Dissecting the genomic regions of selection on the X chromosome in different cattle breeds.

Mammalian X and Y chromosomes independently evolved from various autosomes approximately 300 million years ago (MYA). To fully understand the relationship between genomic composition and phenotypic diversity arising due to the course of evolution, we have scanned regions of selection signatures on the X chromosome in different cattle breeds. In this study, we have prepared the datasets of 184 individuals of different cattle breeds and explored the complete X chromosome by utilizing four within-population and two between-population methods. There were 23, 25, 30, 17, 17, and 12 outlier regions identified in Tajima's D, CLR, iHS, ROH, FST, and XP-EHH. Bioinformatics analysis showed that these regions harbor important candidate genes like AKAP4 for reproduction in Brown Swiss, MBTS2 for production traits in Brown Swiss and Guernsey, CXCR3 and CITED1 for health traits in Jersey and Nelore, and BMX and CD40LG for regulation of X chromosome inactivation in Nelore and Gir. We identified genes shared among multiple methods, such as TRNAC-GCA and IL1RAPL1, which appeared in Tajima's D, ROH, and iHS analyses. The gene TRNAW-CCA was found in ROH, CLR and iHS analyses. The X chromosome exhibits a distinctive interaction between demographic factors and genetic variations, and these findings may provide new insight into the X-linked selection in different cattle breeds.

Comprehensive selection signature analyses in dairy cattle exploiting purebred and crossbred genomic data.

The main objective of the current research was to locate, annotate, and highlight specific areas of the bovine genome that are undergoing intense positive selection. Here, we are analyzing selection signatures in crossbred (Bos taurus X Bos indicus), taurine (Bos taurus), and indicine (Bos indicus) cattle breeds. Indicine cattle breeds found throughout India are known for their higher heat tolerance and disease resilience. More breeds and more methods can provide a better understanding of the selection signature. So, we have worked on nine distinct cattle breeds utilizing seven different summary statistics, which is a fairly extensive approach. In this study, we carried out a thorough genome-wide investigation of selection signatures using bovine 50K SNP data. We have included the genotyped data of two taurine, two crossbreds, and five indicine cattle breeds, for a total of 320 animals. During the 1950s, these indicine (cebuine) cattle breeds were exported with the aim of enhancing the resilience of taurine breeds in Western countries. For this study, we employed seven summary statistics, including intra-population, i.e., Tajima's D, CLR, iHS, and ROH and inter-population statistics, i.e., FST, XP-EHH, and Rsb. The NCBI database, PANTHER 17.0, and CattleQTL database were used for annotation after finding the important areas under selection. Some genes, including EPHA6, CTNNA2, NPFFR2, HS6ST3, NPR3, KCNIP4, LIPK, SDCBP, CYP7A1, NSMAF, UBXN2B, UGDH, UBE2K, and DAB1, were shown to be shared by three or more different approaches. Therefore, it gives evidence of the most intense selection in these areas. These genes are mostly linked to milk production and adaptability traits. This study also reveals selection regions that contain genes which are crucial to numerous biological functions, including those associated with milk production, coat color, glucose metabolism, oxidative stress response, immunity and circadian rhythms.

Changes in m6A RNA methylation of goat lung following PPRV infection.

Peste des petits ruminants (PPR) is an acute, highly contagious viral disease of goats and sheep, caused by the Peste des petits ruminants virus (PPRV). Earlier studies suggest the involvement of diverse regulatory mechanisms in PPRV infection. Methylation at N6 of Adenosine called m6A is a type RNA modification that influences various physiological and pathological phenomena. As the lung tissue represents the primary target organ of PPRV, the present study explored the m6A changes and their functional significance in PPRV disease pathogenesis. m6A-seq analysis revealed 1289 m6A peaks to be significantly altered in PPRV infected lung in comparison to normal lung, out of which 975 m6A peaks were hypomethylated and 314 peaks were hypermethylated. Importantly, hypomethylated genes were enriched in Interleukin-4 and Interleukin-13 signaling and various processes associated with extracellular matrix organization. Further, of the 843 differentially m6A-containing cellular transcripts, 282 transcripts were also found to be differentially expressed. Functional analysis revealed that these 282 transcripts are significantly enriched in signaling by Interleukins, extracellular matrix organization, cytokine signaling in the immune system, signaling by receptor tyrosine kinases, and Toll-like Receptor Cascades. We also found m6A reader HNRNPC and the core component of methyltransferase complex METTL14 to be highly upregulated than the m6A readers - HNRNPA2B1 and YTHDF1 at the transcriptome level. These findings suggest that alteration in the m6A landscape following PPRV is implicated in diverse processes including Interleukin signaling.

