brassica genomes that are all extensively replicated. Comparative mapping showed that a chromosomal region from the top of Arabidopsis thaliana chromosome 5 corresponded to three homoeologous copies in each of the diploid species Brassica nigra, B. oleracea, and B. rapa and six copies in the amphidiploid B. juncea. QTLs were detected in two of the three replicated segments in each diploid genome and in three of the six replicated segments in B. juncea. These results indicate that, for the studied trait, multiple QTLs resulting from genome duplication is the rule rather than the exception. Brassica homologues to two candidate genes (CO and FLC) identified from the corresponding A. thaliana region were mapped. CO homologues mapped close to the QTL peaks in eight of nine QTLs, while FLC homologues mapped farther away in those cases where the mapping resolution allowed a comparison. Thus, our data are consistent with the hypothesis that all the major QTLs we detected in the different species of Brassica could be the result of duplicated copies of the same ancestral gene, possibly the ancestor of CO.     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

Naturally occurring indel variation in the Brassica nigra COL1 gene is associated with variation in flowering time

Previous QTL mapping identified a Brassica nigra homolog to Arabidopsis thaliana CO as a candidate gene affecting flowering time in B. nigra. Transformation of an A. thaliana co mutant with two different alleles of the B. nigra CO (Bni COa) homolog, one from an early-flowering B. nigra plant and one from a late one, did not show any differential effect of the two alleles on flowering time. The DNA sequence of the coding region of the two alleles was also identical, showing that nucleotide variation influencing flowering time must reside outside the coding region of Bni COa. In contrast, the nucleotide sequence of the B. nigra COL1 (Bni COL1) gene located 3.5 kb upstream of Bni COa was highly diverged between the alleles from early and late plants. One indel polymorphism in the Bni COL1 coding region, present in several natural populations of B. nigra, displayed a significant association with flowering time within a majority of these populations. These data indicate that a quantitative trait nucleotide (QTN) affecting flowering time is located within or close to the Bni COL1 gene. The intergenic sequence between Bni COL1 and Bni COa displayed a prominent peak of divergence 1 kb downstream of the Bni COL1 coding region. This region could contain regulatory elements for the downstream Bni COa gene. Our data suggest that a naturally occurring QTN for flowering time affects the function or expression of either Bni COL1 or Bni COa.     

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 radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats

A single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. A map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687-amino-acid protein with a predicted mitochondrial targeting pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis, although a smaller and related PPR gene is found about 40 kb from this site. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.