Brassica rapa, which is closely related to
Arabidopsis thaliana, is an important crop and a
model plant for studying genome evolution via
polyploidization. We report the current understanding of the
genome structure of B. rapa and efforts for the
whole-genome sequencing of the species. The tribe
Brassicaceae, which comprises ca. 240 species,
descended from a common hexaploid ancestor with a basic genome
similar to that of Arabidopsis. Chromosome
rearrangements, including fusions and/or fissions, resulted in
the present-day “diploid” Brassica
species with variation in chromosome number and phenotype.
Triplicated genomic segments of B. rapa are
collinear to those of A. thaliana with InDels.
The genome triplication has led to an approximately 1.7-fold
increase in the B. rapa gene number compared to
that of A. thaliana. Repetitive DNA of B.
rapa has also been extensively amplified and has
diverged from that of A. thaliana. For its
whole-genome sequencing, the Brassica rapa Genome
Sequencing Project (BrGSP) consortium has developed suitable
genomic resources and constructed genetic and physical maps.
Ten chromosomes of B. rapa are being allocated to
BrGSP consortium participants, and each chromosome will be
sequenced by a BAC-by-BAC approach. Genome sequencing of
B. rapa will offer a new perspective for plant
biology and evolution in the context of polyploidization.
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.
comparative genomics; synteny; CoGe; Brassica rapa; syntenic dotplot; Arabidopsis; TOC1; conserved non-coding sequences
Whole genome duplication (WGD) and tandem duplication (TD) are both important modes of gene expansion. However, how WGD influences tandemly duplicated genes is not well studied. We used Brassica rapa, which has undergone an additional genome triplication (WGT) and shares a common ancestor with Arabidopsis thaliana, Arabidopsis lyrata, and Thellungiella parvula, to investigate the impact of genome triplication on tandem gene evolution. We identified 2,137, 1,569, 1,751, and 1,135 tandem gene arrays in B. rapa, A. thaliana, A. lyrata, and T. parvula respectively. Among them, 414 conserved tandem arrays are shared by the three species without WGT, which were also considered as existing in the diploid ancestor of B. rapa. Thus, after genome triplication, B. rapa should have 1,242 tandem arrays according to the 414 conserved tandems. Here, we found 400 out of the 414 tandems had at least one syntenic ortholog in the genome of B. rapa. Furthermore, 294 out of the 400 shared syntenic orthologs maintain tandem arrays (more than one gene for each syntenic hit) in B. rapa. For the 294 tandem arrays, we obtained 426 copies of syntenic paralogous tandems in the triplicated genome of B. rapa. In this study, we demonstrated that tandem arrays in B. rapa were dramatically fractionated after WGT when compared either to non-tandem genes in the B. rapa genome or to the tandem arrays in closely related species that have not experienced a recent whole genome polyploidization event.
whole genome duplication; tandem duplication; tandem gene evolution; Brassica rapa; Arabidopsis thaliana; Arabidopsis lyrata; Thellungiella parvula
The Brassicaceae family includes the model plant Arabidopsis thaliana as well as a number of agronomically important species such as oilseed crops (in particular Brassica napus, B. juncea and B. rapa) and vegetables (eg. B. rapa and B. oleracea).
Separated by only 10-20 million years, Brassica species and Arabidopsis thaliana are closely related, and it is expected that knowledge obtained relating to Arabidopsis growth and development can be translated into Brassicas for crop improvement. Moreover, certain aspects of plant development are sufficiently different between Brassica and Arabidopsis to warrant studies to be carried out directly in the crop species. However, mutating individual genes in the amphidiploid Brassicas such as B. napus and B. juncea may, on the other hand, not give rise to expected phenotypes as the genomes of these species can contain up to six orthologues per single-copy Arabidopsis gene. In order to elucidate and possibly exploit the function of redundant genes for oilseed rape crop improvement, it may therefore be more efficient to study the effects in one of the diploid Brassica species such as B. rapa. Moreover, the ongoing sequencing of the B. rapa genome makes this species a highly attractive model for Brassica research and genetic resource development.