Biomarker discovery and authentication of cold-slaughtered chicken through classical analytical procedures and mass spectrometry based proteomic approaches.

1. This study evaluated the suitability of routine analytical procedures and used mass spectrometry-based proteomic approaches to distinguish meat from dead chicken/ cold-slaughtered birds (CS), electrically stunned and slaughtered birds, as per standard protocols (ES), and birds slaughtered according to halal guidelines (HS).2. Meat from CS birds had lower (P < 0.05) pH, water-holding capacity and higher (P < 0.05) lipid oxidation, haem iron content, residual blood and total viable counts relative to ES and HS meat indicating poor quality.3. The results demonstrated the presence of unique protein bands on SDS-PAGE only in CS meat that can be used for routine screening.4. Protein analysis using MALDI-TOF mass spectrometry identified haemoglobin subunit alpha-A and alpha-D; Adenylate kinase isoenzyme 1 as reliable and stable marker proteins for authentication of dead chicken meat under raw and cooked conditions and halal slaughtered chicken, respectively.5. The methods used may be employed by the food safety and regulatory agencies for regular screening of meat quality and to authenticate CS or HS chicken.

Uncovering genes underlying coat color variation in indigenous cattle breeds through genome-wide positive selection.

The identification of candidate genes related to pigmentation and under selective sweep provides insights into the genetic basis of pigmentation and the evolutionary forces that have shaped this variation. The selective sweep events in the genes responsible for normal coat color in Indian cattle groups are still unknown. To find coat color genes displaying signs of selective sweeps in the indigenous cattle, we compiled a list of candidate genes previously investigated for their association with coat color and pigmentation. After that, we performed a genome-wide scan of positive selection signatures using the BovineSNP50K Bead Chip in 187 individuals of seven indigenous breeds. We applied a wide range of methods to find evidence of selection, such as Tajima's D, CLR, iHS, varLD, ROH, and FST. We found a total of sixteen genes under selective sweep, that were involved in coat color and pigmentation physiology. These genes are CRIM1 in Gir, MC1R in Sahiwal, MYO5A, PMEL and POMC in Tharparkar, TYRP1, ERBB2, and ASIP in Red Sindhi, MITF, LOC789175, PAX3 and TYR in Ongole, and IRF2, SDR165 and, KIT in Nelore, ADAMTS19 in Hariana. These genes are related to melanin synthesis, the biology of melanocytes and melanosomes, and the migration and survival of melanocytes during development.

Genome-wide detection of copy number variations in Tharparkar cattle.

Copy number variations (CNVs) are major forms of genetic variation with an increasing importance in animal genomics. This study used the Illumina BovineSNP 50 K BeadChip to detect the genome-wide CNVs in the Tharparkar cattle. With the aid of PennCNV software, we noticed a total of 447 copy number variation regions (CNVRs) across the autosomal genome, occupying nearly 2.17% of the bovine genome. The average size of detected CNVRs was found to be 122.2 kb, the smallest CNVR being 50.02 kb in size, to the largest being 1,232.87 Kb. Enrichment analyses of the genes in these CNVRs gave significant associations with molecular adaptation-related Gene Ontology (GO) terms. Most CNVR genes were significantly enriched for specific biological functions; signaling pathways, sensory responses to stimuli, and various cellular processes. In addition, QTL analysis of CNVRs described them to be linked with economically essential traits in cattle. The findings here provide crucial information for constructing a more comprehensive CNVR map for the indigenous cattle genome.

Identification of novel polymorphism in mammary-derived growth inhibitor gene of water buffalo and its expression analysis in the mammary gland.

