Dataset Information

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.

SUBMITTER: Ye Q

PROVIDER: S-EPMC3293878 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Ye Qing Q He Ke K Wu Shao-Ying SY Wan Qiu-Hong QH

PloS one 20120305 3

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, o ...[more]

PMID: 22403630

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Project description:BackgroundThe major histocompatibility complex (MHC) genes are vital partners in the acquired immune processes of vertebrates. MHC diversity may be directly associated with population resistance to infectious pathogens. Here, we screened for polymorphisms in exons 2 and 3 of the IA1 and IA2 genes in 12 golden pheasant populations across the Chinese mainland to characterize their genetic variation levels, to understand the effects of historical positive selection and recombination in shaping class I diversity, and to investigate the genetic structure of wild golden pheasant populations.ResultsAmong 339 individual pheasants, we identified 14 IA1 alleles in exon 2 (IA1-E2), 11 IA1-E3 alleles, 27 IA2-E2 alleles, and 28 IA2-E3 alleles. The non-synonymous substitution rate was significantly greater than the synonymous substitution rate at sequences in the IA2 gene encoding putative peptide-binding sites but not in the IA1 gene; we also found more positively selected sites in IA2 than in IA1. Frequent recombination events resulted in at least 9 recombinant IA2 alleles, in accordance with the intermingling pattern of the phylogenetic tree. Although some IA alleles are widely shared among studied populations, large variation occurs in the number of IA alleles across these populations. Allele frequency analysis across 2 IA loci showed low levels of genetic differentiation among populations on small geographic scales; however, significant genetic differentiation was observed between pheasants from the northern and southern regions of the Yangtze River. Both STRUCTURE analysis and F-statistic (F ST ) value comparison classified those populations into 2 major groups: the northern region of the Yangtze River (NYR) and the southern region of the Yangtze River (SYR).ConclusionsMore extensive polymorphisms in IA2 than IA1 indicate that IA2 has undergone much stronger positive-selection pressure during evolution. Moreover, the recombination events detected between the genes and the intermingled phylogenetic pattern indicate that interlocus recombination accounts for much of the allelic variation in IA2. Analysis of the population differentiation implied that homogenous balancing selection plays an important part in maintaining an even distribution of MHC variations. The natural barrier of the Yangtze River and heterogeneous balancing selection might help shape the NYR-SYR genetic structure in golden pheasants.

Project description:BACKGROUND:Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC) plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC) library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes. RESULTS:A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1) PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2) DNA sequencing validation of positive clones, and (3) restriction digest fingerprinting verification of inter-clone overlapping. CONCLUSION:The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a powerful tool for further studies on the giant panda MHC class II genes.

Project description:Intraframe motion blurring, as a major challenge in free-breathing dynamic MRI, can be reduced if high temporal resolution can be achieved. To address this challenge, this work proposes a highly accelerated 4D (3D + time) dynamic MRI framework with subsecond temporal resolution that does not require explicit motion compensation. The method combines standard stack-of-stars golden-angle radial sampling and tailored GRASP-Pro (Golden-angle RAdial Sparse Parallel imaging with imProved performance) reconstruction. Specifically, 4D dynamic MRI acquisition is performed continuously without motion gating or sorting. The k-space centers in stack-of-stars radial data are organized to guide estimation of a temporal basis, with which GRASP-Pro reconstruction is employed to enforce joint low-rank subspace and sparsity constraints. This new basis estimation strategy is the new feature proposed for subspace-based reconstruction in this work to achieve high temporal resolution (e.g., subsecond/3D volume). It does not require sequence modification to acquire additional navigation data, it is compatible with commercially available stack-of-stars sequences, and it does not need an intermediate reconstruction step. The proposed 4D dynamic MRI approach was tested in abdominal motion phantom, free-breathing abdominal MRI, and dynamic contrast-enhanced MRI (DCE-MRI). Our results have shown that GRASP-Pro reconstruction with the new basis estimation strategy enables highly-accelerated 4D dynamic imaging at subsecond temporal resolution (with five spokes or less for each dynamic frame per image slice) for both free-breathing non-DCE-MRI and DCE-MRI. In the abdominal phantom, better image quality with lower root mean square error and higher structural similarity index was achieved using GRASP-Pro compared with standard GRASP. With the ability to acquire each 3D image in less than 1 s, intraframe respiratory blurring can be intrinsically reduced for body applications with our approach, which eliminates the need for explicit motion detection and motion compensation.

Dataset Information

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

Publications

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

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