Sheep and goat production is a predominant livestock activity in harsh climatic regions of the country particularly in hilly areas and arid zones with the little arable agricultural land. Both these species are well adapted to the wide variation of agro-climatic conditions and extensive pasture grazing system. The goat and sheep population in Himachal Pradesh is 1.12 and 0.81 million respectively (
Livestock Census, 2012) constituting 39.8% of state’s total livestock of 4.85 million. Gaddi, also known as “White Himalayan” goat, is the predominant goat breed constituting 60-65% of total goats in the state, while Chegu goat breed, known for pashmina producti on, has economic importance in higher, cold arid regions of the state. Gaddi and Rampur-Bushair are the two indigenous sheep breeds of the state well adapted to the prevalent migratory production system
(Sankhyan et al., 2018). Rapid growth is important in meat animals including sheep and goats, as it influences the net economic returns from the sale of animals. The physiological regulation of muscle growth in animals is under the control of multiple genes. Growth traits of animals are the major consideration during breeding due to its determinant economic value in returns from animal industry
(Zhang et al., 2008a). The somatotrophic axis (GH/IGF-I axis) is considered as the key in postnatal growth and metabolism in different mammals including farm animals
(Shoshana et al., 2000; Sahoo et al., 2018). One of the most important component of the somatotrophic axis is Insulin-like Growth Factor I (
IGF-I) which has a remarkable variation of its biological effect like protein synthesis and skeletal growth (
Baxter 1985;
Clemmons et al., 1987). The
IGF-I gene has been considered to be a candidate marker associated with growth traits in livestock (
Thue and Buchanan, 2002;
Zhang et al., 2008; Wu-Jun et al., 2010). Some researchers had investigated the association between polymorphisms of IGF gene and livestock production traits. The technique of PCR-RFLP was used to analyze polymorphism of
IGF-1 gene in sheep and goat breeds of Himachal Pradesh and to study association with body measurements in Gaddi goats. The experimental animals for the present study were taken from breeding tracts of different recognized sheep and goat breeds native to state of Himachal Pradesh. For polymorphism studies, genomic DNA was isolated from 63, 68, 197 and 73 animals belonging to Gaddi sheep, Rampur- Bushair sheep, Gaddi goat and Chegu goats, respectively. Samples were collected from true to type, unrelated animals of these breeds in their respective breeding tract and institutional farms. The genomic DNA was isolated by phenol-chloroform extraction procedure
(Sambrook and Russsel, 2001).
PCR-RFLP analysis of IGF-1 gene: Region of IGF-1
gene was amplified by using the forward and reverse primers (5’-3’):CACAGCGTATTATCCCAC and GACACTATGAGCCAGAAG, respectively
(Liu et al., 2012). For amplification, 25 µl of PCR reaction was prepared by adding 10pM of each primer, 100µM of each dNTPs, 1.5mM MgCl2, 10 × PCR assay buffer, 100 ng DNA template and 0.5 Unit Taq DNA polymerase. The amplification was carried out using thermal cycler with the following conditions: initial denaturation of 5 min at 94°C followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 2 min and extension at 72°C for 1 min and lastly the final extension of 5 min at 72°C. An aliquot of 20 µl of PCR product was digested overnight digestion with 1.5 Units of HaeIII restriction at 37°C. Representative samples of different genotypes as revealed by PCR-RFLP were got sequenced after purification of respective PCR product.
Association of growth related genes in Gaddi goats
For Gaddi goat, the data for the study was collected from adopted migratory flocks under All India Coordinated Research Project on Goat Improvement (Gaddi field unit) at this university. Biometrical measurements of Gaddi goats were collected for association and statistical analysis. Migratory tract of different adopted flocks are presented in Fig 1. For growth, body weight at different ages and biometry (body length, chest girth and body height) were recorded.
The following fixed effects model was employed for analysis of growth traits in Gaddi goats and least squares mean was used for multiple comparisons for body weight and body measurements for different genotypes.
Y
ijkl= µ + b
i +B
j + X
k +G
l + e
ijkl
Where,
Y
ijk is the phenotypic value of growth trait (body weights, body length, heart girth and body heights) measured on ijkl
th animal. µ is population mean, B
j is fixed effect associated with i
th herd, X
k is fixed effect associated with season and sex, G
l is the ûxed effect of the l
th genotype (l = 1, 2, 3) and e
ijkl is the random residual effect of each observation. Analysis was performed using the general linear model procedure of SAS (Ver 9.3) (SAS Institute Inc, Cary, NC, USA). Mean separation procedures were conducted using a least significant difference test.
