SNPs identification and allele frequencies
The GH1, GH2, and GH6 polymorphisms were examined at three different loci within the GH gene. The HaeIII restriction enzyme was accountable for the digestion of the SNP located at GH1-HaeIII. In both breeds, restriction digestion yielded two alleles, designated A (an uncut segment of 422 bp) and B (366 bp and 56 bp segments). Genotypes BB (366 bp & 56 bp) and AB (422 bp, 366 bp & 56 bp) were identified with no homozygous genotype AA detected in the studied breeds. All of the tested breeds were found to have a preponderance of the AB genotype and the B allele.
Concerning the GH2 gene, the SNP at GH2-HaeIII was digested by the HaeIII restriction enzyme. This fragment generated two alleles in the tested breeds: C (an uncut fragment of 116 bp) and D (fragments of 88 and 28 bp). At this locus, the homozygous genotype CC (116 bp) and heterozygous genotype CD (116, 88, and 28 bp) were detected without the homozygous genotype DD in both goat breeds (Fig. 2a).
Regarding the GH6 gene, the restriction site was revealed in the GH6-HaeIII site, and following restriction digestion with HaeIII, only one allele (B) was generated, while allele A was absent. Among the investigated breeds, only genotype BB was found (108, 78, 70, 48, 44, 40, and 17 bp) (Fig. 3a). According to our results, the studied goat breeds showed no polymorphisms in the GH6 gene, indicating monomorphism.
Genetic Parameters Of Populations And Hardy Weinberg Equilibrium (Hwe)
In the studied breeds, two polymorphic nucleotide alterations were found. Assam hill and Sirohi goats have a heterozygous and homozygous genotype at GH1-HaeIII and GH2-HaeIII loci and only one genotype at GH6-III locus. At GH1-HaeIII and GH2-HaeIII loci, displayed two genotypes (homozygous and heterozygous) but only one genotype was at GH6-III locus. Allele A of locus GH1-HaeIII was shown to have the lowest frequency (0.36), whereas allele B of the same locus was revealed to have the highest frequency (0.64) in the Assam hill goat. Similarly, it was found that allele A of locus GH1-HaeIII was present at a minimum frequency (0.28), whereas allele B was present at a maximum frequency (0.72), in Sirohi goats (Table 2). The population stability at the GH1-HaeIII and GH2-HaeIII loci was tested using a Hardy-Weinberg equilibrium (HWE) analysis. Hardy Weinberg Equilibrium significantly deviated in all SNPs loci among studied breeds (P < 0.05). It is possible for the population to become significantly differ from HWE as a result of genetic drift, selection pressure at a specific locus, and small sample size.
Table 2
Genetic parameters of the GH1, GH2, and GH6 genes in Assam Hill and Sirohi goats
Assam Hill goat |
Gene | Genotype frequency | Allele frequency | Ne* | I* | Nei* | Obs_Hom | Obs_Het | Exp_Hom* | Exp_Het* | Ave_Het | PIC* | χ2* |
GH1-HaeIIII | AA AB BB 0.32 0.82 0.00 | A B 0.36 0.64 | 1.85 | 0.65 | 0.46 | 0.28 | 0.71 | 0.5374 | 0.46 | 0.46 | 0.35 | 35.45 |
GH2-HaeIIII | CC CD DD 0.48 0.66 0.00 | C D 0.71 0.29 | 1.69 | 0.60 | 0.41 | 0.42 | 0.57 | 0.58 | 0.41 | 0.41 | 0.32 | 18.58 |
GH6 | AA AB BB 0.00 0.00 1.00 | A B 0.0 1.0 | - | - | - | - | - | - | - | - | - | - |
Sirohi goat |
GH1-HaeIIII | AA AB BB 0.10 0.37 0.00 | A B 0.28 0.72 | 1.91 | 0.67 | 0.47 | 0.21 | 0.78 | 0.51 | 0.48 | 0.47 | 0.36 | 19.19 |
GH2-HaeIIII | CC CD DD 0.18 0.28 0.00 | C D 0.70 0.30 | 1.74 | 0.61 | 0.42 | 0.38 | 0.61 | 0.56 | 0.43 | 0.42 | 0.32 | 8.97 |
GH6 | AA AB BB 0.00 0.00 1.00 | A B 0.0 1.0 | - | - | - | - | - | - | - | - | - | - |
The symbols ne*, I*, Nei*, Obs Hom*, Obs Het*, Exp Hom*, Exp Het*, Ave Het*, PIC*, and χ2 stand for the following terms: effective number of alleles, Shannon's information value, observed homozygosity, probable heterozygosity, expected homozygosity, expected heterozygosity, average heterozygosity, polymorphic information content, and chi-square value. |
Polymorphic Information Content
All of the loci's PIC values in the current investigation were moderate, ranging between 0.32 to 0.35 and 0.32 to 0.36 in Assam hill and Sirohi goats, respectively. In both breeds, the GH2-HaeIII locus had the lowest PIC value (0.32), while the GH1-HaeIII locus had the highest PIC value (0.36). A marker’s PIC value is used to calculate its informativeness from a statistical perspective. Allelic frequencies of each SNP locus were used in calculating the PIC. The PIC values ranged from 0 to 1, where 0 indicates that there is only one allele for that marker and 1 indicates that there could be an infinite number of alleles for that marker. The PIC value should be at least 0.77 to be considered highly informative. However, it can reach a maximum value of 0.38 when using biallelic markers (Botstein et al. 1980). PIC values for the two breeds were found to be comparable in the current investigation.