Mammary-derived growth inhibitor (MDGI), a member of the lipophilic family of fatty acid-binding proteins, plays an important role in the development, regulation, and differentiation of the mammary gland. The aim of the study was to identify polymorphism in the MDGI gene and its expression analysis in the mammary gland at various stages of lactation, in Indian buffalo. Nucleotide sequence analysis of MDGI gene in different breeds of riverine and swamp buffaloes revealed a total of 16 polymorphic sites and one Indel. Different transcription factor binding sites were predicted for buffalo MDGI gene promoter sequence, using online tools and in-silico analysis indicating that the SNPs in this region can impact the gene expression regulation. Phylogenetic analysis exhibited the MDGI of buffalo being closer to other ruminants like cattle, yak, sheep, and goats. Further, the expression analysis revealed that buffalo MDGI being highly expressed in well-developed mammary glands of lactating buffalo as compared to involution/non-lactating and before functional development to start the milk production stage in heifers. Stage-specific variation in expression levels signifies the important functional role of the MDGI gene in mammary gland development and milk production in buffalo, an important dairy species in Southeast Asia.

Temporal dysregulation of genes in lamb testis cell during sheeppox virus infection.

The present study was aimed to elucidate the host-virus interactions using RNA-Seq analysis at 1 h and 8 h of post-infection of sheeppox virus (SPPV) in lamb testis cell. The differentially expressed genes (DEGs) and the underlying mechanisms linked to the host immune responses were obtained. The protein-protein interaction (PPI) network analysis and ingenuity pathway analysis (IPA) illustrated the interaction between the DEGs and their involvement in cell signalling responses. Highly connected hubs viz. AURKA, CHEK1, CCNB2, CDC6 and MAPK14 were identified through PPI network analysis. IPA analysis showed that IL-6- and ERK5-mediated signalling pathways were highly enriched at both time points. The TP53 gene was identified to be the leading upstream regulator that directly responded to SPPV infection, resulting in downregulation at both time points. The study provides an overview of how the lamb testis genes and their underlying mechanisms link to growth and immune response during SPPV infection.

Selection and validation of suitable reference gene for qPCR gene expression analysis in lamb testis cells under Sheep pox virus infection.

Virus infection alters host gene expression, therefore ideal and stable reference housekeeping genes are required to normalise the expression of other expressed host genes in quantitative real-time PCR (qRT-PCR). The suitable reference gene may vary in response to different viral infections in different hosts or cells. In the present study, we cultured primary lamb testis cells (LTC) and assessed the expression stability of seven widely used housekeeping genes (B2M, HMBS, HPRT1, HSP-90, POLR2A, 18s_RNA, GAPDH) as reference genes in Sheeppox virus (SPPV) infected and control (uninfected-0h) LTC at 0.5h, 4.0h, 8.0h, and 12.0h post-infection) using NormFinder, Bestkeeper, geNorm, and the comparative ΔCT method in RefFinder based on their expression levels. Analysis revealed that HSP90, 18s_RNA, HPRT, POLR2A, and B2M were the most stable genes from the panel in the individual analysis group in 0h, 0.5h, 4.0h, 8.0h, and 12.0h, respectively. Furthermore, B2M was shown to be the most stable reference gene in the combined control with the respective and overall infected groups, except the control group of 4.0hpi of SPPV infection. In this study, we selected the most suitable reference genes in LTC for particular time points of SPPV infection. The identified most suitable housekeeping gene can be used during normalization of expression of other targeted genes at aspecific time point of SPPV infection.

Psychological Resilience, Coping, and Distress in Admitted Patients With COVID-19 Infection.

Objective: To assess psychological resilience, coping, and related psychological distress in admitted COVID-19 patients. Predictors of subsequent development of posttraumatic stress symptoms (PTSS) and disability were also studied.Methods: Stable inpatients with COVID-19 (aged > 18 years with mild symptoms) admitted to a tertiary care hospital from April 2020 to December 2020 were recruited for the study. During admission, the patients were assessed for resilience, coping, and psychological distress using the 10-item Connor-Davidson Resilience Scale (CD-RISC-10), Brief COPE (Coping Orientation to Problems Experienced), and 4-item Patient Health Questionnaire (PHQ-4). Similarly, they were assessed at 4 weeks after discharge using the PTSD Checklist for DSM-5 and World Health Organization Disability Assessment Schedule.Results: A total of 176 patients were recruited for the study and assessed during their admission, and 102 were reassessed during follow-up. Of the patients, 17.6% during admission and 58.8% at follow-up had significant psychological distress (PHQ-4 score > 2). The mean ± SD CD-RISC-10 score was 27.94 ± 5.82. The most used coping strategies were emotional support, religion, and acceptance. Increased resilience was associated with better education (rs[100] = 0.265, P = .007), less psychological distress (r[100] = -0.596, P = .001), and healthy coping strategies. PHQ-4, PCL-5, and disability scores at follow-up were positively correlated (Pearson correlation). The multiple regression model statistically significantly predicted PTSS (F7, 94 = 2.660, P < .015, adjusted R2 = 0.103).Conclusions: COVID-19 patients with better resilience are associated with reduced psychological distress. Better resilient traits and reduced psychological distress may prevent ensuing PTSS and disability.