PCR-RFLP analysis
PCR amplification of
IGF-1 yielded 363 bp amplicon. The PCR-RFLP digestion of amplified products with HaeIII revealed three fragments of 363 bp, 264 bp and 99 bp after digestion (Fig 2). Thus, the samples had 3 genotypes
viz genotype AA, AB and BB. Allele frequencies for A allele were 0.66 0.59, 0.65 and 0.70 and for B allele were 0.34, 0.41, 0.35 and 0.20 in Gaddi sheep, Rampur-Bushair sheep, Gaddi goat and Chegu goat, respectively. The frequency of AA, AB and BB genotypes ranged from 0.38 to 0.55, 0.30 to 0.42 and 0.14 to 0.19 in different breeds studied.
Similar to present investigation,
Deng et al., (2010) reported frequency for GG and GA genotype as 0.85 and 0.15 respectively in Chinese dairy goats. The allele frequencies of G and A alleles were 0.92 and 0.08, respectively in Guanzhong goats and the frequencies of GG and GA genotypes were 0.79 and 0.21 in Xinong Saanen goats.
Wu-Jun et al., (2010) observed frequency of A and B alleles as 0.395 and 0.61, respectively while frequencies of AA, AB and BB genotypes were 0.277, 0.237 and 0.486, respectively in Chinese goat breeds.
Alakilli et al., (2012) also observed similar results in different Egyptian goat breeds with allelic frequencies of A and B allele as 0.731 and 0.269 in Barki, 0.432 and 0.568 in Zaribi, 0.615 and 0.385 in Ardi and 0.473 and 0.527 in Masri goat breeds.
Othman et al., (2016) also observed similar type of polymorphism at
IGF-1 locus in Egyptian small ruminant breeds. Allele frequency for C and G alleles ranged from 0.69 to 0.72 and 0.28 to 0.31, respectively while genotypic frequency for CC, GC and GG genotypes ranged from 0.09 to 0.18, 0.22 to 0.38 and 0.53 to 0.59, respectively in Egyptian sheep and goat breeds.
Rasouli et al., (2017) reported polymorphic pattern in
IGF-1 gene in Markhoz goat population and found GG genotype to be most frequent (83%).
Darwish et al., (2017) also reported polymorphic pattern of
IGF-I in Egyptian Barki sheep with 3 genotypes ranging in frequency from 0.17 to 0.58. Similar to present investigation,
Negahdary et al., (2013) also documented polymorphic nature of
IGF-1 gene in Iranian Makooei sheep. Other studies by
Zhang et al., (2008b) in Chinese goats;
Tahmoorespur et al., (2009) in Iranian Baluchi sheep,
Kazemi et al., (2011) in Zel sheep;
Qiong et al., (2011) in Chinese goat breeds;
Kurdistani et al., (2013) in Kurdish goat of Iran;
Sharma et al., (2013) in Indian goat breeds;
Darwish et al., (2017) in Egyptian Barki sheep;
Grochowska et al., (2017) in Polish Merino sheep and
Rasouli et al., (2017) in Markhoz goats also reported polymorphic pattern at
IGF-1 locus.
Genetic diversity analysis and test for Hardy-Weinberg Equilibrium (HWE)
The amplified fragment of
IGF-1 gene showed polymorphism in all the populations investigated. The N
e, H
obs and H
exp ranged from 1.80 to 1.99, 0.30 to 0.43, 0.42 to 0.48 and 0.33 to 0.37, respectively in different populations. The N
e and H
obs was highest in Rampur-Bushair sheep followed by Gaddi sheep, Gaddi goat and Chegu goat. This indicates that at the studied locus polymorphism and genetic variation was highest in Rampur-Bushair sheep and lowest in Chegu goat.
