BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping

Abstract

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.

Figure 1. Overview of the BAP mapping method.

(a) A genomic BAC library is pooled in a 3-dimensional fashion (1^st D for plate ID; pool all unique clones for each plate and create overlapping superpools of plate pools; 2^nd D for row ID: pool all clones in identical rows over several plates [3.5 plates for Arabidopsis] and create overlapping ‘superpools’ of row pools to allow row identification; 3^rd D pool for column ID: pool all clones in identical columns over several plates [3.5 plates for Arabidopsis] and create overlapping ‘superpools’ of column pools to allow column identification) to generate the BAC-range panel. (b) Of 96 wells of a plate, 88 contain random aliquots of a BAC library (8 wells are reserved for controls), each BAC aliquot contains DNA corresponding 0.6–0.7 fold coverage of the genome. The presence of single copy STS markers (M1-My) is indicated by various colours in the aliquots. (c) The presence of markers found after MT-PCR-HRM. (d) The BAC range panel mapping results allow creation of short linkage maps, and (e) at the same time, to establish a corresponding BAC tiling path. (f) To link the shorter contigs obtained from the BAC panel and to close the gaps between contigs, markers chosen from the ends of the contigs are mapped by the long-range (large size) DNA panel. (g) The merged linkage and physical map as the final result.

Figure 2. Marker typing in the BAC-range panel by MT-PCR-HRM.

Each individual marker, which is uniquely amplified from genomic DNA, is mapped by genotyping in the mapping panel of 88 aliquots (Fig. 1b,f). This MT-PCR-HRM typing identifies the aliquots within the panel that are positive (green) or negative (red) for a particular marker by melting curve analysis using a fluorescent dye. If the PCR product is present in aliquots, the ‘positive’ green trace is detected and scored as 1. The absence of PCR product in aliquot (red line) is scored as 0. With a series of 1 and 0 for all aliquots in the panel, the marker is scored.

Figure 3. The physical map of FCA locus in comparison to its sequence.

The sequence position indicated by the first nucleotide of the 40 markers belonging to the FCA locus (a) is reflected by the physical map (b – enframed and c) after BAP mapping. With the BAC range panel, the BACs (blue rectangles) harbouring the 40 markers are sorted into 8 contigs (c) and assembled by means of the long-range panel into a single linkage map (b) spanning the entire region of 1.8 Mbp.