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Animal Science Journal (2015) 86, 125–131
doi: 10.1111/asj.12260
ORIGINAL ARTICLE Effect of VRTN gene polymorphisms on Duroc pig production and carcass traits, and their genetic relationships Hikaru NAKANO,1,2 Shuji SATO,2 Yoshinobu UEMOTO,2 Takashi KIKUCHI,2 Tomoya SHIBATA,3 Hiroshi KADOWAKI,3 Eiji KOBAYASHI4 and Keiichi SUZUKI1 1
Graduate School of Agricultural Science, Tohoku University, Sendai, 2National Livestock Breeding Center, Nishigo, 3Miyagi Prefecture Animal Industry Experiment Station, Oosaki, and 4National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, Japan
ABSTRACT The thoracic vertebral number is associated with body length and carcass traits, and represents one of the most important traits in the pig industry. Recent studies have shown that vertnin (VRTN) gene is associated with variations in the vertebral number in commercial European pigs. However, the genetic relationships and effect of this VRTN gene in pig production and carcass traits remain uncertain. Therefore, we investigated the genetic relationships among traits such as vertebral numbers, carcass weight and length-related traits, and meat production traits, and the effect of VRTN gene polymorphisms on these traits in a Duroc purebred population selected for its meat production traits. Highly positive genetic correlations were obtained between the thoracic vertebral numbers and length-related traits (0.56 to 0.84), whereas low correlations were obtained with production traits and carcass weight (−0.16 to 0.05). VRTN gene polymorphisms indicated that the number of thoracic vertebrae and length-related traits were significantly associated with the VRTN genotype, but had no significant effect on production traits and carcass weight. The results indicate that VRTN gene may be used as an effective selection marker to obtain pigs with high thoracic vertebral numbers and length-related traits, without adversely affecting meat production traits.
Key words: genetic parameter, pig, thoracic vertebral number, VRTN gene.
INTRODUCTION Carcass traits, such as carcass and loin lengths, are economically important traits in pig breeding programs, with these traits being associated with the number of vertebrae. King and Roberts (1960) showed that carcass length increases by an average of about 0.8 cm per additional vertebra. In general, wild pigs have 19 thoracic and lumber vertebrae and European breeds have 20 to 23, with the number of dorsal vertebrae varying considerably (King & Roberts 1960). An increase in vertebral number is expected to improve carcass traits, with these traits potentially being genetically correlated with other economically important traits, such as production traits. However, detailed studies of the genetic relationships among the number of vertebrae, carcass traits and production traits have yet to be reported. Recently, several studies have reported the detection of two quantitative trait loci (QTLs) for vertebral number on SSC1 and SSC7 in crossbred pig popula© 2014 Japanese Society of Animal Science
tions (Rohrer & Keele 1998; Wada et al. 2000; Sato et al. 2003; Mikawa et al. 2005; Ren et al. 2012; Fan et al. 2013) and on SSC7 in a Duroc purebred population (Uemoto et al. 2008). One is a nuclear receptor Germ Cell Nuclear Factor (NR6A1) gene on SSC1 (Mikawa et al. 2007), while the other is a vertnin (VRTN) gene on SSC7 (Mikawa et al. 2011). The NR6A1 gene influences the number of both dorsal and lumbar vertebrae; however, genetic variation caused by NR6A1 gene polymorphisms was not detected in commercial European breeds. The haplotype of VRTN gene polymorphism takes part in the number of thoracic vertebra, and the heterozygote of this VRTN gene genotype was detected in the commercial European
Correspondence: Keiichi Suzuki, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 981-8555, Japan. (Email: [email protected]) Received 30 January 2014; accepted for publication 17 April 2014.
