ГЕНЕТИКА, 2010, том 46, № 8, с. 1108-1114
ГЕНЕТИКА ^^^^^^^^^^^^^^^^ ЖИВОТНЫХ
УДК 575.17:599.735.5
SHORT TANDEM REPEAT BASED ANALYSIS OF GENETIC VARIABILITY IN KANARESE BUFFALO OF SOUTH INDIA
© 2010 P. Kathiravan, B. P. Mishra, R. S. Kataria, S. Goyal, K. Tripathy, D. K. Sadana
National Bureau of Animal Genetic Resources, P.B. No 129, GTRoad Bypass, Karnal-132001, Haryana, India;
e-mail: kathirvet@yahoo.co.in Received October 13, 2009
The goal of the present study was assessing genetic diversity within Kanarese buffalo, the dual purpose breed of South India. A total of 48 unrelated animals were genotyped at 23 short tandem repeat (STR markers) loci. The total number of observed alleles was 180 with a mean of 7.83 per locus, which varied from 3 to 12 across different loci. The mean observed and expected heterozygosity in South Kanara buffaloes was estimated to be 0.518 and 0.712 respectively. Within population inbreeding estimate (FIS) was significantly positive in most of the investigated loci which resulted in significant deviation from Hardy—Weinberg equilibrium at 19 of 23 loci analyzed. Evaluation of South Kanara buffalo population for mutation drift equilibrium revealed no significant heterozygosity excess under three different models of evolution viz. infinite alleles model (IAM), stepwise mutation model (SMM) and two phase model (TPM), thus indicating the absence of any recent genetic bottleneck. The results of the present study will help in formulating rational breeding strategies as well as conservation of this important germplasm.
India stands first in the world with the largest population of river buffaloes numbering more than 94 million heads (Livestock census, 2003). Although milk is the primary product, buffaloes are reared as multipurpose farm animals for their meat, draught power, hide skin, dung, etc. There are about 10 well defined breeds apart from 15 distinct yet to be characterized buffalo populations adapted to different agro-climatic conditions. Among different river buffalo breeds, Kanarese buffalo of South India is the only dual purpose animal being used for milk production as well as for draught power. South Kanara buffaloes are moderate milk yielders with a good productive life span [1]. These buffaloes are suitable for agricultural operations especially for ploughing and puddling the wet fields for paddy cultivation. They are better than the local cattle as they are active, fast moving and can work continuously for four to six hours in wet fields. Also these buffaloes are famous for the "Kambla racing", a traditional sport in the region. Kanarese buffalo was developed by a sect of Hindus known as "Jain Bants", who owned and developed this hardy breed of buffaloes [2]. Morphologically, they are more similar to Marathwa-da buffalo of Central India especially in their horn pattern except being comparatively compact in size. Kanarese buffalo is the only well defined buffalo breed of South India other than Toda, the unique but endangered buffalo reared by the aboriginal tribe under semi-wild conditions [3, 4].
Understanding the genetic architecture and relationship among different breeds assume significance from conservation stand point particularly for prioriti-
zation of breeds and optimal utilization of available genetic variability. Highly polymorphic markers like microsatellites have been utilized to generate genetic information on population differentiation [5]. The genetic relationship between different Indian river buffalo breeds have been reported [6, 7]. However, the important South Kanara buffalo population was not included in these studies. We earlier reported the physical characteristics, body biometry, management practices, production and reproduction performance and utility of South Kanara buffalo [1]. However, the genetic variability within South Kanara buffalo is poorly understood. Hence, the present study was undertaken with the objectives of evaluating within breed diversity of South Kanara buffaloes using short tandem repeat (STR) markers.
MATERIALS AND METHODS
Microsatellite genotyping. Blood samples were collected from a total of 48 unrelated buffaloes belonging to South Kanara breed. Samples were collected from different villages covering the entire native tract of each breed and the farmers were interviewed in detail in order to ensure unrelatedness among the sampled individuals. Genomic DNA was extracted by standard phenol-chloroform method [8]. A total of 23 hetero-logus bovine microsatellite markers [9] were utilized to genotype the sampled individuals. The forward primer for each locus was labeled with one of the four fluorescent dyes FAM, HEX, NED and PET (Applied Biosystems, USA) (Table 1). Polymerase chain reaction was performed with a total reaction volume of 25 pi,
Table 1. Annealing temperature, allele size range and polymorphism information content of different microsatellite loci in South Kanara buffaloes
Locus Dye Annealing temperature, °C Allele size range PIC
BM1818 FAM 55 255-279 0.767
ILSTS19 VIC 55 173-183 0.243
ILSTS25 FAM 55 110-126 0.649
ILSTS56 PET 55 142-172 0.573
CSSM33 PET 65 157-175 0.651
ILSTS36 NED 59 134-170 0.708
ILSTS89 FAM 64 112-120 0.749
ILSTS95 VIC 58 187-215 0.767
HEL13 VIC 55 168-190 0.683
ILSTS28 PET 55 143-173 0.648
ILSTS58 NED 55 120-152 0.805
ILSTS61 FAM 55 119-143 0.492
CSSM19 NED 55 127-161 0.776
CSSM57 FAM 60 109-127 0.645
ILSTS52 PET 55 141-179 0.748
CSSM47 NED 55 125-165 0.850
ILSTS30 PET 55 142-170 0.556
ILSTS33 FAM 55 126-146 0.585
ILSTS60 VIC 64 162-198 0.610
CSSM45 FAM 60 86-116 0.685
CSSM66 VIC 55 164-210 0.820
ILSTS26 NED 55 137-153 0.759
ILSTS29 PET 55 150-180 0.628
Mean - - - 0.669
Note: PIC, polymorphism information content.
