The first generation of a BAC-based physical map of Brassica rapa

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

An example of correspondence between a single locus RLFP marker, BAN245, and its corresponding contig containing all positive BAC clones. (A), Southern blot analysis of seven positive BAC clones picked from the KBrH library. HindIII digestion of BAC DNA followed by BAN245 hybridization shows a single hybridization band for the BAN245 marker. (B), view of fingerprint contig containing all seven positive BAC clones for the BAN 245 marker. The blue highlighted clones are those screened by Southern hybridization.

Figure 2

An example of a BAC physical contig anchored to the R9 chromosome of the B. rapa genome. This contig consists of 68 BAC clones from three source BAC libraries (Table 1) and is estimated to cover approximately 1.3 Mb. The clones prefixed with KBrH were from the HindIII library, with KBrB from the BamHI library, and with KBrS from the Sau3AI library. This contig was anchored to the region around 86–91 cM of the R9 genetic map using two SSR markers, KS31203 and KS31191, and one RFLP marker, BAN235. The violet-highlighted clones represent the corresponding BAC clones containing the respective DNA markers. All the highlighted BAC clones are in the genome sequencing pipeline and their sequencing phases are indicated.

Figure 3

An example of correspondence between four different types of multiple loci genetic marker, BAN2, and their distinct physical contigs containing corresponding BAC clones. (A), Southern blot analysis showing four different hybridization patterns of BAN2 markers for 22 BAC clones picked from the KBrH library. I-VI represent the grouping of the BAC clones according to their main hybridization bands from EcoRV digestion followed by BAN2 hybridization and fingerprint contig information. *Three clones were excluded from the fingerprint assembly due to failed fingerprinting. ^sFive clones remained as singletons after contig assembly. (B), view of six different fingerprint contigs containing the corresponding groups of 14 BAC clones for respective marker types. The blue highlighted clones are those screened by hybridization.