Page

5.0 DISCUSSION

5.1 Origin and rationale of the probe design 64

5.2 Hybridisation profile of conserved repeat 66 sequences in the bubaline genome

5.3 Somatic and germ line stability of the 70 fingerprinting probe

5.4 Relevance of pedigree analysis 72

5.5 Significance of breed affiliation 73

5.6 Genetic divergence with respect to genomes 75 of other species

5.7 Conclusion 77

DISCUSSION

Way back in 1980, Wyman and White made a landmark contribution by developing

useful genetic markers in humans which they envisaged would be useful for genome

mapping. They realised the potential of this marker because the site had several

variants (alleles). Until then, almost all the polymorphic loci identified in humans

were based on the biochemical variants with fewer alleles. Almost at the same time,

Botstein eta/. (1980) reported that a large number of variations exist at DNA sequence

level in the human population which may be detected as restriction fragment length

polymorphisms (RFLP) using appropriate restriction enzyme(s) and DNA probes.

Today, several genetic markers useful for RFLP are available in animal species also

and are widely used in the marker assisted selection and breeding programs of farm

animals (Beckmann and Soller, 1987~ Hallerman et a/., 1987~ David and Deutch,

1992).

Genetic markers, based on repetitive sequences, detecting multilocus RFLPs which

form the basis of DNA fingerprinting have found a variety of applications (Jeffreys et

a/., 1985b~ 1985c~ Yam eta/., 1987~ Bruford and Wayne, 1993~ Aggarwal eta/., 1994)

in several areas of biology and medicine including propagation of desired germ plasm

in farm animals (Jeffreys eta/., 1987a~ Georges eta/., 1988~ Kuhnlein eta/., 1989~

1990~ Ellegren eta/., 1992; Trommelen eta/., 1993). A great deal of infonnation is

available on the genome analysis of several domestic species but the same is lacking

on the bubaline genome (Nadeau eta/., 1995), although this is an impmiant species for

our dairy industry.

The present study deals with the genetic analysis of the bubaline genome and is

envisaged to be useful in the conservation and propagation of desired germplasm and

better management of this species in our daily industiy. In addition, all the other

beneficial points associated with the DNA fingerptinting approach may also be scored

64

Discussioll

here.

5.1 Origin and rationale of the probe design:

Initially, DNA fmgerprinting was demonstrated using genome derived cloned probe

based on one of the four different repeat units of 33 base long intronic repeats of

myoglobin gene (Jeffreys et a/., 1985b; 1985c). Subsequently, synthetic

oligonucleotides complemental)' to simple repetitive sequences had been reported to be

equally infmmative for DNA fingerprinting (Ali eta/., 1986). Extensive studies in the

past have revealed that many of the di-, tri-, tetra- and hexanucleotide repeats are

evolutionarily conserved in most of the eukaryotic genomes although they show

organizational variation in the number of their repeat units. It is this aspect of the

organization of the repeat motifs that is taken into consideration to design specific

synthetic DNA, based probe(s). Since the organisational status of repeat motifs varies

even within the same species and several types of repeat motifs are present in a

species, it is obvious that not only one but many informative probes may be developed.

The purpose and species for which the probe is to be used are other points to be taken

into consideration before attempting to have a highly specific probe. An advantage of

synthetic DNA probe is that it offers ample working flexibilities to adjust sequence

length and complexities and allows us to circumvent some of the arduous steps

associated with the use of cloned probes (Ali and Epplen, 1991). We wanted to

establish complete DNA profiling of the buffalo genome using synthetic DNA

probe(s). A perusal of the literature revealed that incidentally, there is no well­

documented probe available either for DNA fmgerprinting or for establishing breed

affiliation of the buffalo genome. It may be mentioned that a true DNA fingerprinting

probe has to fulfill several technical criteria before it may be put to use. In view of

this, "empirical.optimisation approach" was adopted and a total of eight different

65

Discussion

synthetic DNA based probes in combination with 18 different restriction enzymes

were used. All the probes reported here are based on the short repeat motifs ranging

from 2-6 base residues. The most conserved dinucleotide repeat motif poly ( dC­

dA).(dG-dT) (Litt and Luty, 1989) has been used for DNA fingerp1inting in species

like dog, cattle, goat, sheep and horse (Epplen et a/., 1991) and varying degrees of

polymorphisms were documented (Kashi et a/., 1990a). Since the (CA)n repeat is

conserved in other species also, we envisaged that the same may be useful in assessing

the genetic distance of bubalines with other related species. The commercially

available long stretch of this PuPy repeat, henceforth designated as (CA)n, failed to

detect high level of genetic variation in the bubaline genome.

