Brassica Genomes Research Articles

Molecular characterization of the flowering time gene FRIGIDA in Brassica genomes A and C

An important determinant of flowering time variation in Arabidopsis, the FRIGIDA (FRI) gene has not been until recently investigated in economically important Brassica species. In diploid Brassica species, this gene exists as two paralogous loci on chromosomes A3 and A4 (B. rapa; A genome), and C3 and C9 (B. oleracea; C genome). Each locus is represented by several genome-specific alleles, which are discerned primarily by polymorphisms in C- and especially N-terminal regions. Locus- and genome-specific sequences of two FRI paralogues are conserved almost completely in the subgenomes A and C of tetraploid B. napus. The phylogenetic analysis of available FRI sequences presumes that the duplication of FRI loci preceded speciation in the genus Brassica.

Characterization and evolutionary analysis of Brassica species-diverged sequences containing simple repeat units

Brassica species, B. napus (canola), B. rapa and B. oleracea, are important sources of nutritionally valuable vegetable oil and protein-rich meal for animals and humans. Sequencing of the model plant Arabidopsis thaliana, has opened the way for investigations into the complex structure of the Brassica genomes, offering important insight into their evolution and composition. We use this sequence information for the characterization and functional analysis of SSR sequences that have diverged between the Brassica species. A total of 56 species-diverged sequences containing simple repeat units (SDS-SSR) of B. napus and its diploid progenitor species B. rapa and B. oleracea were isolated and characterized. Of these, 40 sequences showed homology with other Brassica sequences. Using the SSR Locator software, only 23 sequences were found to have SSRs, possibly due to the loss of SSR units in the process of species divergence. Sequence alignments with A. thaliana revealed that these species-diverged SSR sequences were responsible for Brassica divergence for differences between Brassica species in several genomic regions. Six active genes related to transferase, protein, transcription factor and retroelements were found in the SDS-SSRs. These results will further improve our understanding of the characteristics of species-diverged SSR fragments and their contribution to genome differentiation.

The Brassica genome

BRASSICA GENOME RESEARCH TOPIC Brassica species include important crops and provide unique materials for the study of genome evolution. These crops include six important vegetables and oilseed crops, which have been classically described by “U’s triangle’. The three diploid species B. rapa (A genome), B. nigra (B genome), and B. oleracea (C genome) have formed the amphidiploid species B. juncea (A and B genomes), B. napus (A and C genomes), and B. carinata (B and C genomes) by hybridization. Moreover, the three diploid species themselves are evolved from a paleohexaploid, that is old enough to allowing the species being evolved into diploid species and young enough to maintain significant synteny with its ancestors. These make the Brassica species of the uniqueness of polyploidy in botanical evolution. Brassicas are closely related to the model plant Arabidopsis thaliana, one of the most extensively studied species in the world. Sequencing of the genome of Brassica rapa provided a great opportunity to bridge the rich knowledge obtained from Arabidopsis to be transferred to a cultivated species. Yet, tools and resources need to be established to accomplish the knowledge transfer. The release of the B. rapa var. Chiifu genome is not only of importance for genome evolution research, but also facilitates the gene discovery and breeding of the Brassica crops. Conversely, using the duplicated Brassica species as “deletion machines” to better understand the cis/trans-relationships of ENCODE-like features that are accumulating all over the arabidopsis genome is an additional, fundamental reason for continued study of the Brassica species. This Research Topic—The Brassica Genome—gathers contributions that report establishment of novel tools and methods, comparative genomics, gene discovery, molecular marker development, and genetic dissection of important traits. We hope this modest compendium marks the beginning of a vibrant future for Brassica comparative genome biology, and points the way toward how the Brassica lineage of crucifers, with arabidopsis as the outgroup, can and will revolutionize studies of eukaryotic gene and genome regulation and the phenotypes they specify.

