Molecular Characterization of Assam Hill Goat

A total of 23 polymorphic microsatellite markers were used to evaluate genetic diversity and population structure in Assam Hill Goat (AHG). All the loci studied were polymorphic in nature. The number of observed alleles (Na) detected ranged from 2 to 10 with an overall mean of 4.9±2.220. A total of 114 alleles were observed across all the loci. The effective number of alleles (Ne) ranged from 1.035 to 7.127 with a mean of 2.68±1.590. The allele frequency ranged from 0.013 to 0.982. The overall mean observed (HO) and expected (He) heterozygosity were 0.43 and 0.48 respectively and this population was in Hardy-Weinberg equilibrium at most of the loci studied. The overall mean of within-population inbreeding estimate (FIS) was 0.085. The population was stable with respect to size and was non-bottlenecked. The observed normal L-shaped curve indicated no mode shift in the population.


INTRODUCTION
Genetic diversity is necessary for the long-term survival of the species and populations because it provides the raw material for adoption and evolution, especially when environmental conditions have changed (Rajora and Mosseler, 2001). The genetic markers are playing important role in measuring genetic diversity. These have been used to find out evolutionary relationship within and between species, genera or higher taxonomic categories (Paterson et al., 1991). Goat is one of the significant food sources, because it can convert feed dry matter into milk as efficiently as other ruminants. The goat population in North East India was approximately 3.51% of the total India population (Feroze et al., 2010).
The Assam Hill goat (AHG) is an important meat type animal with high prolificacy from the North Eastern region of India. Most common colours of this goat is white, however, brown, black and mixed colour are not uncommon ( Fig. 1). They are distributed in the hilly terrain of North Cachar hill, Karbi Anglong districts of Assam and also in the adjoining hilly tract of Meghalaya state.
The Network Project on Animal Genetic Resources-Core laboratory, Department of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam, India has undertaken molecular characterization of livestock through microsatellite markers. Onto date no studies are conducted in AHG population from North East India using microsatellites.
In view of this the present study has been planned to investigate genetic variation and population structure within AHG population using 23 polymorphic microsatellite markers.

Sample Collection and DNA Isolation
A total of 40 blood samples of AHG were collected randomly from genetically unrelated individuals from their native breeding tract (Fig. 2).
Blood was collected aseptically into BD vacutainers (6 mL) containing K2 EDTA (10.8 mg) and samples were transported to the laboratory on ice and were stored at 4°C until use.
Genomic DNA was extracted from the blood samples using standard phenol-chloroform method (Sambrook et al., 1989) with few modifications. All extracted samples were conformed through horizontal electrophoresis on 0.8% agarose gel containing ethidium bromide. The quantification of DNA was done by Nano-drop spectrophotometer at 260 nm. The concentrated samples were diluted to reach appropriate concentrations (20-50 ng µL −1 ) for the purpose of PCR amplification.

Microsatellite Analysis
All the 23 microsatellite markers were selected from the list recommended by International Society for Animal Genetics (ISAG) and FAO's (DAD-IS) for Caprine, based on their level of polymorphism, allele size range and reliability of allele calling. The forward primer of each marker was fluorescently labeled with either FAM, NED, PET or VIC dye. All microsatellite markers were first checked under single locus amplification conditions to evaluate their performance in the multiplex and accordingly multiplex panels were prepared. Details of markers used in the present study are shown in Table 1.
Multiplex PCR has been used for multicolor fluorescence genotyping. Based on the guide lines of (Henegariu et al., 1997) the initial parameters of Science Publications AJAVS multiplex PCR were set up. The basic PCR solution (15 µL) containing 20-50 ng of template DNA; 1.5 mM MgCl 2 ; 5 picomoles each of forward and reverse primers; 1 unit of taq DNA polymerase and 200 mM dNTPs was prepared. Amplification was carried out with initial denaturation at 95°C for 2 min followed by 30 cycles of denaturation (95°C for 30 sec), annealing (54 to 60°C for 30 sec) and extension (72°C for 45 sec). PCR conducted on an Applied Biosystems (Model #: 9902) Veriti TM 96-well thermal cycler.
After conformation of magnified PCR products on 2% agarose gel, genotyping was carried out on automated DNA Sequencer (ABI PRISM 3130 XL). The resulting data were analyzed using standard software Gene Mapper TM version 4.0 (Applied Biosystems Inc., California, USA) to generate genotype calls for each locus by using GS 500 (-250) LIZ as size standard.

Information Analysis
POPGENE version 1.31 (Yeh et al., 1999) was used to calculate the allele frequencies, effective number of alleles (N e ), observed (H o ) and expected (H e ) heterozygosity, F-statistics, Shanon's information index (I) and to test of Hardy-Weingberg Equilibrium (HWE). Nei's formula (Nei, 1978) (Cornuet and Luikart, 1996) analysis was performed to know whether this goat population exhibits a significant number of loci with excess of heterozygosity.

