BREEDING AND GENETICS Evaluation of Genetic Variability and Genetic Distances Between Eight Chicken Lines Using Microsatellite Markers T. VANHALA,1 M. TUISKULA-HAAVISTO,2 K. ELO, J. VILKKI, and A. MÄKI-TANILA Agricultural Research Centre MTT, Animal Production Research, Animal Breeding, 31600 Jokioinen, Finland ABSTRACT The genetic variability and divergence of eight chicken lines were evaluated using nine microsatellite markers. The chicken lines included three White Leghorn hybrids, three Finnish Landrace lines, a Rhode Island Red line, and a broiler hybrid line. All the microsatellite loci were found to be polymorphic, the number of alleles varying from 4 to 13 per locus and 1 to 10 per line, respectively. Observed heterozygosities ranged from 0.00 to 0.91. The highest (0.67) and lowest (0.29) mean heterozygosity per line was observed in the broiler and in White Leghorn of Mäkelä, respectively. Three of the microsatellite loci deviated from the HardyWeinberg equilibrium in some populations. F statistics indicated clearly the subdivision of the total population into different lines. The genetic distances confirmed the classification of Finnish Landraces into different lines. A phylogenetic consensus tree was constructed from resampled data (1,000 times) using the neighbor-joining method. According to the phylogenetic tree, the lines were grouped into three clusters, in which the White Leghorns formed one group, two Landraces a second group, and a Landrace, the Rhode Island Red, and the broiler lines a third group. Allele distribution at the loci does not support either the stepwise or the infinite alleles mutation model, but the distribution pattern was quite irregular at different loci. (Key words: microsatellites, chicken, genetic variability, genetic divergence, phylogenetic tree) 1998 Poultry Science 77:783–790 to mutate according to the SMM, whereas the microsatellites with smaller motifs (1 to 2 bp) show variation towards the IAM (Shriver et al., 1993). The mutation model for microsatellites should then be somewhere in between the SMM and IAM. DiRienzo et al. (1994) developed a two-phase mutation model that assumes primarily single-step changes in allele size but also takes mutations larger than the basic repeat motif into account. The validity of this model has not yet been tested in larger scale. Most of the genetic distance measures are based on the IAM (Cavalli-Sforza and Edwards, 1967; Nei, 1978; Reynolds et al., 1983). Goldstein et al. (1995) developed a genetic distance strictly based on SMM. However, according to previous studies (Shriver et al., 1993) the pure SMM holds only for a certain type of microsatellites. The genetic distance measures can be used to construct a phylogenetic tree. Takezaki and Nei (1996) INTRODUCTION Microsatellites are tandem repeat loci with a core motif of 1 to 6 bp repeated several times. They are highly polymorphic (Tautz, 1989) and considered to be evenly distributed in the genome. They have proven to be very useful in determining genetic variation and phylogenies of organisms, especially between populations of the same species (Buchanan et al., 1994; MacHugh et al., 1994). Although microsatellites have been widely used in molecular genetics, their function in the genome have not yet been identified. The most widely accepted hypothesis is that they function in packaging and condensing DNA into eukaryotic chromosomes (Stallings et al., 1991). Microsatellites have been expected to follow the stepwise mutation model (SMM) of Ohta and Kimura (1973). However, it has become clear that microsatellites do not evolve purely by SMM, or according to the infinite alleles model (IAM; Kimura and Crow, 1964). Microsatellites that have a repeat motif of 3 to 5 bp seem Abbreviation Key: BRO = Ross broiler line; DS = standard genetic distance; F = fixation; FIS = fixation coefficient of an individual within the subpopulation; FIT = fixation coefficient of an individual within the total population; FST = fixation coefficient of a subpopulation within the total population; FJ = Finnish Landrace line of Jokioinen; FK = Finnish Landrace line of Kiuruvesi; FS = Finnish Landrace line of Satakunta; HWE = Hardy-Weinberg equilibrium; IAM = infinite alleles model; J = a synthetic White Leghorn line of Jokioinen; LSL = Lohmann Selected Leghorn; M = a White Leghorn line of Mäkelä; RAPD = random amplified polymorphic DNA; RIR = Rhode Island Red line; SMM = stepwise mutation model. Received for publication April 7, 1997. Accepted for publication February 21, 1998. 