126 H. NAKANO et al.
Table 1 Descriptive statistics of selection traits and carcass traits
Selection traits Daily gain Loin eye muscle area Backfat thickness Intramuscular fat content Carcass traits Carcass weight Carcass length Back loin length 1 Back loin length 2 Loin length Carcass width Thoracic vertebrae number Lumber vertebrae number Total vertebrae number
Abbreviation
n
Unit
Mean
SD
DG LEA BF IMF
1642 1639 1642 545
g/day cm2 cm %
873.6 37.0 2.4 4.2
109.3 4.1 0.4 1.5
77.1 90.5 75.0 65.3 48.9 35.4 14.9 6.0 20.9
2.7 2.6 2.1 2.2 2.1 1.4 0.6 0.3 0.6
CWT CL BL1 BL2 LL CW TVN LVN TotalVN
population (Fan et al. 2013; Hirose et al. 2013a). In addition, Fan et al. (2013) showed that g.19034A>C and g.20311_20312ins291 in the haplotype are the most likely causal mutations in the VRTN gene. The discovery of the genes responsible for the numbers of vertebrae would facilitate the improvement of carcass and production traits that are related to the number of vertebrae, by using a simple and unambiguous selection process with a DNA marker. Recently, Hirose et al. (2013a) demonstrated the significant effect of VRTN gene polymorphisms on intramuscular fat content (IMF) and body length in a Duroc pig population. However, a subsequent report by Hirose et al. (2013b) indicated that there was no significant association between VRTN gene polymorphisms and IMF in the same population. Therefore, further study is required to evaluate the effect of VRTN gene polymorphisms on production traits and other economically important traits related to the number of vertebrae in different pig populations. This study aimed to estimate the genetic relationship among the number of vertebrae, production traits and carcass traits, and to evaluate the effect of VRTN gene polymorphisms on these traits by using a Duroc breed selected for meat production and meat quality traits.
METHODS Animal and phenotype measurement The Duroc purebred population used in the this experiment was selected over seven generations for daily gain (DG) of 30 to 105 kg body weight (BW), loin eye muscle area (LEA), backfat thickness (BF) at 105 kg BW measured using ultrasound technology, and IMF. Details of the selection and measurement methods are described by Suzuki et al. (2005). DG, LEA and BF were measured in all pigs (1639 to 1642 pigs), while IMF was measured in slaughtered sibtested pigs (545 pigs). These slaughtered sib-tested © 2014 Japanese Society of Animal Science
545 545 545 545 545 545 545 545 545
kg cm cm cm cm cm
Duroc pigs were also measured for carcass weight (CWT), carcass length (CL), carcass width (CW), back loin length 1 (BL1), back loin length 2 (BL2), loin length (LL), the number of thoracic and lumbar vertebrae (TVN and LVN, respectively), and the number of total vertebrae (TotalVN) as carcass traits. CL was measured from the first cervical spine to the anterior extremity of the pubic bone; BL1, BL2 and LL are the measurements from the leading edge of the first thoracic vertebra to the anterior extremity of the pubic bone, from the leading edge of the first thoracic vertebra to the posterior border of the last lumbar vertebra, and from the leading edge of the fifth thoracic to the leading edge of the last lumbar vertebra, respectively. TotalVN is the total number of thoracic and lumber vertebrae. The descriptive statistics of these traits are shown in Table 1.
Genetic parameter estimates First, the genetic parameters of selection and carcass traits were estimated by the following multiple trait animal model: Yijkl = μ i + G ij + Sik + a il + eijkl ,
where Yijkl = observation for trait i, μi = common constant for trait i and Gij = fixed effect of selection generation j (7 classes) for trait i. The effect of selection on the generations included the genetic effect of selection and the environmental effect on each generation. Sik = fixed effect of sex k (three classes) for trait i, ail = random additive genetic effect of animal l for trait i and eijkl = random residual effect for trait i. Pedigree information was obtained for 1642 animals across seven generations, along with production trait data from 152 ancestors born before the fourth generation (totalling 1794 animals) being included in this analysis. In total, 547 pigs (394 barrows and 153 gilts) were selected as sib-tested pigs, which were then Animal Science Journal (2015) 86, 125–131
GENETIC ASSOCIATIONS OF VRTN IN PIGS 127
Table 2 Number of male and female pigs by generation of selection
Male
Female
Total
Generation
All
Sibtested
All
Sibtested
All
Sibtested
1 2 3 4 5 6 7 Total
91 109 119 118 100 101 135 773
44 59 69 68 52 51 51 394
109 149 120 122 114 121 134 869
18 30 14 16 24 21 30 153
200 258 239 240 214 222 269 1642
62 89 83 84 76 72 81 547
slaughtered to measure meat quality and carcass traits. The number of pigs in each generation is shown in Table 2. The ASREML 3.0 program (Gilmour et al. 2009) was used to estimate the (co)variance components and their respective standard errors.
