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ANIMAL GENETICS |
* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada;
and
Lacombe Research Centre, AAFRD, Lacombe, Alberta T4L 1W1, Canada;
and
ARS, USDA, U.S. Meat Animal Research Center, Clay Center, NE 68933-0166;
and
Agriculture and Agri-Food Canada, Lethbridge Research Center, Alberta TIJ 4B1, Canada
Abstract
Backfat thickness is one of the major quantitative traits that affects carcass quality in beef cattle. In this study, we identified and fine-mapped QTL for backfat EBV on bovine chromosomes 2, 5, 6, 19, 21, and 23 using an identical-by-descent haplotype-sharing analysis in a commercial line of Bos taurus. Eleven haplotypes were found to have significant associations with backfat EBV at the comparison-wise P-value threshold, and one at the chromosome-wise P-value threshold on bovine chromosomes 5, 6, 19, 21, and 23. On average, the 12 significant haplotypes had an effect of 0.62 SD on backfat EBV, ranging from 0.38 SD to 1.33 SD. The 12 significant haplotypes spanned nine chromosomal regions, one on chromosome 5 (65.4 to 70.0 cM), three on 6 (8.2 to 11.8 cM, 63.6 to 68.1 cM, and 81.5 to 83.0 cM), three on 19 (4.8 to 15.9 cM, 39.4 to 46.5 cM, and 65.7 to 99.5 cM), one on 21 (46.1 to 53.1 cM), and one on 23 (45.1 to 50.9 cM). Among the nine chromosomal regions, six were new QTL regions and three showed remarkable agreement with QTL regions that were previously reported. Eight of the nine QTL regions were localized to less than or close to 10 cM in genetic distance. The results provide a useful reference for further positional candidate gene research and marker-assisted selection for backfat.
Key Words: Backfat • Cattle • Haplotype Analysis • Quantitative Trait Loci
Introduction
The amount and distribution of fat has an important impact on carcass and meat quality in beef cattle (Powell and Huffman, 1973
; Wheeler et al., 1994; Lozeman et al., 2001). Beef carcasses can be stratified by using subcutaneous fat thickness alone or in combination with marbling (Jeremiah, 1996). Breeding for optimal fat is therefore one of the major goals toward better profitability in the beef industry. Mapping of QTL and identification of causative genes that affect fat metabolism will enhance the progress toward this goal. Quantitative trait loci mapping for quantitative traits related to fat has been reported in a number of studies (Stone et al., 1999; Casas et al., 2001; MacNeil and Grosz, 2002). Fine mapping and verification of those QTL for fat are therefore necessary for further candidate gene research.
Individuals within a semiclosed population, such as a commercial line of cattle, are expected to be derived from one or a limited number of founders. Some common haplotypes originating from the common ancestors should therefore carry on and segregate among the individuals of the breeding line, particularly when selection is applied. These common haplotypes may harbor QTL of interest and make it possible to locate QTL segregating in the line. This identical-by-descent (IBD) haplotype-sharing QTL mapping strategy avoids the generation of a well-designed mapping population. Obtaining such well-designed mapping populations is costly and may be impractical in commercial herds. In our previous studies, we have successfully identified and fine-mapped QTL for growth on bovine chromosome (BTA) 5 (Li et al., 2002a,b), as well as for backfat EBV on BTA14 (Moore et al., 2003) in commercial lines of Bos taurus using the IBD haplotype-sharing analysis method. We report here the identification and fine mapping of QTL for backfat EBV on BTA2, 5, 6, 19, 21, and 23 in a commercial population of Bos taurus.
Materials and Methods
Animals and Phenotypic Data
Animals were from the M1 line of Beefbooster Inc. (Calgary, Canada) and were born in 1998. The M1 line was developed from an Angus base and has been under selection for over 30 yr. The selection criteria for the lines are based on indices described by MacNeil and Newman (1994). A 10-mL blood sample was collected by venipuncture from each male calf and the potential sires, and the DNA from each blood sample was extracted and kept for later parentage identification. Sire identification was carried out by the Saskatchewan Research Council, Canada, using DNA microsatellite markers. The backfat EBV of each male calf was calculated based on a BLUP procedure by Beefbooster Inc.