Heterozygosity And Genic Variation Statistics
It was observed that all polymorphic markers had two alleles, whereas the effective number of alleles (ne) in Assam hill goats varied from 1.69 (GH2-HaeIII) to 1.85 (GH1-HaeIII). It was determined that the number of effective alleles in Sirohi goats ranged from 1.74 for GH2-HaeIII to 1.91 for GH1-HaeIII. The effective number of alleles is the number of alleles that, if equally prevalent, would contribute to the same level of heterozygosity as found in the investigated population. Selective pressure may be exerted on these SNPs, as indicated by their moderate to high effective allele frequencies. The average heterozygosity values for all loci varied between 0.41 (GH2-HaeIIII) to 0.46 (GH1-HaeIIII) in Assam hill goat and between 0.42 (GH2-HaeIIII) to 0.47 (GH1-HaeIIII) in Sirohi goat.
For all loci, Information indexes for Shannon ranged from 0.60 (GH2-HaeIIII) to 0.65 (GH1-HaeIIII) in the Assam hill goat population and from 0.61 (GH2-HaeIIII) to 0.67 (GH1-HaeIIII) in the Sirohi goat population (Table 2). In the Assam hill goat population, the average homozygosity value was 0.35, while for the Sirohi goat population, it was 0.29. Despite this, Assam hill goats had an expected homozygosity value of 0.55 and Sirohi goats had an expected homozygosity value of 0.53 (Table 2). Based on the genic variation statistics and genotypic arrays, both populations were found to be heterozygote deficient.
Analyzing The Linkage Disequilibrium (Ld) Between Snps In The Assam Hill And Sirohi Goat Populations
It was determined that SNPs loci in the studied population had exceptionally high pairwise LD. This was demonstrated by the fact that more than 0.50 and 0.20 were found for pairwise Lewontin's D' and r2 values, respectively. The SNPs' D' and r2 values in both breeds are quite similar to one another (Fig. 4).
Relationship Between Genotypes And Growth Traits In Assam Hill And Sirohi Goats
Associations of different variation loci with the four growth characteristics such as weight, height at wither, length, and chest girth of different goat breeds are given in Tables 3–6. At GH1-HaeIII locus, Assam hill goats with genotype AB were found to be significantly (P < 0.05) superior in body length compared to those with genotype AA at 6 months of age (Table 3). AB genotype had slightly higher values in most of the growth traits as compared to the AA genotype, though the superiority was statistically not significant (P > 0.05). Similarly, it was found that genotype AB performed better than genotype AA in Sirohi goats at GH1-HaeIII locus at all ages; however, none of them showed a statistically significant difference (Table 4; P > 0.05).