Genotyping of VRTN gene polymorphisms Genomic DNA was collected from 759 Duroc pigs. Of these pigs, 314 were selected as candidate pigs to contribute to the next generation, with DG, LEA and BF being measured. The other 445 were sib-tested pigs, from which IMF and carcass traits were measured. Genomic DNA was isolated from ear tissue or blood samples using a phenol-chloroform method. Mikawa et al. (2011) reported that nine polymorphic sites have been clearly divided between the Q haplotype of a European type, resulting in the VRTN polymorphism of a gene increasing the number of vertebrae and the Wt haplotype of a wild type. Fan et al. (2013) showed that out of the nine polymorphic sites, g.19034A>C and g.20311_20312ins291 were the most likely causal mutations in the VRTN gene. Therefore, the insertion (Q) or deletion (Wt) of g.20311_20312ins291 in the intron region was genotyped. The primer array used for PCR was designed following Mikawa et al. (2011). PCR was performed in a total volume of 15 μL containing 20 ng genomic DNA, 6.25 pmol of each primer, 0.2 mmol/L deoxynucleoside triphosphate (dNTP), 10 mmol/L Tris–HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2 and 0.375 U Taq DNA polymerase (Takara, Kyoto, Japan). The PCR conditions were as follows: denaturation at 94°C for 10 min, 30 cycles of amplification at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final extension step at 72°C for 5 min. The PCR product sizes were measured using a 3130 DNA Sequencer (Applied Biosystems Japan, Tokyo, Japan) and analyzed with GENESCAN and GENOTYPER software (Applied Biosystems, Tokyo, Japan). The allele of Q (insertion: 411bp) and Wt (deletion: 120 bp) of g.20311_20312ins291 was determined. Animal Science Journal (2015) 86, 125–131
A mixed-inheritance animal model was used to evaluate the effects of genotypes on the traits in this population. The snp_ad option of Qxpak software (Pérez-Enciso & Misztal 2004) was used for VRTN gene polymorphism. The assumed model for the phenotypic data of each trait was as follows: y ijk = sex i + generation j + Ca a + Cd d + u k + eijk ,
where yijk is the ijk-th observation for these traits, sexi is the fixed effect of sex i (three classes), generationj is the fixed effect of generation year j (seven classes), uk is the infinitesimal genetic effect of animal k, which is distributed as N ( Aσ 2u ), where A is the numerator relationship matrix and eijk is the residual effect. The additive effect (a) is a covariate coefficient with Ca having values of −1, 0 and 1, and the dominance effect (d) is a covariate coefficient with Cd, with values of 0, 1 and 0 for genotypes Wt/Wt, Wt/Q and Q/Q, respectively. Pedigrees of base population animals were traced back to the parental population to create the numerator relationship matrix. A total of 1004 animals were used in this study. Likelihood ratio test statistics (LRT) were calculated by excluding the additive and dominance effects from the model. Nominal P-values were obtained by assuming a chi-square distribution (d.f. = 2) of the LRT. To account for multiple testing, we considered the false discovery rate (FDR), and calculated q-values by the R procedure (http://www.rproject.org/) using the method of Benjamini and Hochberg (1995). The FDR procedure was run for 13 tests (selection and carcass traits). The proportion of additive genetic variance accounted for by this genotypic effect was calculated as: 2pq {a + d (q − p )} Va
2
Variance percentage =
where p and q were the frequencies of alleles Q and Wt at the VRTN polymorphism, and Va was the additive genetic variance of the trait obtained from an animal model analysis that did not take this genotypic effect into account (Falconer & Mackay 1996). In this study, some individuals had a missing genotype, with the allele frequencies in this population being estimated using the Sequential Oligogenic Linkage Analysis Routines package (SOLAR; Almasy & Blangero 1998), which accounted for pedigree structure.
