Descriptive statistics
In the present study, 45 samples (Sahiwal: 13, Tharparkar: 17, Gir: 15) were considered after excluding 7 samples having less than 10% missing genotypes. Out of 777962 SNPs in the HD chip, 42669 unmapped, X, Y and MT SNPs were excluded. Approximately, 27765 and 278372 SNPs were also excluded owing to low SNP call rate (<95%) and MAF (<0.05) value. A total of 434519 SNPs were filtered to calculate locus wise FST values. After excluding the negative FST values from data set, SNPs that passed quality control were 236989, 229556 and 251653 for SW-GR, SW-TP and TP-GR breed pairs, respectively.
Vineeth et al., (2020) identified 258231 genome-wide SNPs related to milk production and reproduction in Sahiwal cattle through sequence alignment to
Bos indicus reference genome.
Genome-wide distribution of FST values for each SNP locus were evaluated among these three breed pairs (Fig 1). In total, 94.74%, 96.42% and 94.03% of SNPs in SW-GR, SW-TP and TP-GR breed pairs had very low FST value ranged from 0 to 0.1. Approximately 0.37%, 0.22% and 0.50% of SNPs showed high FST value ranged from 0.2 to 0.3, where as only a few SNPs (0.05%, 0.03% and 0.07%) had very high FST value ranged from 0.3 to 0.4 in these respective breed pairs. Similar to this study,
Makina et al., (2015) also observed a less proportion of SNPs (31%) with FST<0.05 among South African cattle breeds.
SNP windows and Selection signatures
FST analysis identified 86419, 86452 and 86706 sliding windows after including 5 consecutive SNPs into a single window in order to identify the genome-wide selection signatures among the Indian cattle breeds. The average FST value in each SNP window was estimated and plotted as Manhattan plot for all the chromosomes in three breed pairs. The Manhattan plot identified 122, 81 and 191 SNP windows (FST>0.25) for SW-GR, SW-TP and TP-GR, respectively (Fig 2, 3 and 4). These most differentiated SNP windows with FST>0.25 represented the top 0.14%, 0.09% and 0.22% of the total SNP windows in these three respective breed pairs.
Makina et al., (2015) reported the top 2% SNPs with the highest (FST<0.25) as the selection signature regions in South African cattle.
Functional annotation of selection signature regions
These genomic regions showing evidences of positive selection are further investigated to identify the underlying candidate genes and their association with milk performance traits in our indigenous cattle.
SW-GR pair
A total of 41 genome-wide selection signature regions were identified in SW-GR breed pair on FST statistics and these selection signatures contained 48 candidate genes. Notable candidate genes controlling milk performance traits were identified as ACADL, SLC26A2, PLCB1, SYT9 on chromosome 2, 7, 13 and 15, respectively (Table 1).
Dias et al., 2015 reported that Acyl-Coenzyme A dehydrogenase (ACADL) gene involved in metabolism of lipid and lipoproteins and plays a key role in the regulation of channeling of fatty acids towards copious milk fat synthesis in the mammary gland. Similarly, the putative selection signature region bearing solute carrier family 26-member 2 (SLC26A2) gene was responsible for carbohydrate metabolism and trans-membrane transport of sulfate like macronutrients in cell
(Brenig et al., 2003). Saravanan et al., (2021) revealed Phospholipase C beta 1 (PLCB1) gene related to milk production trait as a candidate gene among the indicine cattle breeds using FST method. Furthermore, chromosome 15 had a strong differentiating region (FST> 0.35) between 45986438-45998876 bp containing synaptotagmin 9 (SYT 9) gene, which was found in association with calcium binding and phospholipid binding in the present investigation (Table 1).
In addition to these candidate genes, a number of positively selected genes were associated with muscle and carcass traits (GAS2L3), amino acid and protein metabolism (TBC1D20), calcium binding and signal transduction (ATP2C1, GRM5) in the present study. A strong putative selection signature region on chromosome 7 includes TNFAIP8L1 gene which is involved in immune function and in the acute inflammatory response in cattle. Moreover, HSPB6 gene (Heat shock protein family B member 6) mapped on chromosome 18 suggests a strong selection signature in cattle which was associated with thermo-tolerance (Table 1).
Kumar et al., (2015) reported the variants of HSPB6 gene in Sahiwal cattle for better thermo-tolerance capacity.
Makina et al., (2015) detected one heat shock protein gene (HSPB9) under selection on BTA19 between 42.8-42.8 Mb, which was associated with adaptation to tropical environments in South African Zebu cattle.
