Assessment of Bandsharing Values in RAPD-PCR Analysis of Dwarf Cattle of Kerala

Random amplified polymorphic DNA (RAPD-PCR) analysis of 56 animals of four different genetic groups of dwarf cattle in Kerala was done as a single step analysis. Bandsharing (BS) values were calculated for animals of each group and between groups as an analytical tool to find out genetic variation among animals. The different factors affecting BS values were estimated using Harvey’s Least squares analysis. The effects of genetic group, Guanine-cytosine (GC) content of primer and gel on BS values were found significant. Bandsharing values of Kasargode-Highrange dwarf animals were significantly different from Vechur, Vatakara and their combinations. The Vechur, Vatakara and Vechur-Vatakara combinations were found to be more uniform (high BS value) compared with other combinations. The bandsharing value was lowest with primers of GC content 90% and highest with 80% GC content. The effect of gel on BS value points to the need of adjustments of gel factor for calculation of BS values. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 9 : 1217-1220)


INTRODUCTION
Random amplified polymorphic DNA markers (RAPD) were first described by Williams et al. (1990).The technique is simple, fast and relatively cheaper, permitting rapid generation of almost unlimited number of polymorphisms.It uses random oligonucleotide primers at low annealing temperature for amplification.The polymorphisms are due to the difference in spacing between primer binding sites or due to point mutations, which allow or abolish primer binding.The greatest advantage of this technique is its ability to obtain DNA polymorphisms without prior knowledge of genomic sequence.As the marker scans the entire genome, it is used in characterization studies of animals and plants.Bandsharing (BS) values were calculated from the RAPD fingerprints as an analytical measure to estimate the genetic divergence of animals, which is very well needed for characterization of animals.One of the disadvantages of RAPD-PCR technique is the influence of different factors in band formation, which will affect bandsharing values.Standardization of the amplification conditions needed for RAPD-PCR analysis have been done for repeatability.The present study was an investigation towards the factors influencing BS value in RAPD-PCR analysis.

MATERIALS AND METHODS
Genetic divergence in dwarf cattle of Kerala was studied using RAPD-PCR technique.Animals belonging to four native dwarf cattle groups of Kerala viz.Vechur, Vatakara, Highrange dwarf and Kasargode formed the subjects of the study.Ten oligonucleotide primers were used for individual RAPD-PCR analysis of DNA samples, which were extracted by standard phenol: chloroform isolation procedure.PCR amplifications were carried out by single primer method (Gwakisa et al., 1994).
The PCR reactions were carried out in 0.2 ml thin wall PCR reaction tubes using a programmable thermal cycler (MJ Research Inc., USA).Twenty microlitre reaction was standardized comprising 25 ng of template DNA, 5 pM primer, 200 µM each dNTPs, 0.5 unit Taq DNA polymerase, 1.5 mM MgCl 2 and 1× PCR buffer.The PCR amplification condition was standardised as follows.After an initial denaturation of 94°C for 1 min, a 35 cycle reaction was performed, with each cycle having 1 min denaturation (94°C), 1 min annealing (38°C) and 1 min extension (72°C).This was followed by a final extension for 10 min at 72°C.The PCR products were electrophoresed at 100 V in 2% agarose gel containing ethidium bromide.The banding patterns were visualized under a UV transilluminator and documented in gel documentation system (Biorad Laboratories, USA).
The RAPD-PCR products were run on 2% agarose gel in two rows as upper and lower.Thirty samples were loaded in each row.The polymorphic patterns produced by RAPD primers were identified as presence or absence of bands.The average Bandsharing value for each primer and for 10 primers were estimated as given by Gwakisa et al. (1994).The bandsharing was calculated by pair wise comparison method, as an expression of similarity of RAPD fingerprints of animals within or between genetic groups (Dunnington et al., 1990;Gwakisa et al., 1994;Aravindakshan and Nainar, 1998;Islam et al., 2005)  The different factors affecting bandsharing values such as genetic group, GC content of primer and the different combinations of comparison of gel were analysed using least squares analysis as described by Harvey (1986).The mixed model least squares and maximum likelihood computer programme PC-1 was used.The following mathematical model was constructed.[upper, lower and combination] were analysed for finding their effect on bandsharing value.