Genotyping
One hundred seventy-six male calves and their 12 respective sires (nine to 30 calves of each sire) of the M1 line were genotyped using 76 microsatellite markers, 13 from BTA2, 16 from BTA5, 16 from BTA6, 14 from BTA19, 8 from BTA21, and 9 from BTA23. The microsatellite markers genotyped on each chromosome (Figure 1) were chosen at an approximately even genetic distance and spanned on average 93.4% of the chromosomes, with a range of 83.6 (chromosome 6) to 99.7% (chromosome 23). Marker locations and primers designed for genotyping were based on the microsatellite marker information available on the USMARC Website (http://www.marc.usda.gov/genome/genome.html).
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,b). Genotypes of the genotyped microsatellite markers of each of the 176 male calves were checked against the calf’s sire to verify the sire inheritance. Alleles of each locus contributed by the sire, as well as by the dam, were identified for each calf by examining the genotype of their sires. The haplotypes (allele linkage phases) of each male calf were then established along chromosomes 2, 5, 6, 19, 21, and 23. The GLM procedure of SAS (SAS Inst., Inc., Cary, NC) was used to test the association between each of the most commonly observed haplotypes and the backfat EBV. The linear model was: Yij = µ + Hi + Eij, where Yij = the backfat EBV of animal j for haplotype i, µ = overall experimental mean, Hi = fixed effect corresponding to the haplotype effect under test (1 when the individual has the haplotype or 0 when the individual is without the haplotype), and Eij = residual error. Because the number of animals carrying two copies of a haplotype were small in the data set, animals carrying two copies of a haplotype were grouped with animals carrying one copy of a haplotype as haplotype class "1." Animals with uncertain haplotypes were considered to be missing values and were deleted from the analysis.
Type III sums of squares were used in all F-tests. The haplotype effect in standard deviations was estimated by dividing the difference of backfat EBV least squares means between haplotype classes "1" and "0" by the standard deviation of the trait. The comparison-wise and chromosome-wise thresholds of the P-value were generated empirically from the permutation method outlined by Churchill and Doerge (1994) and as described by Li et al. (2002b). Type I error rates of 0.05 and 0.10 were used for calculating comparison-wise and chromosome-wise P-value thresholds, respectively.
Results
On average, 6.5 alleles were detected for each locus on the chromosomes, with a range of 2 to 14 alleles per locus. Associations between a haplotype and backfat EBV were analyzed only for the haplotypes of two adjacent loci with frequencies of 8% or higher. Haplotypes that extended more than two loci were not included in the final haplotype association analysis because of their low frequencies in the dataset compared with those of haplotypes of two adjacent loci. Table 1 lists the haplotypes that had significant associations with backfat EBV above the comparison-wise and chromosome-wise thresholds, and Figure 1
depicts the haplotypes of adjacent loci with the lowest P-values along BTA2, 5, 6, 19, 21 and 23.
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). On average, the 12 significant haplotypes had an effect of 0.62 SD on the backfat, ranging from 0.38 to 1.33 SD (Table 1). None of the haplotypes on bovine chromosome 2 showed significant effects on the backfat EBV at the comparison-wise threshold. On BTA5, haplotype BMS490-4, ETH10-2 had a significant positive effect on the backfat EBV at the comparison-wise threshold. The haplotype represented the chromosomal region of 65.4 to 70.0 cM. Animals with the haplotype had a backfat EBV 0.62 SD higher than animals without the haplotype. In the same chromosomal region, an alternative haplotype, BMS490-2, ETH10-3, had a negative effect on the backfat EBV at the comparison-wise threshold, decreasing the backfat EBV by 0.72 SD.