RESULTS AND DISCUSSION Descriptive statistics and genetic parameter estimates Table 3 shows the genetic parameter estimates for the selection and carcass traits. High heritability was estimated for CL, BL1, BL2 and LL, with values of 0.56, 0.65, 0.63 and 0.70, respectively. In addition, the heritability estimate for TVN and TotalVN was also high © 2014 Japanese Society of Animal Science
128 H. NAKANO et al.
Table 3 Genetic parameter estimates on the selection traits and carcass traits
Trait†
DG
LEA
BF
IMF
CWT
CL
BL1
BL2
LL
CW
TVN
LVN
TotalVN
DG
0.51 (0.05) −0.08 (0.03) 0.28 (0.03) 0.06 (0.05) 0.01 (0.05) −0.23 (0.05) −0.16 (0.05) −0.16 (0.05) −0.11 (0.05) −0.16 (0.05) 0.04 (0.05) −0.07 (0.05) −0.01 (0.05)
−0.08 (0.10) 0.50 (0.06) −0.29 (0.03) −0.23 (0.05) 0.11 (0.05) 0.08 (0.05) 0.03 (0.05) 0.05 (0.05) 0.04 (0.05) 0.03 (0.05) −0.05 (0.05) 0.03 (0.05) −0.03 (0.05)
0.35 (0.08) −0.43 (0.08) 0.72 (0.04) 0.18 (0.05) 0.21 (0.05) −0.43 (0.04) −0.42 (0.04) −0.41 (0.04) −0.36 (0.04) 0.10 (0.05) −0.06 (0.05) −0.08 (0.05) −0.11 (0.05)
0.19 (0.15) −0.27 (0.15) 0.14 (0.14) 0.44 (0.11) 0.12 (0.05) 0.00 (0.05) −0.03 (0.05) −0.02 (0.05) −0.05 (0.05) 0.04 (0.05) 0.08 (0.05) 0.00 (0.05) 0.07 (0.06)
0.11 (0.19) −0.03 (0.20) 0.39 (0.19) 0.29 (0.25) 0.24 (0.10) 0.11 (0.05) 0.06 (0.05) 0.07 (0.05) 0.05 (0.05) 0.32 (0.04) −0.06 (0.05) 0.04 (0.05) −0.04 (0.05)
−0.07 (0.14) 0.07 (0.15) −0.53 (0.10) 0.33 (0.19) −0.21 (0.25) 0.56 (0.12) 0.86 (0.01) 0.82 (0.02) 0.71 (0.03) 0.03 (0.05) 0.25 (0.05) 0.12 (0.05) 0.32 (0.05)
0.01 (0.14) 0.10 (0.14) −0.53 (0.10) 0.18 (0.19) −0.30 (0.23) 0.93 (0.04) 0.65 (0.12) 0.88 (0.01) 0.79 (0.02) −0.01 (0.05) 0.40 (0.05) 0.11 (0.05) 0.47 (0.04)
−0.01 (0.13) 0.14 (0.14) −0.51 (0.10) 0.13 (0.19) −0.22 (0.23) 0.97 (0.03) 0.93 (0.04) 0.63 (0.12) 0.88 (0.01) 0.02 (0.05) 0.45 (0.04) 0.23 (0.04) 0.59 (0.04)
−0.05 (0.13) 0.10 (0.14) −0.42 (0.10) 0.11 (0.20) −0.18 (0.24) 0.91 (0.05) 0.95 (0.03) 0.96 (0.02) 0.70 (0.12) 0.11 (0.20) 0.55 (0.04) 0.25 (0.04) 0.70 (0.03)
−0.02 (0.16) −0.31 (0.16) 0.13 (0.16) 0.27 (0.22) 0.30 (0.25) 0.04 (0.21) 0.07 (0.20) 0.03 (0.20) 0.06 (0.05) 0.37 (0.11) 0.01 (0.05) 0.05 (0.05) 0.03 (0.05)
−0.03 (0.13) 0.05 (0.13) −0.16 (0.12) −0.07 (0.18) −0.12 (0.22) 0.56 (0.14) 0.67 (0.11) 0.70 (0.10) 0.84 (0.07) 0.26 (0.19) 0.77 (0.12) −0.30 (0.05) 0.85 (0.01)
−0.28 (0.25) 0.23 (0.26) −0.14 (0.23) 0.34 (0.39) 0.39 (0.40) 0.54 (0.35) 0.38 (0.33) 0.41 (0.30) 0.31 (0.29) −0.27 (0.33) 0.19 (0.33) 0.13 (0.09) 0.24 (0.05)
−0.08 (0.12) 0.11 (0.13) −0.19 (0.11) −0.09 (0.17) −0.04 (0.22) 0.62 (0.12) 0.70 (0.10) 0.75 (0.08) 0.86 (0.05) 0.13 (0.19) 0.98 (0.02) 0.37 (0.27) 0.91 (0.12)
LEA BF IMF CWT CL BL1 BL2 LL CW TVN LVN TotalVN
†Heritability on the diagonal, upper diagonal is genetic correlation, lower diagonal is phenotypic correlation. Standard errors are shown in prarentheses. BF, backfat thickness; BL1, back loin length 1; BL2, back loin length 2; CL, carcass length; CW, carcass width; CWT, carcass weight; DG, daily gain; IMF, intramuscular fat content; LEA, loin eye muscle area; LL, loin length; LVN, lumber vertebrae number; TotalVN, total vertebrae number; TVN, thoracic vertebrae number.