1Present address: C. T. de Wit Graduate School of Production Ecology, Laboratory of Plant Breeding, Wageningen Agricultural University, P. O. Box 386, 6700 A J Wageningen, The Netherlands. 2To whom correspondence should be addressed: maria.tuiskulahaavisto@mtt.fi 783 784 VANHALA ET AL. tested the validity of different mutation models with simulated data. They concluded that for phylogeny construction, the details of the mutation model are not important. Minisatellites (Hillel et al., 1989), DNA fingerprinting (Kuhnlein et al., 1989; Siegel et al., 1992), random amplified polymorphic DNA markers (RAPD; Smith et al., 1996), and microsatellites (Groen et al., 1994) have been used in previous studies of estimating genetic variation in chicken lines. The microsatellite markers are a less laborious and more accurate and efficient method for estimating genetic variation than the other methods that have been used previously. Finnish Landraces have not been studied genetically; but they were classified into different lines as recently as 1993 (Koskivainio, 1993). In this study, we describe microsatellite polymorphisms in eight chicken lines. From the genotyped data, the measures of genetic variability and genetic divergence were estimated and a phylogenetic tree constructed to visualize the results. MATERIAL AND METHODS Chickens The lines were Lohmann Selected Leghorn (LSL),3 a Synthetic White Leghorn line of Jokioinen (J),4 a White Leghorn line of Mäkelä (M),5 Rhode Island Red (RIR),6 Finnish Landraces of Jokioinen (FJ),3 Satakunta (FS),7 and Kiuruvesi (FK),6 and a Ross broiler line (BRO).8 The sample size per line ranged from 12 to 31. In total, 207 chickens from eight lines were included in this study. The White Leghorns. The LSL commercial hybrid is a line from Lohmann Tierzucht GmbH, Germany. The line is distributed in Finland through LSK Ltd. The line J was formed in 1987 for the use of the Agricultural Research Centre in Finland. The line is a cross between two White Leghorn lines. It was selected for feed conversion ratio and egg mass production for seven generations. The M line originates from a White Leghorn line Derby that was imported to Finland from US in the 1950s. Since then, the line has been closed at Mäkelä Ltd. The Finnish Landraces. The first chickens came to Finland in the Middle Ages with Swedish and German immigrants. There were more importations in the subsequent centuries and the present Finnish Landrace lines descend from these chickens. There has been very little selection for production traits. Selection has mainly been based on plumage color (black, brown, or variegated 3LSK Ltd., Laitila, Finland. 4Agricultural Research Centre, Jokioinen, 5Mäkelä Ltd., Vilppula, Finland. 6Pettersson Ltd., Yttersse, Finland. 7Ojanteen Siitoskanala, Vihti, Finland. 8Suomen Broiler Ltd., Masku, Finland. 9Finnzymes, Helsinki, Finland. Finland. coloring). The FJ line was brought to the Agricultural Research Centre in 1986. The primer population was relatively small: 151 hens and 38 cockerels. There has been no selection in this line. Lines FS and FK can be distinguished from each other, with FS being smaller and mostly black, whereas FK is bigger and more variegated. The Finnish Landrace lines have only recently been classified into different lines according to morphological criteria. Rhode Island Red. This line has the characteristics of RIR but it has been subject to heavy interbreeding. The following lines have been crossed into it: Hisex Brown, ISA Brown, and RIR lines from Denmark and The Netherlands. The Broiler Line. Ross is a commercial line of Ross Breeders Ltd. in Scotland, UK. The line is distributed in Finland through Suomen Broiler Oy. DNA Isolation Venous blood samples were taken from the ulnar vein with a 2-gauge 1.5-in injection needle. The amount of blood was approximately 2 mL. Blood was stored at –20 C in vacuum tubes containing EDTA. DNA was extracted from 100 mL of whole EDTA-blood. Four hundred microliters of lysis buffer (10 mM Tris-HCl pH 7.8, 10.95 % saccharose, 1% Triton X-100, 5 mM MgCl2) was