Three haplotypes on BTA6 were found to have significant effects on the backfat EBV at the comparison-wise thresholds. The three haplotypes, INRA133-208, ILSTS090-149; BMS470-68, BMS360-8; and OAREL03-4, ILSTS087-2 were located in three chromosomal regions of 8.2 to 11.8 cM, 63.6 to 68.1 cM, and 81.5 to 83.0 cM, respectively (Figure 1). All three haplotypes showed significant positive effects on backfat, increasing the backfat EBV by 0.38, 0.43, and 0.42 SD, respectively. Similarly, three chromosomal regions on BTA19 were identified as having significant effects on the backfat EBV. The three chromosomal regions were represented by five haplotypes (Figure 1). In the chromosomal region of 4.8 to 15.9 cM, haplotype BM6000-6, BMS745-2 had a significant negative effect on the backfat EBV at the comparison-wise threshold, decreasing it by 0.67 SD. In the chromosomal region of 39.4 to 46.5 cM, haplotype RM222-0, BP20-3 also had a negative effect on the backfat EBV, and the significance level reached the chromosome-wise threshold. Animals with the haplotype had 1.33 SD lower backfat EBV than the animals without the haplotype. In the chromosomal region of 65.7 to 99.5 cM, three haplotypes were found to have significant effects on the backfat EBV at the comparison-wise threshold. Haplotypes CSSM65-2, BMS1069-3 and BMS1069-3, RM388-2 had significant negative effects on the backfat EBV, decreasing it by 0.38 and 0.57 SD, respectively. Haplotype RM388-4, BMS601-3, however, showed a significant positive effect on the backfat EBV, increasing it by 0.47 SD.
On BTA21, haplotype BMS2815-99, ILSTS092-174 in the chromosomal region of 46.1 to 53.1 cM was the only haplotype that showed a significant effect on the backfat EBV at the comparison-wise level. The haplotype had a negative effect on the backfat EBV, decreasing it by 0.73 SD.
Haplotype RM185-103, BM1818-263 was the only haplotype on BTA23 that showed a significant association with backfat at the comparison-wise threshold. The haplotype spanned the chromosomal region of 45.1 to 50.9 cM and had a significant positive effect on the backfat EBV. Animals with the haplotype had a backfat EBV 0.69 SD higher than the animals without the haplotype.
Discussion
The successful application of marker-assisted selection in commercial animal populations will depend on a number of factors. Among these are the ability to identify the genes or closely linked markers to the genes underlying the QTL, the ability to test whether allelic variations at these loci are segregating in the population, and an understanding of how these genes interact with the environment or with other genes affecting economic traits. All this must be done in an efficient and cost-effective manner in order for the technology to be adopted by the livestock industries.
Identity-by-descent QTL mapping using haplotype sharing has been successfully demonstrated in humans (de Vries et al., 1996; Fallin et al., 2001) and cattle (Riquet et al., 1999). The method takes advantage of linkage disequilibrium in populations with limited outbreeding, in which common chromosome segments are shared by individuals in populations that originated from a few common founders. Thus, chromosome segments that house the QTL can be identified through direct haplotype comparison.
The feasibility of using haplotype-mapping methods depends on the extent of the linkage disequilibrium. Farnir et al. (2000) reported that linkage disequilibrium in a Holstein-Friesian dairy cattle population extended over several tens of centimorgans. In this study, we observed a level of linkage disequilibrium similar to that seen in dairy cattle, and some haplotypes between two adjacent markers had much higher frequencies than others in the M1 line (data not shown). Such a phenomenon may be attributed to the introduction of a limited number of founders and artificial selection over generations, a common breeding practice in beef cattle as well as in dairy cattle. In a commercial breeding line, selection may play an even more important role in maintaining linkage disequilibrium. Selection that is in favor of desired traits increases the percentage of IBD haplotypes housing the corresponding genes, and thus makes IBD mapping based on haplotype sharing analysis even more feasible.