Tetranucleotide repeats GAT NGACA detected originally in the sex specific satellite

DNA of female snake and subsequently reported to be present in all other eukaryotic

genomes (Epplen, 1988) were also used in the present study. The length of the oligo

probe was adjusted to 24 base residues to acheive stable duplexes between the target·

DNA and the probe sequences.

The other probe is related to the most commonly occmnng, somatically stable

trinucleotide repeat, (CAC)n, that was first used as a synthetic oligonucleotide probe

for DNA fingerprinting in humans (Schafer et a/., 1988b ). A total of three probes,

OAT15, OATIS and OAT21 based on 15, 18 and 21 base residues respectively were

included in the study to select the most appropriate one which may detect highest

level of polymorphism.

Another quadruplet repeat sequence (GGAT)n also reported to be useful for DNA

fmgerprinting in fishes (Epplen et a!., 1991 ), and some frum animals (Buitkamp et a!.,

199la; 1991b) was also included in the study.

Telomeric repeat sequence (TTAGGG)n ( Moyzis eta/., 1988; Meyne eta!., 1989)

66

Discussion

became an obvious choice because the same is conserved evolutionarily, known to be

involved in recombinatorial activities more frequently than the interspersed repeat loci

and reported to reveal DNA fmgerprinting in several species (Morin and Cech, 1986;

Pluta and Zakian, 1989; Kipling and Cooke, 1990; Epplen et a/., 1991). The

telomeric repeat (TTAGGG) motif comprising four units was used for hybridisation

polymorphism in the buffalo genome. The ultimate aim of using all these probes in

combination with different restriction enzymes independently, was to find the most

appropriate probe/enzyme combination useful for buffalo DNA fingerprinting and for

breed affiliation.

5.2 Hybridisation profile of conserved repeat sequences in the

bubaline genome:

Several types of repeat motifs varying from 2-3 3 base or longer have been reported to

be ubiquitously or preferentially localised in a typical eukaryotic genome. This is

more true for highly evolved species. Buffalo genome is also e~dowed with various

short repeat motifs. It is therefore obvious to expect that a DNA probe specific to any

of these repeat motifs would detect hybridisation polymorphism in conjunction with

appropriate restriction enzyme. With this background, we attempted to study DNA

fingerprinting of buffalo genome using poly ( dC-dA) repeat. Although this repeat has

been reported to be polymorphic in several species, a 16 base long oligo probe based

on this repeat was used to establish DNA fingerprinting in several animal species (

· Epplen eta/., 1991; Ellegren eta/., 1992). In the present study, however, the extent of

polymorphism detected by (CA)n probe was found to be insufficient for genome

indivdualisation.

As mentioned ·earlier, the quadruplet repeats (GATA)n and (GACA)n have been

reported to detect DNA fingerprint patte1n in several animal species (Epplen, 1988)

67

Discussion

including man (Ali et a/., 1986). (GACA)n failed to detect sequence polymorphism

with any of the enzyme combinations used in the present study whereas (GATA)n

probe did not even cross hybridise (see also table 2) with the bubaline DNA (Fig. 3).

In another independent dot blot hybridisation with DNA from tilapia genome

Oreochromis mossambicus, GATA sequences failed to cross-hybridise (unpublished

data). The absence of cross-hybridisation of (GATA) sequences with buffalo and

tilapia genomes contradicts the hypothesis that these sequences are evolutionarily

conserved in all eukaryotes (Epplen, 1988). Incidentally this appears to be the first

report where GATA repeat is found to be absent in these species.

The GC rich, GACA repeat sequence had been reported to reveal isomorphic species

specific hybridisation pattern in cattle and buffaloes (Ali et a/., I993). A shmter

version of the probe used in the present study detected mostly isomorphic bands in the

bubaline genome with all the I8 different enzymes (Fig. 12). The detection of

isomorphic bands suggests that this repeat is arranged in clusters as closely linked

alleles and probably does not act as or pruticipate in the recombinatorial hot spots in

Bubalus bubalis genome. Thus, 24 base long GAT NGACA repeats are not

informative for ·DNA the fingerprinting of the bubaline genome. The 2I base long

probe OAT21 based on trinucleotide repeat detected sufficiently high level of

polymorphism in buffalo (John and Ali, 1994) and in human (Kumar et a!., 1994)

genomes.