Genome Wide Scan to Identify Potential Genomic Regions Associated With Milk Protein and Minerals in Vrindavani Cattle.

In this study, genome-wide association study (GWAS) was conducted for identifying significantly associated genomic regions/SNPs with milk protein and minerals in the 96 taurine-indicine crossbred (Vrindavani) cows using 50K SNP Chip. After quality control, a total of 41,427 SNPs were retained and were further analyzed using a single-SNP additive linear model. Lactation stage, parity, test day milk yield and proportion of exotic inheritance were included as fixed effects in GWAS model. Across all traits, 13 genome-wide significant (p < 1.20 x 10-06) and 49 suggestive significant (p < 2.41 x 10-05) SNPs were identified which were located on 18 different autosomes. The strongest association for protein percentage, calcium (Ca), phosphorus (P), copper (Cu), zinc (Zn), and iron (Fe) were found on BTA 18, 7, 2, 3, 14, and 2, respectively. No significant SNP was detected for manganese (Mn). Several significant SNPs identified were within or close proximity to CDH13, BHLHE40, EDIL3, HAPLN1, INHBB, USP24, ZFAT, and IKZF2 gene, respectively. Enrichment analysis of the identified candidate genes elucidated biological processes, cellular components, and molecular functions involved in metal ion binding, ion transportation, transmembrane protein, and signaling pathways. This study provided a groundwork to characterize the molecular mechanism for the phenotypic variation in milk protein percentage and minerals in crossbred cattle. Further work is required on a larger sample size with fine mapping of identified QTL to validate potential candidate regions.

Identification of important genomic footprints using eight different selection signature statistics in domestic cattle breeds.

In the present study, the population genomic data of different cattle breeds were explored to decipher the genomic regions affected due to selective events and reflected in the productive, reproductive, thermo-tolerance, and health-related traits. To find out these genomic deviations due to selective sweeps, we used eight different statistical tools (Tajima's D, Fu & Li's D*, CLR, ROH, iHS, FST, FLK, and hapFLK) on seven indigenous and five exotic cattle breeds. We further performed composite analysis by comparing their covariance matrix. Several candidate genes were found to be related to milk production (ADARB, WDR70, and CA8), reproductive (PARN, FAM134B2, and ZBTB20), and health-related traits (SP110, CXCL2, CLXCL3, CXCL5, IRF8, and MYOM1). The outcome of this investigation provides a basis for detecting selective sweeps that explain the genetic variation of traits. They may possess functional importance for multiple cattle breeds in different subcontinents. However, further studies are required to improve the findings using high-density arrays or whole-genome sequencing with higher resolution and greater sample sizes.

Genome-wide assessment of genetic diversity, linkage disequilibrium and haplotype block structure in Tharparkar cattle breed of India.

Knowledge about genetic diversity is very essential for the management and sustainable utilization of livestock genetic resources. In this study, we presented a comprehensive genome-wide analysis of genetic diversity, ROH, inbreeding, linkage disequilibrium, effective population size and haplotype block structure in Tharparkar cattle of India. A total of 24 Tharparkar animals used in this study were genotyped with Illumina BovineSNP50 array. After quality control, 22,825 biallelic SNPs were retained, which were in HWE, MAF > 0.05 and genotyping rate >90%. The overall mean observed (HO) and expected heterozygosity (HE) were 0.339 ± 0.156 and 0.325 ± 0.129, respectively. The average minor allele frequency was 0.234 with a standard deviation of ± 0.131. We identified a total of 1832 ROH segments and the highest autosomal coverage of 13.87% was observed on chromosome 23. The genomic inbreeding coefficients estimates by FROH, FHOM, FGRM and FUNI were 0.0589, 0.0215, 0.0532 and 0.0160 respectively. The overall mean linkage disequilibrium (LD) for a total of 133,532 pairwise SNPs measured by D' and r2 was 0.6452 and 0.1339, respectively. In addition, we observed a gradual decline in effective population size over the past generations.

