Genetic Variation in Composite and Purebreed Hereford Populations

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1 BioSMART ISSN: X Volume 1, Nomor 2 Oktober 1999 Halaman: 8-12 Genetic Variation in Composite and Purebreed Hereford Populations SUTARNO 1, A.J. LYMBERY 2,3 1 Jurusan Biologi FMIPA UNS Surakarta 2 Agriculture Western Australia. PO BOX 1231, Bunbury, WA 6231, Australia 3 Vet. Biology, Murdoch University, Murdoch WA 6150 Australia ABSTRACTS Crossbreeding systems have been widely applied in commercial animal production, due to their many advantages over purebreeding, especially in exploiting heterosis. Purebred Hereford and synthetic (composite) breeds were used in this project. The objectives of the project were to investigate polymorphism in the growth hormone gene and mitochondrial DNA in the composite and purebred Hereford herds from the Wokalup selection experiment, and compare genetic diversity in the growth hormone gene and mitochondrial DNA in the both herds. Polymorphisms were found both in growth hormone gene and mitochondrial DNA. Extensive genetic diversity was found in both breeds of cattle in the growth hormone gene and mitochondrial DNA. As expected, restriction site variation in the two growth hormone regions or in the regions of mitochondrial DNA surveyed was not independent, confirming restricted recombination between sites. Although there was a trend for greater expected heterozygosity at the growth hormone locus in the composite than in the Hereford population, this trend was not statistically significant. Key Words: genetic diversity, composite, hereford INTRODUCTION In commercial animal production, crossbreeding systems have been widely applied due to their many advantages over purebreeding, especially in exploiting heterosis. The theoretical and empirical effects of crossbreeding are well established, particularly in the exploitation of non-additive genetic effects to improve mean levels of performance (Gregory & Cundiff, 1980; Koch et al., 1985). One drawback of continuous crossbreeding schemes is the complexity of management required to crossbreed every generation. Gregory and Cundiff (1980) suggested that composite or synthetic breeds of livestock provide an attractive alternative to continuous crossbreeding systems, since they are expected to combine ease of management with the utilization of heterosis and additive genetic differences between breeds. Many studies in beef cattle have shown that advanced generations of composite breeds retain heterosis for many quantitative production traits at about the expected theoretical level of retained heterozygosity (Gregory et al., 1991a; 1991b; 1991c; 1991d; 1992). There have been no empirical studies, however, of single gene heterozygosity in composite breeds. Crossbreeding has been applied in many livestock industries, including beef cattle, dairy cattle, pigs, meat sheep and wool sheep. In beef cattle, Hammond et al. (1992) suggested that the best possible formation of composites may be between Bos taurus x Bos indicus breeds. Bos taurus cattle generally have poor adaptation to tropical environments, but high growth potential in stress-free conditions, while Bos indicus have the reverse characteristics. The formation of kind of this composite has been successfully achieved, e.g. Brangus, Braford, Simbrah, Charbray and Belmont Red (Hammond et al., 1992). Composite breeds are expected to respond more rapidly to selection than purebreeds because of a greater selection intensity permitted by retained heterosis in reproduction rate, and increased additive genetic variance associated with differences in gene frequencies between the parental breeds (Barker, 1989; Dickerson, 1973). The beef cattle selection experiment carried out by Agriculture Western Australia at Wokalup research station involved a comparison of additive genetic variation and response to selection between a composite multibreed (approximately comprised of ¼ Brahman, Charolais and Friesian, and 1/8 Angus and Hereford) and a purebred Hereford population. Quantitative genetic analyses by Meyer et al. (1993; 1994) and Meyer (1995) showed the composite breed to have consistently high phenotypic and genetic variation for a range of growth and mature size traits. The aims of this part of the study were therefore to: 1. Investigate polymorphism in the growth hormone gene and mitochondrial DNA in the composite and purebred Hereford herds from the Wokalup selection experiment; 2. Compare genetic diversity in the growth hormone gene and mitochondrial DNA in the composite and purebred Hereford herds. This project was part of a big project with the final objective of finding gene markers for production traits in beef cattle. MATERIAL AND METHODS Cattle A total of 194 Hereford and 235 composite breed cattle from Wokalup Research Station were used in this study. They were part of a selection experiment described in detail by Meyer et al. (1993) and maintained at Agriculture Western Australia s Wokalup 1999 Jurusan Biologi FMIPA UNS Surakarta

