Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.     

Genome-wide characterization and expression analysis of pseudo-response regulator gene family in wheat

Molecular Biology Reports - Pseudo-response regulator (PRR) gene family members play a significant role in plant circadian clocks, flowering time inflorescence architecture development during...     

Anthocyanin biosynthetic genes in Brassica rapa

Background

Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level.

Results

In total, we identified 73 genes in B. rapa as orthologs of 41 anthocyanin biosynthetic genes in A. thaliana. In B. rapa, the anthocyanin biosynthetic genes (ABGs) have expanded and most genes exist in more than one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosynthetic pathway have been retained than downstream. More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa.

Conclusions

These results will promote an understanding of the genetic mechanism of anthocyanin biosynthesis, as well as help the improvement of the nutritional quality of B. rapa through the breeding of high anthocyanin content varieties.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-426) contains supplementary material, which is available to authorized users.     

Glucosinolate biosynthetic genes in Brassica rapa

Glucosinolates (GS) are a group of amino acid-derived secondary metabolites found throughout the Cruciferae family. Glucosinolates and their degradation products play important roles in pathogen and insect interactions, as well as in human health. In order to elucidate the glucosinolate biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses of Arabidopsis thaliana and B. rapa on a genome-wide level. We identified 102 putative genes in B. rapa as the orthologs of 52 GS genes in A. thaliana. All but one gene was successfully mapped on 10 chromosomes. Most GS genes exist in more than one copy in B. rapa. A high co-linearity in the glucosinolate biosynthetic pathway between A. thaliana and B. rapa was also established. The homologous GS genes in B. rapa and A. thaliana share 59-91% nucleotide sequence identity and 93% of the GS genes exhibit synteny between B. rapa and A. thaliana. Moreover, the structure and arrangement of the B. rapa GS (BrGS) genes correspond with the known evolutionary divergence of B. rapa, and may help explain the profiles and accumulation of GS in B. rapa.     

Comparative analysis of S haplotypes with very similar SLG alleles in Brassica rapa and Brassica oleracea

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (the S locus) which harbors at least two highly polymorphic genes, SLG and SRK. SRK is a putative transmembrane receptor kinase and its amino acid sequence of the extracellular domain of SRK (the S domain) exhibits high homology to that of SLG. The amino acid sequences of the SLGs of S8 and S46 haplotypes of B. rapa are very similar and those of S23 and S29 haplotypes of B. oleracea were also found to be almost identical. In both cases, SLG and the S domain of SRK of the same haplotype were less similar. This seems to contradict the idea that SLG and SRK of the same haplotype have the same self-recognition specificity. In the transmembrane-kinase domain, the SRK alleles of the S8 and S46 haplotypes had almost identical nucleotide sequences in spite of their lower homology in the S domain. Such a cluster of nucleotide substitutions is probably due to recombination or related events, although recombination in the S locus is thought to be suppressed. Based on our observations, the recognition mechanism and the evolution of self-incompatibility in Brassica are discussed.     

Characterization of DNA methyltransferase genes in Brassica rapa

DNA methylation is essential for normal development and plays important roles in regulating gene expression in plants. Analysis of the key enzymes catalyzing DNA methylation is important to understand epigenetic phenomena. In this study, three putative methyltransferase genes, BrMET1a, BrMET1b, and BrCMT, were isolated from a genome library of Brassica rapa. Structural conservation of the amino acid sequence between BrMET1a/BrMET1b and AtMET1 and that between BrCMT and AtCMT3 suggests that they may function as DNA methyltransferase. BrMET1a was expressed in vegetative and reproductive organs, while BrMET1b was expressed only in pistils, indicating that these two genes have different functions. BrCMT was expressed especially in stamens at the stage of 2-4 days before anthesis. We isolated three DNA methyltransferase genes in Brassica rapa and indicated differences of expression patterns of these DNA methyltransferase genes and expression levels in different tissues and developmental stages, suggesting that these genes might play important roles in epigenetic gene regulation in B. rapa.     

