780 Asian Australas. J. Anim. Sci. Vol. 26, No. 6 : 780-787 June 2013 http://dx.doi.org/10.5713/ajas.2012.12514 www.ajas.info pISSN 1011-2367 eISSN 1976-5517 Genetic Polymorphisms of the Bovine NOV Gene Are Significantly Associated with Carcass Traits in Korean Cattle B. S. Kim, S. C. Kim, C. M. Park, S. H. Lee, S. H. Cho1, N. K. Kim2, G. W. Jang, D. H. Yoon3, B. S. Yang, S. K. Hong, H. H. Seong and B. H. Choi* Animal Genome and Bioinformatics Division, National Institute of Animal Science, Rural Development Administration, Chuksan gil 77, Kwonsun-gu, Suwon, Korea ABSTRACT: The objective of this study was to investigate single nucleotide polymorphisms (SNPs) in the bovine nephroblastoma overexpressed (NOV) gene and to evaluate whether these polymorphisms affect carcass traits in the Korean cattle population. We resequenced to detect SNPs from 24 unrelated individuals and identified 19 SNPs within the full 8.4-kb gene, including the 1.5-kb promoter region. Of these 19 SNPs, four were selected for genotyping based on linkage disequilibrium (LD). We genotyped 429 steers to assess the associations of these four SNPs with carcass traits. Statistical analysis revealed that g.7801T>C and g.8379A>C polymorphisms in the NOV gene were associated with carcass weight (p = 0.012 and 0.008, respectively), and the g.2005A>G polymorphism was associated with the back fat thickness (BF) trait (p = 0.0001). One haplotype of the four SNPs (GGTA) was significantly associated with BF (p = 0.0005). Our findings suggest that polymorphisms in the NOV gene may be among the important genetic factors affecting carcass yield in beef cattle. (Key Words: NOV Gene, Single Nucleotide Polymorphism, Carcass Weight, Back Fat Thickness, Korean Cattle) INTRODUCTION of beef cattle production. The bovine nephroblastoma overexpressed (NOV) gene Carcass weight and back fat thickness are economically is positioned near RM192 on bovine chromosome 14 (Lee important cattle characteristics in the Korean beef industry. et al., 2011). This region has also been reported to harbor a The retail carcass price reflects the sum of the quality and quantitative trait locus (QTL) for carcass weight and back yield grades, and back fat thickness negatively affects the fat thickness in Angus cattle (http://www.animalgenome.org/ yield grade (Moon et al., 2003). Two factors that affect the cgi-bin/gbrowse/cattle/#search). growth rate of cattle are genetic potential, which regulates a The NOV gene was first identified as an overexpressing complex of hormone and growth factors and their gene in virus-induced avian nephroblastoma (Joliot et al., associated interactions, and environmental conditions such 1992), and orthologs were later isolated from Xenopus, rat, as nutrition, climate, disease and management mouse, and human (Joliot et al., 1992; Martinerie et al., (Hermesmeyer et al., 2000). Genetic potential has long been 1992; Snaith et al., 1996; Ying and King, 1996; Liu et al., considered to be an important factor in the competitiveness 1999; Oberst et al., 1999). NOV encodes a putative secretory protein of 343 to 357 amino acids that contains * Corresponding Author: Bong-Hwan Choi. Tel: +82-31-290- four conserved modular domains with sequence similarity 1592, Fax: +82-31-290-1792, E-mail: [email protected] to insulin-like growth factor-binding protein (Collet and 1 Animal Production Division, National Institute of Animal Candy, 1998). The NOV protein is structurally related to a Science, RDA, Suwon, Korea. 