Construction Of Contig In The Class Ⅱb Extension Region Of Ovine Major Histocompatibility Complex Region

Object:The sheep is one of the best economic valued domestic animals in the world and China. Especially the Chinese fine-wool Merino sheep is an excellent genetic germplasma resource and economic breed. Same to other all jawed vertebrates, the major histocompatibility complex (MHC) of ovine genome, also designated as Ovine Leukocyte Antigen (OLA), harbors clusters of tightly linked immune response/regula-tion genes involved in overall resistance/susceptibility of an animal to various pathogenic viruses, bacteria and parasites. So it is very important to research the characters and functions of MHC. A BAC Library of Chinese Fine-Wool Merino Sheep was constructed, and the MTP pysical map of The Ovine MHC Region had been done, but there is a big gap between MHC ClassⅡa and ClassⅡb. Genetic linked studies shows that OLA is similar to BoLA in structure in that the classⅡis split into two discontinuous subregions,'Ⅱa' and'Ⅱb',separated by a large piece of non-MHC autosomal inversion. The aim of this study is to screen the genomic BAC library previously constructed of Chinese fine-wool Merino sheep using PCR strategy based on bovine genomic sequence and comparatively genomics theory, ultimately assemble BAC clone-based MTP physical map of OLA ClassⅡb extended region utilizing effective positive BAC clones selected. The MTP physical map OLA ClassⅡb extended region is a part of OLA inversion region MTP physical map, improving the research on inversion region and improving the construction of the whole OLA MTP physical map.Methods:Firstly, we selected some effective Markers/Symbols in BoLA ClassⅡb extended region based on bovine genomic sequence published and designed corresponding primer pairs for PCR. Then we screened the genomic BAC library previously constructed of Chinese fine-wool Merino sheep using PCR strategy and selected some candidated positive BAC clones. Primary contig was constructed by analyzing enzymic fingerprints of candidated positive BAC clones. Subsequently, we sequenced the ends of effective BAC clones among primary contig and designed corresponding primers-end for PCR in order to determine the overlapping relationships and orders of these clones. Finally, we constructed the MTP physical map of OLA ClassⅡb extended region.Results:(1) A total of 26 pairs of PCR primers weie designed and optimized 23 pairs of primers were used for PCR screening. (2) BAC DNA mixture pools (primary 384-well plate pool and secondary row pool) of genomic BAC library previously constructed were prepared for screening. (3)A total of 58 positive BAC clones were selected utilizing PCR strategy. The overlapping relationships and orders of the positive BAC clones were determined by BAC DNA fingerprinting analysis, BAC-end sequencing and sequence-specific PCR. A total of 25 effective BAC clones were selected to cover the OLA ClassⅡb extended region. Therefore, a complete BAC-based MTP physical map of the OLA ClassⅡb extended region was constructed.Conclusions:(1) we designed corresponding primer pairs for PCR screening through Markers/Symbols of BoLA ClassⅡb extended region. Then we screened the BAC DNA mixture pools of genomic BAC library previously constructed and selected positive BAC clones. The results show that this region of OLA has somewhat greater sequence identity to that of BoLA and it is rapid and effective to screen genomic BAC library utilizing PCR strategy. (2) For three primer pairs designed based on bovine genomic sequence published, the PCR product was not detected when PCR amplification was performed with ovine genome DNA as template. Moreover, an identical BAC clone was selected with two or three distinct markers as probes in several subregions. The results give a hint that local sequence in OLA ClassⅡb extended region may arise from parallel divergence subsequently. (3) The MTP physical map constructed in this study is not only enriched the information of ovine genome, will also facilitate strongly the sequencing of ovine entire genome and comparative studies of genome in ruminants.