using the following thermal conditions, 94°C for 2 min, followed by 30 cycles of 94°C for 1 min, specific annealing temperature (Table 1) for 1 min and 72°C for 1 min and a final extension at 72°C for 10 min. The amplified PCR products containing different dyes were then electrophoresed together after multiplexing in six sets in an automated DNA sequencer along with GS500LIZ (Applied Biosystems, USA) internal lane control. The allele size data for each sample was then extracted using GENEMAPPER software.
Statistical analysis. Basic diversity indices like observed and effective number of alleles, observed and expected heterozygosity and allele frequency were calculated using POPGENE software [10]. Polymorphism information content of different microsatellite markers used in the present study was estimated as per the formula reported [11]. Estimation of FIS and exact test for Hardy Weinberg equilibrium and linkage disequilibrium were performed using GENEPOP software. Ewens-Watterson neutrality test based on Manly [12] was performed using POPGENE. South Kanara buffalo popu-
Table 2. Basic diversity measures and test for Hardy—Weinberg equilibrium in the spectrum of microsatellite loci in South Kanara buffaloes
Locus na ne Ho He Fis HWE P-value
BM1818 9 4.83 0.537 0.803 0.329 0.0000
ILSTS19 3 1.35 0.205 0.264 0.222 0.0616
ILSTS25 5 3.23 0.727 0.698 -0.048 0.9570
ILSTS56 8 2.53 0.605 0.612 0.006 0.2786
CSSM33 7 3.12 0.543 0.687 0.205 0.0003
ILSTS36 9 3.93 0.477 0.754 0.365 0.0000
ILSTS89 5 4.64 0.583 0.793 0.261 0.0099
ILSTS95 9 4.90 0.756 0.805 0.056 0.0000
HEL13 10 3.53 0.542 0.724 0.249 0.0041
ILSTS28 8 3.23 0.646 0.697 0.069 0.0519
ILSTS58 10 5.77 0.708 0.836 0.148 0.0136
ILSTS61 5 2.13 0.109 0.536 0.797 0.0000
CSSM19 10 4.99 0.452 0.813 0.442 0.0000
CSSM57 8 3.14 0.532 0.689 0.225 0.0000
ILSTS52 11 4.40 0.702 0.781 0.097 0.0027
CSSM47 12 7.33 0.636 0.884 0.274 0.0234
ILSTS30 7 2.64 0.511 0.628 0.184 0.0128
ILSTS33 5 2.74 0.303 0.645 0.528 0.0000
ILSTS60 5 2.94 0.149 0.666 0.776 0.0000
CSSM45 7 3.72 0.478 0.739 0.351 0.0000
CSSM66 10 6.25 0.643 0.855 0.243 0.0006
ILSTS26 8 4.75 0.841 0.799 -0.059 0.0342
ILSTS29 9 2.92 0.229 0.667 0.656 0.0000
Mean 7.83 3.87 0.518 0.712 0.277 -
Notes: na, observed number of alleles; ne, effective number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; FIs, Within population inbreeding estimate; HWE, Hardy—Weinberg equilibrium.
lation was tested for the occurrence of recent genetic bottleneck using BOTTLENECK program [13].
RESULTS AND DISCUSSION
The present study presents the first comprehensive genetic analysis of South Kanara buffaloes using a battery of short tandem repeat markers. A total of 1104 genotypes of 48 South Kanara buffaloes across 23 microsatellite loci were generated in the present
study in order to assess the within breed genetic variability. The polymorphism information content of different microsatellite markers analyzed in the present study ranged from 0.243 (ILSTS19) to 0.850 (CSSM47) with a mean of 0.669 (Table 1). All the investigated marker loci except ILSTS19 and ILSTS61 were highly informative with PIC values above 0.5 [11]. The total number of observed alleles was 180 which varied from 3 (ILSTS19) to 12 (CSSM47) across different loci (Table 2). The effective number of
alleles was lower than the observed number of alleles at all the studied loci as expected and ranged from 1.35 (ILSTS19) to 7.33 (CSSM47) with a mean of 3.87. The genetic diversity analysis of South Kanara buffaloes at 23 microsatellite loci revealed the presence of reasonable allelic polymorphism with a mean of 7.83 per locus. This is comparable to earlier reports on other Indian buffalo breeds [6, 7]. The mean observed and expected heterozygosity in South Kanara buffaloes was estimated to be 0.518 and 0.712 respectively (Table 2). The observed heterozygosity varied from 0.109 (ILSTS61) to 0.841 (ILSTS26) while the expected heterozygosity varied between 0.264 (ILSTS19) to 0.884 (CSSM47). The observed heterozygosity was lower than the expected heterozygosi-ty in most of the loci except two (ILSTS25 and ILSTS26). The observed heterozygosity in South Kanara buffaloes was found t
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.