Seeds from the diploid Brassica A genome species, B. rapa were treated with ethyl methane sulfonate (EMS) to produce a TILLING (Targeting Induced Local Lesions In Genomes) population for reverse genetics studies. We used the B. rapa genotype, R-o-18, which has a similar developmental ontogeny to an oilseed rape crop. Hence this resource is expected to be well suited for studying traits with relevance to yield and quality of oilseed rape. DNA was isolated from a total of 9,216 M2 plants and pooled to form the basis of the TILLING platform. Analysis of six genes revealed a high level of mutations with a density of about one per 60 kb. This analysis also demonstrated that screening a 1 kb amplicon in just one third of the population (3072 M2 plants) will provide an average of 68 mutations and a 97% probability of obtaining a stop-codon mutation resulting in a truncated protein. We furthermore calculated that each plant contains on average ~10,000 mutations and due to the large number of plants, it is predicted that mutations in approximately half of the GC base pairs in the genome exist within this population.
We have developed the first EMS TILLING resource in the diploid Brassica species, B. rapa. The mutation density in this population is ~1 per 60 kb, which makes it the most densely mutated diploid organism for which a TILLING population has been published. This resource is publicly available through the RevGenUK reverse genetics platform http://revgenuk.jic.ac.uk.
Euchromatic regions of the Brassica rapa genome were sequenced and mapped onto the corresponding regions in the Arabidopsis thaliana genome.
Brassica rapa is one of the most economically important vegetable crops worldwide. Owing to its agronomic importance and phylogenetic position, B. rapa provides a crucial reference to understand polyploidy-related crop genome evolution. The high degree of sequence identity and remarkably conserved genome structure between Arabidopsis and Brassica genomes enables comparative tiling sequencing using Arabidopsis sequences as references to select the counterpart regions in B. rapa, which is a strong challenge of structural and comparative crop genomics.
We assembled 65.8 megabase-pairs of non-redundant euchromatic sequence of B. rapa and compared this sequence to the Arabidopsis genome to investigate chromosomal relationships, macrosynteny blocks, and microsynteny within blocks. The triplicated B. rapa genome contains only approximately twice the number of genes as in Arabidopsis because of genome shrinkage. Genome comparisons suggest that B. rapa has a distinct organization of ancestral genome blocks as a result of recent whole genome triplication followed by a unique diploidization process. A lack of the most recent whole genome duplication (3R) event in the B. rapa genome, atypical of other Brassica genomes, may account for the emergence of B. rapa from the Brassica progenitor around 8 million years ago.
This work demonstrates the potential of using comparative tiling sequencing for genome analysis of crop species. Based on a comparative analysis of the B. rapa sequences and the Arabidopsis genome, it appears that polyploidy and chromosomal diploidization are ongoing processes that collectively stabilize the B. rapa genome and facilitate its evolution.
Miniature inverted-repeat transposable elements (MITEs) are expected to play important roles in evolution of genes and genome in plants, especially in the highly duplicated plant genomes. Various MITE families and their roles in plants have been characterized. However, there have been fewer studies of MITE families and their potential roles in evolution of the recently triplicated Brassica genome.