The putative phytocyanin genes in Chinese cabbage (Brassica rapa L.): genome-wide identification, classification and expression analysis

Phytocyanins (PCs) are a plant-specific family of small copper-containing electron transfer proteins. PCs may bind with a single copper atom to function as electron transporters in various biological systems, such as copper trafficking and plant photosynthesis. Evidence indicates that PCs may also be involved in plant developmental processes and stress responses. Many PCs possess arabinogalactan protein-like regions and are therefore termed chimeric arabinogalactan proteins (CAGPs). Previously, 38 and 62 PC genes have been identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), respectively. The recent release of the Chinese cabbage genome (B. rapa ssp. Pekinensis line Chiifu-401-42) enabled us to perform a genome-wide identification and analysis. In this study we identified 84 putative PC genes in the B. rapa genome. All of the Brassica rapa phytocyanins (BrPCs) described here could be divided, based on motif constitution, into the following three main subclasses: 52 early nodulin-like proteins (ENODLs), 16 uclacyanin-like proteins (UCLs), and 11 stellacyanin-like proteins (SCLs). A structural analysis predicted that 71 BrPCs contained N-terminal secretion signals and 45 BrPCs may be glycosylphosphatidylinositol-anchored to the plasma membrane. Glycosylation prediction revealed that 48 BrPCs were CAGPs with putative arabinogalactan glycomodules, and 57 BrPCs had N-glycosylation sites. Additionally, gene duplication analysis demonstrated that almost all of the duplicated BrPC genes shared the same conserved collinear blocks and that segmental duplications play an important role in the diversification of this gene family. Surprisingly, all BrUCL genes were duplicated except for BrUCL16. Expression analyses indicated that BrENODL22/27 and BrSCL8/9 were highly expressed in reproductive organs; BrUCL6/16 was strongly expressed in roots and even more strongly expressed in stems. The genome-wide identification, classification and expression analysis of BrPCs will provide a fundamental basis for the evolution and modification of the gene family after a polyploidy event and enable the functional study of PC genes in a polyploid crop species.     

cDNA cloning, genomic structure, chromosomal mapping, and expression analysis of parotid secretory protein in pig

A novel cDNA has been isolated from pig parotid glands by 3' and 5' rapid amplification of cDNA ends and designated parotid secretory protein (PSP). The open reading frame of this cDNA covers 714 bases, encoding 238 amino acids, which show 56% identity with human PSP at the level of the primary protein structure. The PSP genomic sequence comprises eight exons and seven introns, is approximately 22 kb in size, determined by sequencing, and maps to pig chromosome 17q21-q23. RT-PCR, dot blot, and Northern blot analyses demonstrated that PSP is strongly expressed in parotid glands, but is not present in heart, liver, lung, kidney, muscle, or stomach. A search for functionally significant protein motifs revealed consensus sequences for casein kinase II phosphorylation and N-myristoylation. We observed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu-X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is similar to the leucine zipper.     

Fine mapping of the clubroot resistance gene, Crr3, in Brassica rapa

A linkage map of Chinese cabbage (Brassica rapa) was constructed to localize the clubroot resistance (CR) gene, Crr3. Quantitative trait loci analysis using an F₃ population revealed a sharp peak in the logarithm of odds score around the sequence-tagged site (STS) marker, OPC11-2S. Therefore, this region contained Crr3. Nucleotide sequences of OPC11-2S and its proximal markers showed homology to sequences in the top arm of Arabidopsis chromosome 3, suggesting a synteny between the two species. For fine mapping of Crr3, a number of STS markers were developed based on genomic information from Arabidopsis. We obtained polymorphisms in 23 Arabidopsis-derived STS markers, 11 of which were closely linked to Crr3. The precise position of Crr3 was determined using a population of 888 F₂ plants. Eighty plants showing recombination around Crr3 locus were selected and used for the mapping. A fine map of 4.74 cM was obtained, in which two markers (BrSTS-41 and BrSTS-44) and three markers (OPC11-2S, BrSTS-54 and BrSTS-61) were cosegregated. Marker genotypes of the 21 selected F₂ families and CR tests of their progenies strongly suggested that the Crr3 gene is located in a 0.35 cM segment between the two markers, BrSTS-33 and BrSTS-78.     

Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (Brassica rapa L.)