Two smaller versions of this probe OATIS and OATIS also detected high level of

polymorphism giving rise to individual specific band pattern. However, the overall

band patterns detected independently by all the three probes OATIS, OATIS and

OAT2I were found to be distinctly different. Thus, as expected an alteration in the

lengths of these oligos changed the overall band pattern. This result is in accordance

68

Discussion

with the earlier observation where an oligo probe of 20 base residues was suggested to

be optimal for establishing DNA fingerprint pattern in the human genome (Ali et a/.,

1986). The high level of polymorphism detected using OATIS probe and Hinfl

enzyme under high stringent conditions, compared to OAT21 or OAT15 probes

(Table 3) is a clear indication that more 18mer stretches of trimeric repeats are

distributed in the buffalo genome as compared to 15 or 21 mer stretches. This result

also reflects a classical example of copy number variation in the repeat units. The

probability of identical band profile between any two unrelated animals calculated,

using OAT18/Hinfl combination was found to be several orders of magnitude lower

than the estimated 130-150 million world population of buffaloes of which half the

number is from India (Mahadevan, 1992).

Earlier studies showed that heterogeneity detected in most of the domestic livestock is

usually very low (Georges eta/., 1988; 1990). This is due to several factors like loss

of wild type alleles during domestication, fixation of alleles in the population due to

inbreeding, increased homozygosity due to intensive marker assisted selection and less

effective population size due to lower number of breeding males in the herd (Ellegren

eta/., 1992; Trommelen eta/., 1993). It has also been repmted that minisatellites are

usually clustered in the genomes of domestic animals (Jeffreys and Mmton, 1987).

Inspite of all these factors, OAT18/Hinfl probe/enzyme combination revealed a very

high level of polymorphism sufficient enough for DNA fingerprinting in the buffalo

genome.

Tetranucleotide repeat GGA T, though less polymorphic, detected a strong mutant band

in the high molecular weight region (-20 kb) in one of the offsprings of a Smti half-sib

family which is a very rare event (Fig. 14). It has been repmied that mutations to new

length alleles are more frequently detected with a highly polymorphic probe and that

69

Discussion

most of these mutant alleles appear in the low molecular weight regions with four base

recognizing enzymes (Jeffreys eta/., 1988a). Our results with OMS1 probe (GGAT)

contradicts this observation because the probe did not reveal very high level of

polymorphism and mutant allele was detected in about 20 kb range though the genomic

DNA was digested with four base recognition enzyme Mbol. The average mutation

rate· at this locus, using more family samples was not studied because the probe did

not reveal sufficient level of polymorphism that could eventually be used as

fmgerprinting probe.

Telomeric repeat sequence (TT AGGG)n had been repmied to be conserved during the

course of evolution in most of the eukaryotic species from yeast to bony fish, reptiles,

amphibians, birds and mammals including humans (Meyne et a/., 1989). In several of

the mammalian species, this repeat is known to be over-represented as a major

component in the genome (Fanning, 1987; Amason et a/., 1988). Based on the

observations in different animal models, it was speculated that the presence of

TTAGGG stretches in the sites other than the telomeric regions may be conelated to

the evolutionary status of the species (Meyne eta/., 1989; 1990). This is conoborated

by the fact that in the avian species (chicken), these sequences are present in the

interstitial and centromeric regions of several macrochromosomes (Nanda and Schmid,

1994).

In the mouse genome, this telomeric repeat was found to be tenninally located in the

chromosome (Kipling and Cooke, 1990) but by pulse field electrophoresis of Mboi

digested genomic DNA, multiple discrete polymorphic bands in the range of 20-140 kb

were detected upon hybridisation. Interestingly, several novel bands were also

detected with this repeat even in a highly inbred mouse strain. These observations, on

the localisation of this repeat (Nanda and Schmid, 1994) and the probable

70

Discussion

recombinations or rearrangements with other DNA termini (Pluta and Zakian, 1989),

motivated us to use it as probe in southern blot hybridisations with Mbol digested

buffalo genomic DNA to detect DNA fmgerprint. We included DNA samples from

other domestic species also for a comparison (Fig. 15).

As compared to signals detected in other domestic animals, the intensity was weak in

bubaline genome though equal amount of DNA (5 J.tg) was used for restriction

digestion with Mbol enzyme from each species. The slight smeruy signal below 23 kb

in the bubaline genome indicates that the (TTAGGG)n repeat does not always occur in

a clustered manner, instead may be interspersed with other repetitive sequences

harbouring restriction site for Mbol enzyme.