We identified a new MITE family, BRAMI-1, belonging to the Stowaway super-family in the Brassica genome. In silico mapping revealed that 697 members are dispersed throughout the euchromatic regions of the B. rapa pseudo-chromosomes. Among them, 548 members (78.6%) are located in gene-rich regions, less than 3 kb from genes. In addition, we identified 516 and 15 members in the 470 Mb and 15 Mb genomic shotgun sequences currently available for B. oleracea and B. napus, respectively. The resulting estimated copy numbers for the entire genomes were 1440, 1464 and 2490 in B. rapa, B. oleracea and B. napus, respectively. Concurrently, only 70 members of the related Arabidopsis ATTIRTA-1 MITE family were identified in the Arabidopsis genome. Phylogenetic analysis revealed that BRAMI-1 elements proliferated in the Brassica genus after divergence from the Arabidopsis lineage. MITE insertion polymorphism (MIP) was inspected for 50 BRAMI-1 members, revealing high levels of insertion polymorphism between and within species of Brassica that clarify BRAMI-1 activation periods up to the present. Comparative analysis of the 71 genes harbouring the BRAMI-1 elements with their non-insertion paralogs (NIPs) showed that the BRAMI-1 insertions mainly reside in non-coding sequences and that the expression levels of genes with the elements differ from those of their NIPs.
A Stowaway family MITE, named as BRAMI-1, was gradually amplified and remained present in over than 1400 copies in each of three Brassica species. Overall, 78% of the members were identified in gene-rich regions, and it is assumed that they may contribute to the evolution of duplicated genes in the highly duplicated Brassica genome. The resulting MIPs can serve as a good source of DNA markers for Brassica crops because the insertions are highly dispersed in the gene-rich euchromatin region and are polymorphic between or within species.
Miniature Inverted-repeat Transposable Element (MITE); MITE insertion polymorphism (MIP); Brassica species; Evolution; BRAMI-1
Completion of the sequencing of the Brassica rapa genome enabled us to undertake a genome-wide identification and functional study of the gene families related to the morphological diversity and agronomic traits of Brassica crops. In this study, we identified the auxin response factor (ARF) gene family, which is one of the key regulators of auxin-mediated plant growth and development in the B. rapa genome. A total of 31 ARF genes were identified in the genome. Phylogenetic and evolutionary analyses suggest that ARF genes fell into four major classes and were amplified in the B. rapa genome as a result of a recent whole genome triplication after speciation from Arabidopsis thaliana. Despite its recent hexaploid ancestry, B. rapa includes a relatively small number of ARF genes compared with the 23 members in A. thaliana, presumably due to a paralog reduction related to repetitive sequence insertion into promoter and non-coding transcribed region of the genes. Comparative genomic and mRNA sequencing analyses demonstrated that 27 of the 31 BrARF genes were transcriptionally active, and their expression was affected by either auxin treatment or floral development stage, although 4 genes were inactive, suggesting that the generation and pseudogenization of ARF members are likely to be an ongoing process. This study will provide a fundamental basis for the modification and evolution of the gene family after a polyploidy event, as well as a functional study of ARF genes in a polyploidy crop species.
Electronic supplementary material
The online version of this article (doi:10.1007/s00438-012-0718-4) contains supplementary material, which is available to authorized users.
Brassica rapa; Auxin response factor; Genome organization; mRNA sequencing; Evolution
Brassica rapa (AA) contains very diverse forms which include oleiferous types and many vegetable types. Genome sequence of B. rapa line Chiifu (ssp. pekinensis), a leafy vegetable type, was published in 2011. Using this knowledge, it is important to develop genomic resources for the oleiferous types of B. rapa. This will allow more involved molecular mapping, in-depth study of molecular mechanisms underlying important agronomic traits and introgression of traits from B. rapa to major oilseed crops - B. juncea (AABB) and B. napus (AACC). The study explores the availability of SNPs in RNA-seq generated contigs of three oleiferous lines of B. rapa - Candle (ssp. oleifera, turnip rape), YSPB-24 and Tetra (ssp. trilocularis, Yellow sarson) and their use in genome-wide linkage mapping and specific-region fine mapping using a RIL population between Chiifu and Tetra.