RESULTS
The various parameters of genetic differentiation in AHG, such as allele number, effective number of allele, PIC, observed and expected heterozygosity, withinpopulation inbreeding estimate (F IS ) and Shanon's information index are furnished in Table 2.
All the 23 loci investigated were polymorphic in nature. The number of observed alleles (N a ) detected ranged from 2 (ETH225, ILSTS065, OarJMP29 and ILSTS34) to 10 (OarFCB304), with an overall mean of 4.90±2.220 and a total of 114 alleles were observed at these loci in the population. However, the effective number of alleles (N e ) ranged from 1.035 to 7.127 with a mean of 2.68±1.590. Overall allele frequency ranged from 0.013 (at locus ILSTS33) to 0.982 (at locus ETH225). The PIC value ranged from 0.033 (ETH225) to 0.843 (OMHC1) with a mean of 0.44±0.263. The overall means for observed (H O ) and expected (H e ) heterozygosities were 0.43±0.285 and 0.48±0.281, respectively which ranged from 0.034 (ETH225) to 0.862 (ILSTS002) and 0.033 (ETH225) to 0.859 (OMHC1) respectively. The chi-square (χ 2 ) test for HWE revealed that 10 out of 23 loci deviated from equilibrium. Shannon's information index (I) (Lewontin, 1995), which measures the level of diversity, was sufficiently high with a mean of 1.00±0.606. The within population inbreeding estimates (F IS ) observed at 10 loci were positive which ranged from 0.012 (OarHH64) to 0.771 (OARE129). Only 13 loci revealed negative F IS values indicating the absence of inbreeding in these loci. The mean F IS value observed was 0.085. Though positive F IS values were observed at 10 loci, only 8.5% of inbreeding was recorded in AHG.
Three mutation models namely, Infinite Allele Model (IAM), Two Phase Model (TPM), Stepwise Mutation Model (SMM) were used for Bottleneck analysis ( Table 3). In AHG population, under Sign test, the expected number of loci with heterozygosity excess were 8.93 (TPM) and 9.07 (SMM) which are respectively higher than the observed number of loci 6 (TPM) and 4 (SMM) with heterozygosity excess. The expected number of loci (8.67) with heterozygosity excess was not significantly (p>0.05) higher than the observed number of loci (9)

DISCUSSION
The present study revealed that the most of the studied loci were highly informative, indicating high polymorphism. Thus these markers strongly signified genetic diversity investigations of AHG. The number and sizes of microsatellite alleles observed in this study fall within the range mentioned in the Secondary Guidelines for Development of National Farm Animal Genetic Resource Management Plans of FAO. The mean number of alleles observed (4.90) in the present investigation was less than the mean number of alleles reported in Ganjam (6.29) goat (Sharma et al., 2009) and Gohilwari (10.12) goat .
The PIC value in the present investigation ranged from 0.033 to 0.843 which is in close agreement with the reports of (Sharma et al., 2009) in Ganjam goat and  in Gohilwari goat. The low observed heterozygosity 0.034 (ETH225) was observed in the present study may be due to the presence of more homozygote individual in the samples analyzed. Though few loci exhibited lower heterozygosity values, most of the loci showed relatively higher expected heterozygosity, which reflects the existence of differentiation in the Science Publications AJAVS population (Karthickeyan et al., 2008). The chi-square (χ 2 ) test revealed that 13 microsatellite loci in the AHG population are in equilibrium. These results established that the samples were drawn from the large random mating population (Karthickeyan et al., 2008).
The overall mean F IS (0.085) observed in the present study indicated a 8.5% shortfall of heterozygosity in AHG population which is not significant as compared to heterozygote deficiency reported in Ganjam goat 21.7% (Sharma et al., 2009); Gohilwari goat 26.4% ; Kutchi goat 26%, Mehsana goat 14% and Sirohi goat 36% . The present findings of F IS value supports random mating in the studied population. The main reasons for the random mating are wide range of native breeding tract and sufficient availability of breeding bucks in the population.
Bottleneck analysis revealed that the breed is nonbottelnecked where the mode-shift for the frequency distribution of alleles had a normal L-shaped curve stating that there was no recent and/or sudden reduction in the population.

CONCLUSION
The PIC values observed in the present study is indicative of the fact that the markers used are highly informative for characterization of AHG diversity. The significant level of variability in this population reflects that the AHG population contains a valuable genetic diversity. The population has not undergone any reduction at least in the recent past. Hence, this population could provide a valuable source of genetic material that may be used for meeting the demands of future breeding programmes.

ACKNOWLEDGEMENT
The researchers wish to extend their gratitude to the Indian Council of Agricultural Research, New Delhi, India for the financial assistance for molecular characterization work through the Network Projects of the National Bureau of Animal Genetic Resources, Karnal, India.