ABshire eta/., (1989) reported three different types of repeats in the human telomere

with non-random distributions but the number of units, types and anangements of

these repeats were different in the mouse telomere. It is likely that bubaline telomeres

have other repeats also with frequent sites for Mbol enzyme generating small

fragments giving rise to smeruy signals in the lower molecular weight regions. Other

enzyme combinations were not tried since it was known that (TTAGGG)n repeat

containing fragments are usually devoid of recognition sites for all restriction enzymes

tested so far (Kipling and Cooke, 1990). In the bubaline genome, presence of

polymorphic fragments of very high molecular weight, positive to telomere sequences

cannot be ruled out but such fragments are difficult to resolve on conventional agru·ose

gel.

5.3 Somatic and germ line stability of the fingerprinting probe:

Repeat sequence instability is known in many of the diseases of connective tissues like

muscles and nerves (Numberg eta!., 1991; Richards and Sutherland, 1992) and in

neoplastic conditions (Hastei et a!., 1990). The reliability of a fingerprint probe

71

Discussion

depends on its stability and consistent reproducibility of band profiles throughout the

life of an ~dividual with the DNA obtained from any part of the body (Nurnberg et

a/., 1989). In the present study, the probe OAT18 revealed considerable somatic

stability with respect to band profile with Mbol digested DNA from different tissues of

the same individual animal.

Since the methylation sensitivity of the Hinfl enzyme is known (Kessler and Holtke,

1986) and since DNA from tissues is more prone to methylation (Bmford et a/.,

1992), somatic stability was studied with the equally informative Mbol enzyme.

The instability of the repeat length in tumour tissues or any other cell line of buffalo

origin could not be studied due to the non-availa\>ility of such samples. However, a

major reshuffle in the repeat copy number, either an increase or decrease, may be

expected in the tumour tissues of buffalo also. If such somatic mutation in the tumour

tissues can be established, the marker would be useful for early detection of such

neoplastic conditions in this species.

A high level of heterozygosity reflects the increased level of mutation events and the

probes specific for such hypervariable regions would detect more mutant alleles

compared t~ less polymorphic probes (Jeffreys et a!., 1988a). Under such

circumstances, -the chances of false parentage exclusions will also be high when a

hypervariable single locus probe is used. Therefore, a multilocus polymorphic marker

that does not detect loci undergoing frequent mutation will be an ideal fingerprint

probe. In the present study, the mutation rate detected with OAT 18 probe is very low

and the hybridisation pattem also showed vety strong signal intensity, indicating

specificity of the alleles. The estimated frequency of 0.029 of mutant alleles given

here using a single oligo probe is significantly lower than 0.07 repot1ed in cattle using

a panel of six microsatellite probes (Trommelen eta/., 1993). However, this rate of

72

Discussion

mutation was much higher than the repmted frequency (0.004) in the human at

microsatellite loci (Jeffreys et a!., 1985a). If more parent offspring samples are

included in the study, the rate of occurrence of mutant alleles would be even lower

than the observed value since the average number of variable offspring bands would be

high. The results obtained suggest that OATIS fulfills the above mentioned criteria of:,

being an informative probe for DNA fingerprinting.

5.4 Relevance of pedigree analysis:

The important aspects of pedigree analysis are to identify the successful breeders

(mating partners) in a population, to have better assessment of effective population size

and to estimate the long term losses in genetic variability. When the level of

polymorphisms between Munah and Smti breed families were compared, the latter

showed a low level of heterogeneity. This reflects the extensive inbreeding in the

Surti herd since only two breeding bulls have been used as the source of semen for the

past ten years. The dams of some of the calves under study are offsprings of either of

these two bulls. This observation is pa1ticularly important to asce1tain the extent of

inbreeding in this confined group and to take appropriate measures to introduce new

·alleles into the population in order to purge the undesired level of homozygosity. This

aspect should be looked into more seriously, especially when the herd is maintained

for superior germplasm conservation of this pruticular breed. The present study

reveals that in the process of bringing a superior and elite gene pool into the herd for

genetic improvement of the breed, inbreeding and genetic depression had been going

on for the past several years.

