Abstract: Umblachery cattle was assessed genetically using 25 microsatellite markers, as recommended by FAO. The number of alleles was ranging from 2 to 6 with a mean of 4.0±0.11. The mean number of effective alleles were 2.91±0.09. The allele sizes were ranging from 94 to 300 bp with the frequency distribution of 0.0111 to 0.9375. The estimated heterozygosity value was high 0.6139±0.02 and the PIC was 0.5625±0.03 and the loci screened were polymorphic and overall mean FIS value (-0.0487) suggested the excess of heterozygosity in the population.
INTRODUCTION
Umblachery is a reputed draught breed of Thanjavur, Thiruvarur and Nagapattinam districts in eastern parts of Tamilnadu state in south India. This breed is the outcome of selection for short stature, suitable for work in marshy rice fields of Cauvery deltaic region (Thangaraju et al., 2001). As per the 1998 estimate, a total of 2.83 lakhs of Umblachery cattle were available in the breeding tract. However, the breeding tract of this breed has shrunken over the years due to mechanization. Introduction of crossbreeding and lack of concerted efforts for improvement and conservation have deteriorated the status of this breed. Umblachery bullocks are used for ploughing, carting, thrashing and paddling. The bullocks are capable of doing work continuously for 6 to 7 h under hot sun. A pair of bullocks can haul a load of 2 to 2.2 tonnes (including the cart weight) over a distance of 20 km in about 7 h.
The phenotypic, biochemical and cytogenetic characteristics have been studied and documented already (Sivaselvam et al., 2003; Kumarasamy et al., 2003). Hence, the present study was carried out with a view to characterize the breed in respect of microsatellite markers as microsatellites are the most powerful genetic markers for biodiversity evaluation. In addition, this study would bring out the variability at DNA level and genetic structure of the breed.
MATERIALS AND METHODS
Genomic DNA for Analysis
Blood samples were collected from 48 unrelated Umblachery cattle in its
breeding tract. Genomic DNA was extracted from the samples using a routine high
salt method as described by Miller et al. (1988). Quantity and quality
assessment of isolated DNA samples were done by spectrophotometer.
Microsatellite Analysis
As per the suggestions of FAO (2004) in the Secondary Guidelines for Development
of National Farm Animal Genetic Resources Management Plans for global management
of cattle genetic resources using reference microsatellites, a panel of 25 sets
of microsatellite markers (Table 1) were selected to screen
the population of Umblachery cattle during the year 2006. These markers were
amplified using thermal cycler (MJ Peltier Thermal Cycler 200) in a PCR reaction
mixture volume of 20 μL, containing 50-100 ng of template DNA; 1.5 mM MgCl2;
5 picomoles each of forward and reverse primers; 0.75 units of Taq DNA polymerase
and 100 mM dNTPs. Amplification was carried out with different annealing temperature
(51 to 58°C for 45 sec) for various primers for 30 cycles.
Amplified PCR products were checked on one per cent agarose gel and visualized through UV illumination after staining with ethidium bromide. The samples which showed amplification were resolved through 6% denaturing polyacrylamide gel at a voltage of 1200 to 1400 for a period of 2-3 h (depending upon the size of PCR products) with 10 bp DNA ladder (Invitrogen, USA) as a molecular weight marker. The gel was subjected to silver-staining procedure (Cominicini et al., 1995) for genotyping.
Statistical Analysis
The basic statistics such as mean and standard error were calculated as
per Snedecor and Cochran (1989). Scoring of alleles and sizing of fragments
were done using Diversity Database (BioRad, USA) software followed by manual
verification. Allele frequencies were estimated by direct counting. Polymorphism
Information Content (PIC) was estimated using the formula developed by Bostein
et al. (1980). The observed heterozygosity, Hardy-Weinberg equilibrium
proportion, expected heterozygosity and Wright’s fixation index were calculated
by using the software POPGENE 32 (http://www.ualberta.ca/~fyeh).
RESULTS AND DISCUSSION
The parameters estimated out of microsatellite analysis in Umblachery cattle such as number, size and frequency of microsatellite alleles, Chi-square value, polymorphism information content and heterozygosity for different microsatellite loci are furnished in Table 1.
Number, Size and Frequency of Microsatellite Alleles
The number and size of alleles were ranging from 2 to 6 and 94 to 300 bp,
respectively in 25 microsatellite loci in Umblachery cattle. The mean number
of alleles was found to be 4.00±0.11 per locus. A total of 100 numbers
of alleles were found distributed in these polymorphic loci in the breed. The
loci, ETH152 and INRA063 exhibited lowest number of two alleles each and ETH225
and INRA005 loci, each possessed 6 number of alleles. However, the effective
number of alleles (ne) was ranging from 1.13 to 4.89 with a mean of 2.91±0.09.
The mean number of alleles observed in the study is lesser than the number reported
in Sahiwal (5.2) and Deoni (5.9) breeds of cattle of India (Mukesh et al.,
2004). Prabhu (2004) and Kumar (2006) studied the microsatellite pattern, respectively
in Amritmahal and Hallikar cattle breeds in Karnataka, a neighbouring state
of Tamilnadu, using the same sets of microsatellites. The respective number
and sizes of alleles were ranging from 2 to 8 and 89 to 302 bp and 3 to 9 and
102 to 294 bp in Amritmahal and Hallikar breeds. In another study (Karthickeyan
et al., 2006), the Krishna Valley breed of south India revealed 3 to
7 number and 94 to 300 bp size of alleles in these loci, which corroborate with
the findings of the present study. In general, the number and sizes of microsatellite
alleles observed fall within the range mentioned in the Secondary Guidelines
for Development of National Farm Animal Genetic Resources Management Plans,
published by FAO.