Characterisation and comparison of immune response mechanisms in an indigenous and a commercial pig breed after classical swine fever vaccination.

The live attenuated classical swine fever (CSF) vaccine has been successfully used to prevent and control CSF outbreaks for 6 decades. However, the immune response mechanisms against the vaccine remain poorly understood. Moreover, very few reports exist regarding the breed differences in the response to CSF vaccine. In this study, we generated the peripheral blood mononuclear cell transcriptomes of indigenous Ghurrah and commercial Landrace pig breeds, before and 7 days after CSF vaccination. Subsequently, between and within-breed differential gene expression analyses were carried out. Results revealed large differences in pre-vaccination peripheral blood mononuclear cell transcriptome profiles of the two breeds, which were homogenised 7 days after vaccination. Before vaccination, gene set enrichment analysis showed that pathways related to antigen sensing and innate immune response were enriched in Ghurrah, while pathways related to adaptive immunity were enriched in Landrace. Ghurrah exhibited greater immunomodulation compared to Landrace following the vaccination. In Ghurrah, cell-cycle processes and T-cell response pathways were upregulated after vaccination. However, no pathways were upregulated in Landrace after vaccination. Pathways related to inflammation were downregulated in both the breeds after vaccination. Key regulators of inflammation such as IL1A, IL1B, NFKBIA and TNF genes were strongly downregulated in both the breeds after vaccination. Overall, our results have elucidated the mechanisms of host immune response against CSF vaccination in two distinct breeds and revealed common key genes instrumental in the global immune response to the vaccine.

Estimation of linkage disequilibrium levels and allele frequency distribution in crossbred Vrindavani cattle using 50K SNP data.

The objective of this study was to calculate the extent and decay of linkage disequilibrium (LD) in 96 crossbred Vrindavani cattle genotyped with Bovine SNP50K Bead Chip. After filtering, 43,821 SNPs were retained for final analysis, across 2500.3 Mb of autosome. A significant percentage of SNPs was having minor allele frequency of less than 0.20. The extent of LD between autosomal SNPs up to 10 Mb apart across the genome was measured using r2 statistic. The mean r2 value was 0.43, if pairwise distance of marker was less than10 kb and it decreased further to 0.21 for 25-50 kb markers distance. Further, the effect of minor allele frequency and sample size on LD estimate was investigated. The LD value decreased with the increase in inter-marker distance, and increased with the increase of minor allelic frequency. The estimated inbreeding coefficient and effective population size were 0.04, and 46 for present generation, which indicated small and unstable population of Vrindavani cattle. These findings suggested that a denser or breed specific SNP panel would be required to cover all genome of Vrindavani cattle for genome wide association studies (GWAS).

Genome-wide expression analysis reveal host genes involved in immediate-early infections of different sheeppox virus strains.

This study explored the transcriptome of lamb testis cells infected with sheeppox virus (SPPV) wild strain (WS) and vaccine strain (VS) at an immediate-early time. Most of the differentially expressed genes (DEGs) and differentially expressed highly connected (DEHC) gene network were found to be involved in SPPV-VS infection compared to SPPV-WS. Further, the signaling pathways were mostly involved in SPPV-VS infection than SPPV-WS. SPPV modulates the expression of several important host proteins such as CD40, FAS, ITGß1, ITGα1, Pak1, Pak2, CD14, ILK leading to viral attachment and entry; immune-related DEGs such as MAPK, JNK, ERK, NFKB, IKB, PI3K, STAT which provide optimal cellular condition for early viral protein expression; and FOXO3, ATF, CDKNA1, TCF, SRF, BDNF which help in inducing apoptosis and MPTP, BAD and Tp53 inhibits apoptosis or cell death at the immediate-early time. The results captured the specific genes and enabled to understand distinct pathogenic mechanisms employed by VS and WS of SPPV.

Identification of genes affecting milk fat and fatty acid composition in Vrindavani crossbred cattle using 50 K SNP-Chip.

