Analysis of Genetic Variability in the Caprine Class II MHC CLA DRB3.2(Cahi DRB3.2) gene locus of the Sangamneri goat breed of India

The objective of this study was to assess the genetic variability present across the Class II MHC DRB 3.2 gene locus in the Sangamneri goat breed of India. Sixty three single nucleotide variations were observed in CLA-DRB3.2 gene of eleven Sangamneri animals. Sixteen haplotypes with Haplotype diversity of 0.974 were found. Besides the snp(s) having two alleles, both triple and tetra allelic single nucleotide variations were present. Thus, the Class II DRB 3.2 gene of the Sangamneri goat breed animals (CLA-DRB3.2/ Cahi DRB3.2) was exhibiting a very high degree of genetic polymorphism. Of the sixty three single nucleotide variations, fty variations were non-synonymous i.e. they resulted in a change in the corresponding amino acid encoded by the triplet codon in which they were existing. Both conservative and non-conservative amino acid changes were observed to occur. Rich diversity of the DRB3.2 gene reected well on the ability of the Sangamneri animals to survive in the harsh climatic condition(s), exposed to all kinds of pathogen(s) existing in the environment. statistical the eleven sequences obtained, for the genetic polymorphism in the Sangamneri goat breed animals, by Dnasp 5.0 that; of these 63 snp(s)observed to occur in the present study, fty two were parsimony informative sites and the other eleven were singleton variable sites that had two variants. These eleven singleton variable sites that had two variants these two parsimony informative sites, forty ve sites two variants while 6 parsimony informative sites position(s) variants triple alleles; one parsimony informative site position 34 four variants Site positions of forty ve parsimony informative sites had two variants 35, 38, 41, 42,


Introduction
The MHC of goat, also named as Caprine Lymphocyte Antigen (CLA)/Cahi encompasses a large chromosomal region and maps to chromosome 23. Class II MHC gene of goats is similar to that of cattle and sheep wherein it has been extensively characterized. In goats, however, only four class II MHC genes viz. Cahi-DRA, Cahi-DRB, Cahi-DYA and Cahi-DIB, have been identi ed ( Amills et al., 2004). The Class II MHC region in ruminants in contrast to that of humans and mice is split into two subregions which are separated by atleast 15 cM (centi Morgans). The Class IIa subregion comprises two clusters of genes, DR and DQ (Miyasaka et al., 2011, Behl et al., 2012. Peptide binding site (PBS) in goat which has several pockets and is highly variable is partly coded by DRB and DQB genes. DRB is the most polymorphic Class II gene and analysis of DRB polymorphism is particularly useful in inferring evolutionary history of MHC in ruminant species. A non synonymous change in the nucleotide sequence of the MHC DRB or DQB1 gene can substantially substitute the coding amino acid and ultimately bring conformational change in the binding groove, so as to affect the e ciency of the protein to present the antigen e ciently for further processing. The second exon of the DRB3 has been widely characterized in cattle, sheep, goats, buffaloes, red deer and other wild ruminant species and has been observed to be highly polymorphic as it encodes the variable portion of the peptide binding site. Peters  Similarly, there is a scarcity of research database for allelic association of DRB and DQB alleles with disease resistance or susceptibility in goats. The immune-polymorphism database (IPD)-MHC has no space dedicated to goat MHC. The CLA-DRB3.2 encodes the beta-1 domain of the DR molecule, which is in close contact with the foreign antigen and displays a very high degree of polymorphism, with more than 25 different sequences identi ed to date (Schwaiger et al., 1993, Amills et al., 1995 The Semi arid region of Maharashtra comprising of Nasik, Ahmednagar and Pune districts forms the habitat of this goat breed. Although the name Sangamneri is after Sangamneri tehsil of Ahmednagar district but it is also spread over the adjoining places like Sinar tehsil of Nasik district, Junar of Pune district and Rahuri, Shri rampur, Ahmednagar district of Maharashtra. The Sangamneri goats are small to medium sized animals. The coat colour is complete white. Animals with admixture of black and brown colour are also observed. Although these goats are reared mainly for meat purpose but some animals show good milch potential (Verma, et. al., 2010).

