International Journal of Applied Sciences and Biotechnology Indexing and Abstracting

Genetic diversity of Sri Lankan goats ( Capra hircus ) was evaluated using Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method to detect polymophism in four candidate genes (LALBA, k-CSN3, GnRHR and BMP4) in a sample of 219 goats representing three phenotypic groups. All studied loci were polymorphic, having three morphs for BMP4, LALBA and two morphs for k-CSN3 and GnRHR in the study population. A significant difference between genotypic frequencies for BMP4 and GnRHR genes was found while PIC criterion revealed an intermediate polymorphism for all analyzed phenotypic groups except for k-CSN3 and GnRHR genes. Cross-bred animals for BMP4 gene and nondescript animals for LALBA gene were comparatively more polymorphic according to the effective allele number criterion. This study provides insight into the genetic diversity of Sri Lankan goats, which can be utilized to develop single nucleotide polymorphism markers to be used in association studies, and marker assisted selection.

The goat population in Sri Lanka consists mainly of a nondescript type local breed and their crosses with imported Jamnapari, Boer and Saanen animals. Among them, nondescript local goats are the highly adapted animals to local environmental conditions and are known to immune for disease and parasitic stresses compared to exotic breeds (Silva, 2010). At present only limited information is available on the genetic diversity of nondescript type local goats in Sri Lanka (Baker et al., 2001;Silva 2010). In this paper, we present the genetic diversity of four candidate genes (LALBA, k-CSN3, GnRHR and BMP4) detected utilizing Single Strand Conformational Polymorphism (SSCP) in goats of Sri Lanka. This is a highly cost effective, simple and rapid method used to detect variations in DNA compared to DNA sequencing technique which is intensively used for detecting mutations within the target sequences. The SSCP protocol is capable of producing different banding patterns on polyacrylamide gels in the presence of single nucleotide variations in the target (Neibergs et al., 1993;Sheffield et al., 1993) allowing rapid identification of genetic diversity of organisms.

Sample Collection
Altogether 219 goats representing phenotypically nondescript, cross-bred and pure Jamnapari animals were included in this study (Table 1). Genomic DNA was extracted from blood samples using either salting-out protocol (Jawdat et al., 2011) or a commercially available kit (Wizard Genomic DNA Purification Kit, Promega). Extracted DNA was quantified using a DNA spectrophotometer (BIOMATE 3) and lambda marker.

DNA amplification by PCR
Four gene fragments representing four candidate genes (LALBA, k-CSN3, GnRHR and BMP4) of the Capra hirucs genome were amplified. The details of the gene fragments, primer pairs used and respective annealing temperatures are given in the Table 2. PCR amplification of LALBA, k-CSN3, BMP4 and GnRHR was done as described by Jain et al., 2009;Kiplagat et al., 2010;Fang et al., 2009 andYang et al., 2011 respectively. PCR reactions were performed in a 25 µl reaction mixture containing 100-50 ng genomic DNA, 0.5 μM of each primer, 10× buffer 2.5 μl, 1.5 mM MgCl2, 200 μM dNTPs, 0.625 units of Taq DNA polymerase and water for each gene. All PCR amplifications were carried out in ABI2720 (Applied Biosystems®) Thermal Cyclers. Amplified PCR products were visualized on 1% agarose gel stained with ethidium bromide (1 µg/ ml) and verified using a 100 bp ladder (VC DNA Ladder Mix, Vivantis).
380 57 * Part of exon 2, intervening intron and part of exon 3

Single strand conformational polymorphism (SSCP) analysis
Number of samples included in SSCP analysis of each gene is presented in the table 3. A sample (4 µl) of each PCR product was mixed with a same amount of denaturation buffer (10 mM/L EDTA, pH = 8, 80 % (m/V) N, N-dimethyl formamide, 1mg/ ml Bromophenol blue, 1mg/ ml Xylene Cyanol) in PCR tubes, denatured at 95 ˚C in a thermo-cycler for 5 minutes, snap chilled on ice and resolved on polyacrylamide gels (12%). The electrophoresis was carried out in with a vertical unit (130 V, 5 W, 6 mA), in 1x TBE buffer at 4 ˚C for 18 hours. Gels were treated with (soaked in) silver staining to visualize the bands (Sun et al., 2002). The above protocol was further modified for polyacrylamide gel concentration, volume of PCR product, volume and concentration of denaturation buffer components and gel running time for LALBA and k-CSN3 genes to get an optimum resolution (Bastos et al., 2001). The gel phenotypes were identified and scored manually to obtain PCR-SSCP allele frequency after ascertained the reproducibility.