(0.77 and 0.91, respectively). In contrast, the heritability estimate for LVN was very low (0.13). The value for TVN is in good agreement with the estimates of Fredeen and Newman (1962), who reported heritability estimates of 0.73 and 0.59 for thoracic number and 0.60 and 0.59 for vertebrae number by offspring on mid-parent regression and full-sib correlation, respectively. Generally, medium values of heritability estimates for CL are reported to be 0.56–0.57 (Sellier 1998). Furthermore, Borchers et al. (2004) estimated a heritability of 0.51 for thoracic number and 0.62 for vertebrae number. Holl et al. (2008) reported a high heritability estimate of 0.88 for thoracic vertebrae and a low heritability estimate of 0.26 for lumber vertebrae. The genetic correlation values of TVN with CL, BL1, BL2 and LL were 0.56, 0.67, 0.70 and 0.84, respectively. LL was particularly highly correlated with TVN. The higher genetic correlation of LL with TVN than BL1 and BL2 was because they were dependent on the shape of the dorsal line. In the carcass sites from the first to fourth thoracic vertebra and from the last lumbar vertebra to the pubic bone, the dorsal lines were curved. The curved dorsal lines affected body length, and the differences in the lengths of the sites among pigs caused variances in BL1 and BL2. On the © 2014 Japanese Society of Animal Science
other hand, the dorsal line in the LL site was straight, and the difference of LL among pigs was very small, if all pigs had the same TVN. Therefore, TVN difference directly affected LL, and the genetic correlation of LL with TVN was the highest in length-related traits. Although the genetic and phenotypic correlations between BF and length-related traits (CL, BL1, BL2 and LL) were moderately negative (−0.53 to −0.42 and −0.43 to −0.36, respectively), the correlations with TVN were very low (−0.16 and −0.06, respectively). The genetic and phenotypic correlations between CWT and length-related traits (CL, BL1, BL2 and LL) were very low (−0.30 to −0.18 and 0.05 to 0.11, respectively), and also had low correlations with TVN (−0.12 and −0.06, respectively). The genetic correlation between BF and CWT was moderately positive (0.39). In summary, the genetic correlations between length-related traits and TVN were highly positive, whereas those with BF were moderately negative. However, the genetic correlation between TVN and BF was almost zero. In addition, the genetic correlation of CWT with length-related traits and TVN was very low. The results indicate that the increase in the thoracic segment number influences length-related traits, and subsequently influences fat accumulation. However, the promoting increases in thoracic segment number Animal Science Journal (2015) 86, 125–131
GENETIC ASSOCIATIONS OF VRTN IN PIGS 129
Table 4 Number of pigs in each thoracic vertebrae number (TVN) by vertnin (VRTN) genotype
VRTN genotype
TVN
Total
13 14 15 16
Total
Q/Q
Q/Wt
Wt/Wt
0 5 46 50 101
0 21 202 9 232
1 90 19 2 112
1 116 267 61 445
had no direct genetic relationship with fat accumulation and weight gain traits.