added to the aliquot. The mixture was centrifuged and the pellet suspended in 7.5 mM NaCl and 2.4 mM EDTA solution. The solution was made up to 2 mg/mL with Proteinase K and 0.5% SDS. After overnight incubation at 37 C, the proteins were removed by phenol and chloroform-isoamyl alcohol extractions and the DNA was precipitated by ethanol. Microsatellite Loci The microsatellite loci (Table 1) were chosen from the MCW markers (Crooijmans et al., 1994) and the HUJ markers (Khatib et al., 1993), and consisted of MCW03, MCW05, MCW07, MCW14, MCW16 and HUJ5, HUJ7, HUJ10, HUJ12. Based on the East Lansing chicken genetic map (Crittenden et al., 1993; Cheng et al., 1995) seven of the loci reside on different linkage groups and two are on one linkage group separated by approximately 25 cM. The microsatellite repeat HUJ10 is situated in the chicken embryonic myosin heavy chain gene and the HUJ12 in the chicken a-smooth muscle actin gene. The Polymerase Chain Reaction The PCR was carried out in a volume of 20 mL comprising 50 ng of template DNA, 20 pmol of each primer, 0.2 mM of each dNTP, 1 unit of DynaZyme II DNA polymerase,9 10 mM Tris-HCl, pH 8.8; 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100. Two pairs of primers were amplified in the same reaction: HUJ05 and MCW16, and MCW03 and MCW14. To these reactions 10 785 GENETIC VARIATION IN CHICKEN LINES TABLE 1. The location of the microsatellite marker in the East Lansing genetic map, core motif, allele size range, total number of alleles in the whole population, and observed mean unbiased heterozygosity [H (unb.)] for each locus Locus1 Location2 Motif1 Size range Number of alleles H (unb.) HUJ5 HUJ7 HUJ10 HUJ12 MCW03 MCW05 MCW07 MCW14 MCW16 E06 E07 E21 E11 E29 E05 E01 E11 E02 (CA)nGAn (AC)nAn An (AC)n (TG)n (TG)nAn3 (TG)n(TA)n4 (TG)nG(TG)n (TG)n (bp) 105 149 245 116 139 212 296 173 135 9 13 8 8 4 12 8 4 7 0.554 0.613 0.617 0.461 0.185 0.651 0.509 0.455 0.460 to to to to to to to to to 118 181 253 140 145 256 322 190 149 1HUJ; Hebrew University of Jerusalem, Khatib et al., 1993; MCW; Microsatellite Chicken Wageningen, Crooijmans et al., 1994. 2Crooijmans et al., 1994; Cheng et al., 1995. 3The actual repeat is (TG) AA(TG) A (GA) (GAA) . n n n n n 4The actual repeat is (TG) (TA) (TG) TACA(TA) TT(TA) . n n n n n pmol of each primer was included. One primer for each locus was labeled with fluorescein (Fluore Prime). 10 The amplification conditions were: 3 min denaturation at 94 C followed by 30 cycles of denaturation at 94 C for 40 s, annealing at either 55 or 61 C for 50 s, and extension at 72 C for 60 s. The PCR products were separated in 6% polyacrylamide gels (Readymix)10 with A.L.F. DNA sequencer.10 For size determination, an internal size standard (Sizer)10 was included in each lane. Results were visualized and the genotyping done with the Fragment Manager Version 1.1.10 Statistical Analysis The expected (according to Hardy-Weinberg equilibrium, HWE) and observed heterozygosities were calculated. An ANOVA F test was used to examine whether the means of direct count heterozygosities were significantly different from each other among lines. Differences between pairs of means were studied using Tukey’s Studentized range (HSD) test (ANOVA procedure in SAS package, SAS Institute Inc. 1985). Deviations from the HWE were tested from the allele frequencies. The alleles were grouped into three groups at the loci because there were loci that had several rare alleles: the first group consisted of common homozygotes, the second group of rare and common heterozygotes, and the third group of rare homozygotes. The x2 test was performed using exact probabilities. The standard genetic distance (DS) of Nei (1978) and the fixation (F) indices (Wright, 1978) were calculated. These analyses were done with the BIOSYS-1 (Swofford and Selander, 1989) program package. The allele frequency data was resampled 1,000 times with a bootstrapping method (Efron, 1982; Felsenstein, 10Pharmacia, Uppsala, Sweden. 