2 SUTARNO and LYMBERY Genetics Variation Research Station. Most of the cattle used in this study were purebred Herefords or a composite breed comprising approximately 1/4 Brahman, Charolais and Friesian, and 1/8 Angus and Hereford. Both of the populations were closed in 1977, and selection lines established from 300 cows and 12 bulls. Approximately 60 cows and 10 bulls were replaced each year, with replacements selected on pre-weaning growth rate. Control lines were regenerated from frozen embryos and semen from 1990 to Calving occurred in April-May each year. Calves were weighed at birth and monthly thereafter until weaning in December-January. Milk production was estimated for all lactating females each year by the weigh-suckle-weigh method (Meyer et al., (1993). Estimated breeding values (EBVs) for birth weight, the calf component of 200-day growth rate and the maternal component of 200-day growth rate were obtained for all animals in the herd each year. Approximately 60 cows and 10 bulls were replaced each year, with replacement selection based initially on pre-weaning growth, and later on an index of increased breeding values for the direct and maternal components of pre-weaning growth, and reduced breeding values for birth weight. Estimated breeding values (EBVs) were provided by Breedplan, the within-herd genetic evaluation system of the Australian National Beef Recording Scheme. In 1989 and 1990 frozen embryos from the foundation populations were implanted to generate control lines for each breed type. DNA Extraction Genomic DNA and mitochondrial DNA were extracted from leucocytes or whole blood using the method as described in detail by Sutarno (1998). PCR-RFLP Analysis Conditions for PCR-RFLP analysis of the growth hormone gene loci 1 and 2 (GH-L1 and GH-L2) and mitochondrial DNA (D-loop and ND-5) are as described by Sutarno (1998). Data Analysis Growth Hormone Gene The locus 1 and 2 fragments were analyzed using the infinite alleles model of the computer program GENESTRUT (Constantine, Hobbs & Lymbery, 1994) treating each fragment as a separate locus with two alternative alleles. Genetic diversity was described by the mean number of alleles per locus (A), mean observed heterozygosity (H o ) and mean expected unbiased heterozygosity (H e ; Nei 1978), with variance calculated by the method of Nei (1978). Differences between mean expected heterozygosities were tested using the method of Archie (Archie, 1985). For each locus, genotypic frequencies expected under Hardy Weinberg equilibrium were calculated from allelic frequencies using Levene s correction (Levene, 1949) for small sample size. Deviation of observed from expected frequencies were tested by X 2, and the extent of deviation expressed by Wright s fixation index, with the approximate variance of Brown (1970). Associations between the loci were examined with Burrow s composite measure of linkage disequilibrium ( AB ), tested for significance by X 2 as outlined by Weir (1990) Mitochondrial DNA Genetic diversity at the mtdna loci was analyzed using the infinite sites model in the program RESTSITE (Miller, 1991). The nucleotide diversity, or average number of nucleotide substitutions per site within breeds (d) was estimated over all loci by the method of Nei and Li (1979), with standard errors calculated by jackknifing (Nei & Miller, 1990). Allelic frequencies within each breed were calculated separately for each locus / restriction site combination. Associations between restriction site polymorphisms at the same or different loci were tested by comparing observed haplotype frequencies with those expected from allelic frequencies, using a X 2 test bp 171bp 52bp 600bp 100bp 329bp 224bp 105bp 600bp 100bp Figure 1. Example of gel photographs showing growth hormone gene polymorphism detected by PCR-RFLP using, 1. AluI in locus I (GH-L1) and 2. MspI in locus 2 (GH-L2).

3 10 RESULTS Growth Hormone Gene The results of the PCR-RFLP in both regions of growth hormone gene (GH-L1 and GH-L2) were presented in Figure 1. Genetic Diversity Standard measures of genetic diversity at the growth hormone loci between composite and Hereford are shown in Table 1. Differences in expected heterozygosity and nucleotide diversity between breeds were not significant. There were no differences in genetic diversity between sexes in either breed (data not shown). Table 1. Standard measures of genetic diversity (+ standard error) over both loci of the growth hormone gene for composite and Hereford cattle. Breed N Observed heterozygosity No of alleles Unbiased expected heterozigosity Composite Hereford Allelic Frequencies Allelic frequencies at the growth hormone loci in composite and Hereford breeds are shown in Table 2. There were no significant differences in allelic frequencies between sexes in either breed for any locus (data not shown). Table 2. Allelic frequencies at both loci of the growth hormone gene for composite and Hereford breeds. Breed N GH-L1 GH-L2 L V MspI (+) MspI (-) Composite Hereford Genotypic Frequencies Table 3 shows the observed and expected heterozygosities at each growth hormone locus in each breed. Fixation indices were generally positive, indicating fewer heterozygotes then expected, but this was significant only in the composites at locus 2. Over both breeds, F is was not significantly different from 0 for either locus. Table 3. Heterozygosities observed (H o) and expected (H e) under Hardy-Weinberg equilibrium, and fixation indices (F i with variances in parentheses) at the two growth hormone loci in Hereford and composite breeds. F is is the weighted mean of values across breeds. Locus Breed Composite Hereford GH-L1 H o H e F (0.005) (0.006) F is.069 GH-L2 H o H e F 0.178* (0.006) (0.009).098* Linkage Disequilibrium