Abstract: RAPD-PCR techniques were applied to study the genetic identity and diversity in two Indian cattle breeds Kankrej and Gir with a battery of 16 decamer primers. The analysis was carried out by using Band Frequency (BF), Band Sharing Frequency (BSF), Genetic Identity (GI), Genetic Distance (GD) and Mean Average Percentage Deference (MAPD). RAPD pattern revealed polymorphism for 163 loci out of 196 loci (83.16%) in Gir and Kankrej breeds. The present study showed that within breed similarity in both the breeds was observed to be greater as compared to similarity between breeds. Within breed genetic similarity was higher in Gir cattle than Kankrej cattle. Primer KMS6 was able to resolve a product of 388 bp that was seen in 11 out of 12 Gir animals. Similarly primer KMS2 revealed Kankrej specific amplicon of 2274 bp that was seen in 11 out of 12 Kankrej animals. The estimate of band sharing frequency between breeds was highest (0.900) with the primer KMS6 and the lowest (0.557) with the primer KMS11. The band sharing frequency pooled over the primer was 0.738±0.2008 between these two breeds. The highest genetic identity estimate (0.943) between the two breeds was obtained with primer KMS3 and the lowest (0.696) with primer KMS10. Similarly the highest genetic distance estimate (0.20) between the two breeds was obtained with KMS2 and the lowest (0.000) with primer KMS12. The highest (44.2) Mean Average Percentage Difference (MAPD) was observed with primer KMS11 and lowest (9.98) with primer KMS6. The phylogenetic tree based on Nei’s formula revealed that the primers used for the study were able to show the genetic similarity and diversity within and between breeds.
INTRODUCTION
India has 30 recognized Zebu cattle breeds that are adapted to the tropical environments (Nivsarkar, 2000). Among them Gir and Kankrej are from Gujarat state. Both the breeds are of great importance in India. Molecular biology virtually gains access to the entire genome of cattle. Whereas the Polymerase Chain Reaction (PCR), which induce a methodological revolution, have been applied for genetic variation studies. Application of randomly amplified polymorphic DNA (RAPD) techniques has greatly increased the ability to understand the genetic relationship within species at molecular level. This technique has achieved a great deal of acceptance due to its simplicity, readability directly on gel, low cost involvement, requirement of little amount of DNA as compared to other methods (Williams et al., 1993) and is highly informative without prior knowledge of sequence information (Crowhurst et al., 1991; Bostock et al., 1993; Kemp and Teale, 1992). RAPD has been used for population analysis in detecting genetic differences in humans and in estimation of inbreeding in cattle (Bhattacharya et al., 2003). The present study was undertaken to determine the genetic identity and diversity within and between Gir and Kankrej (Bos indicus) cattle breeds using RAPD fingerprints.
MATERIALS AND METHODS
The study was carried out on 12 randomly selected male animals of Gir and Kankrej breeds of cattle each. Blood samples were collected into the heparinized blood collecting tubes from the organized farms and sperm stations across the Gujarat State. Genomic DNA was isolated from blood cells by lysis under the condition that degrades protein and preserve the integrity of DNA molecules as described by Shambrook et al. (1989). The quality and quantity of DNA was analyzed by agarose gel electrophoresis and spectrophotometer.
RAPD- PCR technique and analysis of DNA fingerprints: A battery of 16 decamer primers synthesized from Bangalore Genei (P) Ltd. India, were employed in the representative DNA samples of Gir and Kankrej breeds of cattle. The sequence and Guanine and Cytosine (GC) contents are mentioned in Table 1. The standard condition required for PCR amplification was determined empirically to get the reproducible bands. The amplification reaction was carried in 0.2 mL microfuse tubes using programmable thermal cycler (BIOMETRA). Each 25 μL reaction mixture comprised of 50 ng genomic DNA, 400 μM each of dNTPs, 40 ng primers, 1 U Taq DNA polymerase, 2.5 μL of 10X Taq DNA polymerase buffer (500 mM KCl, 100 mM Tris KCl, 1.5 mM MgCl2 and 1% Triton X-100) and 1 mM Magnesium acetate. Amplification of DNA was carried out at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 36°C for 1 min, 72°C for 2 min and final extension at 72°C for 5 min using programmable thermal cycler (BIOMETRA). The randomly amplified PCR products were electrophoresed on 2% agarose gel along with DNA molecular size marker (Phi x 174 DNA/ Hae III digested). The image was captured by DNR imaging system. Using Total lab software from DNR Imaging System, all amplified and reproducible bands were scored and the presence and absence of a band was recorded as 1 and 0, respectively. Comparison of all RAPD fingerprints of the breed was done on the samples run on the same gel.
