научная статья по теме GENETIC DIVERSITY AND BOTTLENECK ANALYSIS OF INDIAN BELLARY SHEEP BY MICROSATELLITE MARKERS Биология

Текст научной статьи на тему «GENETIC DIVERSITY AND BOTTLENECK ANALYSIS OF INDIAN BELLARY SHEEP BY MICROSATELLITE MARKERS»

ГЕНЕТИКА, 2007, том 43, № 9, с. 1198-1208

ГЕНЕТИКА ^^^^^^^^^^^^^^ ЖИВОТНЫХ

УДК 575.17:59975

GENETIC DIVERSITY AND BOTTLENECK ANALYSIS OF INDIAN BELLARY SHEEP BY MICROSATELLITE MARKERS

© 2007 r. D. Kumar1, R. Sharma1, A. K. Pandey1, D. S. Gour1, G. Malik1, S. P. S. Ahlawat1, and A. Jain2

1 Small Ruminant Molecular Genetics Laboratory, DNA Fingerprinting Unit 2 Animal Genetics Resources Division, e-mail: dineshbhu@rediffmail.com National Bureau of Animal Genetic Resources, Karnal-132 001 (Haryana), India

Received May 24, 2006

Bellary sheep population variability and structure was investigated genetically utilizing FAO recommended microsatellite markers. Genetic variation at 20 microsatellite loci, population structure, and genetic bottleneck hypothesis were examined. Estimates of genetic variability such as effective number of alleles and gene diversities revealed substantial genetic variation frequently displayed by microsatellite markers. A total of 133 alle-les were detected. Average polymorphism across the studied loci and expected gene diversity in the population were 1.419 ± 0.405 and 0.684 ± 0.140, respectively. No significant genotypic linkage disequilibrium was detected across population, suggesting no evidence of linkage between loci. The population was observed to be significantly differentiated into different groups, showed fairly high level of inbreeding (f = 0.253 ± 0.050) and global heterozygote deficit. Population structure analysis indicated the intermixing/introduction of unique/rare alleles in these migrating flocks. A normal L-shaped distribution of mode-shift test, non-significant heterozygosity excess on the basis of different models, as revealed from Sign, Standardized differences and Wilcoxon sign rank tests suggested that there was no recent bottleneck. The study revealed that even breed with increasing population trend needs genetic management for the conservation and improvement.

Sheep is one of the most important small ruminants of India. It plays an important role in livelihood of a large proportion of small and marginal farmers and landless laborers. India ranks 4th in the world and has 50.8 million sheep which is 4.57% of the world sheep population. In India, about 169 million kg of mutton, 42.7 million kg of wool and 40 million kg of skin are produced annually. The diversity of geo-climate over the ages has manifested in to diversity of sheep breeds and India has 40 breeds [1]. There is a tendency for worldwide animal production to be based on a few, highly selected breeds, which is leading to reduction in number of local breeds [2]. Maintaining genetic variation is now recognized as a crucial and international need to fulfill all the market demands and to make the progressive improvement of domestic animal populations, so that they are successful in the future also [3].

The name of Bellary sheep is derived from its native breeding region, Bellary district of Karnataka, India. The population is distributed to Bellary, Chitradurga, Davangere, and partly to Haveri districts of Karnataka. The characteristic features of the breed are medium-sized, with body colour ranging from white through various combinations of white and black. Mostly rams are horned and ewes are polled. Ears are medium long, flat and drooping, tail is short and thin. Fleece is extremely coarse, hairy and open. Belly and legs are devoid of wool [1]. This breed is primarily for mutton but also provides 200-400 gm greasy wool per annum.

Studies have not been done to estimate the inbreeding level in the field animals thus how various forces of genetic change are modifying the foundation genetic structure of the population are not known. Therefore, an investigation of genetic variation within breed, and its structure may help to evaluate these factors. This will provide genetic information to be used for conservation and improvement of this population.

The objectives of this study were to estimate genetic variability, population structure and genetic bottleneck hypothesis in Bellary sheep.

MATERIALS AND METHODS

Sample collection. Blood sampling was done as per the guidelines of FAO [4]. Thus, blood samples of fifty genetically unrelated animals were collected from breeding tract of Bellary sheep (25 samples from Bellary district, 15N11 76E54 and 25 from Chitradurga district, 14N14 76E24 of Karnataka, India) to make them representative of population (Fig. 1). As microsatellite markers are co-dominant, 50 samples represent 100 alleles for a single locus. Blood samples (5-6 ml) were obtained from the jugular vein using vacuitaners treated with 15% ethylene diamine tetra acetic acid (EDTA) as an anticoagulant. The sampling was done from two districts of the breeding region.

