Characterization of Krishna Valley breed of cattle (Bos indicus) in south India using microsatellite markers

S M K Karthickeyan, R Saravanan and P Thangaraju

Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai - 600 007, Tamil Nadu, India
kannikarthi@yahoo.co.in

Abstract

With the help of 25 microsatellite markers, a total of 50 Krishna Valley cattle were screened.

The mean number of alleles was found to be 4.72 ± 0.25 per microsatellite locus with the range of 3 to 7. The range in the allele size was from 94 (CSRM060) to 300 (ILSTS006) bp. These microsatellite alleles distributed at a minimum frequency of 0.0116 (ILSTS054) to a maximum of 0.8128 (ILSTS030). The mean polymorphism information content was found to be 0.6209 ± 0.03 ranging from 0.2583 (ILSTS030) to 0.7975 (INRA035). The overall means for observed and expected heterozygosities were 0.6683 ± 0.06 and 0.6569 ± 0.03 respectively.

The markers used in the study were highly informative and the high heterozygosity value is indicative of the higher amount of genetic variability that can be exploited even in populations of small size.

Keywords: Allele frequency, cattle, Krishna Valley, microsatellites, PIC

Introduction

KrishnaValley is the draught breed of cattle, braving extreme hot, humid climatic conditions and is able to work well in the black cotton soil in the valleys of Krishna river in Karnataka state in India. It was reported that the kings of Southern Mahratta country, in the watershed areas of the rivers Krishna, Ghataprabha and Malaprabha, tried to evolve a powerful bullock for agricultural purposes in the sticky black cotton soil during the last two decades of the nineteenth century (Joshi and Phillips 1953). It was claimed that Gir and possibly Kankrej cattle from Gujarat state, Ongole cattle from Andhra Pradesh state and local cattle having Mysore-type blood were used to evolve the Krishna Valley breed. The king of Sangli, a well-known breeder of Krishna Valley, contributed substantially in making judicious use of all these strains to produce the desired type of animal.

Earlier, the distribution of Krishna Valley cattle was wide, including the districts of Satara, Sangali and Solapur in Maharashtra; and Belgaum, Bijapur and Raichur districts of Karnataka states (Nivsarkar et al 2000). But a shift in the breeding tract of this breed from Maharashtra and Karnataka states to northern Karnataka alone was reported in a pilot survey conducted by Ramesha et al (2001). At present, only a few hundred animals true to type are available in and around few villages of Jamkhandi, Mudhol and Athani taluks of northern parts of Karnataka. The reasons for the decline in number are selling of animals of Krishna Valley due to continuous drought in the tract and preference of the farmers for Khillari breed which is more attractive and massive in appearance resulted in lack of Krishna Valley breeding bulls.

The first step towards conservation of livestock genetic resources is the genetic characterization with respect to phenotypic parameters, unique qualities and utility. Subsequently finding out the genetic architecture through molecular means and evolutionary relationship with other related breeds would provide valuable information about the breed for taking up conservation measures. The physical characterization had already been done by Ramesha et al (2001) in the native tract. Considering these facts, the present study has been carried out to characterize the Krishna Valley breed of cattle using the molecular markers, such as microsatellites.

Materials and methods

DNA samples

The microsatellite analysis was carried out in a sample of 50 unrelated Krishna Valley cattle reared in the villages of Jamkhandi Taluk (part of its breeding tract) in Karnataka State, India. The samples were collected at random form different villages of the tract. The DNA for the study was isolated from peripheral blood by a rapid, non-enzymatic method as described by Lahiri and Nurnberger (1991). The quantity and quality of the isolated DNA samples were tested by spectrophotometric measurements for subsequent analysis.

Microsatellite analysis

As per the secondary guidelines of Food and Agriculture Organisation of United Nations (FAO 2004), a total of 25 microsatellite markers were selected to screen the population of Krishna Valley cattle. The amplification of DNA was carried out using thermal cycler (MJ Research) with the PCR reaction mixture of 20ml. The mixture was prepared by adding 50-100ng of template DNA; 1.5mM MgCl₂; 5 picomoles each of forward and reverse primers; 0.75 units of Taq DNA Polymerase and 100mM dNTPs. Amplification was carried out with annealing temperatures ranging from 51°C to 60°C for different primers for 30 cycles. Amplified PCR products were resolved through a 6% denaturing PAGE along with a single stranded 10 bp DNA ladder (Invitrogen, USA) as marker and the PCR products were sized after silver-staining procedure as recommended by Comincini et al (1995).

The microsatellite alleles were sized using Diversity Database software (BioRad, USA) and followed by manual verification. Effective number of alleles, allele frequencies and heterozygosity were estimated using the software POPGENE 32. The polymorphism information content (PIC) was analysed using Nei's formula (1978).

Results and discussion

The percentage of polymorphic loci obtained was 96 since all microsatellite loci, except ILSTS030 (only 2 alleles), screened in Krishna Valley cattle exhibited polymorphism. The microsatellite allele number, size and frequency, polymorphism information content and heterozygosity values are presented in Table 1.