These alleles occurred at a minimum frequency of 0.0111 (140 bp allele in ETH225)
and a maximum frequency of 0.9375 (194 bp allele in ETH152 locus). Similar ranges
in the frequency of alleles were observed in the other Indian breeds of cattle
viz. Amritmahal, Hallikar and Krishna Valley (Prabhu, 2004; Kumar et al.,
2006; Karthickeyan et al., 2006). However, the higher frequency of allele
(in ETH 152) observed in the present study as well as in the other Indian breeds
of cattle indicates that the preponderance of this particular allele (194 bp)
in the Indian zebu cattle population.
| Table 1: | Microsatellite allele frequency, Polymorphism Information Content (PIC) and heterozygosity in Umblachery breed of cattle |
| na-Observed No. of alleles; ne-Effective No. of alleles; HWE-Hardy-Weinberg Equilibrium; He-Expected Heterozygosity; *-Significant; **-Highly significant | |
Hardy-Weinberg Proportion
The Chi-square (χ2) test for Hardy-Weinberg equilibrium
revealed that the Umblachery population is not in equilibrium with respect to
17 out of 25 loci screened. The disequilibrium exhibited in most of loci revealed
that there might be unobserved null alleles (those which could not be amplified)
in those loci which have not been, hitherto, identified by other means. The
deviation of 68% of the loci from equilibrium would be attributed to high mutation
rates and size homoplasty, the inherent qualities of the microsatellites. Further,
the disequilibrium could have resulted from the sampling from a range of distinct
locations, within the breeding tract of the Umblachery, as suggested by Dorji
et al. (2003).
Informativeness of Microsatellite Markers
The Polymorphism Information Content (PIC) value is the statistical assessment
of informativeness of a marker. This value was ranging from 0.1103 (ETH152)
to 0.7652 (INRA005) with a mean PIC of 0.5625±0.03. Except a few loci,
all other loci showed high PIC values of more than 0.5 indicating more polymorphic
information content in the breed. The polymorphism at any locus is created by
increasing dinucleotide repeats and mutation. The higher number of alleles found
in the INRA005 locus has exhibited high polymorphic information content and
the lower number of alleles (2) in ETH152 with a higher frequency of 194 bp
allele resulting in lower PIC value. In Ongole cattle, the range of PIC between
0.15 and 0.79 was reported by Metta et al. (2004) using 10 different
microsatellite markers which is in close agreement with present study. Almost
similar range of PIC (0.13 to 0.80) was observed in the same study for Deoni
cattle of India. While Kumar et al. (2006) observed slightly higher PIC
range of 0.2322 to 0.8654 in Hallikar cattle using 19 microsatellite markers.
Karthickeyan et al. (2006) reported PIC range of 0.2583 (ILSTS030) to
0.7975 (INRA035) with a mean of 0.6209±0.03 in Krishna Valley cattle
for the same set of markers. In general, the population has got high polymorphism
information content of 56% which indicates that these markers are highly informative
for genetic assessment of Umblachery cattle.
With-in Population Genetic Variability
The heterozygosity is an appropriate measure of genetic variability within
a population. In the present study, overall means for observed and expected
heterozygosities were 0.6581±0.03 and 0.6139±0.02, respectively
with the ranges of 0.9773 (HEL009) to 1.000 (ETH225) and 0.1172 (ETH152) to
0.7955 (INRA005). Majority of the loci had relatively higher expected heterozygosity,
reflecting the existence of variation in the breed. The mean expected heterozygosity
value is comparable to that of Sahiwal (0.61) and Hariana (0.66) and lower than
that of Deoni (0.70), the other Indian cattle breeds studied by Mukesh et
al. (2004). Whereas low average heterozygosity (0.46±0.1) was observed
in Ongole cattle (Metta et al., 2004). In Krishna Valley cattle (Karthickeyan
et al., 2006), the heterozygosity value of 0.6569±0.03 with higher
range from 0.3047 (ILSTS030) to 0.8220 (INRA035) was observed. The high heterozygosity
values observed in the present study indicate more number of polymorphic loci
in Umblachery cattle. This implies the higher amount of genetic variability
that can be still exploited in the population.
Within-Breed Genetic Diversity
The within-breed diversity was estimated using the FIS (within
population inbreeding estimate; Wright’s Fixation Index) values as a measure
of heterozygote deficiency. The negative FIS value of -0.0487 over
all loci reveals that the Umblachery breed is having a wide genetic variability
with excess of heterozygotes and outbred in nature even though some of the loci
exhibited the positive values. On the contrary, Metta (2004) reported high FIS
value (0.36) which resulted from small sample size (n = 17) in Ongole breed.
In spite of the general belief that Umblachery breed has originated from Kangayam
breed of cattle, the variability is maintained to a greater extent and their
selection for short stature should have been done from a very wide ancestral
population.
CONCLUSIONS
From the large number of polymorphic loci in Umblachery breed and the overall mean polymorphism information content of 56%, these markers are highly informative and can be utilized for characterization of domestic animal biodiversity. It also opens up the scope for exploiting the genetic variability in the population for conservation. Comparative analysis with other Indian draught cattle breeds will determine the genetic distance and evolutionary relationship of this breed with other zebu cattle of India.
ACKNOWLEDGMENTS
The authors wish to extend their gratitude to the Indian Council of Agricultural Research, New Delhi, India for the financial assistance for molecular characterization work through the Network Projects of the National Bureau of Animal Genetic Resources, Karnal, India.