Microsatellite DNA Polymorphism Studies in Mudhol Hound Dog Native of India

In India, dogs have always been used for hunting, fighting against enemies, companionship and for guarding and has the privilege of having excellent dog populations/breeds like Mudhol Hound,  Caravan,  Rampur  Banjara,  Rajapalayam, Chippiparai, Kombai, Kanni, Alangu, Kaikadi, Bhotia sheep dog, Himalayan sheep dog, Indian Spitz, Bakharwal, Dhangiri Alunk, Kuchi, Patti, Poligar/Palegar hound and Shenkottah dog etc. By nature, all these are hunters, swift, keen and alert. Mudhol Hound is a hardy dog and a keen sight hound but it has an independent temperament with the male dog having the tendency to attack at times. The trademark features of the breed are small head which is flat and moderately wide between the ears. It has long and narrow skull. The muzzle is long and tapering. The eyes are dark brown or hazel in colour, oval shaped and are obliquely placed which give the dog an intelligent expression. The ears are thin, small to medium sized, mobile, triangular and set fairly high carried flat close to the head, rose shaped and semi-erect thrown back and folded except when excited. They have an elegantly arched, long, supple and well-muscled neck. The body is muscular with small powerful loins. The brisket is long and deep, with a roomy thorax and a well tucked in abdomen. The back is fairly broad. They have muscular legs with forelegs long and straight from elbow to knee. Hip bones are set wide apart, stifle moderately bent, hocks low to the ground. The tail is long and set on line with the body. It is strong at the base tapering and slightly curved carried low at times. The coat is smooth, short and fine. The predominant colours are black, grizzle, grey, white, cream, red, fawn, brindle or any of these colors broken with white or in small patches. The average height of adult females ranges from 23 to 25 inches and in adult males it is 26 to 28 inches. The average weight of adult females ranges from 22 to 26 kg, whereas in adult males it ranges from 27 to 32 kg. The gait and movement of the breed is an effortless stride, like flying rather than running with the hind legs under the body giving good propulsion (Bijoor, 2006). The picture of both female and male Mudhol Hound breed of dog is represented in Plate 1 and 2 respectively.
 

 

       
Mudhol Hound being a keen sight hound is very much in demand as a companion, hunting and guard dog. The government and NGOs, considering the historic importance of Mudhol Hound have given a lot of importance in conservation and development of the breed like: releasing of postage stamps and establishing Canine Research and Information Centre (CRIC). Molecular genetic characterization is an essential component in characterization in order to unravel the genetic basis of inheritance of a particular phenotype (FAO, 2011). Molecular markers have been shown to be an efficient tool in quantification of genetic diversity of various populations (Saitbekova et al., 1999; Barker et al., 2001) and to determine evolutionary relationship within and between species, genera or higher taxonomic categories.
       
Microsatellites were a special class of tandem repeat loci having a base motif of 1 to 6 bp repeated up to 100 times (Tautz, 1993). Microsatellites are easy to test, present abundantly on the canine genetic map, can be used for both the genders and the results can be confounded by high mutation rates (Francisco et al., 1996; Landry et al., 2002). The common repeats of microsatellite in the canine genome are (CA)_n, (GATA)_n or (CAG)_n (Ostrander et al., 1992). The microsatellite genotypes could be used to correctly assign 99 per cent of individual dogs to breeds (Parker et al., 2004).
       
In recent days high throughput microsatellite markers offer the best measure to identify genetic diversity among the populations. Recently, some of the Mudhol Hound dogs were taken up by the army as part of its sniffer dog squad. Since most of these animals were kept without genetically tested pedigree records profiling based upon DNA testing for breed characterization is becoming a dire need. Such genetic study has not been taken up in Mudhol Hounds. Hence, the present work was taken up with the objectives of analyzing the microsatellite patterns for Mudhol Hound dog breed using specified primers.

Blood samples were collected from 50 unrelated Mudhol Hound dogs maintained in Canine Research and Information Centre (CRIC), Thimmapur village, Bagalkot district, Karnataka, India and also from the Mudhol Hound dogs maintained by the farmers of villages close to the CRIC. The blood samples were collected with EDTA as anticoagulant and all the samples were stored until analysis at -20°C. Isolation of genomic DNA was done using high salt method as described by Miller et al., (1988). The quality and quantity of isolated genomic DNA was assessed by 0.8 per cent agarose gel electrophoresis and Spectrophotometer (Eppendorf, USA). The DNA samples were stored at -20°C.
       
