Genetic characterization of different lines of meat type chicken by microsatellite markers

M B R Mollah, M Asaduzzaman, F B Islam, M G Azam and M A Ali

Department of Poultry Science, Faculty of Animal Husbandry, Bangladesh Agricultural University, Mymensingh-2202.
mbrmollah@gmail.com

Abstract

A set of chicken microsatellite markers covering different chromosomes were used to characterize four lines of meat type chicken namely Male Line White (MLW), Male Line Color (MLC), Female Line White (FLW) and Female Line Color (FLC). Among the 10 primers initially tested, six primers viz. MCW248, MCW295, MCW183, ADL0112, LEI094 and MCW104 produced high intensity amplification products with minimal smearing. These six primers were used to detect polymorphism among four lines of meat type chicken.

In total, 10 alleles were detected at the 6 microsatellite loci that are distributed on different autosomes.For all loci in four lines, significant deviation from Hardy-Weinberg equilibrium was found. Among the six loci, no line specific allele was found in all lines. The observed homozygosity value varied from 0.667 to 0.867. The highest observed homozygosity (0.867) was found in MLW. On the other hand, the highest observed heterozygosity (0.333) was found in MLC. The percentages of genetic homogeneity in all lines across all loci were 86.67, 76.67, 73.33 and 66.67 in MLW, FLC, FLW and MLC, respectively. The fixation coefficient (F_ST) of six loci varies from -0.008 to 0.238, with the mean being 0.102. The fixation indices (F_IT) ranged from -0.303 to 1.000. Individual’s fixation indices (F_IS) varied from -0.379 to 1. The findings of this study suggest that MLW, FLC and FLW lines reached more than 70% genetic homogeneity.

Key words: chicken line, DNA marker, genetic homogeneity, heterozygosity, polymorphism

Introduction

Inbred lines of chicken are indispensable for harnessing heterosis by two-way and/or three-way crossing, which is an established method for producing commercial broilers and layers. Development of inbred lines requires enormous efforts, huge capital investment and long time selection program. However, integration of recently developed molecular tools with conventional chicken breeding techniques that involves crossing of the best chicken possessing the most desirable traits (e.g., high meat yield or disease resistance, better feed efficiency) along with intensive selection has helped in achieving this target to a considerable extent. In addition, the role of inbred lines in biological research has fundamental importance by providing genetic diversity between lines and constancy over time and place within each individual line (Abplanalp1992, Crooijmans et al 1996, Ponsuksiliet al 1996, Vanhala et al 1998, Hassen et al 2009). Inbred lines, after genetic characterization within and among the lines, can be used as resource populations in genome mapping and linkage analysis between DNA markers and qualitative or quantitative traits. With a view to develop inbred lines of meat type chicken, a long term selection program is continuing at the Department of Poultry Science, Bangladesh Agricultural University (BAU), Mymensingh. In this selection program, phenotypic index selection is practiced; therefore, molecular information related to within and between lines homozygosity, rate of allele fixing and rate of inbreeding is unavailable.

Recently, an increasing number of studies have focused on the genetic characterization of chicken lines by molecular genetic markers. In poultry, different genetic marker systems have been used for estimation of genetic variability and relatedness successfully. They are DNA fingerprints (Lynch 1991), RAPD (Mollah et al 2009; Rahimi et al 2005) and microsatellites (Olowofeso et al 2005a, b; Kaiser et al 2000; Rikimaru et al 2007; Dávila et al 2009 and Tautz 1989). Molecular typing methods provide a powerful and reproducible approach of estimating genetic relatedness within and among chicken lines based on DNA variation. Many studies have been conducted to determine intra- and inter-lines characteristics in chickens. Comprehensive surveys of relatedness, including jungle fowl, layer and broiler strains and white leghorn, based on DNA fingerprints, have been described (Hillel et al 1989, Siegel et al 1992, Kuhnlein et al 1989 and Ponsuksili et al 1998; Kaiser et al 2000). Chen and Lamont (1992) and Plotsky et al (1995) characterized genetic variation in many highly inbred chicken lines by restriction fragment length polymorphism (RFLP), DNA minisatellite fingerprinting and randomly amplified polymorphic DNA (RAPD). Microsatellite polymorphism in commercial broiler and layer lines has been estimated (Crooijmans et al 1996; Kaiser et al 2000). Currently, the determination of heterozygosity and genetic distances based on microsatellite analysis is regarded as most convenient tool because many microsatellite loci are available and distributed throughout in the chicken genome. In addition, it shows a higher degree of polymorphisms, ease of identification and reproducibility than other markers, such as allozyme assay or random amplified polymorphic DNA analysis (Takahashi 1998; Zhang et al 2002a, b). The microsatellite markers are extensively used for estimating genetic structure, diversity and relationships because of many advantages: they are numerous and ubiquitous throughout the genome, show a higher degree of polymorphisms, and have a co-dominant inheritance (Zhou and Lamont 1999, Tautz and Renz 1989). Microsatellite markers are more accurate and efficient method for estimating genetic diversity and relationships among populations (Takezaki and Nei 1996; Romanov and Weigend 2001; Zhang et al 2002a; Hillel et al 2003, Nakamura et al 2006; Kamara et al 2007; Boruszewska et al 2009; Dávila et al 2009 and Tadano et al 2014). Therefore, the objective of this study was to estimate within and between genetic variability and relatedness among the selected lines of chicken using microsatellite markers.