AFLP mapping of quantitative trait loci influencing seven head-related traits in broccoli (Brassica oleracea var. italica)

SummaryThis study identified amplified fragment length polymorphism (AFLP) markers linked to head-related traits in broccoli (Brassica oleracea var. italica) grown in both Winter (W) and Summer (S) in 2008 and in 2009. In total, 227 F2 plants derived from a cross between the heat-tolerant cultivar ‘Shin-Fa 1208’ (SF), and the heat-sensitive cultivar ‘Green Magic’ (GM), were grown in the field. Seven traits including floret height (FH), floret width (FW), head height (HH), head width (HW), head weight (HWT), top weight (TWT), and stem width (SW) were scored. Polymorphic bands were generated by AFLP, and the segregation ratios and distribution of the identified markers over the Brassica genome were compared. Nine linkage groups (LGs), ranging from 75.8 cM to146.5 cM in size, were constructed using 202 informative markers spanning a total of 1,034.5 cM. For the seven traits investigated, 73 quantitative trait loci (QTLs) were dispersed non-randomly in the broccoli genome. Thirty-one unique regions of the genome were detected across the population in Summer trial, and three QTL markers, located on LGs 1, 3, and 5, showed pleiotropic effects on these seven traits. Among these, the QTL linked to the d-64 (S) marker on LG 3 had effects on HH, HWT, and FW, and significant positive correlations existed among these three traits. Thus, the AFLP marker d-64 (S) may have potential for the simultaneous selection of large FW and HH, and increased HWT under high-temperature conditions.

High-density SNP-based genetic map development and linkage disequilibrium assessment in Brassica napus L

BackgroundHigh density genetic maps built with SNP markers that are polymorphic in various genetic backgrounds are very useful for studying the genetics of agronomical traits as well as genome organization and evolution. Simultaneous dense SNP genotyping of segregating populations and variety collections was applied to oilseed rape (Brassica napus L.) to obtain a high density genetic map for this species and to study the linkage disequilibrium pattern.ResultsWe developed an integrated genetic map for oilseed rape by high throughput SNP genotyping of four segregating doubled haploid populations. A very high level of collinearity was observed between the four individual maps and a large number of markers (>59%) was common to more than two maps. The precise integrated map comprises 5764 SNP and 1603 PCR markers. With a total genetic length of 2250 cM, the integrated map contains a density of 3.27 markers (2.56 SNP) per cM. Genotyping of these mapped SNP markers in oilseed rape collections allowed polymorphism level and linkage disequilibrium (LD) to be studied across the different collections (winter vs spring, different seed quality types) and along the linkage groups. Overall, polymorphism level was higher and LD decayed faster in spring than in “00” winter oilseed rape types but this was shown to vary greatly along the linkage groups.ConclusionsOur study provides a valuable resource for further genetic studies using linkage or association mapping, for marker assisted breeding and for Brassica napus sequence assembly and genome organization analyses.

Cytomorphology of Brassica napus × B. rapa hybrids and patterns of variation in the F2 derivatives

The present study was carried out to determine crossability between Brassica napus and B. rapa, to elucidate chromosome homoeology between different Brassica genomes in the F1 hybrids and to estimate genetic variability in F2 progenies of the crosses. Crosses between B. napus (AACC, 2n = 38) and B. rapa (AA, 2n = 20) vars. brown sarson, yellow sarson and toria were attempted and the hybrid derivatives were advanced to F2 generation. Hybrid recoveries were higher when B. napus was used as a female parent. The F1 plants were luxuriant in growth and intermediate to their progenitor parents for morphological attributes. The meiotic studies of F1 plants (AAC, 2n = 29) showed 10 bivalents in the majority of pollen mother cells analyzed. A maximum of 12 II was found in a few cells. The occurrence of multivalent associations as trivalent and quadrivalent at metaphase I, and a bridge-fragment configuration at anaphases were attributed to homoeology between A and C genomes. In the F2 generation, a high percentage o...

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next‐generation sequencing

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B.napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B.napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B.napus. Molecular analyses of the candidate genes using B.napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.

Altered Patterns of Fractionation and Exon Deletions in Brassica rapa Support a Two-Step Model of Paleohexaploidy

The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This "two-step" hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short--single or two-gene--deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity--a hallmark of the Brassica genus.

Screening of core simple sequence repeat primer pairs and establishment of a multiplex polymerase chain reaction system for Brassica genomes A and C

With 2 figures and 1 table Abstract Brassica species are important industrial materials. Recently, simple sequence repeat (SSR) markers have become preferred molecular markers because of their simplicity, high levels of polymorphism and codominant nature. Numerous SSR markers have been developed in Brassica crops, but not every SSR marker has an equal value, and they must be tested individually for each application. To develop a set of standardized, highly efficient core SSR primers, we screened 190 primer pairs from among over 2353 SSR primer pairs that have been mapped for Brassica species. Ten different primer pairs were selected for each of the 19 linkage groups that corresponded with the chromosomes of Brassica genomes A and C. We also established 19 multiplex polymerase chain reaction (PCR) combinations, one per linkage group. Twenty accessions from six Brassica species were used to test these primer combinations, and an average of 15 clear bands were amplified successfully in each reaction. It is clear that multiplex PCR is a practical method for germplasm identification, genetic diversity analysis and backcross breeding in Brassica crops.