The aim of this study was to identify candidate genes associated with milk fat per cent and fatty acid (FA) composition in Vrindavani cattle using the Illumina 50 K single-nucleotide polymorphism (SNP) array. After quality control, a total of 41,427 informative and high-quality SNPs were used for a genome-wide association study (GWAS) for milk fat percentage and 16 different types of fatty acids. Lactation stage, parity, test day milk yield, and proportion of exotic inheritance were included as fixed effects in the GWAS model. A total of 67 genome-wide significant (P < 1.20 × 10-06) SNPs and 176 suggestive significant (P < 2.41 × 10-05) SNPs were identified. Out of these, 15 SNPs were associated with more than one trait. The strongest associations were found on BTA14 for milk fat percentage and on BTA2 and BTA16 for polyunsaturated fatty acids. Several significant SNPs were identified close to or within the genes ELOVL6, FABP4, PMP2, PLIN1, MFGE8, GHRL2, and LDLRAD3 which are known to be associated with fat percentage and FA composition in dairy cattle breeds. This study is a step forward to better characterize the molecular mechanisms of phenotypic variation in milk fatty acids in a taurine-indicine composite cattle breed reared in tropical environments.

Genomic scans for selection signatures revealed candidate genes for adaptation and production traits in a variety of cattle breeds.

Domestication and selection are the major driving forces responsible for the determinative genetic variability in livestock. These selection patterns create unique genetic signatures within the genome. BovineSNP50 chip data from 236 animals (seven indicine and five taurine cattle breeds) were analyzed in the present study. We implemented three complementary approaches viz. iHS (Integrated haplotype score), ROH (Runs of homozygosity), and FST, to detect selection signatures. A total of 179, 56, and 231 regions revealed 518, 277, and 267 candidate genes identified by iHS, ROH, and FST methods, respectively. We found several candidate genes (e.g., NCR3, ARID5A, HIST1H2BN, DEFB4, DEFB7, HSPA1L, HSPA1B, and DNAJB4) related to production traits and the adaptation of indigenous breeds to local environmental constraints such as heat stress and disease susceptibility. However, further studies are warranted to refine the findings using a larger sample size, whole-genome sequencing, and/or high density genotyping.

SNPs with intermediate minor allele frequencies facilitate accurate breed assignment of Indian Tharparkar cattle.

Tharparkar cattle breed is widely known for its superior milch quality and hardiness attributes. This study aimed to develop an ultra-low density breed-specific single nucleotide polymorphism (SNP) genotype panel to accurately quantify Tharparkar populations in biological samples. In this study, we selected and genotyped 72 Tharparkar animals randomly from Cattle & Buffalo Farm of IVRI, India. This Bovine SNP50 BeadChip genotypic datum was merged with the online data from six indigenous cattle breeds and five taurine breeds. Here, we used a combination of pre-selection statistics and the MAF-LD method developed in our laboratory to analyze the genotypic data obtained from 317 individuals of 12 distinct breeds to identify breed-informative SNPs for the selection of Tharparkar cattle. This methodology identified 63 unique Tharparkar-specific SNPs near intermediate gene frequencies. We report several informative SNPs in genes/QTL regions affecting phenotypes or production traits that might differentiate the Tharparkar breed.

Comparative analysis of five different methods to design a breed-specific SNP panel for cattle.

Single nucleotide polymorphisms (SNPs) have now replaced microsatellite markers in several species for various genetic investigations like parentage assignment, genetic breed composition, assessment for individuality and, most popularly, as a useful tool in genomic selection. However, such a resource, which can offer to assist breed identification in a cost-effective manner is still not explored in cattle breeding programs. In our study, we have tried to describe methods for reducing the number of SNPs to develop a breed-specific panel. We have used SNP data from Dryad open public access repository. Starting from a global dataset of 178 animals belonging to 10 different breeds, we selected five panels each comprising of similar number of SNPs using different methods i.e., Delta, Pairwise Wright's FST, informativeness for assignment, frequent item feature selection (FIFS) and minor allele frequency-linkage disequilibrium (MAF-LD) based method. MAF-LD based method has been recently developed by us for construction of breed-specific SNP panels. The STRUCTURE software analysis of MAF-LD based method showed appropriate clustering in comparison to other panels. Later, the panel of 591 breed-specific SNPs was called to their respective breeds using Venny 2.1.0 and UGent web tools software. Breed-specific SNPs were later annotated by using various Bioinformatics softwares.