RNA-seq was carried out on the RNA isolated from young inflorescences containing unopened floral buds, floral axis and small leaves, using Illumina paired-end sequencing technology. Sequence assembly was carried out using the Velvet de-novo programme and the assembled contigs were organised against Chiifu gene models, available in the BRAD-CDS database. RNA-seq confirmed the presence of more than 17,000 single-copy gene models described in the BRAD database. The assembled contigs and the BRAD gene models were analyzed for the presence of SSRs and SNPs. While the number of SSRs was limited, more than 0.2 million SNPs were observed between Chiifu and the three oleiferous lines. Assays for SNPs were designed using KASPar technology and tested on a F7-RIL population derived from a Chiifu x Tetra cross. The design of the SNP assays were based on three considerations - the 50 bp flanking region of the SNPs should be strictly similar, the SNP should have a read-depth of ≥7 and no exon/intron junction should be present within the 101 bp target region. Using these criteria, a total of 640 markers (580 for genome-wide mapping and 60 for specific-region mapping) marking as many genes were tested for mapping. Out of 640 markers that were tested, 594 markers could be mapped unambiguously which included 542 markers for genome-wide mapping and 42 markers for fine mapping of the tet-o locus that is involved with the trait tetralocular ovary in the line Tetra.
A large number of SNPs and PSVs are present in the transcriptome of B. rapa lines for genome-wide linkage mapping and specific-region fine mapping. Criteria used for SNP identification delivered markers, more than 93% of which could be successfully mapped to the F7–RIL population of Chiifu x Tetra cross.
Brassica rapa; RNA-seq; Next generation sequencing; Single nucleotide polymorphism (SNP); Paralog specific variation (PSV); Coding DNA Sequences (CDS); KASPar assays
Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00438-009-0492-0) contains supplementary material, which is available to authorized users.
Brassica rapa; NBS-encoding gene; Disease resistance; Genome diploidization; Evolution
Brassica rapa is an important crop species that produces vegetables, oilseed, and fodder. Although many studies reported quantitative trait loci (QTL) mapping, the genes governing most of its economically important traits are still unknown. In this study, we report QTL mapping for morphological and yield component traits in B. rapa and comparative map alignment between B. rapa, B. napus, B. juncea, and Arabidopsis thaliana to identify candidate genes and conserved QTL blocks between them. A total of 95 QTL were identified in different crucifer blocks of the B. rapa genome. Through synteny analysis with A. thaliana, B. rapa candidate genes and intronic and exonic single nucleotide polymorphisms in the parental lines were detected from whole genome resequenced data, a few of which were validated by mapping them to the QTL regions. Semi-quantitative reverse transcriptase PCR analysis showed differences in the expression levels of a few genes in parental lines. Comparative mapping identified five key major evolutionarily conserved crucifer blocks (R, J, F, E, and W) harbouring QTL for morphological and yield components traits between the A, B, and C subgenomes of B. rapa, B. juncea, and B. napus. The information of the identified candidate genes could be used for breeding B. rapa and other related Brassica species.
Brassica rapa; quantitative trait loci (QTL); morphological traits; single nucleotide polymorphism (SNP); conserved genome blocks
Chinese cabbage (Brassica rapa ssp. pekinensis) is a member of one of the most important leaf vegetables grown worldwide, which has experienced thousands of years in cultivation and artificial selection. The entire Chinese cabbage genome sequence, and more than forty thousand proteins have been obtained to date. The genome has undergone triplication events since its divergence from Arabidopsis thaliana (13 to 17 Mya), however a high degree of sequence similarity and conserved genome structure remain between the two species. Arabidopsis is therefore a viable reference species for comparative genomics studies. Variation in the number of members in gene families due to genome triplication may contribute to the broad range of phenotypic plasticity, and increased tolerance to environmental extremes observed in Brassica species. Transcription factors are important regulators involved in plant developmental and physiological processes. The AP2/ERF proteins, one of the most important families of transcriptional regulators, play a crucial role in plant growth, and in response to biotic and abiotic stressors. Our analysis will provide resources for understanding the tolerance mechanisms in Brassica rapa ssp. pekinensis.