Characterization of interploid hybrids from crosses between Brassica juncea and B. oleracea and the production of yellow-seeded 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.

Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae

The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.

Unleashing the Genome of Brassica Rapa

The completion and release of the Brassica rapa genome is of great benefit to researchers of the Brassicas, Arabidopsis, and genome evolution. While its lineage is closely related to the model organism Arabidopsis thaliana, the Brassicas experienced a whole genome triplication subsequent to their divergence. This event contemporaneously created three copies of its ancestral genome, which had diploidized through the process of homeologous gene loss known as fractionation. By the fractionation of homeologous gene content and genetic regulatory binding sites, Brassica’s genome is well placed to use comparative genomic techniques to identify syntenic regions, homeologous gene duplications, and putative regulatory sequences. Here, we use the comparative genomics platform CoGe to perform several different genomic analyses with which to study structural changes of its genome and dynamics of various genetic elements. Starting with whole genome comparisons, the Brassica paleohexaploidy is characterized, syntenic regions with A. thaliana are identified, and the TOC1 gene in the circadian rhythm pathway from A. thaliana is used to find duplicated orthologs in B. rapa. These TOC1 genes are further analyzed to identify conserved non-coding sequences that contain cis-acting regulatory elements and promoter sequences previously implicated in circadian rhythmicity. Each “cookbook style” analysis includes a step-by-step walk-through with links to CoGe to quickly reproduce each step of the analytical process.

Physical Mapping in a Triplicated Genome: Mapping the Downy Mildew Resistance Locus Pp523 in Brassica oleracea L.

We describe the construction of a BAC contig and identification of a minimal tiling path that encompass the dominant and monogenically inherited downy mildew resistance locus Pp523 of Brassica oleracea L. The selection of BAC clones for construction of the physical map was carried out by screening gridded BAC libraries with DNA overgo probes derived from both genetically mapped DNA markers flanking the locus of interest and BAC-end sequences that align to Arabidopsis thaliana sequences within the previously identified syntenic region. The selected BAC clones consistently mapped to three different genomic regions of B. oleracea. Although 83 BAC clones were accurately mapped within a ∼4.6 cM region surrounding the downy mildew resistance locus Pp523, a subset of 33 BAC clones mapped to another region on chromosome C8 that was ∼60 cM away from the resistance gene, and a subset of 63 BAC clones mapped to chromosome C5. These results reflect the triplication of the Brassica genomes since their divergence from a common ancestor shared with A. thaliana, and they are consonant with recent analyses of the C genome of Brassica napus. The assembly of a minimal tiling path constituted by 13 (BoT01) BAC clones that span the Pp523 locus sets the stage for map-based cloning of this resistance gene.

Trigenomic Bridges for Brassica Improvement

We introduce and review Brassica crop improvement via trigenomic bridges. Six economically important Brassica species share three major genomes (A, B, and C), which are arranged in diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) species in the classical triangle of U. Trigenomic bridges are Brassica interspecific hybrid plants that contain the three genomes in various combinations, either triploid (ABC), unbalanced tetraploid (e.g., AABC), pentaploid (e.g., AABCC) or hexaploid (AABBCC). Through trigenomic bridges, Brassica breeders can access all the genetic resources in the triangle of U for genetic improvement of existing species and development of new agricultural species. Each of the three Brassica genomes occurs in several species, where they are distinguished as subgenomes with a tag to identify the species of origin. For example, the A subgenome in B. juncea (2n = AABB) is denoted as Aj and the A subgenome in B. napus (2n = AACC) as An. Trigenomic bridges have been used to increase genetic diversity in allopolyploid Brassica crop species, such as a new-type B. napus with subgenomes from B. rapa (Ar) and B. carinata (Cc). Recently, trigenomic bridges from several sources have been crossed together as the ‘founders’ of a potentially new allohexaploid Brassica species (AABBCC). During meiosis in a trigenomic bridge, crossovers are expected to form between homologous chromosomes of related subgenomes (for example Ar and An), but cross-overs may also occur between non-homologous chromosomes (for example between A and C genome chromosomes). Irregular meiosis is a common feature of new polyploids, and any new allotetraploid or allohexaploid Brassica genotypes derived from a trigenomic bridge must achieve meiotic stability through a process of diploidisation. New sequencing technologies, at the genomic and epigenomic level, may reveal the genetic and molecular basis of diploidization, and accelerate selection of stable allotetraploids or allohexaploids. Armed with new genetic resources from trigenomic bridges, Brassica breeders will be able to improve yield and broaden adaptation of Brassica crops to meet human demands for food and biofuel, particularly in the face of abiotic constraints caused by climate change.