Molecular techniques. Genomic DNA was isolated as per the method described by Sambrook et al. [5] with minor modifications. After checking the quality and

Fig. 1. Sampling localities of the Bellary sheep population.

quantity DNA was diluted to a final concentration of 50 ng/|l in water and stored at 4°C. A battery of 20 microsatellite markers (Table 1) was selected based on the guidelines of ISAG & FAO's DADIS (Domestic Animal Diversity Information System) MoDAD (Measurement of Domestic Animal Diversity Information System) programme for generating data in a panel of 50 animals (A combination of 20 co-dominant loci and 50 samples are expected to generate 2,000 allelic data for the population under study). Polymerase Chain Reaction (PCR) was carried out on about 50 ng genomic DNA in a 25 | l reaction volume using PTC-200 PCR machine (MJ Research Inc., USA). The reaction mixture consisted of 200 |M each of dATP, dCTP, dGTP and dTTP, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl2, 0.75 unit Taq DNA polymerase and 4 ng/| l of each primer (Sigma

Genosys). The "touchdown" PCR protocol used with initial denaturation of 95°C for 1 min, 3 cycles of 95°C for 45 s and 60°C for 1 min, 3 cycles of 95°C for 45 s and 57°C for 1 min, 3 cycles of 95°C for 45 s and 54°C for 1 min, 3 cycles of 95°C for 45 s and 51°C for 1 min, 20 cycles of 95°C for 45 s and 48°C for 1 min. At the end of the reaction, 5.0 |l of stop dye (95% formamide, 0.25% bromophenol blue and 0.25% xylene cyanol) was added and 6 | l of PCR products were loaded on 2% agarose gel, electrophoresed and visualized over UV light after ethidium bromide staining to detect the amplification.

The PCR products were resolved on 6% denaturing polyacrylamide gels (Sequi GT System, Bio-Rad), 10 bp ladder (Invitrogen, Life Technologies, USA) was used as a size standard for sizing PCR products. To visualize the PCR products, gels were stained using silver staining

Table 1. Microsatellite markers, their sequences, type of repeat, location and accession numbers

S.N. Loci Primer sequences Type of repeats Ch. No. Gene Bank Accession Number

1 OMHC1 atctggtgggctacagtccatg, gcaatgctttctaaattctgaggaa - 20 228

2 OarJMP29 gtatacacgtggacaccgctttgtac, gaagtggcaagattcagaggggaag (CA)21 24 U30893

3 OarHH41 tccacaggcttaaatctatatagcaacc, ccagctaaagataaaagatgatgtgggag (CA)23 10 L12555

4 BM827 gggctggtcgtatgctgag, gttggacttgctgaagtgacc (CA)B 3 U06763

5 OarHH35 Aattgcattcagtatctttaacatctggc, atgaaaatataaagagaatgaaccacacgg (CA)17 4 AF394447

6 BM6526 catgccaaacaatatccagc, tgaaggtagagagcaagcagc (CA)22 26 G818454

7 OarHH64 cgttccctcactatggaaagttatatatgc, cactctattgtaagaatttgaatgagagc (GT)n 4 L12558

8 BM8125 ctctatctgtggaaaaggtggg, gggggttagacttcaacatacg (CA)16 17 G18745

9 RM4 cagcaaaatatcagcaaacct, ccacctgggaaggccttta (CA)13 15 U32910

10 ILSTS005 ggaagcaatgaaatctatagcc, tgttctgtgagtttgtaagc (nn)39 5 L23481

11 OarHH47 tttattgacaaactctcttcctaactccacc, gtagttatttaaaaaaatatcatacctcttaagg (CA)22 18 L12557

12 BM757 tggaaacaatgtaaacctggg, ttgagccaccaaggaacc (GT)14 9 BE603866

13 OarCP34 gctgaacaatgtgatatgttcagg, gggacaatactgtcttagatgctgc (GT)16 3 U15699

14 OarFCB48 gagttagtacaaggatgacaagaggcac, gactctagaggatcgcaaagaaccag (GT)10 17 M82875

15 OarJMP8 cgggatgatcttctgtccaaatatgc, catttgctttggcttcagaaccagag (GT)18 6 U35059

16 Oar AE 129 aatccagtgtgtgaaagactaatccag, gtagatcaagatatagaatatttttcaacacc (CA)14 5 L11051

17 HUJ616 ttcaaactacacattgacaggg, ggacctttggcaatggaagg (GT)21 13 M88250

18 OarVH72 ctctagaggatctggaatgcaaagctc, ggcctctcaaggggcaagagcagg (GT)14 25 L12548

19 OarFCB128 cagctgagcaactaagacatacatgcg, attaaagcatcttctctttatttcctcgc (GT)15 2 L01532

20 BM6506 gcacgtggtaaagagatggc, agcaacttgagcatggcac (CA)23 1 G18455

Notes: Accession number of Arkdb data base (http://www.thearkdb.org), Ch. No., chromosome number.

[38] and dried between sheets of cellophane paper. The genotypes were scored manually. Size of the alleles was calculated online using INCHWORM programme which estimates the length of the molecule, based on the electrophoretic mobility (http://www. molecular-workshop.com/programs/inchworm.html).

Statistical analysis. The original microsatellite allelic data is available from the authors upon request. For 20 microsatellite loci, observed and expected heterozygosity estimates were calculated after Levene [6] and Nei [7] as implemented in POPGENE software [8]. The observed and effective numbers of alleles [9] were also calculated using POPGENE software.

Allelic frequencies were utilized for the calculation of the Polymorphic Information Content (PIC) values [10]. The PIC value was estimated as

k k -1 k

PIC = 1 - X x2- X I 2 x2 x2,

i=1 i=1 j = i +1

where k is the number of alleles and xt and Xj are the frequencies of the i allele and j allele.

The tests for deviation from Hardy-Weinberg equilibrium were derived using the exact tests of POPGENE. Heterogeneity of deviations from Hardy-Wein-berg equilibrium among the microsatellite loci was ex-

amined by treating the deviations as correlation coefficient and tested accordingly [11]. As samples were obtained from two different localities, deviations from Hardy-Weinberg equilibrium in the population could be due to genetic differences

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