The PIC ranged from 0.35 to 037 in different populations suggesting usefulness of marker in population studies, since in bi-allelic marker system the maximum possible value for PIC is 0.375
(Shete et al., 2000). The PIC revealed median level of polymorphism in studied breeds. In study of Chinese goat breeds,
Wu-Jun et al., (2010) reported H
obs, H
exp and PIC values of 0.52, 0.47 and 0.36, respectively which were in close agreement with present study. The positive FIS estimate indicates that mild heterozygous deficiency exists at studied locus for studied breeds. The F
IS estimates were 0.14, 0.10, 0.27 and 0.28, respectively in Gaddi sheep, Rampur-Bushair sheep, Gaddi goat and Chegu goat, respectively. The test for HWE indicated that Gaddi goat and Chegu goat populations were deviating significantly from equilibrium (P<0.01), while Gaddi sheep and Rampur-Bushair sheep populations are in HWE.
Wu-Jun et al., (2010) in Chinese goat breeds and
Negahdary et al., (2013) in Iranian Makooei sheep reported genotypic distribution at IGF-1 loci in agreement with HWE.
Sanger sequencing of PCR products
Purified PCR products of different genotypes were got sequenced. The sequences so generated were subjected to BLAST analysis so as to ascertain that whether they belong to IGF gene and to check accuracy of PCR-RFLP detection. The chromatogram representing the substitution is depicted in Fig 3. Alignment of sequences also confirmed presence of C/G substitution at 264 bp. Nucleotide variability was found at 17 places between different breeds.
Association of IGF-1 polymorphism with growth traits in Gaddi goats
In Gaddi goat population, the IGF-1 genotypes of 108 individuals were screened along with phenotypic data on growth traits. The mean squares at different ages are presented in Table 1 respectively. Among genotypes of
IGF-1 gene the body weight (kg) at 3M, 6M, 9M, and 12M ranged from 14.72±0.18 to 15.10±0.21, 19.40±0.36 to 19.92±0.31, 24.96±0.26 to 25.46±0.23 and 27.73±0.56 to 28.88±0.31, respectively. The body height (cm) at 3M, 6M, 9M, and 12M ranged from 54.70±0.77 to 55.33±0.67, 60.94±0.52 to 62.19±0.54, 61.79±0.61 to 62.44±0.93 and 62.50±0.36 to 64.66±0.49, respectively for different genotypes. The body length (cm) at 3M, 6M, 9M, and 12M ranged from 56.53±0.82 to 56.98±0.93, 62.06±0.52 to 62.54±0.45, 62.41±1.35 to 64.25±0.62 and 65.39±0.38 to 66.53±0.37, respectively for different genotypes. The corresponding body girth (cm) ranged from 55.34±0.67 to 55.72±0.74, 63.50±0.55 to 65.38±0.47, 70.41±0.57 to 71.00±0.80 and 73.75±0.42 to 74.66±0.50, respectively for different genotypes.
No significant association could be detected between
IGF-1 genotypes and growth traits in Gaddi goats. The earlier reports also showed that the results vary in association between
IGF-1 polymorphism and growth traits in different breeds. The results of present investigation were similar to those reported by
El-Hanafy et al., (2009) in Egyptian sheep breeds.
Gholibeikifard et al., (2013) also did not observe the effects of SNP, located in the third exon of the
IGF-I gene, on growth traits in Baluchi sheep. The present findings were in agreement with those of
Rasouli et al., (2017) in Markhoz goat who reported that
IGF-1 genotypes had no significant effect on birth weight and body weight at 6, 9 and 12 months.
However,
Zhang et al., (2008b) showed significant (P<0.05) association of
IGF-I gene polymorphism with birth weight, body weight at 6 and 12 months in Nanjing Huang goats.
Wu-Jun et al., (2010) also reported similar HaeIII digestion pattern as in current study along with significant association of IGF genotypes with body weight, cashmere yield and fiber diameter in cashmere goats.
Sharma et al., (2013) reported significant association of SNP of
IGF-1 gene with growth traits in Sirohi breed of goats.
Naicy et al., (2017) reported significant association of
IGF-1 genotype with Attappady Black goats of Kerala and differences in body weight, body height and chest circumference between genotypes were significant (P< 0.05).
Grochowska et al., (2017) also reported association of
IGF gene with carcass trait in Chinese Merino sheep. The literature reviews further showed that results vary in association of
IGF-1 polymorphism and growth traits in different breeds. Linkage disequilibrium of the
IGF-1 gene with QTLs could be one of the main reasons for varied results. Quantitative traits are regulated by large number of genes and are also affected by the interaction of these genes, so varied effect of a candidate gene associated with a particular trait in a population is observed.