1985), then DS; (Nei, 1978) were calculated and a phylogenetic consensus tree was formed using the neighbor-joining method (Saitou and Nei, 1987). These analyses were done with the PHYLIP (Felsenstein, 1993) program package. The phylogenetic tree was edited with TREEVIEW (Page, 1996). RESULTS Genetic Variation The allele size ranges showed large variation across loci. For example, in locus MCW05, the allele range was from 212 to 256 bp (44 bp) and in locus MCW03 from 139 to 145 (6 bp) (Table 1). The numbers of alleles varied from 4 to 13 per locus and 1 to 10 per line. The allele frequency distribution was uneven in the allele size classes (Figure 1). Six loci were polymorphic in all lines: MCW03 was monomorphic in the three White Leghorn lines and in FJ, MCW07 in LSL, and MCW14 in FK (Table 2). Estimates for the mean heterozygosity per line expected under the HWE were in the range of 0.38 to 0.67 (Table 3). All the expected heterozygosities were larger than the observed heterozygosities. The total average heterozygosity was 0.50. The estimates of heterozygosity at different loci between lines showed large variation. The variation was greatest at the locus MCW03, where all the White Leghorns and FJ were monomorphic, whereas the heterozygosity of FS was 0.52. The heterozygosity of the locus MCW05 was above 0.50 in all the lines (Table 2). The means of observed heterozygosities among lines (Table 3) were significantly different from each other, F = 3.37, P = 0.004. Differences between all pairs of means were tested and two comparisons, BRO vs M and BRO vs FJ, showed significant differences at the 5% level (Table 3). 786 VANHALA ET AL. FIGURE 1. Distribution of allele frequencies at loci HUJ5 (a), HUJ7 (b), HUJ10 (c), HUJ12 (d), MCW03 (e), MCW05 (f), MCW07 (g), MCW14 (h), and MCW16 (i). In graph b there is a break in the x-axis. In each allele size class (base pairs), each bar represents one of the studied populations. 787 GENETIC VARIATION IN CHICKEN LINES TABLE 2. For each locus-line combination, the expected heterozygosities [H(exp.)] according to Hardy-Weinberg equilibrium (HWE), the observed heterozygosities [H(obs.)], and the probability values for deviations from HWE Line1 Locus HUJ7 HUJ5 HUJ10 HUJ12 MCW03 MCW05 MCW07 MCW14 MCW16 H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P H(exp.) H(obs.) P LSL (30) M (31) J (29) RIR (30) FJ (19) FS (27) FK (13) BRO (30) 0.488 0.000 *** 0.499 0.600 NS 0.515 0.033 *** 0.364 0.467 NS 0.000 0.000 NS 0.705 0.700 NS 0.000 0.000 NS 0.589 0.767 NS 0.499 0.667 NS 0.554 0.387 NS 0.647 0.355 NS 0.627 0.000 *** 0.444 0.581 NS 0.000 0.000 NS 0.589 0.600 NS 0.643 0.129 *** 0.236 0.258 NS 0.135 0.143 NS 0.512 0.280 NS 0.724 0.621 NS 0.593 0.240 * 0.440 0.345 NS 0.000 0.000 NS 0.665 0.630 NS 0.409 0.182 ** 0.668 0.586 NS 0.354 0.448 NS 0.728 0.633 NS 0.747 0.690 NS 0.747 0.276 *** 0.253 0.207 NS 0.339 0.217 NS 0.737 0.862 NS 0.729 0.320 *** 0.509 0.360 NS 0.687 0.483 NS 0.478 0.263 NS 0.152 0.053 NS 0.569 0.105 *** 0.563 0.526 NS 0.000 0.000 NS 0.602 0.737 NS 0.496 0.053 *** 0.522 0.526 NS 0.149 0.152 NS 0.815 0.731 NS 0.621 0.704 NS 0.662 0.500 NS 0.706 0.667 NS 0.604 0.522 NS 0.477 0.519 NS 0.752 0.593 NS 0.453 0.593 NS 0.642 0.615 NS 0.467 0.417 NS 0.518 0.583 NS 0.554 0.250 NS 0.239 0.520 NS 0.148 0.154 NS 0.706 0.909 NS 0.304 0.167 NS 0.000 0.000 NS 0.464 0.500 NS 0.863 0.867 NS 0.521 0.567 NS 0.653 0.500 * 0.676 0.667 NS 0.387 0.345 NS 0.787 0.862 NS 0.737 0.633 NS 0.662 0.724 NS 0.753 0.867 NS 1The lines are: LSL, Lohmann Selected Leghorn; M, White Leghorn of Mäkelä; J. Synthetic White Leghorn line of Jokioinen; RIR, Rhode Island Red; FJ, Finnish Landrace of Jokioinen; FS, Finnish Landrace of Satakunta; FK, Finnish Landrace of Kiuruvesi; and BRO, Ross. Number of studied individuals shown in parentheses. *P ≤ 0.5. **P ≤ 0.01. ***P ≤ 0.001. TABLE 3. The estimates of genetic variation in eight chicken lines, the mean number of alleles across loci, and the expected (according to Hardy-Weinberg equilibrium) and observed average heterozygosities. The standard errors are indicated in the parentheses. Means of direct count heterozygosities were significantly different (P < 0.05) between M and BRO and FJ and BRO lines1 Three of the microsatellite loci deviated from the HWE (HUJ10, MCW07, and HUJ7). Two of them deviated more consistently across lines; HUJ10 deviated from HWE in all other lines except in FK and FS. Locus MCW07 deviated from HWE in half of the lines (M, J, RIR, and FJ) (Table 2). Genetic Divergence Line The mean number of alleles across loci Direct count Expected LSL M J RIR FJ FS FK BRO 2.4 3.3 3.4 4.6 3.3 4.6 2.7 5.7 