Kanniadu goats of Tamilnadu, India: genetic characterisation through microsatellite markers

K Thilagam, J Ramamoorthi, S N Sivaselvam, S M K Karthickeyan and P Thangaraju

Department of Animal Genetics and Breeding, Madras Veterinary College
Tamilnadu Veterinary and Animal Sciences University, Chennai - 600 007, India
snsivaselvam@yahoo.com

Abstract

Characterisation of Kanniadu goats was done using microsatellite markers. The genomic DNA from 50 unrelated Kanniadu goats were PCR-amplified with a panel of 20 microsatellite markers and resolved through 6 per cent denaturing polyacrylamide gel electrophoresis followed by silver staining.

The number of alleles ranged from 5 to 14 with allele sizes ranging from 90 to 222bp. The allele frequencies ranged from 0.0106 to 0.4480. Polymorphism information content ranged from 0.5710 to 0.8570. Except four loci, the population was not in Hardy-Weinberg equilibrium. The observed heterozygosity ranged from 0.7142 to 0.9778 while the expected heterozygosity ranged from 0. 6390 to 0.8702, indicating the heterogenous nature of the population distributed in the breeding tract.

Key words: genetic characterisation, heterozygosity, Kanniadu goats, microsatellite markers, PIC

Introduction

In the world of animal production, a good method to identify animals by using faster and more reliable methods is of utmost importance in order to obtain accurate selection. Because of the negative effects of indiscriminate crossbreeding programmes, there is urgent need to prevent rapid erosion of animal genetic resources. This is especially true for breeds in developing countries where many will be lost without ever having been adequately characterised and studied. However, such breeds may still be a valuable source of major genes such as for disease resistance, tolerance to extreme climatic conditions and thriving well in low input system of management. Genetic characterisation of native breeds is the first step in conservation programme, as it will help the decision makers to identify genetically unique breeds so that they may be prioritized for the purpose of breed conservation.

In genetic characterisation, though identification methods such as typing of blood groups and biochemical polymorphisms had proved their usefulness to some extent, the discriminating power of these techniques is less than that of DNA markers. The DNA markers are more powerful and not affected by environmental influences. The markers should be selectively natural, so that observed similarities are attributable to common ancestry and not convergence. At present microsatellites are being used successfully to define genetic structures and genetic relationships among different breeds. Microsatellite analysis of genetic diversity provides two distinct levels of information. In addition to allele frequency differences among populations, it also provides information about the cladistic relationships between alleles on the basis of differences in allelic repeat units.

Goats are widely distributed and adaptable ungulate species. They are very important and economical animals for the rural poor. India has 20 recognised breeds of goats, of which Kanniadu is one among them; a native breed of Tamilnadu State of southern India. The Kanniadu goat is a prolific meat breed thriving well in the tropical draught conditions. They are tall animals, predominantly black in colour with white or brown stripes on either side of the face and white or brown under belly and inner sides of legs. The special features of Kanniadu goats are early sexual maturity, high prolificacy (67 per cent of multiple births) and low adult mortality. They are reared under sedentary system of management. The estimated population was 98,000 in the breeding tract (Thiruvenkadan et al 2000). With this background, the present study was carried out to know the genetic structure of Kanniadu goats of Tamilnadu with the help of microsatellite markers.

Materials and methods

The Food and Agriculture Organization of United Nations (FAO) has proposed an integrated programme for the global management of genetic resources (Project MoDAD, http://www.fao.org/dad-is) using microsatellite methodology for breed characterization. Twenty microsatellite markers were used in the present study. Detailed information on these markers is presented in Table 1. Blood samples were collected at random from 50 Kanniadu goats (aged above one year), distributed in different villages of the breeding tract. Genomic DNA was isolated from five ml of blood by a rapid non-enzymatic method (Lahiri and Nurnberger 1991). The amplification reactions were carried out using a programmable thermal cycler (MJ Research, USA) in reaction volume of 20µl. Each 20µl PCR reaction contained 50 to 100ng template DNA; 5pmol of each primer; 500µM each of dNTPs; 0.75 unit of Taq DNA polymerase; 1µl of 1.5mM MgCl₂. The primer annealing for 45 sec at the desired temperature of 50, 53, 55 or 58°C was adopted. The amplified PCR products were electrophoresed in 6% denaturing urea polyacrylamide gel at a constant voltage of 1300 volts for a period of 1 to 2 h, depending upon the size of PCR products and then silver-stained (Comincini et al 1995). The gels were blotted and dried at 80°C for 1h. The alleles were scored manually and analysed by software-aided gel documentation system (Bio-Rad, USA). The exact allele sizes were determined by direct comparison with adjacent PCR bands and 10bp ladder. Allele frequencies were estimated by direct counting from the genotypes of the goats. The polymorphism information content (PIC) was calculated using the individual frequencies in which the alleles occur at each locus (Nei 1978). The heterozygosity at the locus was estimated (Nei 1973). The expected number of genotypes was compared with the observed genotypes in a χ² test for goodness of fit to assess whether the study population was in Hardy-Weinberg equilibrium (HWE).

