Evaluation of Genetic Diversity in Goats of Telangana and Andhra Pradesh States of India

India is a rich repository of goat genetic resources with 34 registered goat breeds distributed in different parts of the country (NBAGR, 2019). These breeds/populations have evolved through natural selection and selective breeding and are adapted to different agro-ecological conditions. In addition to these well-defined breeds there are several goat populations which are yet to be categorized as a breed. Some states like Bihar, Jharkhand, Telangana, Andhra Pradesh, Haryana, Chhattisgarh and many states of NEH region although have a good goat population but no registered goat breed. The goat population of undivided Andhra Pradesh was 90,71,221 (LC, 2012).  As per the latest livestock census (LC, 2018) this population is 55, 22,133 in new Andhra Pradesh and 49,34,673 in Telangana. It is not known whether these populations have any uniform population. Some reports are available on the characterization of Mahbubnagar goats (Ekambaram et al., 2010, Dasari et al., 2018, Raghavendra et al., 2017) which exist in areas adjoining the Srisailam Hydel Project and the Nallamala forest of Mahbubnagar district of Telangana state. Mahbubnagar goats are also known by local name of “Palamuru”. A study on the goats of Telangana and Andhra Pradesh states has been carried out to estimate the genetic diversity among them and to establish the unique genetic identity if it exists. Morphobiometric markers (Radhika et al., 2018) and microsatellite markers that are commonly used for unraveling population diversity and differentiation in goats (Jayashree et al., 2019) were selected to unravel structure of investigated populations. The blood samples were collected from different locations of both the states for genetic diversity estimation.

Research work was planned and executed at ICAR-NBAGR, Karnal during 2017-2019. Diversity status of three lesser known populations; two Telangana goat populations- Telangana Black (TB, n=26) and Telangana Mixed (TM, n=49) and one Andhra Pradesh goat population (AP, n=45) was established. Blood samples were collected from flocks of different villages of Mahabubnagar, Nagarkurnool, Jogulamba Gadwal, Adilabad, Bhupapally district, Nallamalla forest area of Telangana and villages of district East Godavari and Vizinagram. The sampled animals included black, admixtures and white phenotypes. Information on phenotypic and biometric attributes were also recorded on goats of different flocks. Blood samples were processed to isolate DNA following standard procedure of Sambrook et al., (1989). A battery of 22 microsatellites was selected to estimate the genetic variability. Forward primer of each marker was 5' labeled with fluorescent dye. PCR amplification was performed and multiplexed samples were genotyped on an automated ABI-3100 DNA sequencer. DNA fragment size details were estimated from the electropherograms using Gene mapper software (version 3.0) of Applied Biosystem, USA. Allele numbers,  heterozygosities (observed and expected), Hardy Weinberg Equillibrium test and Shanon information index were calculated using Pop Gene software, version 1.32 (Yeh et al., 1999). Polymorphic information content for each locus was calculated according to Botstein et al., (1980). F statistics were determined using FSTAT software (Goudet, 2002). Tests for deviations from Hardy Weinberg equilibrium were conducted. To detect the genetic bottle neck in the long hair goat population, two tests namely ‘Sign test’ and ‘Wilcoxon sign rank test’ were employed under three microsatellite evolution models like Infinite allele model (IAM), stepwise mutation model (SMM) and two phase model (TPM) of mutation. The graphical presentation of mode shift indicator of Luikart and Cornuet (1998) was also attempted. A comparative diversity for differentiation from geographically closely distributed registered breeds viz. Bidri (n=28) and Nandidurga (n=48) of Karnataka and Ganjam (n=48) of Odisha was also elucidated.

The goat populations of Telangana and Andhra Pradesh comprises of mainly black, white, brown and admixture animals (Fig 1). The  average body measures of Telangana goats (Verma et al., 2018) and of Andhra Pradesh (Verma et al., 2020) have been studied and observed better growth in  Andhra goats, where bucks upto 50 kg body weight and long horns were recorded.
 

 
Genetic variability
 
The estimated values for number of alleles (observed and effective), heterozygosity, expected heterozygosity and fixation index for three goat flocks is given in Table 1. All the markers were polymorphic and a total of 344 alleles were detected across the 22 loci in six goat groups. An exact test for genotypic linkage disequilibrium yielded no significant P values across the population and therefore independent assortment of all the loci was assumed. Reasonable polymorphism is evident from the allele frequency data with 6.59 (TB), 7.27 (TM) and 8.36 (AP) mean number of alleles in lesser known populations (Table 1). Expected number of alleles varied from 3.33 in (TB) to 3.69 in AP goats. The use of microsatellites with a range of polymorphism reduced the risk of overestimating genetic variability, which might occur with microsatellite exhibiting only high polymorphism. Shannon’s information Index (I) is a parameter indicative of the informative degree of a marker and most of the markers had high I values thus can potentially be used for diverse genetic applications including linkage mapping, individual identification and parentage testing.
 