Materials and methods

Experimental populations

Female chickens from four lines namely Male Line White (MLW), Female Line White (FLW), Male Line Color (MLC) and Female Line Color (FLC) as described previously (Ali et al 2013) were used in this study. These lines of chicken were developed at BAU Poultry farm by index selection for rapid early growth and better egg production. The major characteristics these chickens are shown in Table 1.

Collection of blood samples and isolation of DNA

Blood was collected from five to seven female chickens of each line and prepared for DNA isolation by using the procedure suggested by Hoelzel (1992) with little modifications. Approximately 1-1.25ml blood was collected by 5ml disposable syringe from wing vein. The collected blood was immediately transferred into the 1.5ml microfuge tube. The blood containing tubes were kept in ice box, transported to the laboratory and centrifuged at 5000 rpm for 10 minutes and the serum was discarded. Genomic DNA was extracted from clotted blood cell using the standard Phenol: Chloroform: Isoamyl alcohol method with slight modifications. The isolated DNA samples were diluted with DNAase free water in a new tube and the concentration was measured by using UV-spectrophotometer (Spectronic® GENESIS^TM 5). Finally, the diluted DNA samples were kept at -20°C until further use.

Selection of primers, PCR, electrophoresis and visualization of amplified products

Initially highly polymorphic microsatellite markers were screened from chicken microsatellite primer set (http://poultry.mph.msu.edu/) and published articles based on the allele size, chromosomal location and annealing temperature. The details of the primers that were initially selected are shown in Table 2. These primers were screened on three sub samples from each population to test their suitability for amplifying DNA sequence. These primers were evaluated on the basis of polymorphism and potential for population discrimination. Finally, a subset of six primers, out of ten was retained for further analysis. Each PCR amplification was conducted in a 14μl reaction mixture, which included 10 pmol of each primer, 100μM deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 0.5 U of AmpliTaq Gold (Applied Biosystems, Foster City, CA), and approximately 20 ng of genomic DNA as a template. The PCR procedure, using the GeneAmp PCR System 2700 (Applied Biosystems) was as follows: first denaturation step at 95°C for 10 min, 43 cycles of denaturation at 95°C for 1 min, annealing at primer-specific temperature (58 or 60°C) for 1 min, extension at 72°C for 1 min, followed by final extension at 72°C for 10 min. Then the samples were cooled at 4°C. The amplified DNA was placed into a submarine electrophoresis system (Mupid®-2X, Japan). A 100bp DNA ladder was also run along the amplified fragments. Electrophoresis was carried out at 100V for 45 minutes. Then the gel was stained with ethidium bromide for 15 minutes. The amplified fragments were visualized on UV Transilluminator and photographed by using Bimetra Gel Documentation System (Germany).

Data analysis

The genetic diversity of each line was assessed by calculating the number of alleles per locus and its mean, observed heterozygosity, unbiased expected heterozygosity (Nei 1987). Additionally, F-statistics [fixation coefficient of an individual within a subpopulation (F_IS), fixation coefficient of an individual within the total population (F_IT), and fixation coefficient of a subpopulation within the total population (F_ST)] per locus across four lines were calculated. Nei’s (1972) genetic distance and construction of a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram among lines with 1000 simulated samples which were carried out by using POPGENE (Version 1.31) (Yeh et al., 1999) computer program.

Results and discussion

The quality of isolated DNA is the major determinant in successful and reproducible Polymerase Chain Reaction (PCR), thus, the DNA samples were examined for its integrity through 1% agarose gel electrophoresis and absorption (OD) by spectrophotometer at 260nm and 280nm. The OD ratio (260/280nm) of all DNA samples was in between 1.68 to 1.75, whereas the concentration of extracted genomic DNA of all samples was in between 0.98 and 2.5 mg/ml. The value 1.68–1.75 of 260/280nm OD ratio indicated high quality DNA that was free from dissolved protein (Sambrook et al 1989). The PCR condition reported in the previous study (Cheng, 1997 and Croojimans et al 1996) did not work well with the lines of chicken used in this study. Thus, PCR condition was optimized by the examination of varying annealing temperatures and primer concentrations. Among the 10 primers initially tested, six primers namely MCW248, MCW295, MCW183, ADL0112, LEI094 and MCW104 produced high intensity amplification products with minimal smearing. These six primers were used to detect polymorphism among four lines (MLW, MLC, FLW and FLC). A representative microsatellite profiles generated by primer MCW0183 among the four lines of chickens are shown in Figure 1.

