using faecal DNA 1

Aim: To study the genome profile of Chital deer (A.axis) by microsatellite markers using faecal DNA & to assess its usefulness as non invasive method of genotyping. 
 
Materials and Methods: DNA was isolated from 44 animals from faeces and blood, out of which 26 (3 from blood and 23 from faeces) were subjected for genotyping. The PCR products of all the ten microsatellite loci were subjected to 6 % Urea-PAGE to differentiate allelic size. 
 
Result: Total 57 alleles and 49 genotypes were found with their frequencies ranging from 0.022 to 0.457 and 0.043 to 0.696 respectively. The observed number of alleles ranged from 4 -8, (Av. = 5.7 per locus). The PIC and Shannon\'s information index values for all loci ranged between 0.6645 to 0.8270 (Av. = 0.7442 per locus) and 1.1870 to 1.8852 (Av. = 1.5122 per locus) respectively. 
Conclusion: Faecal DNA can be effectively used in genotyping wild animals, however the method and time of collection & storage as well as the type of faecal material largely influences the quality & quantity of DNA.


Introduction
Microsatellite marker provides an advantage in conservation studies as they are relatively easy to Spotted deer or commonly called as "Chital" are genotype and they require a very less amount of DNA one of the most common wild herbivore found in that can be isolated from epithelial cells sloughed off in forests of India.The chital deer (Axis axis), also called saliva [11], urine [12] or faeces [13].Hence, non the Indian spotted deer and the Axis deer, is native to invasive techniques like utilizing faecal DNA to study India and Sri Lanka and does not have any special the genetic architecture of wild animal population conservation status (Schedule III of Indian Wildlife using microsatellite markers has become the backbone Protection Act, 1972) and is listed as Least Concern of conservation biology, these methods have found (LC) in the IUCN Red List of threatened species [1].
promising role in genetic monitoring [14], study of Being so ubiquitous this deer species can act as a model habitats [15], detection of rare and cryptic species [16], organism for conservation genetic as well as wildlife effect of human interventions on wild animal forensic studies.Till date, there are very few studies on population [17] and genetic monitoring of founder molecular genetics of chital deer in India.Recently, ten population [18].In this study we have targeted a semi novel microsatellite markers were identified in chital captive population of chital deer which is found in a deer and tested for their cross species amplification in natural enclosure of a small protective area, the DNA other cervids in a captive population by Gaur et al. [2].
was isolated from two sources, firstly from blood of the In the current study we used ten microsatellite loci, as animals that were brought to the nearest wildlife center, described by Gaur et al. [2], in a free ranging, semi-Center for Wildlife Forensic and Health (CWFandH, captive population of chital deer.Microsatellites have Jabalpur) for treatment and from faeces of the animals become the genetic markers of choice for many kinds collected from the protected area.Then, the appropriate of molecular applications and they have found their DNA samples were subjected to microsatellite extensive use in conservation biology too, including genotyping to study the genome profile and estimate species identification [3], analysis of population structure [4]  electrophoresis for preliminary analysis and then on 6 % Urea -PAGE for size differentiation between the Sample collection: The samples were collected within alleles [20].The PAGE products were visualized by an hour of defecation, stored on ice then brought to the silver nitrate staining following the procedure of o lab and stored at -21 C and processed rapidly.DNA Gustavo and Gresenhoff [21].from total 44 chital deer was isolated out of which 26