In the present study, 291 putative AP2/ERF transcription factor proteins were identified from the Chinese cabbage genome database, and compared with proteins from 15 additional species. The Chinese cabbage AP2/ERF superfamily was classified into four families, including AP2, ERF, RAV, and Soloist. The ERF family was further divided into DREB and ERF subfamilies. The AP2/ERF superfamily was subsequently divided into 15 groups. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns, and interaction networks of the AP2/ERF transcription factor superfamily were predicted and analyzed. Distribution mapping results showed AP2/ERF superfamily genes were localized on the 10 Chinese cabbage chromosomes. AP2/ERF transcription factor expression levels exhibited differences among six tissue types based on expressed sequence tags (ESTs). In the AP2/ERF superfamily, 214 orthologous genes were identified between Chinese cabbage and Arabidopsis. Orthologous gene interaction networks were constructed, and included seven CBF and four AP2 genes, primarily involved in cold regulatory pathways and ovule development, respectively.
The evolution of the AP2/ERF transcription factor superfamily in Chinese cabbage resulted from genome triplication and tandem duplications. A comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes in Chinese cabbage is required to fully elucidate AP2/ERF, which provides us with rich resources and opportunities to understand crop stress tolerance mechanisms.
Chinese cabbage; AP2/ERF; Stress tolerance; Gene expression; Interaction network; Protein annotation
DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5′-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5′-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.
Brassica species include both vegetable and oilseed crops, which are very important to the daily life of common human beings. Meanwhile, the Brassica species represent an excellent system for studying numerous aspects of plant biology, specifically for the analysis of genome evolution following polyploidy, so it is also very important for scientific research. Now, the genome of Brassica rapa has already been assembled, it is the time to do deep mining of the genome data.
BRAD, the Brassica database, is a web-based resource focusing on genome scale genetic and genomic data for important Brassica crops. BRAD was built based on the first whole genome sequence and on further data analysis of the Brassica A genome species, Brassica rapa (Chiifu-401-42). It provides datasets, such as the complete genome sequence of B. rapa, which was de novo assembled from Illumina GA II short reads and from BAC clone sequences, predicted genes and associated annotations, non coding RNAs, transposable elements (TE), B. rapa genes' orthologous to those in A. thaliana, as well as genetic markers and linkage maps. BRAD offers useful searching and data mining tools, including search across annotation datasets, search for syntenic or non-syntenic orthologs, and to search the flanking regions of a certain target, as well as the tools of BLAST and Gbrowse. BRAD allows users to enter almost any kind of information, such as a B. rapa or A. thaliana gene ID, physical position or genetic marker.
BRAD, a new database which focuses on the genetics and genomics of the Brassica plants has been developed, it aims at helping scientists and breeders to fully and efficiently use the information of genome data of Brassica plants. BRAD will be continuously updated and can be accessed through http://brassicadb.org.
The species Brassica rapa includes important vegetable and oil crops. It also serves as an excellent model system to study polyploidy-related genome evolution because of its paleohexaploid ancestry and its close evolutionary relationships with Arabidopsis thaliana and other Brassica species with larger genomes. Therefore, its genome sequence will be used to accelerate both basic research on genome evolution and applied research across the cultivated Brassica species.
We have determined and analyzed the sequence of B. rapa chromosome A3. We obtained 31.9 Mb of sequences, organized into nine contigs, which incorporated 348 overlapping BAC clones. Annotation revealed 7,058 protein-coding genes, with an average gene density of 4.6 kb per gene. Analysis of chromosome collinearity with the A. thaliana genome identified conserved synteny blocks encompassing the whole of the B. rapa chromosome A3 and sections of four A. thaliana chromosomes. The frequency of tandem duplication of genes differed between the conserved genome segments in B. rapa and A. thaliana, indicating differential rates of occurrence/retention of such duplicate copies of genes. Analysis of 'ancestral karyotype' genome building blocks enabled the development of a hypothetical model for the derivation of the B. rapa chromosome A3.
