Strong evidence exists for polyploidy having occurred during the evolution of the tribe Brassiceae. We show evidence for the dynamic and ongoing diploidization process by comparative analysis of the sequences of four paralogous Brassica rapa BAC clones and the homologous 124-kb segment of Arabidopsis thaliana chromosome 5. We estimated the times since divergence of the paralogous and homologous lineages. The three paralogous subgenomes of B. rapa triplicated 13 to 17 million years ago (MYA), very soon after the Arabidopsis and Brassica divergence occurred at 17 to 18 MYA. In addition, a pair of BACs represents a more recent segmental duplication, which occurred ∼0.8 MYA, and provides an exception to the general expectation of three paralogous segments within the B. rapa genome. The Brassica genome segments show extensive interspersed gene loss relative to the inferred structure of the ancestral genome, whereas the Arabidopsis genome segment appears little changed. Representatives of all 32 genes in the Arabidopsis genome segment are represented in Brassica, but the hexaploid complement of 96 has been reduced to 54 in the three subgenomes, with compression of the genomic region lengths they occupy to between 52 and 110 kb. The gene content of the recently duplicated B. rapa genome segments is identical, but intergenic sequences differ.

Background: 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. Results: 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. Conclusions: 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.

A detailed genetic linkage map of Brassica rapa has been constructed containing 545 sequence-tagged loci covering 1287 cM, with an average mapping interval of 2.4 cM. The loci were identified using a combination of 520 RFLP and 25 PCR-based markers. RFLP probes were derived from 359 B. rapa EST clones and amplification products of 11 B. rapa and 26 Arabidopsis. Including 21 SSR markers provided anchors to previously published linkage maps for B. rapa and B. napus and is followed as the referenced mapping of R1–R10. The sequence-tagged markers allowed interpretation of the pattern of chromosome duplications within the B. rapa genome and comparison with Arabidopsis. A total of 62 EST markers showing a single RFLP band were mapped through 10 linkage groups, indicating that these can be valuable anchoring markers for chromosome-based genome sequencing of B. rapa. Other RFLP probes gave rise to 2–5 loci, inferring that B. rapa genome duplication is a general phenomenon through 10 chromosomes. The map includes five loci of FLC paralogues, which represent the previously reported BrFLC-1, -2, -3, and -5 and additionally identified BrFLC3 paralogues derived from local segmental duplication on R3.

Summary: We report the identification and characterization of the major repeats in the centromeric and peri‐centromeric heterochromatin ofBrassica rapa.The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere‐specific retrotransposons ofBrassica(CRB) and various peri‐centromere‐specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S–25S rDNA sequences. The chromosomal positions of selected repeats were determined usingin situhybridization. These revealed that CRB is a major component of all centromeres in three diploidBrassicaspecies and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed,B. nigra. PCRBr and TR238 were found to be major components in the peri‐centromeric heterochromatin blocks of four chromosomes ofB. rapa.These repetitive elements were not identified inB. oleraceaorB. nigra, indicating that they are A‐genome‐specific.

In view of the immense value of Brassica rapa in the fields of agriculture and molecular biology, the multinational Brassica rapa Genome Sequencing Project (BrGSP) was launched in 2003 by five countries. The developing BrGSP has valuable resources for the community, including a reference genetic map and seed BAC sequences. Although the initial B. rapa linkage map served as a reference for the BrGSP, there was ambiguity in reconciling the linkage groups with the ten chromosomes of B. rapa. Consequently, the BrGSP assigned each of the linkage groups to the project members as chromosome substitutes for sequencing.

Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition. Some glucosinolate‐derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity. To better understand glucosinolate biosynthesis inBrassica rapa, we conducted a comparative genomics study withArabidopsis thalianaand identified total 56 putative biosynthetic and regulator genes. This established a high colinearity in the glucosinolate biosynthesis pathway betweenArabidopsisandB. rapa. Glucosinolate genes inB. rapashare 72–94% nucleotide sequence identity with theArabidopsisorthologs and exist in different copy numbers. The exon/intron split pattern ofB. rapais almost identical to that ofArabidopsis, although inversion, insertion, deletion and intron size variations commonly occur. Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion. At least 12 paralogs of desulfoglucosinolate sulfotransferase were found inB. rapa, whereas only three were found inArabidopsis. The expression of those paralogs was not tissue‐specific but varied greatly depending onB. rapatissue types. Expression was also developmentally regulated in some paralogs but not in other paralogs. Most of the regulator genes are present as triple copies. Accordingly, glucosinolate synthesis and regulation inB. rapaappears to be more complex than that ofArabidopsis. With the isolation and further characterization of the endogenous genes, health‐beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway.