Performance of bovine high density SNPs genotyping array in indigenous Pakistani cattle breeds

The bovine high density SNPs genotyping array has a wide range of applications including detection of selection signatures, identification of copy number variants (CNVs), genome wide association studies, and dendrogram relationships. This array can also be used to improve the accuracy of genomic predictions for low heritable traits viz. reproductive traits. The effectiveness of this array for genomic selection mainly depends on polymorphism level. In this study, we used 136 individuals from ten different Pakistani cattle breeds, include Achi (18), Bhagnari (14), Cholistani (13), Dajal (10), Dhanni (10), Kankraj (12), Lohani (19), Red Sindhi (13), Sahiwal (14), and Tharparkar (13) using high density SNPs genotyping and this array contained approximately 777, 962 SNPs. The results of this study revealed that approximately 500, 939 SNPs were found polymorphic in these breeds. The results indicate that high density SNPs bead chip would offer an informative genotyping platform for quantitative trait loci (QTL) mapping in indigenous Pakistani cattle breeds.


Introduction
The bovine high density SNPs is the most comprehensive genome wide genotyping array.This chip contained more than 777, 962 SNPs markers that are equally distributed across entire bovine genome [1].
This array was first time introduced and became available in 2010 [2].Applications of this array include genome wide association studies, identification of quantitative traits loci (QTLs), genetic merits prediction, and extent of linkage disequilibrium (LD).The potential of this array has been proved in several studies and identified genomic regions, which effects on phenotypic variations [3].These regions are mainly associated with milk traits [4]; feed intake, growth traits, and carcass traits [5].In addition, the prediction of genomic values in an effective breeding program is based on reliable genomic data, which have been extensively used for cattle selection [6].The genomic selection is based on the existence of linkage disequilibrium (LD), SNPs associations, and quantitative traits loci (QTLs) that effect on traits of interest In Pakistan, all genetic improvement programmes for cattle is still based on conventional quantitative genetic methods.There is also limited availability of phenotypic and pedigree data information for estimation of breeding values.However, it has been found that genetic selection has a high potential to increase genetic gain in cattle and also permits more accurate genetic predictions for low heritability traits in farm animals than conventional phenotypic selection [6].The indigenous cattle breeds of Pakistan are still lacking the opportunity for high throughput genomic evaluation for better understanding of domestication process and breed improvement programs.To date, no indigenous Pakistani cattle breed has been included either in training or a validation population using bovine high density SNPs Bead Chip.The evaluation of this high-throughput technique may help to improve the cattle farming.Therefore, the aim of this study was to find the level of informativeness of the bovine high density SNPs bead chip by measuring loci polymorphism in indigenous Pakistani cattle population.

Animal's selection and DNA extraction
In this study, we used 136 individuals from ten different Pakistani cattle breeds; include Achi (18), Bhagnari (14), Cholistani (13), Dajal (10), Dhanni (10), Kankraj (12), Lohani (19), Red Sindhi (13), Sahiwal ( 14), and Tharparkar (13) from different livestock experiment stations (Table 1).A 10 ml jugular blood was collected in falcon tubes containing EDTA and genomic DNA was extracted using modified organic method.Genomic DNA (gDNA) was qualitative and quantitatively checked through gel electrophoresis and Nanodrop spectrophotometer (Nanodrop 1000).Genotyping was performed at the BFGL, ARS/USDA, USA, platform with the Illumina bovine high density SNPs bead chip v2 containing 777, 962 SNPs disseminated across the bovine genome.Approximately, 200 ng genomic DNA was used to performed genotyping for each sample.Genomic DNA was processed according to manufacturer protocol (the Illumina, Inc.San Diego, CA, USA).Amplified sample was incubated at 37⁰C for 20 h.The hybridization of samples was performed on the bovine high density SNPs bead chip at 48⁰C for 20h.Following the hybridization, nonspecifically hybridized loci were stained and imaged on an Illumina iScan Reader after processing for single base extension reactions.

Data analysis
Genotypic data were generated from the iScan system.Primary data analysis including genotyping calling, clustering and data normalization was performed by using Illumina genome studio version 1.9.0 software.Ped and map file was created for downstream analyses from the genome studio using plink input report 2.

Minor allele frequency (MAF)
A rare variant of minor allele frequency (MAF) estimation is difficult due to the limitation of markers in genome wide association studies.The minor allele frequency (MAF) used in a typical genome wide association studies (GWAS) is below 1%.In this study, we used minor allele frequency for all autosomes in each breed ranged from 0.18 to 0.23 with average value 0.21±0.1552,which was slightly lower than previously reported MAF in Brahman, Gir, and Nellore cattle [1].Simultaneously, the observed minor allele frequency in this study was higher than African zebu [15].The average minor allele frequency (MAF) ranged from 0.18 (BTA 22) to 0.23 (BTA 25).Elimination of low polymorphic loci and monomorphic loci (MAF<0.01), the remaining average minor allele frequency was 0.21±0.1462.

Linkage disequilibrium (LD) Pruning
The linkage disequilibrium method is used to identify linked SNPs to defined genetic variation in genome wide association studies (GWAS) [14].SNPs linkage or in high linkage disequilibrium (LD) genetic information is same in runs of homozygosity, therefore LD pruning is recommended [17].In this study, we used r 2 threshold to detect linked SNPs (r 2 = 0.45).This method prune 446, 715 SNPs distributed across all autosomes.The average distance between the SNPs markers for the Bovine high density SNPs Bead Chip is 3.43 kb (http// www/illumina.com).

Conclusion
In conclusion, the analysis of this study showed that 42, 669 markers in the bovine high density SNPs Bead Chip are limited for use of population genetics or association studies in indigenous Pakistani cattle breeds.The monomorphic loci, which is less than 1% has no genomic coordinates.This study provides 500, 939 informative SNPs, which is 64% of the all autosomes and can be potentially used for further genomic applications.The results of this study is comparable with previously then reported studies in worldwide cattle populations [1].The results of this study encourage that the bovine high density SNPs bead chip is very informative and can be used for further genomic studies in Pakistani indigenous cattle breeds based on different agroecological zones.The use of this array can also improve the understanding of evolutionary processes of Pakistani indicus cattle breeds and future synthetic breed development for improved milk and meat production.Authors' contributions Conceived and designed the experiments: H Mustafa & TS Sonstegard, Performed the experiments: H Mustafa, HJ Heather & K Eui-soo, Analyzed the data: H Mustafa, WA Khan, HJ Heather & K Eui-soo, Contributed materials/ analysis/ tools: Hamid and TS Sonstegard, Wrote H Mustafa, ZH Kuthu, K Eui-soo, A Ajmal & NU Ain.