We report the near-complete chromosome sequence from a dicotyledonous crop species. This provides an example of the complexity of genome evolution following polyploidy. The high degree of contiguity afforded by the clone-by-clone approach provides a benchmark for the performance of whole genome shotgun approaches presently being applied in B. rapa and other species with complex genomes.
Brassica rapa is an economically important crop and a model plant for studies concerning polyploidization and the evolution of extreme morphology. The multinational B. rapa Genome Sequencing Project (BrGSP) was launched in 2003. In 2008, next generation sequencing technology was used to sequence the B. rapa genome. Several maps concerning B. rapa pseudochromosome assembly have been published but their coverage of the genome is incomplete, anchoring approximately 73.6% of the scaffolds on to chromosomes. Therefore, a new genetic map to aid pseudochromosome assembly is required.
This study concerns the construction of a reference genetic linkage map for Brassica rapa, forming the backbone for anchoring sequence scaffolds of the B. rapa genome resulting from recent sequencing efforts. One hundred and nineteen doubled haploid (DH) lines derived from microspore cultures of an F1 cross between a Chinese cabbage (B. rapa ssp. pekinensis) DH line (Z16) and a rapid cycling inbred line (L144) were used to construct the linkage map. PCR-based insertion/deletion (InDel) markers were developed by re-sequencing the two parental lines. The map comprises a total of 507 markers including 415 InDels and 92 SSRs. Alignment and orientation using SSR markers in common with existing B. rapa linkage maps allowed ten linkage groups to be identified, designated A01-A10. The total length of the linkage map was 1234.2 cM, with an average distance of 2.43 cM between adjacent marker loci. The lengths of linkage groups ranged from 71.5 cM to 188.5 cM for A08 and A09, respectively. Using the developed linkage map, 152 scaffolds were anchored on to the chromosomes, encompassing more than 82.9% of the B. rapa genome. Taken together with the previously available linkage maps, 183 scaffolds were anchored on to the chromosomes and the total coverage of the genome was 88.9%.
The development of this linkage map is vital for the integration of genome sequences and genetic information, and provides a useful resource for the international Brassica research community.
Polyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. Brassica napus L. is one of the most economically important polyploid oil crops and has been broadly studied as an example of polyploid crop. RNA-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids.
We examined the global gene expression patterns of the first four generations of resynthesized B. napus (F1–F4), its diploid progenitors B. rapa and B. oleracea, and natural B. napus using digital gene expression analysis. Almost 42 million clean tags were generated using Illumina technology to produce the expression data for 25959 genes, which account for 63% of the annotated B. rapa genome. More than 56% of the genes were transcribed from both strands, which indicate the importance of RNA-mediated gene regulation in polyploidization. Tag mapping of the B. rapa genome generated 19023, 18547, 24383, 20659, 18881, 20692, and 19955 annotated genes for the B. rapa, B. oleracea, F1–F4 of synthesized B. napus, and natural B. napus libraries, respectively. The unambiguous tag-mapped genes in the libraries were functionally categorized via gene ontological analysis. Thousands of differentially expressed genes (DEGs) were identified and revealed the substantial changes in F1–F4. Among the 20 most DEGs are DNA binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs suggested approximately 120 biological pathways.
The systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized B. napus. This information broadens our understanding of the mechanisms of B. napus polyploidization and contributes to molecular and genetic research by enriching the Brassica database.
The genus Brassica includes the most extensively cultivated vegetable crops worldwide. Investigation of the Brassica genome presents excellent challenges to study plant genome evolution and divergence of gene function associated with polyploidy and genome hybridization. A physical map of the B. rapa genome is a fundamental tool for analysis of Brassica "A" genome structure. Integration of a physical map with an existing genetic map by linking genetic markers and BAC clones in the sequencing pipeline provides a crucial resource for the ongoing genome sequencing effort and assembly of whole genome sequences.