2 Division of Genetic Analysis Lab, Experiment Reserch Institute, family of early response proteins that probably play a role National Agricultural Products Quality Management Service of in cell growth regulation. It is a member of the growing Animal Science, 80, Seonyu-dong 1-ro Youngdeungpo-gu, Seoul, family of secreted regulatory proteins termed CCN 150-804, Korea. (CYR61/CTGF/NOV) (Bork, 1993). Some CCN proteins 3 Department of Animal Science, Kyungpook National University, have been demonstrated to possess growth factor-like Sangju, 742-170, Korea. activity and to regulate cell growth and tissue formation. Submitted Sept. 18, 2012; Accepted Dec. 30, 2012; Revised Jan. 4, 2013 Copyright © 2013 by Asian-Australasian Journal of Animal Sciences Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 781 For example, connective tissue growth factor (CTGF) is a Seq.; NOV: NC_007312). Primer information is provided in matrix-associated heparin binding protein that mediates cell Table 1. proliferation, migration, and adhesion (Grotendorst, 1997; Moussad and Brigstock, 2000). Compared with CTGF, the Genotyping by PCR-RFLP and electrophoresis biological activity of the NOV gene remains poorly We applied PCR-RFLP methods to detect four SNPs in understood. To date, no polymorphisms have been reported the NOV gene. Four primer sets flanking DNA fragments of in the NOV gene in farm animals. 372 bp, 823 bp, 941 bp, and 933 bp at the mutation sites in In the current study, we examined the NOV gene as one Exon 3, Intron 4, and the 3′ UTR region were synthesized. of the most promising candidate genes related to meat PCR amplification was performed using 20 ng of genomic production in beef cattle. We performed extensive NOV DNA, the four primer sets (Table 1), and HS Prime Taq gene screening by resequencing to detect polymorphisms polymerase (GENET BIO, Korea). Amplifications were and then examined their genetic associations with carcass performed in a thermal cycler (PT-200, MJ Research, MA, traits. Here, we present four polymorphisms identified in USA) as follows: 94C for 10 min, followed by 35 cycles of the NOV gene as well as an analysis of their associations 94C for 30 s, 55 to 65C for 30 s, and 72C for 1 min, with with carcass yield traits in Korean cattle. a final extension at 72C for 10 min. The first fragment (372 bp) had a restriction site for HpyCH4Ⅳ; the second MATERIAL AND METHODS fragment (823 bp), for SpeI; the third fragment (941 bp), for HincII; and the fourth fragment (993 bp), for HpyCH4Ⅳ. Cattle and phenotypic data For restriction enzyme digestion, 5 l of each PCR product Korean cattle genomic DNA samples were obtained were mixed with 2 units of the appropriate restriction from 429 steers produced from 76 sires used in the progeny enzyme and incubated at 37C for 3 h. The digest mixtures testing program at the National Institute of Animal Science were separated in agarose gels containing ethidium bromide (NIAS) in Korea. All steers were fed for 731.3916.53 d for visualization (Figure 3). under a tightly controlled feeding program at the Daekwanryeong and Namwon branches of NIAS. They Statistical analysis were weaned at a mean age of 3 months and fed with 30% A goodness of fit chi-squared test was used to test for concentrates and 70% roughage until they were 6 months Hardy-Weinberg equilibrium (HWE) by comparing the old. After 6 months of age, they were fed concentrates observed number of subjects for each genotype with the consisting of 15% crude protein (CP) and 71% total expected number of subjects assuming HWE. Genotype digestible nutrients (TDN) until they were 14 months old, distributions at each polymorphic locus were tested for followed by 13% CP and 72% TDN until 20 months, and departure from HWE. Associations between individual 11% CP and 73% TDN until 24 months of age. Roughage SNPs and measured carcass traits were determined by was offered ad libitum, and steers had free access to fresh regression analysis using the nlme library in the R statistical water during the entire period. Live weights (LW) were package (http://www.r-project.org). Trait association determined before slaughter. The mean live weight was analyses were performed using a mixed effect model, 539.9551.96 kg. Yield grades for carcasses were treating “sire” as a random effect and “age” at slaughter and determined by the cold carcass weight (CW). Dressing genotype as a fixed effect in the model. Other covariates were not available for this analysis. We examined a percentage (DP) was determined