0.370 0.293 0.379 0.450 0.304 0.613 0.359 0.670 0.407 0.431 0.485 0.608 0.392 0.637 0.378 0.671 (0.4) (0.6) (0.5) (0.4) (0.6) (0.6) (0.3) (0.8) Average heterozygosity (0.083) (0.082) (0.074) (0.064) (0.075) (0.039) (0.074) (0.048) 1The lines are: LSL, Lohmann Selected Leghorn; M, White Leghorn of Mäkelä; J, Synthetic White Leghorn line of Jokioinen; RIR, Rhode Island Red; FJ, Finnish Landrace of Jokioinen; FS, Finnish Landrace of Satakunta; FK, Finnish Landrace of Kiuruvesi; and BRO, Ross. The fixation coefficients of subpopulations within the total population (FST) for the nine loci varied from 0.204 to 0.602, with the mean being 0.303. The fixation indices of individuals within the total population (FIT) ranged from 0.146 to 0.659, with the mean being 0.389. Individual’s fixation indices within the subpopulation (FIS) varied from –0.130 to 0.479, with the mean being 0.124. The two loci that deviated mostly from HWE (HUJ10 and MCW07) had large FIS values, which also reflects smaller amount of heterozygotes than expected. The unbiased genetic distances (Nei, 1978) are presented in Table 4. The smallest genetic distance was 788 VANHALA ET AL. TABLE 4. The genetic distances (Nei, 1978) of the eight chicken lines1 LSL M J RIR FJ FS FK BRO SL M J FJ RIR FS FR BRO ***** 0.248 0.117 1.170 0.484 0.684 0.563 0.840 ***** 0.241 1.076 0.736 0.570 0.770 0.712 ***** 1.051 0.541 0.436 0.687 0.592 ***** 1.122 0.520 1.123 0.306 ***** 0.476 0.531 0.950 ***** 0.724 0.317 ***** 0.819 ***** 1The lines are: LSL, Lohmann Selected Leghorn; M, White Leghorn of Mäkelä; J, the Synthetic White Leghorn line of Jokioinen; RIR, Rhode Island Red; FJ, Finnish Landrace of Jokioinen; FS, Finnish Landrace of Satakunta; FK, Finnish Landrace of Kiuruvesi; BRO, Ross. between J and LSL (0.117) and the largest between RIR and LSL (1.170). Of the Finnish Landraces the closest were FS and FJ (0.476). All three White Leghorn lines had small distances between each other. The BRO line was closest to RIR and FS (0.306 and 0.317, respectively). Distances between BRO and other lines were much larger (0.592 to 0.950). Phylogenetic Tree The phylogenetic consensus tree constructed using the bootstrapped data and neighbor-joining method grouped the lines into three clusters (Figure 2). The first group was formed of the three White Leghorn lines, the second of the FK and FJ lines, and the third of FS, RIR, and BRO. Of all the 1,000 trees, the occurrence of the first group was 78.6%, the second 65.3%, and the third 88.6%. FIGURE 2. A phylogenetic consensus tree of eight chicken lines. The data was resampled 1,000 times with bootstrapping method and the trees formed using the neighbor-joining method (Saitou and Nei, 1987). The numbers in the figure indicate the occurrence of the groups among the 1,000 trees. The lines are: J, the Synthetic Line of Jokioinen; LSL, Lohmann Selected Leghorn; M, White Leghorn of Mäkelä; FK, Finnish Landrace of Kiuruvesi; FJ, Finnish Landrace of Jokioinen; FS, Finnish Landrace of Satakunta; BRO, Ross; RIR, Rhode Island Red. DISCUSSION The small number of individuals in Finnish Landrace lines has clearly affected their genetic variation. The lowest heterozygosity was observed in the FK and FJ lines. The FJ line has had a small base population and the FK line has a small total population size. Due to small population size, genetic drift has been the strongest force influencing the genetic background of these chicken lines. The line FS has, however, a high heterozygosity (0.637). One explanation to the difference in heterozygosity is that the FS has more individuals than the other Finnish Landrace lines. Thus, genetic drift has not been as strong as in the other lines of Finnish Landrace. Another explanation is that there might have been crossbreeding with some other chicken line(s), because the Finnish Landrace chickens have always been kept more or less free outside in the farm yard with other chickens. Significant differences (P < 0.05) were observed between pairs of heterozygosity means between BRO, M, and FJ lines. These pairwise heterozygosity comparisons indicate that meat line breeding utilizes and maintains genetic variation differently than layer line breeding. The lowest observed heterozygosity values were found in M and FJ lines. This low heterozygosity may be