VRTN gene frequency and its relationship with the number of thoracic vertebrae Table 4 shows the number of pigs with different TVN in relation to VRTN genotype. In the Q/Q genotype, a total of 50, 46, 5 and 0 individuals had 16, 15, 14 and 13 thoracic vertebrae, respectively. In the Wt/Wt genotype, a total of 2, 19, 90 and 1 individuals had 16, 15, 14 and 13 thoracic vertebrae, respectively. A pig with the Wt/Wt genotype had 13 thoracic vertebrae. The proportion of each TVN showed an asymmetric ratio between the Q/Q and Wt/Wt genotype of the VRTN gene. This asymmetric diversity may be caused by the following process. Genes that influence the thoracic segment number act as continuous variants, even though the phenotype of the number of thoracic vertebra is a threshold characteristic. The expression of the wild type (Wt type) of the VRTN gene is of very short duration compared to the mutant type (Q type) in heterozygous embryos (Mikawa et al. 2011). The difference of gene expression influences the number of thoracic segments, leading to an increase in TVN, because the Q type increases VRTN gene expression in the domesticated pig. The genotypic and allelic frequencies of VRTN gene polymorphisms are shown in Table 5. Pigs that had both genotype information and phenotypic value for the traits were used in this analysis. The allelic frequencies of Q and Wt alleles in this population were 0.51 and 0.49, respectively. Hirose et al. (2013a) reported that the allelic frequency of this gene was approximately 0.60 for Q and 0.40 for Wt in a purebred Duroc population. Fan et al. (2013) also reported similar Q allele frequencies (0.54, 0.71 and 0.66 for Duroc, Landrace and Large White, respectively). The results indicate that variant types of the VRTN gene are present in European breeds and that a difference in the allele frequency causes a variety of major TVN types in pig populations. Table 5 also presents the average number of TVN and the number of pigs in each TVN in each selected generation. The average number of pigs with each Animal Science Journal (2015) 86, 125–131
TVN changed with selection for DG, LEA, BF and IMF from the first generation to the seventh generation (15.2, 15.0, 15.0, 15.0, 14.9, 14.5 and 14.7, respectively). In addition, the number of pigs with TVN 14 increased (from four to 29 pigs), with the number of pigs with TVN 16 decreased (from 11 to four pigs). The genotypic and allelic frequencies of VRTN changed from the first generation to the seventh generation, with only 6% of pigs in the seventh generation having the Q/Q genotype. In the Duroc population, selection was conducted for DG, LEA, BF and IMF based on the desired gains, and the aggregate breeding value was used as an index of selection. When the aggregate breeding value and inbreeding coefficient of the pigs showed the same values, pigs with less body length were selected considering the demand of pig farmers, because the purpose of the selection experiment was to ensure a pig population that is useful to the farmer. In such pig populations, the average body lengths of the first and seventh generations were 107.4 cm and 106.0 cm, respectively. Therefore, the selection for body length might have an effect on the genotypic frequencies of VRTN gene polymorphisms.
The effect of the VRTN gene on selection and carcass traits Additive genetic effects, dominance effects and the contribution ratio of VRTN gene to selection and carcass traits are shown in Table 6. Six carcass lengthrelated traits (CL, BL1, BL2, LL, TVN and TotalVN) were significantly related to the VRTN genotype. In particular, the contribution ratio of VRTN gene polymorphisms to TVN was very high (95.3%). Furthermore, this VRTN genotype had significant effects on carcass length-related traits. Although the contribution ratios of VRTN gene polymorphism to these traits exceeded 11.0%, this value was considerably lower compared to that of TVN. The contribution of this VRTN gene to genetic variance in carcass lengthrelated traits was low compared to that of TVN. This result indicates that carcass length-related traits are influenced by both the number of vertebrae and the length of each vertebra. CL includes the cervical, thoracic, lumbar and sacral vertebrae. BL1 includes the thoracic, lumbar and sacral vertebrae, and BL2 includes the thoracic and lumbar vertebrae, respectively. LL includes the fifth to last thoracic and lumbar vertebrae (Suzuki et al. 1991). Therefore, the economically important parts are BL2 and LL, with TVN being highly genetically correlated with BL2 and LL (0.70 and 0.84, respectively) in this population (Table 3). The contribution of the VRTN gene to BL2 and LL was comparatively high (24.7% and 35.3%, respectively). There were large differences in additive effect (1.00 and 1.17 cm) for BL2 and LL between Q and Wt alleles. The difference of additive effect in TVN between the Q and Wt alleles was 0.57. Large additive © 2014 Japanese Society of Animal Science