using CW as a proportion common measure of linkage disequilibrium (LD) between of LW. After the carcasses were chilled for 24 h, CW was all pairs of biallelic loci, Lewontin's D' (Hedrick, 1987) and measured. Then the left side of each carcass was cut r2. Haplotypes and their frequencies were inferred using the between the last rib and the first lumbar vertebrae to algorithm developed by Stephens et al. (2001). A type I determine back fat thickness (BF) (APGS, 1995). The mean error of 5% was used to obtain the Bonferroni corrected P- carcass trait values were 311.4433.20 kg for CW, value. For the haplotype analyses, we fitted the model with 57.641.83% for DP, and 0.700.28 cm for BF. the same covariates in a manner similar to that used for the SNP association test. NOV sequencing analysis We sequenced the full 8.4-kb NOV gene, including the RESULTS AND DISCUSSION promoter region (1.5 kb), to discover variants among 24 unrelated Korean cattle, using an ABI PRISM 3730XL Resequencing and SNP discovery DNA analyzer (Applied Biosystems, Foster City, CA, USA). By resequencing the DNA of 24 unrelated Korean cattle, For amplification and sequencing analysis, 16 primer sets 19 polymorphisms were identified in the NOV gene: four were designed based on GenBank sequences (Ref. Genome SNPs in the promoter and exon, and 15 SNPs in the UTR 782 Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 Table 1. Primer list for SNP discovery and genotyping of NOV gene Primer ID Length (bp) Primer sequences (5'-3') Product size (bp) PCR site NOV-1F 23 catatggctgaatcactttgttg 818 Promoter NOV-1R 21 agcctgtgagggtgtttaaga NOV-2F 20 gaaagtaggtgccaggtgga 852 Promoter NOV-2R 20 ccttcccccaggactaagac NOV-3F 20 gcatcccccatcctaactct 848 Exon 1 NOV-3R 20 gggctttgctcagaaagtga NOV-4F 20 caggtgcctctggtcacttt 803 Exon2 NOV-4R 20 ggttccataggtcccaggag NOV-5F 20 tgagaagcgttggtcacttg 882 Intron 2 NOV-5R 20 gcttggtgggctacagtgat NOV-6F 20 atcagcaaccagatgccttc 1080 Exon3 NOV-6R 18 cagaggagcctggagagcta NOV-E1F 20 tgcaggcaggtgttttaatg 372 Exon 3 (g.1952A>G) NOV-E1R 20 ctgtgctggggctgttaaat PCR-RFLP NOV-7F 20 tatgcacacagcctctcctg 824 Intron 3 NOV-7R 20 ggcacatctcctcccttaca NOV-8F 20 acactggcaggacacagaaa 904 Intron 3 NOV-8R 18 caatgagcaaggccacct NOV-9F 21 caccaacaggaatcctcactg 850 Exon 4 NOV-9R 21 gccttctgctcagcattaaca NOV-10F 20 caccagtacatttgccagga 823 Intorn 4 (g.4590T>G) NOV-10R 20 gccgtataacaatgcaacca PCR-RFL NOV-11F 20 ccctacccagggattgaact 927 Intron 4 NOV-11R 20 ggcacagtccataaatcgtg NOV-12F 20 gcaaattacagggatccaca 995 Intron 4 NOV-12R 20 tgaaatggccatctttcctc NOV-13F 20 cctgtctgaagggcaaagaa 1187 Exon 5 NOV-13R 20 ccagtttacgacaccagtgc NOV-14F 21 tggaatcaaggtaagctcagg 941 3’ UTR (g.7748T>C) NOV-14R 22 agctgaacacatagggtgacaa PCR-RFLP NOV-15F 20 tgactgcagtggcgagatac 931 3’ UTR (g.8326A>C) NOV-15R 20 gaaggcaggagggacaagat PCR-RFLP NOV-16F 20 ctaccccaaaggaggtggac 627 3’ UTR NOV-16R 20 ttaggtgcagcttgcggtat and intron regions. Sequence variants were verified by genotyped on a larger scale, in 429 Korean cattle (Table 2). chromatography (Figure 1). The results showed >99% homology with the sequence reported in GenBank Genotyping and allele frequencies (NC_007312). The locations and allele frequencies of Large-scale genotyping was performed using the PCR- polymorphisms are presented in Table 2 and Figure 2. Pair- RFLP method. The PCR-RFLP analysis verified unique wise linkage analysis with DNA from the 24 unrelated binding patterns, showing DNA fragments of different Korean cattle used for resequencing demonstrated that five mobilities by agarose gel electrophoresis (Figure 3). sets of polymorphisms were in absolute LD (|D'| = 1 and r2 Restriction enzyme digestion of the PCR products with = 1) in the NOV gene (Figure 2). For the subsequent large- HpyCH4IV resulted in fragments of 303 and 69 bp for the G scale association analysis, SNPs were selected based on the allele, and a 372-bp uncut fragment for the A allele in the following criteria: i) location (exon and promoter g.1952A>G polymorphism. The g.4590T>G