A high-density BAC physical map covering the entire MHC region of addax antelope genome

Abstract

Background: The mammalian major histocompatibility complex (MHC) harbours clusters of genes associated with the immunological defence of animals against infectious pathogens. At present, no complete MHC physical map is available for any of the wild ruminant species in the world.

Results: The high-density physical map is composed of two contigs of 47 overlapping bacterial artificial chromosome (BAC) clones, with an average of 115 Kb for each BAC, covering the entire addax MHC genome. The first contig has 40 overlapping BAC clones covering an approximately 2.9 Mb region of MHC class I, class III, and class IIa, and the second contig has 7 BAC clones covering an approximately 500 Kb genomic region that harbours MHC class IIb. The relative position of each BAC corresponding to the MHC sequence was determined by comparative mapping using PCR screening of the BAC library of 192,000 clones, and the order of BACs was determined by DNA fingerprinting. The overlaps of neighboring BACs were cross-verified by both BAC-end sequencing and co-amplification of identical PCR fragments within the overlapped region, with their identities further confirmed by DNA sequencing.

Conclusions: We report here the successful construction of a high-quality physical map for the addax MHC region using BACs and comparative mapping. The addax MHC physical map we constructed showed one gap of approximately 18 Mb formed by an ancient autosomal inversion that divided the MHC class II into IIa and IIb. The autosomal inversion provides compelling evidence that the MHC organizations in all of the ruminant species are relatively conserved.

Keywords: Addax nasomaculatus; BAC; MHC; Physical map.

Fig. 1

Overview of the addax MHC physical map construction. a BAC library construction. Fibroblasts were embedded into low-melting point agarose to form plugs for subsequent genomic DNA isolation. Genomic DNA fragments of 100–300 Kb were ligated into the pCC1BAC vector. White colonies were picked into 384-well plates after transformation. b 3D-PCR screening strategy. To improve screening efficiency, the entire addax BAC library composed of 500,384-well plates was divided into 50 super-pools. After the positive MHC clones were determined in a certain super-pool by the first dimension of PCR, the second dimension of PCR was further performed on 10 plates. Then, the third dimension of PCR screening was continued on 16 rows and 24 columns. The intersection of a row and column indicated a potential positive clone covering the addax MHC region. c The physical map covering the addax MHC region was assembled by integrating the results from sequence-specific PCR and DNA fingerprinting

Fig. 2

Distribution of insert sizes in BAC clones of the addax (Addax nasomaculatus). A total of 192,000 BAC clones were harvested and stored in 500,384-well plates. Based on an analysis of 172 random BAC clones, the average insert size of the genomic DNA in a BAC was approximately 115 Kb, and the insert size for over 80% of BAC clones was larger than 90 Kb. The percentage of empty vectors was less than 5%

Fig. 3

DNA fingerprintings of the 47 positive BAC clones to determine the overlap. The positive BAC clones identified in the PCR screening were digested with HindIII and then separated on a 1% TAE agarose gel. The gel was stained with ethidium bromide (EB) for imaging with a UVP LabWorks system. M: DNA size standard marker (1 Kb Plus DNA Ladder from Transgene); the base pair (bp) sizes are indicated on both sides. a, b Fingerprints of BAC clones consisting of addax MHC contig 1. c Fingerprints of BAC clones consisting of addax MHC contig 2

Fig. 4

PCR verification of the overlap between pairs of overlapped BAC clones. Pairs of overlapped clones were PCR-amplified using the same primer pair designed based on the BAC-end sequence. The markers above the black lines indicate the primer pairs, and the ones below the lines are labels of positive clones used as PCR templates. M: DNA size standard (DL2000 Plus DNA Ladder from Transgene); the base pair (bp) sizes are indicated on both sides. a-d Validation of the overlap between clones covering the addax MHC class I–class III-class II a region. e Validation of the overlap between clones covering the addax MHC II b region

Fig. 5

Physical map covering the entire MHC region of the addax. The order and orientation of BAC clones (overlapping horizontal bars with clone ID name listed above) were determined based on integration of the results of DNA fingerprinting, BAC-end sequencing, and sequence-specific-PCR. The genes identified by BAC-end sequencing are marked by vertical bars along the horizontal line, with the locus names listed above. The continuous BAC map is represented by two panels with the overlapping regions marked with the same coloured shadows at the ends