130 H. NAKANO et al.
Table 5 Average number of thoracic vertebrae number (TVN), number of pigs in each TVN, and genotypic and allelic frequencies of vertnin (VRTN) gene polymorphisms in each generation of selection
Generation
Mean
1 2 3 4 5 6 7 Mean
15.2 15.0 15.0 15.0 14.9 14.5 14.7 14.9
Number of pigs in each TVN
Genotypic frequency
Allelic frequency
13
14
15
16
Total
Wt/Wt
Wt/Q
Q/Q
Wt
Q
0 1 0 0 0 0 0 1.8
4 17 10 12 11 33 29 16.3
25 49 25 50 40 33 45 35.3
11 16 9 15 4 2 4 9.6
40 83 44 77 55 68 78 63.6
0.25 0.17 0.18 0.16 0.22 0.44 0.33 0.25
0.50 0.49 0.48 0.52 0.55 0.49 0.60 0.52
0.25 0.34 0.34 0.32 0.24 0.07 0.06 0.23
0.50 0.42 0.42 0.42 0.49 0.68 0.63 0.51
0.50 0.58 0.58 0.58 0.51 0.32 0.37 0.49
Table 6 Effects of the vertnin (VRTN) gene polymorphisms on selection and carcass traits
Trait
DG LEA BF IMF CWT CL BL1 BL2 LL CW TVN LVN TotalVN
Unit
g/day cm2 cm % kg cm cm cm cm cm
Descriptive statistics
LRT†
n
Mean
SD
759 756 759 445 445 445 445 445 445 445 445 445 445
874.9 37.0 2.5 4.3 76.9 90.4 74.9 65.2 48.9 35.3 14.9 6.0 20.9
98.3 4.2 0.4 1.5 2.7 2.6 2.1 2.2 2.1 1.4 0.6 0.3 0.6
1.4 4.7 1.7 3.5 1.8 23.0 43.7 57.5 77.1 2.9 233.8 2.0 2.0
P-value
0.50 0.10 0.44 0.18 0.41 1.0 × 10−5 3.3 × 10−10 3.3 × 10−13 1.8 × 10−17 0.24 1.7 × 10−51 0.37 2.7 × 10−49
q-value
0.50 0.19 0.48 0.29 0.48 2.2 × 10−5 8.5 × 10−10 1.1 × 10−12 7.9 × 10−17 0.35 2.2 × 10−50 0.48 1.8 × 10−48
Additive effect‡
Dominance effect‡
Mean
SE
Mean
SE
3.24 −0.45 −0.01 0.19 0.02 0.78 0.87 1.00 1.17 0.12 0.57 −0.03 0.53
4.57 0.22 0.02 0.10 0.18 0.17 0.13 0.14 0.14 0.10 0.03 0.02 0.03
−5.38 −0.09 −0.03 0.01 0.29 0.21 0.18 0.37 0.44 0.14 0.13 0.01 0.14
5.12 0.24 0.02 0.13 0.22 0.21 0.16 0.17 0.17 0.13 0.04 0.03 0.04
Variance§
0.002 0.015 0.000 0.022 0.000 0.112 0.213 0.247 0.353 0.008 0.953 0.015 0.856
†Likelihood ratio test statistics. ‡Additive and dominance effects were genotypic values of (QQ-WtWt)/2 and QWt-(QQ+WtWt)/2, respectively. §The proportion of additive genetic accounted for by the VRTN gene polymorphism. BF, backfat thickness; BL1, back loin length 1; BL2, back loin length 2; CL, carcass length; CW, carcass width; CWT, carcass weight; DG, daily gain; IMF, intramuscular fat content; LEA, loin eye muscle area; LL, loin length; LVN, lumber vertebrae number; TotalVN, total vertebrae number; TVN, thoracic vertebrae number.