Karyotype and genomic in situ hybridization pattern in ×Brassicoraphanus, an intergeneric hybrid between Brassica campestris ssp. pekinensis and Raphanus sativus

The karyotype and genomic in situ hybridization (GISH) of an intergeneric hybrid Baemoochae, ×Brassicoraphanus, which originated from hybridization between Chinese cabbage, Brassica campestris (synonym, rapa) ssp. pekinensis, and radish, Raphanus sativus, were analyzed to determine its chromosome complement. In the karyotype analysis, B. campestris was verified to have 2n = 20 chromosomes, including a particular pair of the subtelomeric chromosomes with the nucleolar organizer; R. sativus to have 2n = 18 chromosomes, including a particular pair of the submetacentric chromosomes with the secondary constriction of nucleolar organizer; and ×Brassicoraphanus to have 2n = 38 chromosomes, including both the subtelomeric chromosomes of Brassica and the secondary constriction chromosome pair of Raphanus. These findings indicate that ×Brassicoraphanus is a polyploid between Brassica and Raphanus. In the GISH analysis using chromosomes of B. campestris and R. sativus as the probe and blocking DNA, respectively, only 20 chromosomes of Brassica had hybridization signals. This result reveals that ×Brassicoraphanus is an intergeneric hybrid consisting of the complete genomes of both Brassica and Raphanus. However, the nucleolar organizers of Brassica and Raphanus were not identified because the hybridization signals appeared to be centering mainly around the centromere, becoming weak at the edges.

A physical map of Brassica oleracea shows complexity of chromosomal changes following recursive paleopolyploidizations

BackgroundEvolution of the Brassica species has been recursively affected by polyploidy events, and comparison to their relative, Arabidopsis thaliana, provides means to explore their genomic complexity.ResultsA genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-information-content fingerprinting (HICF) of Bacterial Artificial Chromosome (BAC) clones with hybridization to sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin, showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5% (shown to contain repetitive sequences) to pericentromeric regions. Overgo probe hybridization data showed that contigs aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal breakpoints during the diversification of B. oleracea and Arabidopsis thaliana, a relatively high level of genomic change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available eudicot genomes showed appreciable 'shadowing' produced by more ancient polyploidies, resulting in a web of relatedness among contigs which increased genomic complexity.ConclusionsA high-resolution genetically-anchored physical map sheds light on Brassica genome organization and advances positional cloning of specific genes, and may help to validate genome sequence assembly and alignment to chromosomes.All the physical mapping data is freely shared at a WebFPC site (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/; Temporarily password-protected: account: pgml; password: 123qwe123.

The Same Regulatory Point Mutation Changed Seed-Dispersal Structures in Evolution and Domestication

It is unclear whether gene regulatory changes that drive evolution at the population and species levels [1-3] can be extrapolated to higher taxonomic levels. Here, we investigated the role of cis-regulatory changes in fruit evolution within the Brassicaceae family. REPLUMLESS (RPL, At5g02030) controls development of the replum, a structure with an important role in fruit opening and seed dispersal [6]. We show that reduced repla resembling the Arabidopsis rpl mutant correlated across the Brassicaceae with a point mutation in a conserved cis-element of RPL. When introduced in Arabidopsis, this nucleotide change specifically reduced RPL expression and function in the fruit. Conversely, Brassica RPL containing the Arabidopsis version of the cis-element was sufficient to convert the Brassica replum to an Arabidopsis-like morphology. A mutation in the same nucleotide position of the same cis-element in a RPL ortholog has been independently selected to reduce seed dispersal during domestication of rice, in spite of its very different fruit anatomy. Thus, single-nucleotide regulatory mutations at the same position explain developmental variation in seed-dispersal structures at the population and family levels and suggest that the same genetic toolkit is relevant to domestication and natural evolution in widely diverged species.