In our previous studies, we successfully mapped QTL for birth weight, preweaning ADG, and ADG on feed in both the M1 and M3 commercial lines of Beefbooster Inc. using the IBD haplotype-sharing analysis, and narrowed down some of the QTL regions to less than 10 cM (Li et al., 2002a,b). The IBD haplotype-sharing analysis detected the same, but better defined, QTL regions in comparison to the interval-mapping method (Li et al., 2002a). In addition to the actual phenotypic data, we have also used the birth weight EBV data for QTL fine mapping on BTA5 and found that the QTL regions for birth weight identified using the primary phenotypic data were in very good agreement with those detected using EBV data (Li et al., 2002a,b). We also mapped a QTL region for backfat on bovine chromosome 14 using backfat EBV data and found that the QTL region was consistent with other studies (Moore et al., 2003).
In this study, we identified a total of nine chromosomal regions, one on BTA5 (65.4 to 70.0 cM), three on 6 (8.2 to 11.8 cM, 63.6 to 68.1 cM, and 81.5 to 83.0 cM), three on 19 (4.8 to 15.9 cM, 39.4 to 46.5 cM, and 65.7 to 99.5 cM), one on 21 (46.1 to 53.1 cM), and one on 23 (45.1 to 50.9 cM) that had significant associations with backfat EBV. Among the nine QTL regions, three QTL regions showed remarkable consistency with those identified by other studies and six were new QTL regions for backfat. Casas et al. (2000) reported a QTL for fat depth in the chromosomal region of 40 to 80 cM on bovine chromosome 5. We confirmed the QTL region in the chromosomal region of 65.4 to 70.0 cM and narrowed it down to about 5 cM. On bovine chromosome 6, Wiener et al. (2000) identified one QTL for milk fat yield in the region of 73 to 91 cM in a Holstein-Friesian family, similar to the QTL region of 81.5 to 83.0 cM that we identified for backfat in this study. Whether the two QTL regions represent the same QTL or separate QTL for milk fat yield and backfat, however, remains to be determined. On BTA19, Taylor et al. (1998) reported that QTL for subcutaneous fat and ether-extractable fat were located in the chromosome region of approximately 60 to 80 cM that harbored the growth hormone 1 gene. In this study, we identified a similar chromosomal region of 65.7 to 99.5 cM that showed a significant association with backfat. Such consistency strongly indicates the effectiveness of identification and fine mapping QTL in commercial lines of livestock using the IBD haplotype sharing method.
The M1 line has been developed as a maternal component of a commercial crossbreeding scheme. Selection is based on an index described by MacNeil and Newman (1994), along with independent culling levels specifying minimum and maximum birth weight, minimum preweaning ADG, and minimum ADG on feed in M1 line individuals. The selection index was constructed based on 18 different traits and selection was in favor of greater fat depth in the M1 line (MacNeil and Newman, 1994). Among the 12 haplotypes with higher frequencies and also showing significant associations with the backfat, six haplotypes had positive effects and six had negative effects on backfat, which suggests that selection in favor of backfat may not be strong in the M1 line. This emphasizes the care that must be taken in implementing marker-assisted selection when only one or a few markers are considered. Selection on a marker may also have negative effects on other traits due to pleiotropic effects of the gene or due to other genes closely linked to the marker affecting the other traits.
Implications
Quantitative trait loci for backfat in beef cattle have been identified and fine mapped on bovine chromosomes 5, 6, 19, 21, and 23 in a commercial line of Bos taurus. In total, nine regions on these five chromosomes were identified to have significant associations with backfat estimated breeding value. The results should provide a valuable reference for further positional candidate gene research and marker-assisted selection.
Footnotes
1 This work was supported through grant No. 2000M624 awarded to S. S. Moore through the Alberta Agricultural Research Institute and grant No. ACC-99AB343 awarded to B. Benkel and S. S. Moore through the Canada-Alberta Beef Industry Development Fund. 
2 Correspondence: 4-10 Ag/For Bldg. (phone: 780-492-0169; fax: 780-492-4265; e-mail: stephen.moore{at}ualberta.ca).
Received for publication August 22, 2003. Accepted for publication November 12, 2003.
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