SW-TP pair
We found 29 selection signatures for SW-TP breed pair based on FST analysis which are distributed over 423.93 kb region across 12 chromosomes. These selection signature regions had 30 candidate genes under positive selection, out of which five genes control the milk performance traits viz., ATPAF1, LEF1, PPARGC1B, EIF6 and ACSS3 mapped on chromosome 3, 6, 7, 13 and 20, respectively (Table 1). The ATPAF1 (ATP synthase mitochondrial F1 complex assembly factor 1) gene encodes an enzyme which involves in energy production through mitochondrial biogenesis in cell.
Zhao et al. (2015) reported a selection signature region around the gene ATPAF1 on chromosome 3 in Angus cattle. The positively selected lymphoid enhancer-binding factor-1 (LEF1) gene is associated with Wnt signaling during the morphogenesis of the mammary gland during embryogenesis
(Raven et al., 2014). The chromosome 7 harbours a strong differentiation region among Tharparkar and Gir cattle containing peroxisome proliferator-activated receptor gamma coactivator 1 beta (PPARGC1B) gene which was associated with oxidation of lipid, energy homeostasis in cattle
(Romao et al., 2014). We found selection signatures for eukaryotic translation initiation factor 6 (EIF 6) gene and Acyl-Coenzyme A synthetase short-chain family member 3 (ACSS3) gene as a candidate gene for milk fat composition in cattle
(Buitenhuis et al., 2014).
In this study, a strongly selected CAPN5 gene was related with proteolytic activity in cell, marbling score and meat tenderness in cattle (Table 1).
Wang et al., (2019) reported CAPN as a potential candidate gene for meat quality in Chinese Wagyu cattle. We detected a candidate region on chromosome 13 which harbours Agouti Signaling Protein (ASIP) gene influencing coat colour in cattle.
Randhawa et al., (2014) reported a strong selection signature for coat colour around ASIP gene in cattle. Furthermore, SLIT3 encoded the inflammatory mediators such as IL-1β, IL-6 and IL-8 and this gene was key regulator of pulmonary immune response during bovine respiratory disease complex in Holstein calves
(Neibergs et al., 2014). One selection signature region was found at TDRD9 which had an important role during spermatogenesis which is essential for germ line integrity in cattle (Table 1).
TP-GR pair
There were 60 selection signature regions in TP-GR breed pair which were spread over 1541.12 kb genomic region across 19 chromosomes and they harbour 60 candidate genes. The candidate genes
viz. PLA2R1, SCP2, ATPAF1, CACNA2D1, LEF1 and SUMF1 mapped on chromosome 2, 3, 4, 6 and 22 were found involving with lipid metabolism, carbohydrate metabolism and morphogenesis of mammary gland (Table 1).
Devadasan et al., (2020) identified 2871 high quality genome-wide SNPs in 383 candidate genes related to milk production, fertility, carcass, adaptability and immune response of economically important traits in Tharparkar cattle.
Our analysis revealed Phospholipase A2 receptor 1 (PLA2R1) gene on chromosome 2 having a strong differentiation (FST> 0.28) between Tharparkar and Gir cattle was known to catalyze hydrolysis of phospholipids
(Balsinde et al., 2002) and involved in pro-inflammatory cytokine production in the mammary gland epithelial cells in cattle. Another gene sterol carrier protein 2 (SCP 2) on chromosome 3 was related with lipid metabolism and beta oxidation of fatty acid in cattle
(Stolowich et al., 2002). This gene was also significantly differentially expressed in Peroxisome Proliferator-Activated Receptors signaling pathway, which was activated by fatty acids and their derivatives. As described earlier, the ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1) and lymphoid enhancer-binding factor-1 (LEF1) gene were identified as candidate gene under selection between Tharparkar and Gir cattle. The variants of CACNA2D1 gene were also found to be associated with somatic cell score (SCS) and mastitis resistance/susceptibility in Sahiwal cattle
(Magotra et al., 2016) of India. The sulfatase modifying factor 1 (SUMF1) gene on chromosome 22 was also found within the selection signature region and this gene is related with the metabolism of lipids and lipoproteins.
The genes within the selection signature region (CCDC141 and CRHR2) were associated with marbling in muscle meat quality traits in cattle. One selection signature region at DNER gene had an important role in regulating puberty and age at first calving, while another gene FSHR (Follicle Stimulating Hormone Receptor) on chromosome 11 controls the reproduction in cattle (Table 1).
Cory et al., (2013) revealed seven SNPs in the coding region of the bovine FSHR gene and suggested that specific alleles of the bovine FSHR gene were associated with the embryo yield and number of unfertilized oocytes in cattle.