Results
DNA samples (3 from blood and 23 from faeces) showed optimum quality and quantity and were The microsatellite analysis was done using Popgene subjected for further genotyping.v1.32 [22].Total 57 alleles and 49 genotypes were found with their frequencies ranging from 0.022 to DNA isolation: DNA isolation from blood was carried 0.457 and 0.043 to 0.696 respectively.The observed out by using method described by John et al. [19].DNA number of alleles ranged from 4 -8, (Average = 5.7 per from freeze dried faeces was isolated by using QIAamp locus).Maximum number of alleles i. e., eight were DNA stool mini kit (Qiagen, Germany) as per seen on loci Ca42 and Ca60.The PIC values for all loci manufacturer's instruction with little modifications.ranged between 0.6645 to 0.8270 (Av.= 0.7442 per The modifications include, crusing the faeces in liquid locus).The observed homozygosity was zero and the nitrogen to make it finely powdered then adding double observed heterozygosity was one.Expected homozythe amount of lysis buffer (supplied with kit) as well as gosity and expected heterozygosity parameters varied addition of 0.5% Triton X 100 and then incubation at o between 0.1546 to 0.3208 (Av.= 0.2392) and 0.6792 to 37 C for about 2 -4 hr. in a water bath after thorough 0.8454 (Av.= 0.7608).The values of Shannon's vortexing, and keeping the tubes in horizontal position information index were also calculated that ranged for atleast 15 min with a little mixing in between during between 1.1870 to 1.8852 with an average of 1.5122 the addition of InhibitEX tablet.Proteinase K ( 25per locus (Table 1: Population genetic parameter for mg/µl) was prepared fresh just before the DNA each microsatellite marker in studied chital deer (axis isolation. axis) population).F values ranged from -0.2091 to -

Quality and quantity check of DNA:
The DNA was 0.5050 (Av.= -0.3319).Chi square value revealed that subjected to 0.8% agarose gel electrophoresis to assess all the loci significantly (P > 0.05) deviated from Hardy its quality; the concentration of DNA in samples was -Weinberg equilibrium.obtained from Nanodrop spectrophotometer (ND -Discussion 1000).The average quantity of DNA from blood ranged from 250ng/µl to 1220.7 ng/µl (OD 260/280 -The results of gene and genotype frequency and 1.7-1.9),this DNA also served as a positive control, heterozygosity parameters were found to be in DNA from faeces was of relatively poor quality and concordance with those of Gaur et al.
from species to species and sometimes within species.
The heterozygosity and number of alleles determine its PCR amplification and urea denaturing PAGE: Ten polymorphic information content and the polymorphic primers specific for microsatellite loci in Chital deer as nature of microsatellite markers makes them marker of described by Gaur et al. [2] were used in the current choice in characterization and genetic diversity study.A typical PCR cycle consisted of initial denatustudies.The PIC obtained in the present study was ration at 94°C for 10 min., denaturation at 94°C for 30 about 0.7442 per locus which was not significantly s, annealing 46.3°C to 64°C for 30 s, extension at 72°C inhibitors is a common problem and hence multiple 862-871, Doi: 10.1111/j.1755-0998.2011.03031.x.10.Dobigny T.R., Doan L.P., Quang N.L., Maillard J.C. and copies are to be set up in PCR.The microsatellite Michaux J. (2009).Species identification, molecular sexing analysis revealed high degree of polymorphism among and genotyping using non-invasive approaches in two wild the studied loci in the targeted population.There was bovids species: Bos gaurus and Bos javanicus, Zoo Biology, no homozygote individual found, it might be because 28 (2) : 127-136.the population is not entirely closed and there is 11.Inoue E., Inoue-Murayama M., Takenaka O. and Nishida T. (2007).Wild chimpanzee infant urine and saliva sampled continuous movement of animals in and out of protected noninvasively usable for DNA analyses, Primates, 48 (2): area as well as the small population size included in the 156-159.study.Faecal DNA can become a good source of 12. Valiere N. and Taberlet P. (2000).Urine collected in the field genomic DNA in wild animals, although time of as a source of DNA for species and individual identification, collection, type of preservative [31] used and processing Mol.Ecol., 9: 2149-2154.13.Brinkman T.J., Person D.K., Chapin III F.S., Smith W. and steps do affect the quality and quantity of DNA.Hundertmarka K. J. (2011).Estimating abundance of Sitka