Statistical analysis
Genotypic and allelic frequencies for LALBA, k-CSN3, BMP4 and GnRHR loci within and between nondescript, cross-bred and pure Jamnapari populations were analyzed using Chi square test. Homozygous dominant, homozygous recessive and heterozygous genotypes were selected according to Hardy-Weinberg equilibrium equation. Gene heterozygosity, gene homozygosity, effective allele numbers and Polymorphism Information Content (PIC) were calculated by Nei methods (Nei and Roychoudhury, 1974;Nei and Li, 1979).

Results and discussion
All four gene fragments were polymorphic. BMP4 and LALBA genes showed three patterns whereas k-CSN3 and GnRHR gene fragments showed only two patterns (Fig. 1) showed all three genotypic patterns whereas Jamnapari showed only two homozygous genotypes (Table 3). For BMP4 gene, AA genotype was predominant among both nondescript and cross-bred animals (66% and 72% respectively). Nondescript animals showed 40%, 38% and 22% genotypic distribution among AA, AB and BB genotypes for LALBA gene while cross-bred animals were represented by AA genotype (100%) only. The k-CSN3 gene had only two genotypes and AA was unique to nondescript ones (100%) and the BB was restricted to crossbred animals (100%). GnRHR gene was limited for homozygous genotypes (AA and BB) where BB genotype (85%) was the most shared among nondescript animals while AA genotype (75%) was shared amongst cross-bred animals (Table 3). A significant difference between genotypic frequencies for BMP4 and GnRHR genes was found (χ 2 = 12.29, df = 4, P = 0.001 and χ 2 = 54, df = 2, P = 0.00001 respectively). Our observed homozygosity values are higher than expected heterozygosity values for all the genes examined (Table 3) except for cross bred animals in BMP4 gene where both values were equal (Ho= 0.5, He=0.5). This could be due to subpopulation structure and inbreeding system of mating (Wahlund, 1928) taking place among the goats in Sri Lanka.  According to the classification of PIC, PIC < 0.25 is considered as low polymorphism, 0.25 < PIC < 0.5 as intermediate polymorphism and PIC > 0.5 as high polymorphism (Ma et al., 2010). However, as described by the PIC criterion all our analyzed breeds possessed intermediate polymorphism for all the genes tested except CSN3 and GnRHR genes. Both nondescript and cross-bred animals showed low polymorphism (PIC=0) for k-CSN3 whereas nondescripts were highly polymorphic (P=0.76) for GnRHR gene (Table 3).
Higher effective allele number (Ne) indicates higher polymorphism (Lan et al., 2007) and it ranges from 1-2 in our studied goat population (Table 3). In this population the most polymorphic group was cross-bred animals for the BMP4 locus with the recorded Ne of 2 and the other two breeds (nondescript and Jamunapari) were equally diverse. For the LALBA gene, nondescript local goats showed the highest diversity (Ne= 1.9168) over the cross breds (Ne=1). Cross bred and non-descript animals for both k-CSN3 and GnRHR gene were also equally polymorphic.
Many authors have suggested that SSCP is a reliable, reproducible (Neibergs et al., 1993, Sheffield et al., 1993 and cost effective method compared to DNA sequencing (Orita et al., 1989) for detecting structural variations in genome due to point mutations. According to studies by Yang et al., (2011) and Kiplagat et al., (2010) respectively on exon 1 of GnRHR gene and exon 4 of k-CSN3 gene indicated that these genes possess only two haplotypes and the results of our study confirm the above finding for Sri Lankan goats by showing only two haplotypes for both genes in Sri Lankan goats as well. Further, findings of this study reveal that both exon 2 of LALBA and intron 2 of BMP4 gene possess three haplotypes that were previously described by Cosenza et al., (2003) and Xing-Tang et al., (2010) respectively. The present study is a part of ongoing genetic characterization project aims at detection and development of genetic markers for marker-assisted selection, intended for improving the quantity and quality of goat production in Sri Lanka. DNA sequencing and association analysis with production traits are progressing for all four gene fragments at present.

Conclusion
All four gene regions analyzed are polymorphic in the sample of goats used in this study. A significant difference between genotypic frequencies for BMP4 and GnRHR genes was found while PIC criterion revealed an intermediate polymorphism for all analyzed phenotypic groups except for k-CSN3 and GnRHR genes. This study provides preliminary evidence for genetic polymorphism of Sri Lankan goats, which can be utilized to develop single nucleotide polymorphism markers to be used in association studies, and marker assisted selection. This will be critical for decision making on suitable management systems at farmers' level especially for low input systems in the country.