For the present study, a total of 26 canine-specific microsatellite primers (AHT121, AHT137, AHTh171, AHTh260, AHTk211, AHTk253, CXX279, FH2054, FH2326, FH2848, INRA21, INU005, INU030, INU055, PEZ03, PEZ08, PEZ11, PEZ12, PEZ17, REN105LO3, REN162C04, REN169D01, REN169018, REN247M23, REN54P11 and REN64E19) were selected from the ISAG (International Society for Animal Genetics) canine panel (ISAG, 2005), as well as from published literature (Eggleston et al., 2002; Cho, 2005 and Gagliardi et al., 2011) and they were procured from Sigma Aldrich Chemical Pvt. Ltd, Bengaluru. The annealing temperature for 26 microsatellite primers was standardized between 53.9°C to 64.5°C by gradient temperature technique. Based on the size of PCR products resolved through Polyacrylamide gel electrophoresis, the product sizes were assessed using 50 bp molecular DNA markers (ladder) and the genotyping of animals was done. Microsatellite variability measures were determined using POPGENE-Version 1.31 software.
       
The PCR master mix used for the amplification was obtained from Sigma Aldrich, Bengaluru and the PCR were carried out in a final reaction volume of 25 μl. The PCR reaction components consisting of 2X PCR Master Mix (12.5 μl), 1.0 μl each of the forward and reverse primer of the individual microsatellite marker were mixed in 9.5 μl of triple distilled water to make a total volume of 25 μl of PCR reaction mixture. PCR tubes containing mixture were tapped gently and quickly spinned at 10,000 rpm for few seconds. The tubes were placed in a Master cycler gradient (Bio Rad S1000, USA) and subjected to PCR. The PCR protocols using the following thermal profile: 3 min of initial denaturation at  94°C followed by 33 cycles of denaturation at 94°C for 45 sec, annealing at the range of 53.9°C to 64.5°C for 45 sec, elongation of primers 72°C for 45 sec and final elongation of primers at 72°C for 5 min. The annealing temperature of each primer set (forward and reverse primers) was standardized by carrying out gradient PCR, based on the melting temperature of the primer set.
       
Microsatellite loci were analyzed by running the PCR products on 6.5 per cent polyacrylamide gels followed by silver staining and visualizing the band patterns. Based on the size of PCR products resolved through Polyacrylamide gels, genotyping was done and the product sizes were assessed using 50 bp molecular DNA markers (DNA ladder). All the samples studied were analyzed using Diversity Database Software (Bio-Rad, USA). Genotypes of animals were scored based on the presence of a single band or double bands in the gel. The genotypes were scored and microsatellite variability measures were determined using POPGENE-Version 1.31 software (Yeh et al., 1999).

Microsatellite alleles, their number, allele sizes and their frequencies at different loci are furnished in Table 1 and  2. A total of 221 alleles were identified in this study, whereas Hisham (2014) observed a total of 297 alleles in four native breeds of Tamil Nadu, viz., Rajapalayam, Chippiparai, Kanni and Kombai using 29 microsatellite markers and lesser allele number were reported in mongrels of Japan and Kinatamani dogs of Bali, Indonesia, South Korean dogs (Oishi et al., 2005, Puja et al., 2005, Kang et al., 2009).
 

 

       
The number of alleles ranged from 5 (AHTk211) to 12 (INU055, PEZ12) with a mean of 8.5±1.9442 alleles. The effective number of alleles ranged from 3.6792 (AHTk211) to 9.5785 (PEZ12) with the mean of 6.3032±1.5018. The allele sizes ranged from 80 bp (INRA21) to 342 bp (AHTK253). These microsatellite alleles were distributed between the frequencies of 0.01 (INU055) and 0.37 (CXX279). The allelic diversity of Mudhol Hound dogs was found to be lower than that reported in other breeds (Morera et al., 1999, Koskinen and Bredbacka., 2000) but was similar to that of the studies in Tetra Shepherd Dogs (Anna Radko et al., 2015).
       
The Polymorphism Information Content (PIC), PIC values for the 26 micro satellite value ranged between 0.6844 (AHTk211) and 0.8757 (FH2326) with a mean value of 0.8117. In the present study, the percentage of polymorphic loci obtained is 100 as all the 26 microsatellite loci studied were found to be polymorphic having the PIC values more than 0.5. Therefore, these microsatellite markers can be effectively used for molecular characterization and genetic diversity studies in Mudhol Hound dog breed. Similar observation was reported by Hisham (2014) in different breeds of Indian origin and Tahir et al., (2015) in German Shepherd and Labrador Retriever dog breeds. 
       