Band frequency (BF): Band frequency of RAPD fingerprints were calculated by using the formula:
BF = n/N |
Band sharing frequency (BSF) Band Sharing frequency were calculated in pair wise comparisons as described by Gwakisa et al. (1994).
BSF (between breeds) = 2 BGK/[
BG+BK] |
Where BGK is the number of bands common to Gir and Kankrej for a
primer. BG is the total number of bands for Gir for a particular
primer. Similarly BK is the total number of bands for Kankrej for
a primer. Average of band sharing frequency was calculated by dividing the sum
of BSF value of pair wise comparison by the total number of pairs compared.
| Table 1: | The sequence and guanosine-cytosine contents of the oligoes |
Within breed band sharing frequency was calculated as the average of band sharing frequencies within the breeds pair wise using the formula:
BSF (within Gir breeds ) = 2 B Gi.
Gii/[BGi + BGii] and BSF (within Kankrej breeds)= 2BKi Kii/[BKi + Bkii] |
Where B Gi.Gii is the band sharing frequency within a pair of Gir and BKi Kii is the band sharing frequency within a pair of Kankrej. BGi, BGii, BKi and BKii are the total bands observed in the individual animal of Gir and Kankrej breeds, respectively.
Genetic identity (GI): Genetic identity between two breeds was obtained as follows:
Where BFiG and BFiK are the frequency of occurrence of ith band in two different breeds respectively and N is the total number of bands scored as per Apuya et al. (1988).
Genetic distance (D): The genetic distances were obtained by the formula of Lynch (1991).
DGK = -ln BSFGK /√(BSFG
x BSFK) |
Where BSFGK is the Band sharing frequency between Gir and Kankrej breeds of cattle. BSFG is the Band sharing frequency within Gir breeds. BSFK is the Band sharing frequency within Kankrej breeds.
Mean average percentage difference (MAPD): MAPD was calculated by using the following formula (Gilbert et al., 1990; Yukhi and O’Brien, 1990).
Percentage Difference (PD) = NGK/NG+NK]
x100 |
Where NGK are the number of fragment that differed between two animals, for a single primer. NG and NK are the number of bands observed in individual breeds. C is the number of interbreeds pair wise comparison and R is the number of random primers used.
RESULTS AND DISCUSSION
DNA analyzed by agarose gel electrophoresis followed by observation on UV transilluminator revealed sharp high molecular weight bands. The visual estimation revealed good concentration of DNA. The OD 260 nm : OD 280 nm ratio ranged between 1.7-2.0 indicating a good quality and purity of DNA.
Analysis of randomly amplified polymorphic DNA (RAPD) fingerprints: Sixteen primers were screened to amplify DNA samples of Gir and Kankrej. The numbers of RAPD bands observed in the range of 7-17 with molecular size ranging from 241-2941 bp. 163 out of 196 loci (83.16%), were polymorphic. The amplification patterns of representative samples of Gir and Kankrej breeds with two primers have been shown in Fig. 1-2, respectively.
Band frequency: Most of the primers showed polymorphic bands in both
the breeds. KMS1, KMS2, KMS7 and KMS8 primers exhibited polymorphic bands of
1537, 2274, 691 and 345 bp with a band frequency of 1.0, 0.92,0.75 and 0.75,
respectively only in Kankrej breed. Similarly KMS2, KMS4, KMS6, KMS7, KMS9,
KMS10, KMS11, KMS13 and KMS14 showed polymorphic bands of 431, 704, 388, 753,
2226, 1990, 2096, 928 and 644 bp with a band frequency of 1.0, 0.58, 0.92 0.67,
0.75, 0.75, 0.92, 0.75 and 0.58, respectively only in Gir breed. Similar findings
in buffalo were reported by Arvndakshan and Nainar (1998) and Singru (1998).
A few publications also demonstrated breed specific RAPD fingerprints (Gwakisa
et al., 1994; Shivakumar, 1997; Ahn et al., 1999). Primers KMS1
and KMS2 produced breed specific bands in 92-100% animals of Kankrej breed.