Table 3
Association of GH1-HaeIII genotypes of GH gene with growth and morphometric attributes (Mean ± S.E.) in Assam Hill goat
Growth traits | Genotypes | P value |
AA | AB |
1-month body weight (kg) | 1.92 ± 0.43 | 2.01 ± 0.07 | 0.827 |
1-month body length (cm) | 23.13 ± 0.89 | 23.13 ± 0.89 | 0.316 |
1-month height at withers (cm) | 28.49 ± 0.73 | 28.49 ± 0.73 | 0.289 |
1-month chest girth (cm) | 33.45 ± 1.19 | 33.45 ± 1.19 | 0.956 |
3-month body weight (kg) | 5.37 ± 0.11 | 4.78 ± 0.06 | 0.312 |
3-month body length (cm) | 26.77 ± 0.53 | 26.77 ± 0.53 | 0.411 |
3-month height at withers (cm) | 33.60 ± 0.76 | 33.60 ± 0.76 | 0.203 |
3-month chest girth (cm) | 42.11 ± 1.09 | 42.11 ± 1.09 | 0.178 |
6-month body weight (kg) | 7.50 ± 0.12 | 7.14 ± 0.08 | 0.266 |
6-month body length (cm) | 32.35 ± 1.45 | 34.08 ± 1.23 | 0.043 |
6-month height at withers (cm) | 38.55 ± 0.86 | 38.55 ± 0.86 | 0.518 |
6-month chest girth (cm) | 48.03 ± 2.33 | 48.03 ± 2.33 | 0.910 |
Table 4
Association of GH2-HaeIII genotypes of GH gene with growth and morphometric attributes (Mean ± S.E.) in Assam Hill goat
Growth traits | Genotypes | P value |
CC | CD |
1-month body weight (kg) | 1.87 ± 0.11 | 1.93 ± 0.25 | 0.612 |
1-month body length (cm) | 22.01 ± 0.66 | 23.20 ± 0.06 | 0.210 |
1-month height at withers (cm) | 28.11 ± 0.82 | 27.84 ± 0.43 | 0.139 |
1-month chest girth (cm) | 34.21 ± 1.07 | 34.07 ± 1.40 | 0.671 |
3-month body weight (kg) | 5.10 ± 0.79 | 5.22 ± 0.11 | 0.393 |
3-month body length (cm) | 26.03 ± 0.07 | 27.18 ± 0.60 | 0.508 |
3-month height at withers (cm) | 35.48 ± 0.23 | 34.28 ± 0.46 | 0.189 |
3-month chest girth (cm) | 41.66 ± 1.23 | 43.09 ± 1.51 | 0.049 |
6-month body weight (kg) | 8.07 ± 0.37 | 9.12 ± 0.47 | 0.451 |
6-month body length (cm) | 34.07 ± 1.68 | 34.12 ± 1.10 | 0.638 |
6-month height at withers (cm) | 38.04 ± 0.92 | 39.11 ± 0.30 | 0.482 |
6-month chest girth (cm) | 48.03 ± 2.33 | 47.66 ± 2.05 | 0.721 |
Table 5
Association of GH1-HaeIII genotypes of GH gene with growth and morphometric attributes (Mean ± S.E.) in Sirohi goat
Growth traits
|
Genotypes
|
P value
|
AA
|
AB
|
1-month body weight (kg)
|
4.06 ± 0.14
|
4.13 ± 0.23
|
0.152
|
1-month body length (cm)
|
35.10 ± 0.58
|
35.37 ± 0.41
|
0.434
|
1-month height at withers (cm)
|
36.08 ± 0.23
|
37.11 ± 0.31
|
0.170
|
1-month chest girth (cm)
|
36.21 ± 0.09
|
35.04 ± 0.72
|
0.542
|
3-month body weight (kg)
|
10.81 ± 0.18
|
11.12 ± 0.07
|
0.319
|
3-month body length (cm)
|
44.32 ± 1.41
|
46.51 ± 1.33
|
0.110
|
3-month height at withers (cm)
|
50.66 ± 1.18
|
49.05 ± 1.84
|
0.270
|
3-month chest girth (cm)
|
50.12 ± 1.30
|
51.07 ± 1.62
|
0.188
|
6-month body weight (kg)
|
15.07 ± 0.42
|
16.11 ± 0.53
|
0.506
|
6-month body length (cm)
|
52.07 ± 1.17
|
51.09 ± 1.08
|
0.741
|
6-month height at withers (cm)
|
58.63 ± 0.72
|
59.12 ± 0.53
|
0.302
|
6-month chest girth (cm)
|
57.84 ± 0.90
|
58.06 ± 0.32
|
0.690
|
Table 6
Association of GH2-HaeIII genotypes of GH gene with growth and morphometric attributes (Mean ± S.E.) in Sirohi goat
Growth traits | Genotypes | P value |
CC | CD |
1-month body weight (kg) | 4.10 ± 0.32 | 4.34 ± 0.10 | 0.134 |
1-month body length (cm) | 34.98 ± 0.13 | 35.04 ± 0.27 | 0.367 |
1-month height at withers (cm) | 36.11 ± 0.64 | 37.08 ± 0.82 | 0.210 |
1-month chest girth (cm) | 35.82 ± 0.16 | 35.01 ± 0.90 | 0.340 |
3-month body weight (kg) | 10.13 ± 0.55 | 10.84 ± 0.21 | 0.312 |
3-month body length (cm) | 44.05 ± 1.70 | 45.98 ± 1.01 | 0.215 |
3-month height at withers (cm) | 50.12 ± 1.07 | 49.66 ± 1.12 | 0.210 |
3-month chest girth (cm) | 50.34 ± 1.12 | 51.02 ± 1.50 | 0.171 |
6-month body weight (kg) | 14.99 ± 0.16 | 15.28 ± 0.69 | 0.423 |
6-month body length (cm) | 52.13 ± 1.99 | 52.02 ± 1.07 | 0.599 |
6-month height at withers (cm) | 58.02 ± 0.69 | 59.20 ± 0.17 | 0.168 |