        
These three goat populations (TB, TM, AP) had moderate genetic variation based on its gene diversity in addition to the average number of alleles per locus. Observed heterozygosity was lowest in the TM (0.474) followed by TB (0.504) and was highest in the AP goats (0.569). Observed heterozygosity in the AP goats is equivalent to that existing in the neighboring goat breeds (Table 2). Observed heterozygosity was less than the expected heterozygosity indicating non-random mating prevalent among the populations/breeds. F_IS value reinforced that the populations were not in the Hardy Weinberg equilibrium. Heterozygote deficiency ranged from 0.4% in Nandidurga goat breed to 25.1% in the Ganjam goat (Table 2). AP goat population had 6.3% heterozygote deficiency, whereas, both TB (15.4%) and TM (17.5%) had very high inbreeding coefficients.  Raghavendra et al., (2017) reported average observed and effective means of allele number, heterozygosities as 8.80, 7.71, 0.69, 0.86, respectively in Mahbubnagar goats. The inbreeding estimate showed mild to moderate inbreeding with F_IS value of 0.196.
 

 
Differentiation of goat populations
 
Four lines of evidence suggest that AP goats are distinct from two Telangana goat populations as well as from other registered breeds of Indian goats having geographic closeness.
        
Firstly, F-statistics for each of the loci across populations was computed (Table 3). The global deficit of heterozygotes across populations (F_IT) amounted to 29.4%. An overall significant (P<0.001) deficit of heterozygotes (F_IS) of 13.9% occurred in the analyzed loci because of inbreeding within populations. The multi-locus F_ST values of breed differentiation indicated that moderately high value of 19.1% of the total genetic variation was due to unique allelic differences between the breeds, with the remaining 80.9 % corresponding to differences among individuals within the breed/ population.
 

 
Secondly, the pair-wise Nei’s genetic distance values of groups (Table 4) revealed that the least distance (0.033) was observed between the two Telangana goat populations (TB and TM) and  the highest divergence was recorded between Ganjam and all the five population groups followed by the AP goats and its distance from all other five groups. Visualization of breed relationship was done by constructing Neighbor joining (NJ) tree on the basis of Nei’s genetic distance (Fig 2). As expected, Ganjam and AP goats separated from all other populations, whereas TB and TM were closely associated. 
 

 

        
Thirdly, assignment test could correctly assign individuals of four groups except for TB and TM goat groups. The assignment test based on likelihood method with the leave one out procedure assigned 89% of the individuals correctly to their respective populations. All the individuals of Bidri, Ganjam and AP and all except three of Nandidurga were assigned correctly. The isolation of AP goats as a distinct population is indicated by PCA (Fig 3).
 

        
Bottleneck influences the distribution of genetic variation within and among populations. In recently bottlenecked populations, the majority of loci will exhibit an excess of heterozygotes, exceeding the heterozygosity expected in a population at mutation drift equilibrium. To estimate the excess of such heterozygosity Sign, Standardized differences and Wilcoxon sign rank tests were utilized. The actual mutation model of evolution followed by our microsatellites is not known, thus all the three models; Infinite allele model (IAM), stepwise mutation model (SMM) and two-phase model of mutation (TPM) were applied. Non-significant heterozygote excess on the basis of different models, as revealed from Wilcoxon rank test and under IAM model of Sign and Standardized differences tests (Table 5) suggested that there was no recent bottleneck in the existing AP goat population.
 

        
The Mode-shift indicator test was also utilized as a method to detect potential bottleneck. The non-bottleneck populations that are near mutation-drift equilibrium are expected to have a large proportion of alleles with low frequency. A graphical representation utilizing allelic class and proportion of alleles showed a normal ‘L’ shaped distribution (Fig 4). The L shaped curve indicated the abundance of low frequency (<0.10) alleles. This finding suggested the absence of any detectably large, recent genetic bottleneck (last 40-80 generations) in this population.
 

Microsatellite data confirms AP goat to be a unique germplasm of indigenous goats. Heterozygote deficiency in this population warrants immediate attention. Moderate allele and gene diversity suggests existence of enough genetic variation in the AP goat population for scientific improvement or conservation programs.

The authors are thankful to the Director, ICAR-NBAGR for providing the facilities to carry out this work. We convey our sincere thanks to the AH Departments of Telangana and Andhra Pradesh states for their support and cooperation. Thanks are due to the goat keepers who permitted for blood sampling from their animals.