A genome-wide physical map of the B. rapa genome was constructed by the capillary electrophoresis-based fingerprinting of 67,468 Bacterial Artificial Chromosome (BAC) clones using the five restriction enzyme SNaPshot technique. The clones were assembled into contigs by means of FPC v8.5.3. After contig validation and manual editing, the resulting contig assembly consists of 1,428 contigs and is estimated to span 717 Mb in physical length. This map provides 242 anchored contigs on 10 linkage groups to be served as seed points from which to continue bidirectional chromosome extension for genome sequencing.
The map reported here is the first physical map for Brassica "A" genome based on the High Information Content Fingerprinting (HICF) technique. This physical map will serve as a fundamental genomic resource for accelerating genome sequencing, assembly of BAC sequences, and comparative genomics between Brassica genomes. The current build of the B. rapa physical map is available at the B. rapa Genome Project website for the user community.
We have developed DNA-based genetic markers for rapid cycling Brassica rapa (RCBr), also known as Fast Plants. Although markers for B. rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP) based markers and 14 variable number tandem repeat (VNTR)-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a non-genic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology.
Fast Plants; rapid cycling Brassica rapa; marker; SNP; education; DNA fingerprinting; genetic mapping
Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined. The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E. coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens. The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P. rapae and P. brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H. virescens. When each of these three genes was expressed individually in E. coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes xptB1 and xptC1 were expressed in the same E. coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P. rapae and P. brassicae. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H. virescens. Individual gene disruptions in X. nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E. coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes. The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.
In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) gene CRb is effective against Plasmodiophora brassicae isolate No. 14, which is classified as pathotype group 3. Although markers linked to CRb have been reported, an accurate position in the genome and the gene structure are unknown. To determine the genomic location and estimate the structure of CRb, we developed 28 markers (average distance, 20.4 kb) around CRb and constructed a high-density partial map. The precise position of CRb was determined by using a population of 2,032 F2 plants generated by selfing B. rapa ‘CR Shinki.’ We determined that CRb is located in the 140-kb genomic region between markers KB59N07 and B1005 and found candidate resistance genes. Among other CR genes on chromosome R3, a genotype of CRa closest marker clearly matched those of CRb and Crr3 did not confer resistance to isolate No. 14. Based on the genotypes of 11 markers developed near CRb and resistance to isolate No. 14, 82 of 108 cultivars showed a strong correlation between genotypes and phenotypes. The results of this study will be useful for isolating CRb and breeding cultivars with resistance to pathotype group 3 by introducing CRb into susceptible cultivars through marker-assisted selection.
clubroot; Plasmodiophora brassicae; CRb; fine mapping; marker-assisted selection; Brassica rapa
The Multinational Brassica rapa Genome Sequencing Project (BrGSP) has developed valuable genomic resources, including BAC libraries, BAC-end sequences, genetic and physical maps, and seed BAC sequences for Brassica rapa. An integrated linkage map between the amphidiploid B. napus and diploid B. rapa will facilitate the rapid transfer of these valuable resources from B. rapa to B. napus (Oilseed rape, Canola).
In this study, we identified over 23,000 simple sequence repeats (SSRs) from 536 sequenced BACs. 890 SSR markers (designated as BrGMS) were developed and used for the construction of an integrated linkage map for the A genome in B. rapa and B. napus. Two hundred and nineteen BrGMS markers were integrated to an existing B. napus linkage map (BnaNZDH). Among these mapped BrGMS markers, 168 were only distributed on the A genome linkage groups (LGs), 18 distrubuted both on the A and C genome LGs, and 33 only distributed on the C genome LGs. Most of the A genome LGs in B. napus were collinear with the homoeologous LGs in B. rapa, although minor inversions or rearrangements occurred on A2 and A9. The mapping of these BAC-specific SSR markers enabled assignment of 161 sequenced B. rapa BACs, as well as the associated BAC contigs to the A genome LGs of B. napus.
