Polymorphism of microsatellite loci in MHC complex for three populations of local cattle in Albania

Anila Hoda, Ylli Biçoku, Lumturi Papa and Alfred çaushi

Department of Animal Production, Agricultural University of Tirana, (AUT), Albania
hodanila@yahoo.com

Abstract

Cattle is the most important livestock species for Albanian farmers. Major Histocompatibility Complex (MHC) plays a key role in immune response. We intend to study the polymorphism in two microsatellite loci of MHC complex, for three populations of local cattle breed located in North of Albania. The study was carried out in 75 unrelated individuals.

 Both markers were highly polymorphic. The analysis revealed a great number of alleles 15 and 31 per BoLaMHC1 and BoLaDRB respectively. The level of gene diversity was very high: 83% for BoLAMHC1 and 94% BoLADRB. This can be explained with the participation of the molecules encoded by MHC complex in the immune response, and with the balanced selection which act on antigen recognition site. PIC values were higher than 0.5. Therefore, both loci can be used as genetic markers.  

Keywords: BoLaMHC1 and BoLaDRB loci, selection, heterozygosity, genetic variability

Introduction

The cattle is the most important livestock species in Albania. Since years 60, are applied different breeding programs in order to improve the local cattle breed. There are different exotic cattle breed in Albania and many crossbred individuals. There is a high level of risk for local cattle breed to be extinct and a very limited number of pure bred individuals can be found in North of Albania, in the mountainous area. The individuals are adapted to harsh environment conditions, to poor pasture and the low level of concentrate in feeding.

Microsatellite are short tandem repeats, that are widely used as genetic markers, since they exist in a high number are distributed throughout the genome and can be easily genotyped. There are several studies regarding the diversity of microsatellite loci in MHC region of different species (Ammer et al 1992; Ellegren et al 1993; Schwaiger et al 1993; Gruszczynska et al 2002). In this study we intend to compare the level of polymorphism in two microsatellite loci, BoLAMHC1 of MHC Class I, and BoLADRB of MHC Class II, in three populations of local cattle breed, from North of Albania in the mountainous area of breed location.

Material and Methods

Sample collection and microsatellite markers

A total of 75 randomly sampled animals representing three populations of local cattle breed, in Albania were analyzed. For each population, Nerlys, Bogë and Xhan were sampled maximum three unrelated individuals (two females and one male) per flock. The populations are marginally farmed and indigenous. There are used 2 microsatellite markers, of MHC region: BoLAMHC1 and BoLADRB.

PCR condition and fragment length analysis

Blood samples (5 ml) were collected in EDTA vacutainer tubes. Buffy coat of leucocytes was used for DNA isolation according to phenol – chloroform extraction, proteinase K digestion and ethanol precipitation of DNA (Sambrook et al 1989). All samples were genotyped for 2 microsatellite markers of MHC complex: BoLAMHC1 and BoLADRB.

The Polymerase chain reaction (PCR) amplifications were carried out using a programmable thermal cycler (Biometra). The reactions were performed in 20 ml reaction volumes with 1-2 ng of template DNA, 22 pmol of each primer, 200µM dNTP mix, 2 U of Taq DNA polymerase, buffer supplied by the enzyme manufacture. The set of primer used for the amplification of B0LAMHC1 locus was: primer 1 5’-ATCTGGTGGGCTACAGTCCATG-3’ (Groth and Wetherall 1993) and primer 2 5’GCA ATG CTT TCT AAA TTC TGA GGAA-3’ (Groth and Wetherall 1993). The set of primer used for the amplification of BoLADRB locus on MHC Class II region was primer 1 5’-GAGAGTTCACTGTGCAG-3’ and primer 2 5’-CGTACCCAGAKTGAGTGAAGTATC-3’. The amplifications were performed as follows: an initial denaturation step at 94^oC (3 min) was followed by 32 cycles at 94^oC (1 min), and at 60^o (1 min) and at 72^oC (1 min). The final extension step was at 72^oC for 5 min. 

The PCR products (0.3-0.1 ml) were labeled with fluorescent labeled primers and detected using 6% denaturing sequencing gel. PCR products were separated by an electrophoresis machine, (LI-COR, model 4300 DNA analyzer).

Statistical analyses

Allele and genotype frequencies were calculated using Genalex program (Peakall & Smouse 2006). Genetix version 4.05.2 was used for measuring genetic diversity within population, by the calculation of observed heterozygosity (H_O) and mean unbiased estimates of gene diversity (H_e), (Nei 1978), mean number of alleles (MNA) per locus, the number of private alleles (PA, alleles found in only one breed). The program FSTAT was used for the calculation of corrected allele diversity (allelic richness) and inbreeding coefficient (F_IS). The polymorphic information content (PIC) index for each marker was calculated according to Botstein et al 1980.