With ongoing frequent inbreeding, more and more alleles would be fixed resulting in

an average genotypic frequency of one, under extreme conditions. This may be easily

determined by the genetic vruiability of the founder population (breeding stock) which ., ,

73

Discussion

in tum is dependent on the average number of alleles per locus distinguishable by the

DNA fmgerprint approach. Thus, the present work seems to have immediate relevance

in the dairy industry of our country.

In case of highly inbred populations, the overall discriminating power of even the most

polymorphic probe will decrease and the individual animals will have more chance of

sharing an allele (band). Similarly, a decrease in the paternity index also lowers the

exclusion probability of false paternity. This aspect is well explained from our work

where even by using the highly polymorphic probe OAT18, paternity of the calf from

Surti herd could not be conclusively confi1med.

The dependence of allelic frequency on inbreeding is well established by the earlier

workers through a linear approximation (Kuhnlein eta/., 1990). Using OAT18/Hinfl

probe/enzyme combination reported here, it is possible to determine the extent of

inbreeding in the buffalo population in each isolated fa1m. This would provide

information on the genetic variability of the breeding group enabling one to assess the

response to selection while carrying out marker assisted selection (MAS) in the

breeding herd. Paternity index of the fingerprinting probe OAT 18, in the Surti breed

was found to be much lower due to less number of paterpal specific bands for reasons

described earlier. The analysis of the phylogenetic trees of these families suggests that

female buffaloes ranking very close to the breeding bulls must be prevented from

mating with the latter to reduce inbreeding and to maintain the heterogeneity in the

gene pool.

5.5 Significance of breed affiliation:

The OAT15/Mbol pro~e/enzyme combination was useful for the grouping of the

OTUs according to their breed characters, although this probe was found to be

relatively less polymorphic for DNA fingerprinting compared with OAT 18. This

74

Discussion

observation was in accordance with an earlier report mentioning that the markers

which have higher F st values tend to produce phenograms where clusters are more

defmed (Nakamura eta/., 1987). On the contrary, when the probe detects a locus with

high heterozygosity (low F sJ, there is extreme level of diversity indicating presumably

higher mutation rate and such probe is non-informative for a cluster analysis or for

inter-population studies. This explains why clustering of breed groups could not be

obtained using a relatively more polymorphic probe OAT 18 with Hinfl digested DNA

samples.

Of the four breeds ~tudied, the semi-wild type Toda resembled swamp buffaloes in its

phenotypic appearance but the karyological pattern resembles that of Munah breed

(Nair et a/., 1986; Sethumadhavan et a/., 1987). The phylogenetic relationship of

Toda breed, based on sequence variability detected at microsatellite loci, showed that

the degree of sequence divergence of this breed is significantly greater as compared to

the other domestic breeds. This implies the need for gennplasm conservation and

management of this breed as a separate unit.

The phenogram obtained using OAT15/Mbol probe/enzyme combination is in

accordance with the earlier karyological report where close relationship of the Toda

breed with that of Murrah was shown. The indices of genetic distance (GD) at two

different loci also confirmed the proximity of the Toda to Munah breed. Among the

domestic breeds, Murrah and Sm1i were genetically closer with respect to genetic

distance (GD). The breed evolved from the cross between Munah and Sm1i is

Mehsana, which was found to be close to Sm1i breed based on clustering result

obtained using OAT15/Mbol but when the genetic distance at (TGG) and (GGAT)

repeat loci was estimated, using Nei's genetic distance method, Mehsana was found to

be equally close to both Toda and Munah breeds. The exact degree of relationship can

75

Discussion

be seen in Fig. 34. It appears that the Mehsana breed would have derived favourable

·alleles from both Murrah and Surti for adaptation to its immediate habitat in the given

geographical location. However, its relatedness with the semi- wild Toda breed cannot

be explained from this preliminary data . . , :

We envisage that the OAT15/Mbol would prove to be useful probe/enzyme

combination in the extensive cross breeding programs of the buffalo species with a

view to propagating desired quantitative trait loci. The high yielding MuiTah breed is

widely used for upgrading the inferior getmplasm but has some disadvantages like

massive body size and lower feed convertibility compared to Surti. In cross-breeding ·

program, assessment of overall allelic contribution in the F 2 and in the subsequent

generations is important. Thus, information gathered on the degree of .relatedness

between individuals will help in limiting the exotic gene flow into a population upto.

the desired level of 50-62.5%.