6-month chest girth (cm) | 57.02 ± 0.81 | 58.11 ± 0.27 | 0.610 |
In terms of the GH2-HaeIII locus, it was revealed that Assam hill goats with the genotype CD had significantly (P < 0.05) higher girth of the chest with genotype CC at the age of three months (Table 5); nevertheless no statistical differences (P > 0.05) were observed between different genotype variants with rest of the records of growth traits (Table 5). In Sirohi goat at the GH2-HaeIII locus, CD genotype individuals exhibited superior body weight and biometric features at all ages than AA genotype, but no significant differences were detected (P > 0.05; Table 6). These outcomes align with those found by Singh et al. (2015) in Sirohi and Barbari goats regarding body length across the lifespan.
AB and CD genotypes had slightly higher values in most of the growth traits studied as compared to AA and CC genotypes, though the superiority was statistically significant (P < 0.05) with only body length and chest girth. The results of Hua et al. (2009) indicated that genotypes did not significantly affect birth weight, height, length, chest circumference, or body weight at 11 months of age; however, they did affect significantly chest circumference at birth and weight during weaning. A similar study by Zhang et al. (2008) in Nanjiang Huang goat and Deng et al. (2010) in Xinong Saanen goats found that body weight and GH gene expression were substantially associated at different ages. Sarmah et al. (2020) found no significant influence of GH polymorphism on body weights among the various genotypes in Assam hill goats, which is consistent with the findings of the present investigation. Similarly, Aradhana et al. (2021) showed the AB genotype was connected to higher means on morphometric features in the Ganjam and Baigani goat populations of Odisha; however, the differences were not statistically significant. Findings that are comparable to these were found by Wickramaratne et al. (2010) in Sangamneri goats for various regions of the GH gene. In contrast to our observations, Pandya et al. (2021) found that at six months of age, goats with BB genotypes had a superior body mass (P < 0.05) than those with AB genotypes in Surti Goats. Singh et al. (2015) observed similar findings for body length in Sirohi and Barbari goats across all ages; however, statistical analysis failed to reveal significantly differ (P > 0.05) in body mass across the various genotypes.
Numerous observations indicate that genetic polymorphisms in the GH1 gene influence livestock economic characteristics (Carter-Su et al. 2016; Liu et al. 2016; Wang et al. 2022; Abdelhafez et al. 2022). It was recently demonstrated by Yan et al. (2022) that two indel polymorphism loci, P49-bp indel (ID) and P1410-bp indel (II) were correlated with chest girth and chest width in Shaanbei white cashmere goats (SBWC). The same findings were reported by Esen and Elmac (2022), who found that yearling lambs with the P1 variant of the growth Hormone Exon-5 (GHE5) gene have a higher body length (BL), wider leg girth, and lean cannon bone periphery (CBP) (P > 0.05), in comparison to lambs with the other variant (P2), whose body length is shorter and CBP is thicker. Selionova et al. (2022) revealed a favourable connection between the molecular marker among MSTN, HEG1, FGF10, FGF14, GHRH, and SLAIN genes linked to live body mass at age four months old. It was also demonstrated by SNP genotyping and their use for discovering selection signatures and applying them as part of GWAS for identifying polymorphic loci associated with various biological aspects and body size in sheep and goats (Gu et al. 2022; Saleh et al. 2022). As a result, this research led to the finding that the polymorphisms in the GH gene in goat populations might be responsible for the superior growth traits.