Results and Discussion

The allele and genotype frequencies of two microsatellite loci were determined in three populations of local cattle breed from North Albania. In table 1 are given the alleles and allelic frequencies for both loci in three populations of local cattle breed. For all population the most frequent allele of BoLAMHC1 locus, is the allele 186, with frequency of 0.271, 0.417 and 0.292 in Nërlys, Xhan and Bogë respectively. For the BOLADRB microsatellite locus of MHC Class II region the most frequent allele for all populations is the allele 190 with a frequency of 0.125, 0.174 and 0.104in each population.  

 The total number of alleles and allele size range for each locus are presented in Table 2. Allelic size for BoLAMHC1 ranged from 168 to 206. Fragment length of different alleles of BoLADRB locus varied from 148 to 228.  

Summary statistics of allelic distribution of both loci is presented in table 3, and the histogram of allelic frequencies is presented in Figure 1 and 2. A total of 46 alleles were detected over two loci in 75 individuals. In all populations of cattle breed we detected a high level of polymorphism. MHC microsatellite loci were found to be highly polymorphic, also in other species (Paterson 1998; van Haeringen et al 1999, Radwan et al 2007, Rahimnahal et al 2010). It is already well known that the MHC loci are highly polymorphic, since the proteins by MHC genes play an important role in initiating immune response to parasites (Gaudieri et al 2000). Allelic richness for each of microsatellite loci in both populations is presented in table 2. Allelic richness was 11 and 21 for BoLAMHC1 and BoLADRB loci respectively. Both markers have very high PIC values.

Figure 1. Histogram of allele frequencies at the microsatellite locus in MHC Class I in each populations

Figure 2. Histogram of allele frequencies at the microsatellite locus in MHC Class II in each populations

We have identified 15 alleles for BoLAMHC1 locus. For the locus BoLADRB we have identified 31 alleles. Table 3 shows the observed and expected heterozygosity at each locus for three populations. Observed heterozygosity is lower than expected heterozygosity. 

The mean number of alleles (MNA) and expected heterozygosity (H_E) are useful in estimating the genetic diversity of a breed. All populations indicated a high level of genetic diversity. The balancing selection is the primary cause of the great genetic variability seen in the major histocompatibility complex Allelic richness showed high values for each locus, in each population and in total as well. The mean expected heterozygosity (H_E) was similar in all populations, having a very high value. The level of gene diversity was very high: 83% for BoLAMHC1 and 95% BoLADRB. The level of heterozygosity is high in different mammal species, since these loci play an important role in the immune response to infections. One reason of maintenance of MHC genetic diversity is heterozygote advantage, The individuals that are heterozygote in any MHC locus have a better immune response than homozygote individuals (Doherty and Zinkernagel 1975; Hughes & Nei 1988; Penn et al. 2002 Wegner et al. 2004). The breeds live in extensive condition, grazing in the pasture. This may be the explanation for the high degree of genetic variability. Observed heterozygosity (H_O) and the mean number of alleles per locus (MNA) were similar in all populations. These populations represented a reservoir of allelic and genetic diversity, that have to be in consideration in breeding programs and in husbandry.

 Inbreeding values for all populations were positive, but not very high, showing heterozygotes deficiency within breeds. The values are given in table 5. F_IS values are 0.058, 0.038 and 0.031 in each population. There were found 15 breed specific alleles (Table 4), but their frequency was lower than 5%. A feasible reason might be the presence of population substructure within the breed, which may lead to Wahlund’s effect. Sampling was carried out in several flocks per populations. The average genetic differentiation between all breeds (F_ST value) was 0.018 (Table 2), showing that about 2% of the total genetic variation was explained by population differences and 98% correspond to the differences among individuals within each breed.

 In three populations we found a high level of polymorphism for both microsatellite loci. We found eleven alleles of BoLAMHC1 gene in Nërlys, 12 in Xhan and 10 in Bogë, and for the BoLADRB locus we found 24, 19 and 23 in each population respectively. Both markers had high heterozygosity and PIC values displaying to be highly informative. Therefore these markers were appropriate for measuring genetic variation. MHC genes are used as markers in animal breeding. Major histocompatibility complex (MHC) genes are the most polymorphic genes found in vertebrates. MHC proteins play a crucial role in vertebrate immunity. Natural selection favors MHC heterozygous hosts because heterozygotes display a higher parasite resistance This is the first effort to characterize the local cattle breed on molecular level and the research has to go further, increasing the number of the markers. 

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Received 12 October 2010; Accepted 10 January 2011; Published 6 March 2011

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