Tau class glutathione S-transferases candidates from Brassica rapa: Genetic mapping and sequence analysis

Glutathione S-transferases (GSTs) have medically and agriculturally important roles in plants such as herbicide detoxification, responses to biotic and abiotic stress and catalytic reaction of anticancer compounds. The fully sequenced Arabidopsis thaliana and rice (Oryza sativa) genome revealed identification of 52 and 61 members of GSTs, and 28 and 39 of those belong to the Tau class of GSTs (GSTUs), respectively. Based on the sequences of AtGSTUs, 14 BAC clones derived from Brassica rapa that contain similar sequences of AtGSTUs were identified and 17 unique sequences of open reading frames were detected in the 14 BAC clones using blast search and sequence alignment. Those sequences were designated the Tau class candidate of GST derived from B. rapa (BrGSTUs) and it is confirmed that BrGSTUs are also clustered in tandem as it has been known to be common feature in plant. They were mapped on BrR5, BrR7, BrR8, BrR9, or BrR10, and their nucleotide and amino acid sequences were highly similar to those of AtGSTUs. In addition, in silico analysis of BrGSTUs was performed using Korea Brassica Genome Project 24 K (KBGP-24K) oligochip and microarray database for cold, salt, and drought stresses. The results of this study will facilitate further researches for breeding of Chinese cabbage containing medically and agriculturally important traits.

BraSto, a Stowaway MITE from Brassica: recently active copies preferentially accumulate in the gene space

We characterized a Brassica miniature inverted repeat transposable element (MITE) from the Stowaway superfamily, designated BraSto (Bra ssica Sto waway). BraSto copy number was assessed using real-time quantitative PCR in the two diploid species B. rapa (genome A) and B. oleracea (genome C) and the corresponding allotetraploid species B. napus (genome AC). Phylogenetic relationships among a set of 131 BraSto copies were then analyzed. BraSto appears to have been only moderately amplified in the Brassica genome and was still active recently with marks of proliferation in both diploid Brassica species, which diverged 3.75million years ago, but also in the allotetraploid species after reuniting of the two differentiated genomes. We characterized insertion sites for low-divergence BraSto copies among the gene space of the B. rapa genome using bioinformatics approaches. For BraSto copies localized nearby or within genes, we observed frequent associations of BraSto with putative promoters and regulatory regions of genes, but exclusion from coding regions. In addition, BraSto was significantly similar to several Brassica expressed sequence tags (ESTs), including stress-induced ESTs. We also demonstrated the enrichment of BraSto sequences in binding sites for transcription factors and other regulatory elements. Our results lead to the question of a role for BraSto in the regulation of gene expression: this putative role, if further confirmed experimentally, would help to obtain a new insight into the significance of MITEs in the functional plant genome.

Integration of linkage maps for the Amphidiploid Brassica napus and comparative mapping with Arabidopsis and Brassica rapa

BackgroundThe large number of genetic linkage maps representing Brassica chromosomes constitute a potential platform for studying crop traits and genome evolution within Brassicaceae. However, the alignment of existing maps remains a major challenge. The integration of these genetic maps will enhance genetic resolution, and provide a means to navigate between sequence-tagged loci, and with contiguous genome sequences as these become available.ResultsWe report the first genome-wide integration of Brassica maps based on an automated pipeline which involved collation of genome-wide genotype data for sequence-tagged markers scored on three extensively used amphidiploid Brassica napus (2n = 38) populations. Representative markers were selected from consolidated maps for each population, and skeleton bin maps were generated. The skeleton maps for the three populations were then combined to generate an integrated map for each LG, comparing two different approaches, one encapsulated in JoinMap and the other in MergeMap. The BnaWAIT_01_2010a integrated genetic map was generated using JoinMap, and includes 5,162 genetic markers mapped onto 2,196 loci, with a total genetic length of 1,792 cM. The map density of one locus every 0.82 cM, corresponding to 515 Kbp, increases by at least three-fold the locus and marker density within the original maps. Within the B. napus integrated map we identified 103 conserved collinearity blocks relative to Arabidopsis, including five previously unreported blocks. The BnaWAIT_01_2010a map was used to investigate the integrity and conservation of order proposed for genome sequence scaffolds generated from the constituent A genome of Brassica rapa.ConclusionsOur results provide a comprehensive genetic integration of the B. napus genome from a range of sources, which we anticipate will provide valuable information for rapeseed and Canola research.