The genetic mapping of SSR markers derived from sequenced BACs in B. rapa enabled direct links to be established between the B. napus linkage map and a B. rapa physical map, and thus the assignment of B. rapa BACs and the associated BAC contigs to the B. napus linkage map. This integrated genetic linkage map will facilitate exploitation of the B. rapa annotated genomic resources for gene tagging and map-based cloning in B. napus, and for comparative analysis of the A genome within Brassica species.
Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.
Arabidopsis; Brassica rapa; full-length cDNA; jasmonic acid; salicylic acid
Gene duplication is an important mechanism for the origination of functional novelties in organisms. We performed a comparative genome analysis to systematically estimate recent lineage specific gene duplication events in Arabidopsis thaliana and further investigate whether and how these new duplicate genes (NDGs) play a functional role in the evolution and adaption of A. thaliana. We accomplished this using syntenic relationship among four closely related species, A. thaliana, A. lyrata, Capsella rubella and Brassica rapa. We identified 100 NDGs, showing clear origination patterns, whose parental genes are located in syntenic regions and/or have clear orthologs in at least one of three outgroup species. All 100 NDGs were transcribed and under functional constraints, while 24% of the NDGs have differential expression patterns compared to their parental genes. We explored the underlying evolutionary forces of these paralogous pairs through conducting neutrality tests with sequence divergence and polymorphism data. Evolution of about 15% of NDGs appeared to be driven by natural selection. Moreover, we found that 3 NDGs not only altered their expression patterns when compared with parental genes, but also evolved under positive selection. We investigated the underlying mechanisms driving the differential expression of NDGs and their parents, and found a number of NDGs had different cis-elements and methylation patterns from their parental genes. Overall, we demonstrated that NDGs acquired divergent cis-elements and methylation patterns and may experience sub-functionalization or neo-functionalization influencing the evolution and adaption of A. thaliana.
The Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level.
We developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events.
Our results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions of genes in B. rapa based on the locations of orthologues in A. thaliana can be misleading. Our results will be of relevance to a wide range of plants that have polyploid genomes, many of which are being considered according to a paradigm of comprising conserved synteny blocks with respect to sequenced, related genomes.
The species Brassica rapa includes various vegetable crops. Production of these vegetable crops is usually impaired by heat stress. Some microRNAs (miRNAs) in Arabidopsis have been considered to mediate gene silencing in plant response to abiotic stress. However, it remains unknown whether or what miRNAs play a role in heat resistance of B. rapa. To identify genomewide conserved and novel miRNAs that are responsive to heat stress in B. rapa, we defined temperature thresholds of non-heading Chinese cabbage (B. rapa ssp. chinensis) and constructed small RNA libraries from the seedlings that had been exposed to high temperature (46 °C) for 1 h. By deep sequencing and data analysis, we selected a series of conserved and novel miRNAs that responded to heat stress. In total, Chinese cabbage shares at least 35 conserved miRNA families with Arabidopsis thaliana. Among them, five miRNA families were responsive to heat stress. Northern hybridization and real-time PCR showed that the conserved miRNAs bra-miR398a and bra-miR398b were heat-inhibitive and guided heat response of their target gene, BracCSD1; and bra-miR156h and bra-miR156g were heat-induced and its putative target BracSPL2 was down-regulated. According to the criteria of miRNA and miRNA* that form a duplex, 21 novel miRNAs belonging to 19 miRNA families were predicted. Of these, four were identified to be heat-responsive by Northern blotting and/or expression analysis of the putative targets. The two novel miRNAs bra-miR1885b.3 and bra-miR5718 negatively regulated their putative target genes. 5′-Rapid amplification of cDNA ends PCR indicated that three novel miRNAs cleaved the transcripts of their target genes where their precursors may have evolved from. These results broaden our perspective on the important role of miRNA in plant responses to heat.
Brassica rapa; heat response; miRNA; small RNA