Candidate genes and proteins regulating bull semen quality: a review.

Poor semen profile reflected by suboptimum fertility statistics is a concern in bulls reared for breeding purpose. A critical review of research on candidate genes and proteins associated with semen quality traits will be useful to understand the progress of molecular marker development for bull semen quality traits. Here, we have tabulated and classified candidate genes and proteins associated with bull semen quality based on a literature survey. A total of 175 candidate genes are associated with semen quality traits in various breeds of cattle. Several studies using candidate gene approach have identified 26 genes carrying a total of 44 single nucleotide polymorphisms. Furthermore, nine genome-wide association studies (GWASes) have identified 150 candidate genes using bovine single nucleotide polymorphisms (SNP) chips. Three genes, namely membrane-associated ring-CH-type finger 1 (MARCH1), platelet-derived growth factor receptor beta, and phosphodiesterase type 1, were identified commonly in two GWASes, which, especially MARCH1, are required to explore their regulatory roles in bull semen quality in in-depth studies. With the advancement of high-throughput-omic technologies, more candidate genes associated with bull semen quality may be identified in the future. Therefore, the functional significance of candidate genes and proteins need to be delved further into future investigations to augment bull semen quality.

Genotyping of Novel SNPs in BMPR1B, BMP15, and GDF9 Genes for Association with Prolificacy in Seven Indian Goat Breeds.

Goats form the backbone of rural livelihood and financial security systems in India but their population is showing decreasing trend. Improvement of reproductive traits such as prolificacy offers a solution to stabilize the decreasing goat population and to meet the nutritional needs of growing human population. In the present study, six novel SNPs in three candidate genes for prolificacy (BMPR1B, BMP15, and GDF9) were genotyped in seven breeds of Indian goats to evaluate their association with litter size. Tetra primer ARMS-PCR and PCR-RFLP based protocols were developed for genotyping six novel SNPs, namely, T(-242)C in BMPR1B; G735A and C808G in BMP15; and C818T, A959C, and G1189A in GDF9 gene. The effect of breed was highly significant (p ≤ 0.01) on litter size but the effect of genotype was nonsignificant. The effect of parity on litter size was also significant in the prolific Black Bengal breed. The litter size differences observed between breeds are attributed to breed differences. Novel mutations observed at different loci in GDF9, BMP15, and BMPR1B genes do not contribute to the reproductive capability of the investigated breeds. Further studies with more number of breeds and animals exploring association of these novel SNPs with reproductive traits may be fruitful.

Polymorphism of JY-1 gene is not associated with reproductive traits in goats.

Reproductive traits show either low to moderate heritability and are measured late in life. Thus molecular markers are required to improve the pace of genetic gain in these traits of economic value. Recent publications report the association of polymorphism in JY-1 gene with cattle reproductive traits. In this study, the only known single nucleotide polymorphism (C15329T) of caprine JY-1 gene was explored for its association with reproductive traits; age at first heat, age at first service and age at first kidding. Black Bengal (n = 70), a sexually precocious and prolific goat breed of India was genotyped for the SNP by designing PCR-RFLP. Effect of year of birth and genotype was found to be non-significant at 1% for all three traits. However, the effect of season of birth was highly significant (p ≤ 0.01) on age at first kidding. Polymorphism in 3'UTR of the JY-1 gene is not associated with the reproductive traits of goats. In future, studies involving large number of animals from different goat breeds as well as investigation of other regions of this gene would be exciting to perform.

Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits.

KiSS1 is considered to be a key mediator of molecular mechanism of reproduction (puberty and prolificacy) in mammals. Kisspeptins are a family of structurally related peptides, encoded by KiSS1 gene, with ability to regulate gonadotropin-releasing hormone and hence hypothalamic-pituitary-gonadal axis. The present study investigated the polymorphism of caprine KiSS1 gene in 9 Indian goat breeds differing in sexual precocity and prolificacy. Comparison of KiSS1 amplified sequences of indigenous goats resulted in identification of nine SNPs (intron (1) G296C, T455G, T505A, T693C, T950C and intron (2) T1125C, A2510G, C2540T, A2803G) of which four are novel. These loci were not segregating together (r(2)<0.33). Mutations existed in both, sexually precocious and late-maturing goat breeds as well as low and high prolificacy goat breeds. Three loci reported to be associated with goat litter size (G296C, G2510A and C2540T) were identified in Indian goats as well. Association between loci of KiSS1 gene and age of puberty as well as litter size was explored in Black Bengal (N=158), a sexually precocious and prolific goat breed of India by designing PCR-RFLP. None of the mutations were found to be associated with reproductive traits however, difference in litter size as well age of sexual maturity for different genotypes indicates that the study on additional data based on more number of breeds and animals would be interesting to perform. Considering the importance of the reproductive trait in small ruminants, the results extend the limited information on genetic variation of the caprine KiSS1, which might contribute toward molecular breeding to enhance productivity of goat.

Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes.

New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes.

Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß (transforming growth factor-beta) superfamily, of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus expansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4 gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective of the present study was to detect polymorphism in intronic, exonic and 3' un-translated regions of BMP4 gene in Indian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning>40%), Osmanabadi, Sangamneri (Twinning 20-30%), Sirohi and Ganjam (Twinning<10%)) differing in prolificacy and geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) was used to design primers for the amplification of a targeted region followed by direct DNA sequencing to identify the genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic region and the 3' flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutation resulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534 locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predominant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhrana breed with a genotype frequency of 0.02. A microsatellite was identified in the 3' flanking region, only 20 nucleotides downstream from the termination site of the coding region, as a short sequence with more than nineteen continuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of a non-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is the first report of a mutation in the coding region of the caprine BMP4 gene. But whether the reproduction trait of goat is associated with the BMP4 polymorphism, needs to be further defined by association studies in more populations so as to delineate an effect on it.