The expected heterozygosity was more than 0.5 in all the loci indicating the suitability of these markers for genetic diversity studies in Mudhol Hound dogs. The average observed and expected heterozygosity pooled over different loci were 0.7685±0.2122 and 0.8326±0.0397, respectively. The observed heterozygosity ranged between 0.26 (PEZ11) and 1 (AHTh260, AHTk211, INU005 and REN169D01) and the expected heterozygosity ranged from 0.7282 (AHTk211) to 0.8956 (PEZ12). The results were in agreement with that studied in Golden Retriever, Labrador Retriever, Rottweiler breeds, Japanese Mongrel, Kintamani dogs of Bali, Rajapalayam, Chippiparai and Konbai breeds (Koskinen and Bredbacka., 2000, Altet et al., 2001, Oishi et al., 2005., Puja et al., 2005., Hisham, 2014, Anna Radko et al., 2015).
       
F_IS indicates a measure on inbreeding and this ranged from -0.1988 (INU005) to 0.5664 (PEZ17) with the mean of 0.0955 indicating moderate inbreeding within the population in Mudhol Hounds studied. The negative values of F_IS point towards out breeding i.e., mating of individuals who are less related than the average relationship of the population (Holsinger and Bruce, 2009; Mishra et al., 2010). This may also be attributed to the existence of fewer numbers of males in the area. The positive values of F_IS (inbreeding in individual relative to the sub population) ranged from 0.0222 (AHTh171) to 0.5664 (PEZ17). Hisham (2014) and Tahir et al., (2015) observed lesser F_IS in breeds of Tamilnadu and in German Shepherd and Labrador Retriever dogs respectively.
       
In the present study Shannon’s information index (I) ranged from 1.4285 (AHTk211) to 2.3524 (PEZ12) with a mean of 1.9381±0.232 which was in agreement with Rajapalayam, Chippiparai, Kanni and Kombai dog breeds (Hisham 2014).
       
A departure from HWE (P<0.001) was observed in all the loci (26) studied and were with highly significant χ² values. The disequilibria observed in all the microsatellite loci may be attributed to the number of alleles and their frequencies at the respective loci, which might be due to the moderate influence of both systematic and dispersive forces over the population. Unlike diallelic system, the attainment of equilibrium would take longer time as the nature of inheritance of microsatellites follows multiple allelic fashions. The instability observed, in general, among the population studied could be attributed to the heterogeneity, resulting from immigration and emigration of certain microsatellite alleles into or from the population. The small sample size included in this study could be the reason for the departure from HWE. A similar results were observed in studies conducted elsewhere in other breeds (Kim et al., 2001, Oishi et al., 2005, Puja et al., 2005), however in studies conducted by Lupke and Distl (2004) and  Ciampolini et al., (2011) in other breeds revealed that the population was found to be in HWE at certain loci AHTk211, FH2054, INRA21, INU055 and REN169018 loci and on the other hand showed significant deviation from HWE in six microsatellites deviation (AHTh260, CXX279, RENI05L03, REN54Pll, REN162C04 and AHTH130). Assessment of HWE in four native breeds of Tamil Nadu; Rajapalayam, Chippiparai, Kanni and Kombai by Hisham (2014) revealed that among 29 microsatellite loci studied loci INRA21 in both Rajapalayam and Chippiparai, FH2054 in Kanni and AHTh260 in Kombai were in Hardy-Weinberg disequilibrium. In another study of genetic variation including German Shepherd and Labrador Retriever dogs by Tahir et al., (2015), a moderate trend of deviation from Hardy-Weinberg equilibrium. 
       
The present study on molecular characterization of Mudhol Hound breed of dog revealed high allelic diversity and substantially higher level of genetic variability. The high content of polymorphic information displayed by these loci is suggestive of effective utilization of these 26 markers in the molecular characterization and genetic diversity studies in Mudhol Hound breed. The population in the present study is in disequilibrium at all the loci studied, which might be due to the moderate influence of both systematic and dispersive forces over the population. However, further studies based on a larger sample as well as a greater number of microsatellite markers are desirable to draw more valid conclusions.

The study is part of M.V.Sc Thesis submitted to KVAFSU in the year 2017 by the first author. The article is dedicated in remembrance of Prof. M.R. Jayashankar who has guided the first author during her M.V.Sc research. The study was partly funded as part of the KVAFSU, Staff Research project “Molecular characterization of Mudhol breed of dog” No. DR/KVAFSU/SRP/Anisci-05/2016-17.