· 5.6 Genetic divergence with respect to genomes of other species:

The hypothesis that VNTRs are· enhancers or regulators of transcription (Spandidos

and Holmes, 1987) would mean that hypervariability associated with them should be

conserved during the course of evolution. Many of the microsatellites, together with

their flanking sequences, have been shown to be conserved in closely related species

(Pardue et a/., 1987; Stallings et a/., 1991 ). These microsatellites have been used as

markers to show evolutionary relationship amongst different species and to constmct

genetic map in the species of economic importance and finally to monitor .the

inheritance of desired traits. Multilocus hypervariable microsatellite probes reveal

enormous genetic variability in the fmm of restriction fragment length polymorphism

spread over the entire genome. They evolve rapidly allowing estimation of the overall

genetic variability and providing a more amenable molecular tool for looking at the

76

Discussion

phylogeny of closely related groups. Despite all these, the reliability of using

microsatellites as potential markers measuring the evolutionaty changes depends on

their mutation rate and their evolutionaty conservation between the species (Dover,

1987). In the present study, we could not estimate the mutation rate at these repeat

loci due to non-availability of family samples in all the species tested.

Even though the two species, cattle and buffaloes are reproductively isolated and

classified into two seperate genera, eru·Iier reports show that they have many

homologous loci and are morphologically related (Dung et a/., 1993). Our results

demonstrate the potential applications of the technique of DNA fingerprinting by using

synthetic oligos in deriving the phylogenetic relationships among closely related but

relatively recently diversified domestic animals. Based on the band sharing coefficient

and mean allelic frequencies at the microsatellite loci, the degree of relatedness and

genetic distance between each species were detennined. The bubaline species was

found to be closer to the ovines and caprines at the molecular level and its genome

organisation with respect to the microsatellite repeat loci was found to be different

from that of the bovine (cattle) species. This observation contradicts the similarities . between bubaline and bovine species with respect to morphology (Dung et a/., 1993)

and certain biochemical parameters (Mahadevan, 1992). Our results show the relative

genetic similarity of the bovine species to swine (family suidae). The genetic distance

data obtained by this study also revealed the close relationship of the swines (pig) to

lagomorphs (rabbit). Earlier studies on sequence homology at coding and non-coding

regions had also shown such topology of ungulates with lagomorphs (Easteal, 1990).

This explains that the evolutionary rate of different repeat loci, compared to their

coding sequences, are different in the genomes of different species.

77

Discussion

5. 7 Conclusion:

The present study has been conducted employing empirical approach to first develop '

the most informative probe/enzyme combination for DNA fingerprinting and breed

affiliation and then to demonstrate that hypervariable mini/microsatellite loci can be

used for genome individualisation and for population structuring of the desired species.

To the best of our information, no probe useful for buffalo DNA fingerprinting was

ever reported and this seems to be the fust such detailed infmmation. During the

course of investigation, we also discovered that GAT A quadmplet repeat does not

cross hybridise with buffalo, goat, cattle (Fig. 3) and one strain of tilapia (unpublished

data) genomes . This again, seems to be the first such observation because so far, this

Bkm derived repeat has been thought to be evolutionarily conserved throughout the

eukaryotic genome.

Of all the known short tandem repeats used in the eukruyotic genome for DNA

fingerprinting, OAT18/Hinfl probe/enzyme combination was found to be most

informative for bubaline species. But before using this probe in the actual farm

conditions, it is desirable to develop a reliable database on the genetic variability,

segregation pattern and mutation rates at different repeat loci using a large number of

random individual samples (Lewin, 1989).

The parameters used in assessing the degree of relationships like band shruing

coefficients and· allelic frequency need to be estimated for a large population of each

breed in order to use a probe successfully in actual pedigree analysis and progeny

testing programs in the farms.

Analysis of large families including full sibs and half sibs having both male and female

offsprings would be appropriate in studying the segregation patte1n of the alleles at

these repeat loci as well as for estimating the mutation rate with respect to different

78

· Discussion

length alleles. In humans, more accurate studies are being carried out

with respect to mutation rate by taking into account the rate of appearence of novel

alleles in different molecular weight range at micro-molecular level (Jeffreys et a/.,

1994b}. In the present study, a more precise scoring of bands (alleles) at lower

· molecular weight was difficult due to constraints of not having lengthy DNA

fmgerprinting electrophoretic apparatus for better resolution of the bands. Once

detailed database on the genetic structure .of this species is developed, linkage of any

particular allele/locus with production/adaptability traits can be studied and then the

marker will be of immense use in the breeding programs of bubaline genome. We are

aware of these aspects and we beleive that the present study has paved a path towards

reaching the above mentioned goals.