RESULTS AND DISCUSSION
The RAPD-PCR conditions to get reproducible banding pattern varies between laboratories (Penner et al., 1993;Rothuizen and Wolferen, 1994;Ambady et al., 1996;Parejo et al., 1997;Yoon and Kim, 2004).Standardization of RAPD-PCR conditions was done under this study so that reproducible fingerprints were obtained.The various levels of variables that were best suited were identified after conducting a series of tests.Ten random primers were analyzed on individual DNA samples.Different primers produced different RAPD profiles.Figure 1  In RAPD analysis, minute variations in different factors can affect the product profile.The various variables affecting the bandsharing values were taken into account on least square analysis of variance.The effects of genetic group, GC content of primer and the effect of gel were studied using Harvey's Least squares analysis, Model-1.The least square means for these variables are given in Table 1 and the least squares analysis of variance is presented in Table 2.The effects of GC content of primer, genetic group and the gel on bandsharing values were found significant (p<0.01).
The variations of band sharing values in comparisons of upper gel to upper gel (Upper), lower gel to lower gel (Lower) and upper gel to lower gel (Combination) were analyzed.A significant effect was observed for gel effect on BS value.The upper gel had the highest BS values (0.84±0.00) followed by the lower gel (0.82±0.00) and the combination had the least value (0.78±0.00).These indicate that the comparison of between gel bandsharing values may not yield exactly the same results as that of a single gel.Gwakisa et al. (1994) and Aravindakshan and Nainar (1998) used samples run on same gel for comparison.From this, it was clear that between gel comparisons had considerable effect on BS values.Hence the effect of gel has to be adjusted before analysis of BS values.
Of the four different classes of primers used based on the GC content, the one with 90 percent GC content yielded significantly lowest bandsharing value (0.71) followed by primers with 60 percent (0.79), 70 percent (0.85) and 80 percent (0.91) GC content respectively.This information emphasizes the role of primers and their GC content on BS values.Williams et al. (1990) reported that 10-base random primers with GC content of 60-90 percent was suitable for amplification of genomic DNA segments for studying the polymorphisms between different individuals.

CONCLUSION
The technique of RAPD-PCR in animals has been standardized and the genetic diversity among native cattle groups was found based on Bandsharing values.The different factors affecting bandsharing value were assessed using Harvey's Least squares analysis of variance.The Highrange dwarf and Kasargode animals differed significantly from Vechur, Vatakara and their combinations.The comparisons of animals of VV, VD and DD showed more uniformity.Hence Vechur and Vatakara animals are found as more similar compared with Highrange dwarf and Kasargode animals.These combinations were different from VH, HH, DK, VK and HD combinations.The effects of GC content of primer, genetic group and the gel on BS values were significant.Adjustments for GC content of primer and gel are needed before assessing BS values in RAPD-PCR analysis (p<0.01).
using the formula, BS = 2 B ab /B a +B b Where, B ab is the number of bands shared by individual a and b.B a is the total number of bands for individual a. B b is the total number of bands for individual b.
represents the RAPD profile of animals when amplified with single primer, OPA 18. RAPD fingerprints obtained with individual animals with different primers were used for the calculation of BS values.The bandsharing values within and between four genetic groups were calculated under this study as an indicator of genetic similarities.All possible 15,400 pairwise combinations of different animals were made for ten primers and the average BS value for each comparison of genetic groups were estimated.Pairwise comparison and band sharing value estimates were used as given byGwakisa et al. (1994), Mohd-Azmi et al. (2000);Ramesha et al. (2002);Yoon and Park (2002) andSaifi et al. (2004).

Table 1 .
Least squares means of band sharing values for different genetic groups, GC content of primer and gel in RAPD-PCR analysisThe least square means with same superscript within the same variable does not vary significantly (p≤0.01).

Table 2 .
Least squares analysis of variance for the effects of GC content of primer, genetic group and gel on bandsharing ** p≤0.01.