Collection of Blood samples and Isolation of DNA
Blood was collected from the jugular vein of fteen randomly selected animals of Sangamneri goat breed spread in different pockets of the breeding tract. Care was taken while drawing the blood samples that minimum degree of discomfort is caused to the animal. Guidelines set up by the Institute Animals Ethics Committee (IAEC) of NBAGR, Karnal were followed while collection of the blood. The blood samples were kept in laboratory at -20ºC in deep freezer until being used for DNA isolation. DNA was isolated by the method described by Sambrook et al., (1989).

Sequencing of the ampli ed products
The ampli ed products obtained were treated with EXO-SAP (Bell, R., 2008) and then puri ed by QIAquick PCR puri cation kits (Qiagen) and sequenced on ABI 3100 automated DNA sequencer by using the ABI Prism Big Dye Terminator dideoxy 3.0 Cycle Sequencing kit. Sequencing was done for both the forward and the reverse primers. In order to avoid artifacts in interpretation and to add to the accuracy of the results each sequencing reaction was carried out in triplicate.

4.Analysis of the Sequence(s)
Sequence analysis, and SNP identi cation was done by manual inspection using Chromas Pro 1.

Results And Discussion
Eleven novel gene sequences of the CLA-DRB3.2 gene of the Sangamneri goat breed were obtained after carrying out the present study. These sequences were deposited to Gen Bank, NCBI, and the following Gen Detailed statistical analysis of the eleven sequences obtained, for the study of genetic polymorphism in the Sangamneri goat breed animals, by Dnasp 5.0 revealed that; of these 63 snp(s)observed to occur in the present study, fty two were parsimony informative sites and the other eleven were singleton variable sites that had two variants. These eleven singleton variable sites that had two variants were at position(s) 45, 47, 49, 81, 82, 83, 86, 88, 95, 128 The Tajima's D value was 0.03362, which was statistically insigni cant at P > 0.10 (Tajima, 1989). A positive value of Tajima D, in the present study, indicates balancing selection forces, to be in action, resulting in the maintenance of a large number of alleles in the population. The dynamic selective pressures exerted by pathogens promote balanced polymorphism in the host response genes in several cases. The best documented example is the major histocompatibility complex (MHC) in vertebrates (Hedrick, 1998;Edwards and Hedrick, 1998;Hughes andYeager, 1998, Bernatchez andLandry, 2003 ).
Analysis of the results obtained, for different population indices by the use of the POPGENE software (Yeh et al., 2000), gave the values of observed and expected heterozygosities to be 0.0792 ± 0.0953 and 0.3180 ± 0.1698, respectively. The overall Fis value (Wright, 1951) was observed to be 0.07391. These results where the observed heterozygosity of the population studied was lower than the expected heterozygosity value, and a positive value of Fis -Wrights xation index indicated that the animals that were studied for their genetic polymorphism in the Class II MHC CLA-DRB3.2 gene locus, in the present study, had a certain degree of genetic relatedness amongst themselves. The genetic variability results in the amino acid variability that further leads to the protein being able of attaining varying conformations that aid in its interaction with the varying antigenic peptides being presented by different pathogens and eliciting an appropriate immune response in order to counter the infection of the host by the pathogen and conferring the host with an ability to ght against disease and protect it from the varied plethora of antigens present in the environment, thereby protecting the host from the assaults by the pathogens and contributing to disease resistance/susceptibility of the host. Table 1 List of the nucleotide variation(s) in the Class II MHC CLA-DRB3.2 gene of the Sangamneri goat; and the corresponding amino acid variation(s) occurring as a result of that nucleotide variation.  which has to interact with the plethora of antigenic peptides present in different pathogens present in the environment with which the animal comes into contact. The animals showing the existence of high number of the DRB3.2 gene alleles and the ones having high number of heterozygous snp(s) could be selected as potential candidate animals expected to be possessing a higher degree of resistance to disease(s). However, for this the sample size of the animals taken up for the study shall have to be increased.