Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant

Abstract

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

Figure 1. Schematic representation of the process of (A) 4D-PCR method; and (B) Reverse-4D method.

Figure 2. Distribution of insert size of 128 randomly selected clones.

Figure 3. Gene organization of the 97 kb (97,476 bp) Chpi-MHC-B (JQ440366).

A, Plot of GC content in overlapping 200 bp windows. B, Locations of repeat sequences, as shown in red. C, Genes and CpG islands predicted within Chpi-MHC-B. The orange columns flanking the lines indicate genes, of which the upper and lower ones represent forward and reverse orientations, respectively. The pink triangles depict locations of CpG islands.

Figure 4

A, A dot plot of Chpi-MHC-B itself. B, Inter-species dot plot of the Chpi-MHC-B and Gaga-MHC-B. C, A VISTA plot of multiple alignments from four avian MHC-B sequences. Percentage of identity plots are displayed by pink blocks and the blue regions indicate locations corresponding to exons. The duplicate blocks detected by dot plot and the corresponding genes inside were indicated in red while the inversed genes relative to chicken were shown in green.

Figure 5. Phylogenetic trees of IA (A), IIB (B), NK-Blec (C), DMB (D) and TAP (E) genes.

For the turkey, chicken and quail, the coding regions of genes were extracted from genomic sequences of Mega-MHC (DQ993255), Gaga-MHC (AB268588) and Coja-MHC (AB078884), respectively. The following sequences were also included for tree building: For the IAs: AF033106 (Grus Canadensis, Grca-f51), AY387652 (Anser anser, Anan), and AB115241 (Anas platyrhynchos, Anpl-Du1); For the IIBs: L42335 (Lonchura striata, Lost), DQ490139 (Anas platyrhyncho, Anpl), AJ404372 (Acrocephalus arundinaceus, Acar-c01), AF170972 (Agelaius phoeniceus, Agph-DAB1), and FJ588549 (Pachytila belcheri, Pabe-DAB1); For the DMB: DQ268506 (Xenopus laevis, Xela); For the TAPs: AY885227 (Anas platyrhynchos, Anpl-TAP1 and Anpl-TAP2), XM_003230087 (Anolis carolinensis, Anca-TAP1), XM_003229647 (Anolis carolinensis, Anca-TAP2). The NK and Blec members of lection superfamily were all from the same genomic sequences of DQ993255, AB268588 and AB07888.