Target resequencing for bovine major histocompatibility complex region

ISHIDA A.; AIDA Y.; TAKESHIMA S-N; MURAKAWA Y.; KAWAMURA A.; GIOVAMBATTISTA G.

Lleira

Congreso; 37th International Society for Animal Genetics Conference; 2019

International Society for Animal Genetics

Major histocompatibility complex (MHC) is one of most polymorphic regions in vertebrate genome. Bovine MHC (BoLA) region, embedded at chromosome 23, and constructed by BoLA class IIb, BoLA class IIa, BoLA class III and BoLA class I region. BoLA region contain the genes in high density. Additionally, each quite polymorphic BoLA genes frequently shows copy number variations. Therefore, it is difficult to genotyping BoLA region by genome-wide re-sequencing. For long time, UMD3.1 cattle genome reference was used in worldwide for bovine whole genome study, but recently, newer bovine genome reference called ARS-UCD1.2 was published. In this study, we tried to perform re-sequencing of BoLA region using 2 set of probes designed based on UMD3.1 and ARS-UCD1.2 and compared the result. Genomic DNA were collected from 9 heads of Japanese black cow and 1 Holstein cow. BoLA-DRB3 genotyping were performed by PCR sequence-based typing (SBT). DNA libraries were constructed from each DNA using KAPA HyperPlus Kit, and selected the libraries using SeqCap EZ and custommade probes. Ten cows using for this study were subjected to BoLA-DRB3 typing and the genotypes were DRB3*012:01/014:01, *014:01/*016:01, *016:01/*016:01, *005:03/*012:01, *002:01/*015:01, *007:01/*010:01, *007:01/*009:02, *007:01/*014:01, *011:01/*012:01 and *010:01/*011:01. Next, we constructed 4 kind of probes based on 2 bovine genome references. The target regions were as follows; set 1: BoLA region excluded intron sequences using UMD3.1, set 4: Whole BoLA region using UMD3.1, BoLA1: BoLA region excluded intron sequences using ARS-UCD1.2, and BoLA2; Whole BoLA region using ARS-UCD1.2. All of 4 result were compared with BoLA-DRB3 PCRSBT results and there are no conflicts between the result of PCR-SBT and target resequencing result. The variant call result containing a lot of SNPs which difficult to validated in single position. Therefore, we compared the rate of the succession SNPs and average of sequencing depth in each cow among 4 compared probes. Finally, we determined that BoLA2 probe were most suitable for re-sequencing whole BoLA region, and this result may be useful for study about the association between various economical and disease and of BoLA region.