Figure 1.  A representative microsatellite profile generated by primer MCW0183 from four lines of chicken

The allele frequencies of six microsatellite markers across different lines of chicken are shown in Table 3. In total, 10 alleles were detected at six microsatellite loci distributed on different autosomes in the four lines of chicken. The summary of the observed number of alleles, effective number of alleles and Shannon’s information index are shown in Table 4. For all loci in four lines, significant deviation from Hardy-Weinberg equilibrium was found. Among the six loci, no monomorphic locus was found. As shown in Table 4, there was no line specific alleles were detected in this study.

The result using six microsatellite primers indicated that these lines of chicken show relatively low variation and allele distribution. The low distribution of the number of allele per population may be due to, firstly, the primer selected for this experiment have low polymorphism or use of agarose gel to resolve polymorphisms because agarose gel have low resolution power than acrylamide gel. Further use of acrylamide gel might provide an explanation in this regard. Secondly, the low distribution may be due the developmental strategies of these lines as they were first initiated from a very small base population.

The summary of the observed homo- and heterozygosity value of four lines of chicken are shown in Table 5. The observed homozygosity value varied from 0.667 to 0.867. All values of observed homozygosity were higher than observed heterozygosity. The highest observed homozygosity (0.867) was found in MLW. On the other hand, the highest observed heterozygosity (0.333) was found in MLC.

The estimated genetic homogeneity in all lines across all loci are shown in Table 6. The homozygosity percentages were 86.67, 76.67, 73.33 and 66.67 in MLW, FLC, FLW and MLC, respectively. From the Table 6, it is evident that MLW lines are more homozygous (86.67%) than other lines of chicken. This result seems to reflect the present breeding state of MLW, in which intense selective breeding is carried out among closely related birds in small population to improve growth and reproductive traits. In contrast, relative high heterozygosity was observed in MLC (33.33%). This might be due to the fact that FLC line was introduced later and thus they are undergone for few generation of selection compared to other lines.

The summary of F-statistics and gene flow for all six loci are shown in Table 7. For the genetic divergence of the lines, the fixation coefficient (F _ST) of six loci varies from 0.008 to 0.238, with the mean being 0.102. The fixation indices (F_IT) ranged from -0.303 to 1.000. Individual’s fixation indices (F_IS) varied from -0.379 to 1.

The F_ST represent a degree of nonrandom mating (deviation from Hardy-Weinberg equilibrium). A positive number for F_ST means deviation from Hardy-Weinberg equilibrium. As expected, all loci showed a positive F_ST number, which indicated that nonrandom mating was practised in these lines of chicken. From the breeding strategies of these lines (Ali et al 2013), it is evident that that these lines were produced through inbreeding with selection to improve growth or egg production. The mean F_ST value of 0.102 indicates that approximately 89.80% of the total genetic variation is caused by line differences, whereas the remaining 10.20% is due to differences among individuals within lines. Tadano et al (2007) reported the mean F_ST value of 0.383 from long tailed Japanese breeds, whereas Vanhala et al (1998) reported the mean F_ST value of 0.303 from 8 Finnish chicken lines using 9 microsatellite markers. As compared to other domestic animals, the mean F_ST value in this study was relatively low. For instance, Kim et al (2001, 2005) reported the mean F_ST value of 0.261 and 0.154 for pig breeds and East Asian native dog breeds, respectively. This fact might suggest that the chicken lines used in the present study are genetically subdivided to a higher extent than other chicken breeds/lines because of inbreeding between related individuals or more intensive selection to fix desirable traits.

Table 8 shows the Nei’s original measure of genetic identity and distance between each pair of all four chicken lines, based on 6 microsatellite loci. The genetic identity was ranged from 0.882 (between MLC and FLW lines) to 0.964 (between FLW and FLC lines).

Figure 2. Dendrogram based on Nei’s genetic distance

A phylogenetic relationship of four lines of chicken based on Nei (1972) using UPGMA method with 1000 simulated data is shown in Figure 2. With the limited information we found that the lines were relatively distinct in which FLC and MLC were clustered in one group, whereas MLW and FLW were in separate group.

The findings of this study may suggest that the level of genetic homogeneity is relatively high among these lines of chicken and the lines may be relatively distinct.

Acknowledgment

The research is partly supported by the SPGR NATP: Phase -1 research grant of Bangladesh Agricultural Research Council (BARC), Bangladesh.

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Received 22 May 2015; Accepted 11 June 2015; Published 1 August 2015

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