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), have both predicted AGP-like glycosylated regions and putative fasciclin (FAS) domains, which may function in cell adhesion and communication. Previous studies have identified 21, 27, and 34 FLAs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), respectively. In this study, we identified 33 FLAs in the annotated genome of Chinese cabbage (Brassica rapa ssp. pekinensis line Chiifu-401-42). Sequence analysis indicated that FAS domains each contain two highly conserved regions, named H1 and H2, and that 17 FLAs from B. rapa (BrFLAs) possess both of these regions. Prediction of glycosylphosphatidylinositol (GPI) modification sites suggested that 15 BrFLAs were GPI-anchored to the plasma membrane. Additionally, 25 BrFLAs may have been duplicated during the processes that shaped the triplicated genome of the mesopolyploid B. rapa. Expression analyses indicated that BrFLA1, BrFLA11, BrFLA13, BrFLA28 and BrFLA32 were specifically expressed in inflorescence. Meanwhile, BrFLA9 (homologous to AtFLA12) is specifically expressed in stem, and BrFLA6/22 (homologous to AtFLA11) is also highly expressed in stem, suggesting BrFLA6/9/22 may have the same functions as AtFLA11/12 in A. thaliana. Taken together, the identification and bioinformatic analysis of FLAs in B. rapa will open the way for studying their biological functions in plant growth and development as well as evolutionary history of this gene family from A. thaliana to B. rapa.     

Comparative mapping of loci controlling winter survival and related traits in oilseed Brassica rapa and B. napus

Winter survival is an important characteristic of oilseedBrassica that is seeded in the fall in northern climates,and it may be affected by genetic variation for other cold-regulated traits,such as freezing tolerance and vernalization responsive flowering time. Weanalyzed immortalized populations of oilseed Brassica rapa(recombinant inbred lines) and B. napus (double haploidlines) derived from crosses of annual and biennial types in order to comparethe map positions and effects of quantitative trait loci controlling wintersurvival, nonacclimated and acclimated freezing tolerances, and flowering time.The B. napus population was evaluated in multiple winters,and six of the 16 total significant QTL for winter survival were detected inmore than one winter. Correspondence in the map positions of QTL controllingdifferent traits within species provided evidence that some alleles causinggreater acclimated freezing tolerance and later flowering time also contributedto increased winter survival. Correspondence in the map positions of QTLbetween species provided evidence for allelic variation at homologous loci inB. rapa and B. napus. The potentialrole of some candidate genes in regulating these traits is discussed.     

Comparative genomic analysis of soybean flowering genes

Flowering is an important agronomic trait that determines crop yield. Soybean is a major oilseed legume crop used for human and animal feed. Legumes have unique vegetative and floral complexities. Our understanding of the molecular basis of flower initiation and development in legumes is limited. Here, we address this by using a computational approach to examine flowering regulatory genes in the soybean genome in comparison to the most studied model plant, Arabidopsis. For this comparison, a genome-wide analysis of orthologue groups was performed, followed by an in silico gene expression analysis of the identified soybean flowering genes. Phylogenetic analyses of the gene families highlighted the evolutionary relationships among these candidates. Our study identified key flowering genes in soybean and indicates that the vernalisation and the ambient-temperature pathways seem to be the most variant in soybean. A comparison of the orthologue groups containing flowering genes indicated that, on average, each Arabidopsis flowering gene has 2-3 orthologous copies in soybean. Our analysis highlighted that the CDF3, VRN1, SVP, AP3 and PIF3 genes are paralogue-rich genes in soybean. Furthermore, the genome mapping of the soybean flowering genes showed that these genes are scattered randomly across the genome. A paralogue comparison indicated that the soybean genes comprising the largest orthologue group are clustered in a 1.4 Mb region on chromosome 16 of soybean. Furthermore, a comparison with the undomesticated soybean (Glycine soja) revealed that there are hundreds of SNPs that are associated with putative soybean flowering genes and that there are structural variants that may affect the genes of the light-signalling and ambient-temperature pathways in soybean. Our study provides a framework for the soybean flowering pathway and insights into the relationship and evolution of flowering genes between a short-day soybean and the long-day plant, Arabidopsis.     

Association mapping of leaf traits, flowering time, and phytate content in Brassica rapa.

Association mapping was used to investigate the genetic basis of variation within Brassica rapa, which is an important vegetable and oil crop. We analyzed the variation of phytate and phosphate levels in seeds and leaves and additional developmental and morphological traits in a set of diverse B. rapa accessions and tested association of these traits with AFLP markers. The analysis of population structure revealed four subgroups in the population. Trait values differed between these subgroups, thus defining associations between population structure and trait values, even for traits such as phytate and phosphate levels. Marker-trait associations were investigated both with and without taking population structure into account. One hundred and seventy markers were found to be associated with the observed traits without correction for population structure. Association analysis with correction for population structure led to the identification of 27 markers, 6 of which had known map positions; 3 of these were confirmed in additional QTL mapping studies.     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Sequence and structure of Brassica rapa chromosome A3

Background

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.

Results

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.

Conclusions

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.