Results

The results of the microsatellite analysis of Kanniadu goats in terms of number of alleles observed, alleles size, polymorphism information content and heterozygosity were furnished in Table 1.

In total, 163 alleles were observed for the 20 microsatellite loci with a mean of 8.2 per locus. The number of alleles per locus ranged from 5 (ILSTS078) to 14 (ILSTS040). The allele sizes ranged from 90bp (ILSTS001) to 222bp (ILSTS060). The frequency of the alleles ranged from 0.0106 (ILSTS001) to 0.4480 (ILSTS029). The polymorphism information content (PIC) values ranged from 0.5710 (ILSTS029) to 0.8570 (ILSTS004 and ILSTS087). Except four loci, ILSTS001, ILSTS040, ILSTS044 and ILSTS049, the differences between observed and expected number of genotypes were significant (P≤0.01) at the other remaining 16 loci indicating that these loci were not in Hardy-Weinberg equilibrium. The observed heterozygosity ranged from 0.7142 to 0.9778 while the expected heterozygosity ranged from 0.6390 to 0.8702 across the microsatellite markers studied.

Discussion

Allele numbers and sizes

There is lack of information in Indian goats on the number of alleles, their sizes and frequencies at various microsatellite loci. The mean number of alleles observed in the present study was higher than that reported by Ganai and Yadav (2001) in three Indian goat breeds using heterologous microsatellite markers - Sirohi (4.12), Jamnapari (4.00) and Barbari (3.37). The available literature on exotic goat breeds revealed that the number of alleles was seven for ILSTS008, nine for ILSTS029 and three for ILSTS059 in Swiss goat breeds (Saitbekova et al 1999) and 11 for ILSTS087 in Moroccan and French goat breeds (Ouafi et al 2002). It is reported that there was a positive relationship between the number of dinucleotide repeats and number of alleles at a given locus and that the number of alleles per locus might range from one to 18 (Bishop et al 1994). The allele sizes obtained in the present study are similar to Barbari (88 to 220 bp) goats and lower than Tellicherry goats (92 to 298 bp), the other two Indian goat breeds studied earlier (Ramamoorthi 2003).

Polymorphism information content

An average PIC value of 0.48 was reported in three Indian goat breeds (Sirohi, Jamnapari and Barbari) using cattle microsatellite markers (Ganai and Yadav 2001), which was lower than the values obtained in the present study. However, PIC estimated in the present study are comparable with those values obtained in Chinese goat breeds, which ranged from 0.746 to 0.800 (Yang et al 1999) using ovine microsatellite markers. In contrast, lower PIC values were obtained for Korean (0.350), Chinese (0.620) and Saanen (0.570) goats (Kim et al 2002). Based on the PIC values, the microsatellite markers can be well utilized for molecular characterization of goat breeds.

Hardy-Weinberg equilibrium

The present population of Kanniadu goats was in HWE only at four loci. The deviation of 80 per cent of the loci (16 out of 20) from the HWE might be due to many causes such as existence of "null" alleles, high mutation rate and size homoplasy of microsatellite loci, besides the small study population. Similar disequilibria were also reported in microsatellite analysis of Korean and Chinese goat breeds (Kim et al 2002).

Heterozygosity

The heterozygosity is an appropriate measure of genetic variability within a population. The high observed and expected heterozygosity values obtained in the study are comparable with the values reported in Chinese goat breeds, 0.777 to 0.823 (Yang et al 1999). However, in Korean and Chinese goats, lower heterozygosity values (0.351 and 0.671) were reported for bovine, ovine and caprine microsatellite markers (Kim et al 2002). In indigenous goat breeds of South Africa, the estimated heterozygosity values were 0.63 to 0.69 and in Boer goats, it was 0.49 (Visser et al 2004). The higher heterozygosity observed in the present study might be due to the random sampling from the open population, where there was no chance of mixing or inbreeding, which has resulted in instability of the population at the majority of microsatellite loci studied. Because of higher heterozygosity, there is further scope for improvement of the breed and it can be effectively used to identify quantitative trait loci.

To conclude, the present study shows that all the microsatellite markers used were highly polymorphic and informative for molecular characterization. The population remains unstable and highly heterozygous with respect to the loci screened. The results of the present study have elucidated the genetic structure of the Kanniadu goats.

References

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Received 4 August 2006; Accepted 7 September 2006; Published 4 October 2006

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