Similarly, primers KMS2, KMS6 and KMS11 produced breed specific bands in 92-100
% animals of Gir breed. Such amplicons could be used to develop breed specific
Sequence Characterised Amplified Region (SCAR).
| Fig. 1: | RAPD fingerprints of genomic DNA using primer KMS-2. Lane # 1 : Phi X 174 DNA/Hae 7III digested marker, lane # 2-13: Gir DNA samples, lane# 14-25 : Kankrej DNA samples and lane # 26: Phi X 174 DNA/Hae III digested marker |
| Fig. 2: | RAPD fingerprints of genomic DNA using primer KMS-6. Lane # 1 : Phi X 174 DNA/Hae III digested marker, lane # 2-13: Gir DNA samples, lane# 14-25: Kankrej DNA samples |
| Table 2: | Band sharing frequency within Gir and Kankrej breeds |
Band sharing frequency: The average band sharing frequency within and
between breeds in Gir and Kankrej are given in Table 2. Most
of the primers revealed band sharing within and between breeds. The frequency
varied in Gir from 0.91-0.59 with respect to primers KMS6 and KMS12 while in
Kankrej it varied from 0.96-0.66 for primers KMS6 and KMS11, respectively.
| Table 3: | Band sharing frequency and genetic identity between Gir and Kankrei breeds |
Similarly band-sharing frequency between Gir and Kankrej varied from 0.90-0.55 for primers KMS6 and KMS11 (Table 3). The pooled average band sharing frequency within Gir breed was 0.92±0.08 and Kankrej breed was 0.81±0.08. The obtained data of band sharing frequency on the basis of the primers revealed that Gir breeds are genetically more similar than Kankrej breeds. Sharma et al. (2004) reported similar observation in Rathi and Tharparker cattle. Within breed genetic similarity was higher than between breeds (0.74±0.20). Primers KMS5 and KMS6 generated relatively higher value of band sharing frequencies within and between breeds (Table 2 and 3) showing that these primers could be less informative in differentiating the breeds. Similar finding have also been reported by Kantanen et al. (1995).
Genetic identity: Genetic identity varied with the random primers used
in both the cattle breeds as presented in Table 3. All primers
used showed similarity in (medium to high) genetic identity between the two
breeds. Highest genetic identity (0.9434) was observed in primer KMS3 and the
lowest genetic identity (0.6930) with primer KMS10. The genetic identity (pooled
of all the 16 primers) was 0.77±0.11 between Gir and Kankrej cattle.
Shende and Yadav (2004) reported similar observations in Nagpuri and Murrah
buffaloes. The higher estimates of genetic identity between Gir and Kankrej
cattle in the present study indicated that both are having common descent as
reported by Sharma et al. (2004) on his work with Rathi and Tharparker
breeds. Ramesha et al. (2002) observed high degree of resemblance of
DNA bands between Ongole and Krishnavelly of cattle breed.
| Table 4: | Percentage difference and genetic distance between Gir and Kankrej breeds |
Mean average percentage difference: The MAPD value was calculated from all the averages of two breeds. The average Percentage Difference (APD) estimated for all the primers is presented in Table 4. The highest APD value between these two breeds obtained was 44.21 with primer KMS11 and lowest value of 9.98 with primer KMS6. The MAPD between these two breeds was estimated to be 26.14±9.37% indicating these breeds differed at 26.14% loci amplified by a battery of 16 primers.
Genetic distance: The genetic distance calculated for various primers
are given in Table 4. All the primers used showed genetic
distance. Primer KMS2 showed highest genetic distance (0.20) between these two
breeds while the primer KMS12 indicated lowest genetic distance (0.00). The
over all average genetic distance was 0.08±0.06 between Gir and Kankrej
breeds. No significant genetic distance was found between the breeds with respect
to the primers used. The phylogenetic tree based on Nei’s (Nie, 1978) genetic
distance (Fig. 3) revealed that the primers used for the study
were quite enough to show the genetic similarity and diversity. The animals
were separated into two clads. First cluster containing nine Gir animals and
second cluster containing all twelve Kankrej animals. Remaining three animals
of Gir were out grouped from the cluster that indicates genetic distance within
breed. However, with the inclusion of larger sample of animals and more number
of primers RAPD would correctly group these samples to the respective breeds.
| Fig. 3: | Phylogenetic tree showing genetic similarity and diversity within and between two cattle breeds (Nei’s, 1978) |
ACKNOWLEDGMENT
The authors are thankful to the National Dairy Development Board, Anand, Gujarat, India for providing necessary facilities to conduct the study. The help received from Acharya Nagarjuna University, A.P., India is also duly acknowledged.