| The whole genome SNP breeding chip is a crucial tool for advancing the breeding of livestock and poultry and is widely utilized in the field of molecular breeding of animals and plants.However,the unavailability of a commercial SNP chip with separate intellectual property rights in buffalo currently severely impedes the breeding process.Therefore,in this study,a broadly representative buffalo SNP library was built using the whole genome sequencing data of 188 buffalo from 25 species around the world.SNP sites associated with economic features of buffalo were gathered by comparative genomics study and integration of existing papers.Meanwhile,to enhance the annotation of functional genes in buffalo and screen important SNP sites in functional regions of buffalo,transcriptome sequencing data and known gene information from common cattle were used.The hotspots of substantial fragment sequence variation on the buffalo genome were further discovered in order to eliminate the influence of complex genome sequences on the binding effect of SNP probes and ensure the accuracy of detection and typing of SNP breeding chips.Finally,a screening procedure was constructed for the buffalo genome-wide SNP breeding chip site,and the buffalo genome-wide 50K SNP breeding chip was successfully obtained.This was done in light of the information provided above as well as the density distribution,frequency,and linkage imbalance of SNP sites.The main findings are as follows:（1）Identification of selected SNP in buffalo population and collection of candidate sites of SNP chip188 buffalo from 25 different species across the globe had their second generation whole genome sequenced.There were discovered 55M SNP sites in total,which were widely scattered across chromosomes.SNP were found to be primarily concentrated in non-functional regions like intergenic space and introns when the distribution pattern of SNP in functional buffalo genome elements was examined.The successful separation of the river buffalo and swamp buffalo subgroups was achieved by examining the SNP of buffalo population structure.1117 non-redundant significantly differentiated genes were found to overlap with the top 0.1%of SNP variation sites by computing the FST selection signals of the two subpopulations.The LD attenuation distance and Di selection signals were calculated after the buffalo population was divided into 6 groups based on various regions.There were 8215 significantly different genes that overlapped,and a total of 6639 de-redundant selected sites were found.At the same time,319 SNP loci from 66 genes were associated with significant buffalo traits after 47 literatures and reports were reviewed.The SNP sites obtained through this procedure can be used as SNP breeding chip candidate sites.（2）Further improvement of functional domain annotation of buffalo genomeWe obtained 194 widely representative transcriptome data of buffalo,including 20kinds of organs and tissues such as brain and liver,and successfully identified 17,185 new genes in addition to the currently known genes.Based on whole-genome comparison between buffalo and common cattle,14,139 new genes were successfully converted from common cattle into buffalo through liftover,which more completely presented the gene functional regions in the buffalo genome,ensuring that the selected buffalo SNP chip loci have enough effective and potential functional effects.（3）Detection of large complex DNA sequences of buffalo genome and identification of hot spotsA total of 50K SV loci were detected in the buffalo genome using the same data,including 37,325 deletions,68.94 inversions,62.11 duplicates,and 470 insertions.SV was widely distributed and unevenly on chromosomes,with 11,556 SV hot spots（at least 10 SV in the regions）.SV was mainly concentrated in non-functional regions such as introns and intergenic regions,while it was rarely found in functional regulatory elements such as exons and promoters.Two subgroups of river buffalo and swamp buffalo can also be separated by SV-based population structure analysis.By calculating the FST selection signals of the two subpopulations,2974 non-redundant significantly differentiated genes were detected that overlapped with the top 1%of SNP variation sites.The SV hotspots obtained in this process can provide reference for the selection of SNP breeding chip sites.（4）Creation and assessment of the buffalo genome-wide 50K SNP breeding chip’s precisionA set of site screening procedures for a buffalo genome-wide 50K SNP breeding chip containing 49956 probes was successfully created by carefully taking into account the density distribution,frequency,selection degree,linkage unbalance state,and other information of SNP sites in combination with earlier studies.The average GC content was45.24%,the probe length was 120 bp,and the probe coverage was 100%.A number of detection indicators were assessed after 24 buffalo samples were detected using the device.The SNP chip performed exceptionally well in terms of uniformity,sample detection rate,site detection rate,and typing consistency rate,measuring 98.43%,99.39%,98.53%,and98.43%,respectively.This allowed it to meet the detection needs of many samples.In this study,different SNP sites selected by different subpopulations and regions of buffalo were detected and analyzed.SNPS associated with important characters of buffalo were collected from previous studies.Sites affected by SV hot spots were considered and removed;The functional annotation of the buffalo genome region was further improved by transcriptomics and comparative genomics analysis.A set of site screening procedures for buffalo genome-wide SNP breeding chip was established,and a buffalo genome-wide 50K SNP breeding chip with high quality was successfully developed,which provided a powerful testing tool for subsequent studies in buffalo genomics,molecular breeding and other fields.And lay a solid foundation for the rapid development of Chinese water buffalo industry,enhance the economic value and so on. |