SNP was polymorphisms were preferred); ii) frequency of minor cleaved by SpeI into 621- and 202-bp fragments for the T allele (frequency, 0.05); iii) LD, such that among allele, while a fragment of 823 bp remained uncut for the G polymorphisms in absolute LD (r2 = 1), only one was allele. For the g.7748T>C polymorphism, digestion with selected; and iv) restriction enzyme site consideration. HincII created fragments of 471 and 470 bp for the T allele, Among the 19 polymorphisms in the NOV gene, four SNPs and a 941-bp uncut fragment was observed for the C allele. (g.1952A>G, g.4590T>G, g.7748T>C, g.8326A>C) were For the g.8326A>C polymorphism, digestion with Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 783 g.-349T>C g.-224C>T g.-194G>T g.-44C>A g.1952A>G g.4290T>C g.4590T>G g.6285C>G g.7412G>A g.7748T>C g.7775A>G g.7791C>T g.7974T>C g.8229A>T g.8326A>C g.8471G>A g.8540C>A g.8554T>C g.8661G>T Figure 1. Chromatograms of discovered polymorphisms in NOV gene. HpyCH4Ⅳ gave 622- and 311-bp fragments for the C allele, minor allele frequency of 0.186 and heterozygosity of 0.303. while a 933-bp fragment remained for the A allele. No By comparison, the minor allele frequencies for the other significant departure from HWE was found for any of the SNPs ranged from 0.346 to 0.375, and heterozygosity was alleles (Table 2). The G allele of the g.1952A>G SNP had a 0.453 to 0.469. low frequency, resulting in low genetic variability with a 784 Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 Table 2. Genotype, minor allele frequency (MAF), heterozygosity (He), and Hardy-Weinberg equilibrium (HWE) of 19 polymorphisms in the NOV gene SNP name Position Genotypes and number of animal MAF He HWE g.-349T>C Promoter T(9) TC(10) C(3) 0.364 0.463 0.996 g.-224C>T Promoter C(11) CT(8) T(1) 0.250 0.375 0.957 g.-194G>T Promoter G(11) GT(8) T(1) 0.250 0.375 0.957 g.-44C>A 5'UTR C(9) CA(9) A(1) 0.289 0.411 0.804 g.1952A>G Exon3 A(289) AG(132) G(15) 0.186 0.303 1.000 g.4290T>C Intron4 T(4) TC(4) C(2) 0.400 0.480 0.870 g.4590T>G Intron4 T(190) TG(193) G(59) 0.352 0.456 0.670 g.6285C>G Intron4 C(21) CG(2) G(1) 0.083 0.153 0.084 g.7412G>A 3'UTR G(14) GA(7) A(3) 0.271 0.395 0.440 g.7748T>C 3'UTR T(170) TC(199) C(62) 0.375 0.469 0.955 g.7775A>G 3'UTR A(13) AG(8) G(3) 0.292 0.413 0.639 g.7791C>T 3'UTR C(14) CT(8) T(2) 0.250 0.375 0.862 g.7974T>C 3'UTR T(12) TC(8) C(3) 0.304 0.423 0.693 g.8229A>T 3'UTR A(13) AT(8) T(2) 0.261 0.386 0.895 g.8326A>C 3'UTR A(189) AC(209) C(51) 0.346 0.453 0.838 g.8471G>A 3'downstream G(10) GA(8) A(2) 0.300 0.420 0.978 g.8540C>A 3'downstream C(10) CA(9) A(2) 0.310 0.427 1.000 g.8554T>C 3'downstream T(11) TC(9) C(2) 0.295 0.416 0.997 g.8661G>T 3'downstream G(9) GT(9) T(2) 0.325 0.439 0.993 Association analyses with increased BF (regression coefficient, -0.1) and CW Associations of NOV gene polymorphisms with carcass (regression coefficient, -6.3). This result is in agreement traits were analyzed using the mixed effect model with sire with a study by Ferrell and Jenkins (1984), who and age as covariates. Sire was treated as a random effect demonstrated a correlation between CW and BF. The and age as a fixed effect. The obtained p-values were g.4643T>G genotype, which is intronic, was weakly, but corrected for multiple testing using the Bonferroni significantly, associated with LW (p = 0.029), CW (0.016), correction method (p>P = 0.0125). The associations DP (0.037), and BF (0.029); however, when Bonferroni adjusted between the genotypes and the carcass phenotypes are listed corrections were strictly adopted, the respective p-values in Table 3. did not retain significance. It is possible that intronic Statistical analyses revealed that the g.2005A>G polymorphisms could influence gene function by altering polymorphism showed a strong association with BF (p = donor and acceptor splice sites or nearby regions, as well as 0.0001), a weak association with CW (p = 0.036), and no regulatory motifs within introns. association with LW or DP. The A allele was associated Two of the polymorphisms located in the 3′ UTR Figure 2. Map of SNPs in the NOV gene on bovine chromosome 14. The first base of the translational site is denoted as nucleotide 1. An asterisk (*) indicates polymorphisms genotyped in Korean cattle (n = 429). The minor allele frequency is based on 24 sequencing samples only, which is different from the minor allele frequency of absolutely linked SNPs genotyped in the larger population. Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 785 Figure 3. Sequence chromatograms and PCR-RFLP patterns of four SNPs detected in the bovine NOV gene. The left lane in each gel image is a standard size marker (100 bp DNA ladder). Table 3. Analysis of associations between four polymorphisms in the NOV gene and carcass traits in Korean cattle Genotype Trait Loci Position C/C C/R R/R p R N(LSMEANSE) N(LSMEANSE) N(LSMEANSE) LWT g.2005A>G Exon3 278(538.03.1) 120(544.24.4) 15(550.711.2) 0.067 -8.43 g.4643T>G Intron4 178(543.83.6) 184(540.04.1) 57(527.56.4) 0.029 8.00 g.7801T>C 3'UTR 158(544.03.6) 191(536.64.0) 59(529.76.8) 0.024 8.34 g.8379A>C 3'UTR 177(545.13.4) 200(538.74.1) 49(524.36.7) 0.012* 9.54 HAPLO1 - 146(539.94.3) 192(536.53.5) 91(547.45.1) 0.434 -2.65 HAPLO2 - 191(543.03.4) 191(540.34.1) 47(526.45.1) 0.036 7.84 HAPLO3 - 299(537.13.1) 116(545.74.3) 14(554.311.4) 0.055 -8.86 CW g.2005A>G Exon3 278(309.82.0) 120(314.62.9) 15(322.97.1) 0.036 -6.28 g.4643T>G Intron4 178(314.12.3) 184(311.62.7) 57(302.44.2) 0.016 5.70 g.7801T>C 3'UTR 158(314.72.3) 191(309.92.6) 59(303.24.4) 0.012* 6.02 g.8379A>C 3'UTR 177(314.92.1) 200(311.02.6) 49(298.94.2) 0.008* 6.57 HAPLO1 - 146(311.62.8) 192(309.12.4) 91(316.13.2) 0.452 -1.64 HAPLO2 - 191(313.52.2) 191(312.12.7) 47(300.14.3) 0.021 5.56 HAPLO3 - 299(309.32.0) 116(315.32.9) 14(326.06.8) 0.021 -6.87 DP g.2005A>G Exon3 278(57.50.1) 120(57.80.2) 15(58.60.5) 0.053 -0.30 g.4643T>G Intron4 178(57.80.1) 184(57.60.1) 57(57.30.2) 0.037 0.26 g.7801T>C 3'UTR 158(57.80.1) 191(57.70.1) 59(57.20.2) 0.042 0.26 HAPLO1 - 146(57.70.2) 192(57.60.1) 91(57.70.2) 0.697 -0.04 HAPLO2 - 191(57.70.1) 191(57.70.1) 47(57.00.3) 0.089 0.21 HAPLO3 - 299(57.50.1) 116(57.70.2) 14(58.80.5) 0.023 -0.35 BF g.2005A>G Exon3 278(6.80.2) 120(7.50.3) 15(8.60.9) 0.0001* -0.96 g.4643T>G Intron4 178(7.30.2) 184(6.90.2) 57(6.60.3) 0.029 0.44 g.7801T>C 3'UTR 158(7.40.2) 191(6.90.2) 59(6.60.3) 0.020 0.47 g.8379A>C 3'UTR 177(7.20.2) 200(7.00.2) 49(6.50.3) 0.048 0.41 HAPLO1 - 146(7.20.2) 192(7.00.2) 91(7.00.3) 0.495 0.12 HAPLO2 - 191(7.20.2) 191(7.00.2) 47(6.50.4) 0.041 0.41 HAPLO3 - 299(6.80.2) 116(7.50.3) 14(8.90.9) 0.00005* -1.00 P adj * Significant SNP after Bonferroni correction. N (LSMEANSE): Number of animals with trait, genotype and standard error. C/C = Common homozygote; C/R = Heterozygote; R/R = Rare homozygote. R = Regression coefficient. 786 Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 (g.7801T>C and g.8379A>C) showed very strong Table 4. Linkage disequilibrium coefficients (|D'| and r2) among association with CW. In addition to its significant NOV SNPs association with CW (p = 0.012), the g.7801T>C |D'| SNPs polymorphism had a weak association with LW (p = 0.029), g.2005A>G g.4643T>G g.7801T>C g.8379A>C DP (0.037), and BF (0.029). The regression coefficient r2 g.2005A>G - 0.473 1.000 0.716 indicated that the T allele was related to an increase in CW g.4643T>G 0.070 - 0.798 1.000 (regression coefficient, 6.0). The g.8379A>C polymorphism g.7801T>C 0.137 0.637 - 0.727 was strongly associated with both CW (p = 0.008) and LW g.8379A>C 0.152 0.569 0.891 - (p = 0.012), weakly associated with BF (p = 0.048), and not associated with DP. The A allele of this SNP was related to products of the CCN gene family, which are approximately increased LW and CW (regression coefficients, 9.5 and 6.6, 40 kDa in size, regulate numerous biological processes, respectively). These results are in agreement with a including differentiation, migration, proliferation, and cell previous study on Korean cattle that reported a QTL for CW adhesion (Katsube et al., 2009). Although the mechanisms near the RM192 marker, which is close to the NOV gene on by which alternative genotypes in the UTR and introns BTA14 (Lee et al., 2011). Although the mechanisms by could be associated with CW and BF are not currently which polymorphisms in the UTR would affect traits are understood, it is now widely acknowledged that non-coding not currently understood, our analysis clearly indicates that, portion of genomes play crucial roles. Polymorphisms even after correction for multiple testing, two within introns could influence gene function by affecting polymorphisms in the UTR influence CW in Korean cattle. donor or acceptor splice sites or nearby regions, or For haplotype-based association analysis, three major regulatory motifs within introns. UTRs are involved in haplotypes (frequency, >0.05) were constructed in the NOV many post-transcriptional regulatory pathways that control gene (Tables 4 and 5). Of nine reconstructed haplotypes, mRNA localization, stability, translation efficiency, and three (HAPLO1, HAPLO2, and HAPLO3) were initiation of protein synthesis. For UTR and intronic SNPs, predominant (total frequency, 94%) in the 429 Korean cattle although the allele itself may be functional and directly tested. Other haplotypes were rare and were not analyzed. affect the expression of the phenotype, it is more likely that HAPLO1 was not significantly associated with carcass the allele is in linkage disequilibrium with another allele at traits, whereas HAPLO2 showed significant associations a nearby locus and that allele is the true causal allele. with LW, CW, and BF (p = 0.036, 0.021, and 0.041, The QTL for carcass traits that is located between respectively). HAPLO3 was significantly associated with markers RM192 and BMS947 on bovine chromosome 14 increased BF (p = 0.00005) and weakly significantly contains many functional candidate genes related to body associated with increased LW, CW, and DP (p = 0.055, weight and fat synthesis in beef cattle, as verified with the 0.021, and 0.023, respectively) (Table 3). The estimated NOV gene (Lee et al., 2011). A suggested QTL for CW and two-haplotype effect was larger than the effect of either BF has been reported to occur near 50 cM on BTA14 in locus alone; this suggests that none of these SNPs is the Korean cattle (Wibowo et al., 2008). Other QTL studies causal mutation, but rather that another undetected also have reported that this region on BTA14 is related to polymorphism within this haplotype is causing phenotypic BF (Casas et al., 2000; McClure et al., 2010). variation. In summary, we hypothesized that NOV gene A number of potential candidate genes containing polymorphisms are associated with carcass traits. The genetic variants have been selected to identify associations results of our study demonstrate that four polymorphisms with economically important traits based on physiological (two in the 3′ UTR, one exonic, and one intronic) in the and biochemical mechanisms. In terms of biochemical NOV gene are associated with LW, CW, and/or BF. mechanisms, the NOV gene belongs to the CCN family, Although the mechanisms involved in these associations are which comprises multifunctional secreted proteins that act not currently understood, this study clearly indicates that as cellular matrix regulators. The extracellular protein NOV gene polymorphisms have an effect on LW, CW, and Table 5. NOV gene haplotypes and their frequencies among Korean cattle Haplotype g.2005A>G g.4643T>G g.7801T>C g.8379A>C Frequency HAPLO1 A G T A 0.436 HAPLO2 A T C C 0.332 HAPLO3 G G T A 0.168 HAPLO4 A G C A 0.022 Other(5) - - - - 0.042 Kim et al. (2013) Asian Australas. J. Anim. Sci. 26:780-787 787 BF in beef cattle. These findings represent an important step Lee, S. H., J. H. van der Werf, N. K. Kim, S. H. Lee, C. Gondro, E. for the successful application of the NOV gene in marker W. Park, S. J. Oh, J. P. Gibson and J. M. Thompson. 2011. 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