As expected, there were significant associations between the two growth hormone loci in both breeds (Table 4). Table 4. Estimates of Burrows composite measure of linkage disequilibrium ( AB + SE) for the two growth hormone loci in composite and Hereford breeds of cattle. Breed AB P Composite <0.001 Hereford <0.05 Mitochondrial DNA Genetic Diversity Standard measures of genetic diversity at the mitochondrial D-loop and ND-5 between composite and Hereford are shown in Table 5. There were no differences in genetic diversity between sexes in either breed (data not shown). Table 5. Genetic diversity over mitochondrial D-loop and ND-5 loci for composite and Hereford breeds. d is the nucleotide diversity, or average number of nucleotide substitutions per site (+ standard error) within breeds. Breeds N d (Nei and Li) Composite Hereford Allelic Frequencies Allelic frequencies at the mitochondrial D-loop and ND-5 loci are shown in Table 6. There were no significant differences in allelic frequencies between sexes in either breed for any locus (data not shown). Linkage Disequilibrium Out of 10 possible mtdna haplotypes, only 3 were observed in the composite breed and 3 in the Hereford. The most common haplotypes were significantly overrepresented in both breeds (Table 7), indicating extensive associations within and between loci. Table 7. Frequency of mtdna haplotypes observed and expected (from products of allelic frequencies) in composite and Hereford. Breed Haplotypes Expected Observed P Frequency Frequency Composite AAAAA <0.005 ABBBA <0.005 ABBBB <0.005 Hereford AAAAA <0.005 ABBBA <0.005 BBBBA <0.005

4 SUTARNO and LYMBERY Genetics Variation Table 6. Allelic frequencies at mitochondrial D-loop and ND-5 for composite and Hereford breeds. At each locus the common allele is designated as A, and the less common allele B. Breed D-loop ND-5 TaqI PstI SspI ApaI AvaII HindIII SpeI A B A B A B A B A B A B A B Composite Hereford DISCUSSION Extensive genetic diversity was found in both breeds of cattle in the growth hormone gene and mitochondrial DNA. As expected, restriction site variation in the two growth hormone regions or in the regions of mitochondrial DNA surveyed was not independent, confirming restricted recombination between sites. Populations of livestock often show reduced heterozygosity across all nuclear loci as a consequence of inbreeding arising from small population sizes and artificial selection. This was not evident in the present study. Heterozygote deficiency was found only at GH- L2 in the composite breed. This may have resulted from selection against heterozygotes, perhaps as a consequence of previous artificial selection policies, but more data are needed to confirm this hypothesis. Composite breeds are expected to be genetically more diverse than purebreeds, because of recombination of the loci from different breeds which takes place during breed formation. Greater diversity at the individual locus level in composite breeds is expected to provide greater genetic variance in quantitative traits and more rapid response to selection. Empirical tests of this theory are rare and have invariably involved estimation of quantitative genetic parameters in composite and purebred populations, rather than comparisons of allelic or nucleotide diversity at individual loci. Howe & James (1973) found a greater realized heritability for sternopleural bristle number in composite, than in parental lines of Drosophila melanogaster. Liu et al. (1991) found greater genetic variance for growth traits in a composite, than in purebred beef cattle population. Meyer et al. (1993; 1994), working with the same populations studied here, found greater phenotypic and genetic variance for growth and size traits among composite than among purebred Hereford. Although there was a trend for greater expected heterozygosity at the growth hormone locus in the composite than in the Hereford population, this trend was not statistically significant. For mitochondrial DNA, diversity was actually greater (although not significantly so) in the Herefords. Simulation studies conducted by Gunawan & James (1986) and Mohd-Yusuff & Dickerson (1991) indicate that additive genetic variation may or may not be increased in composite populations, depending on the degree of dominance, diversity in gene frequency among parental lines, and the mean gene frequencies in the composite. Mohd-Yusuff & Dickerson (1991) suggested that favourable dominant alleles at QTLs should be fixed at equilibrium, so that genetic variance for the trait in composite populations will only exceed that in their purebred parental populations when dominance is partial or when frequencies of dominant and overdominant alleles are less than expected at equilibrium. The theoretical point is whether the growth hormone and mitochondrial DNA loci studied here are actually QTLs. This will be addressed in the next publications. ACKNOWLEDGEMENT I am grateful to Patrick Donnelly and the staff at Wokalup Research Station, Agriculture Western Australia, especially Phil Moyle, Bob Jones and Richard Seaward for the phenotypic data and sample collections. REFERENCES Archie, J. W Statistical analysis of heterozygosity data: independent sample comparisons. Evolution 39: Barker, J. S. F Population Structure. In Evolution and Animal Breeding ed. W. G. Hill and T. F. C. 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