explained by founder effect or small population size. The M line is also known to have been a closed population since the 1950s. In this study, two commercial lines were analyzed, a layer line LSL and a meat line BRO. The mean number of alleles across loci in LSL was 2.4 and in BRO 5.7. These numbers were the lowest and the highest observed in this study. Direct count heterozygosities were quite typical (0.37 in LSL) or the highest (0.67 in BRO) in this study. It has been suggested that the biological limits for broiler breeders (Barton, 1994) and layers have not yet been encountered. There is enough genetic variation left to generate further progress in the years ahead (Flock, 1994). The results of this study are in accordance with these earlier observations. The loci in this study were mainly in agreement with HWE. Two loci were more consistently deviating from HWE. There are several explanations for the deviations. GENETIC VARIATION IN CHICKEN LINES These microsatellite loci might be associated with some genes that are of some economic importance. The other locus (HUJ10) is located in the chicken embryonic myosin heavy chain gene. Secondly, there could have been a null allele, but no homozygous null individuals were found. The most likely explanation for the deviation from HWE is wrong genotyping. The gel conditions may not have been accurate enough to separate 1 bp differences between the large fragments of HUJ10 (245 to 253 bp) and thus some heterozygotes may have been ignored. This explanation is supported by the fact that the locus contained several different alleles overrepresented as homozygotes. No line was fixed for a specific allele. MCW07 had also large fragments but it consisted of dinucleotide repeats. The alleles were always clearly separated. The chicken lines included in this study clearly diverged from each other. According to the F statistics, there is very clear differentiation into subpopulations in the total population. This differentiation is expected, as this study includes commercial layer and broiler lines, and Landraces that have not been selected. The allele distributions of the microsatellite loci (Figure 2) do not seem to follow the expectations of SMM. According to SMM, the allele frequencies should be normally distributed. None of the distributions were strictly normal; however, in locus MCW07 two approximately normal distributions were observed (Figure 2g). These normal distributions can be explained by two separate repeat sequences in core motif (see Table 1) and expecting SMM. In addition, locus MCW03 had adjacent allele sizes (139, 141, 143, and 145 bp) and thus it may have mutated by single steps. Some of the loci had long sequences between clusters of alleles, which probably reflects the complexity of the repeat motifs of the selected microsatellites; only four of the microsatellite markers had perfect motifs. No definite conclusions from these data can be made concerning the validity of SMM, because the number of loci was small. There is need for a large-scale study of the mutation model underlying the microsatellite mutations with real data. Although the phylogenetic tree is based on only nine microsatellite markers, the reliability estimates gained through bootstrapping were relatively high. Takezaki and Nei (1996) suggested that for gaining a reliable phylogeny one should use at least 30 markers; however, their simulation study expected lower levels of genetic divergence between populations (or species) than was observed in this study. Relatively high level of divergence (DS varied from 0.117 to 1.170) mostly explains the high reliability in the topology of the tree (Table 4). As expected, White Leghorns formed one cluster. Their grouping together was clear, although their internal grouping was not so clear. The Finnish Landrace lines do not seem to have a uniform background according to their genetic relationships. Why BRO and RIR grouped together is an interesting question that cannot yet be answered. These lines might 789 have common lines in their background, or perhaps were selected for the same traits, such as size or growth. The results of this research confirm the usefulness of microsatellites for the research of genetic variation and divergence. 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