effects were noted for BL2 (1.00 cm) and LL (1.17 cm), which corresponded to the additive effect in TVN (0.57), because LVN was not affected by the VRTN gene. Because the dorsal line in a carcass site from the first to fourth thoracic vertebrae is curved and BL2 has a larger variance than LL, LL shows larger contribution and additive effect of VRTN gene than that by BL2. These results show that VRTN gene polymorphisms could be used as effective DNA markers for the improvement of LL. In contrast, VRTN gene polymorphism had no significant effect on genetic variance in CWT, CW and LVN. Mikawa et al. (2011) also reported that the VRTN gene was effective only in the thoracic vertebrae, and that its relationship to LVN was not statistically significant. The estimation of genetic correlation between TVN and CWT was very low, and this value is in good agreement with the results of the effect of the VRTN gene on CWT. Table 6 also shows the effect of VRTN gene polymorphisms on selection traits. In our population, © 2014 Japanese Society of Animal Science
there was no significant effect of VRTN gene polymorphisms on DG, LEA, BF and IMF. The results of DG and BF were the same as those described by Hirose et al. (2013a). In the present study, the estimate of genetic correlation between TVN and LEA was close to zero (0.05) (Table 3). Moreover, that of LEA and LL was also estimated to be close to zero (0.10). This finding indicates that LL might be improved by constantly maintaining the loin area. The Duroc population used in this study was selected by IMF, with the genotypic and allelic frequencies being changed by the selection process (Table 5). However, VRTN gene polymorphism had no significant effect on IMF. In addition, the genetic correlation between VRTN and IMF was very low (−0.07). Therefore, these results indicate that the VRTN gene is not associated with IMF in this population. Hirose et al. (2013a) reported that IMF content in the Longissimus muscle is significantly associated with the VRTN genotype in a Duroc population. In a subsequent analysis, Hirose et al. (2013b) reported no significant association between the VRTN Animal Science Journal (2015) 86, 125–131
GENETIC ASSOCIATIONS OF VRTN IN PIGS 131
gene and IMF in the same Duroc population. The biological explanation for the result obtained by Hirose et al. (2013a) is uncertain, with further study being needed by using other populations. In this study, we investigated the genetic relationships among the number of vertebrae, carcass traits and production traits, and the effect of VRTN gene polymorphisms on these traits. VRTN gene polymorphism greatly contributed to TVN and carcass lengthrelated traits, while exhibiting an extremely low relationship with other traits. In particular, the VRTN gene was not associated with selection traits. These results also indicate that economically important meat production and meat quality traits were not adversely affected when selection was conducted by using this VRTN gene polymorphism as an index for targeting an increase in the number of dorsal vertebrae. The results of this study indicate that the selection of the Q allele in the VRTN gene increased the number of dorsal vertebra, which, in turn, was effective in increasing the economically important trait of LL, without creating adverse effects on meat production traits.
REFERENCES Almasy L, Blangero J. 1998. Multipoint quantitative-trait linkage analysis in general pedigrees. American Journal of Human Genetics 62, 1198–1211. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society, Series B 57, 289–300. Borchers N, Reinsch N, Kalm E. 2004. The number of ribs and vertebrae in a Pietrain cross: variation, heritability and effects on performance traits. Journal of Animal Breeding and Genetics 121, 392–403. Falconer DS, Mackay TFC. 1996. Introduction to Quantitative Genetics, 4th edn. Longman, Harlow. Fan Y, Xing Y, Zhang Z, Ai H, Ouyang Z, Ouyang J, et al. 2013. A further look at porcine chromosome 7 reveals VRTN variants associated with vertebral number in Chinese and Western pigs. PLoS ONE 8, e62534. Fredeen HT, Newman JA. 1962. Rib and vertebral numbers in swine. II. Genetic aspects. Canadian Journal of Animal Science 42, 240–251. Gilmour AR, Gogel BJ, Cullis BR, Thompson R. 2009. Asreml User Guide Release 3.0. VSN International Ltd., Hemel Hempstead. Hirose K, Ito T, Fukawa K, Arakawa A, Mikawa S, Hayashi Y, Tanaka K. 2013b. Evaluation of effects of multiple candidate gene (LEP, LEPR, MC4R, PIK3C3, and VRTN) on production traits in Duroc pigs. Animal Science Journal 85, 198–206. Hirose K, Mikawa S, Okumura N, Noguchi G, Fukawa K, Kanaya N, et al. 2013a. Association of swine vertnin (VRTN) gene with production traits in Duroc pigs
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improved using a closed nucleus breeding system. Animal Science Journal 84, 213–221. Holl JW, Rohrer GA, Shackelford SD, Wheeler TL, Koohmaraie M. 2008. Estimates of genetic parameters for kyphosis in two crossbred swine populations. Journal of Animal Science 86, 1765–1769. King JW, Roberts RC. 1960. Carcass length in the bacon pig: its association with vertebrae numbers and prediction from radiographs of the young pig. Animal Production 2, 59–65. Mikawa S, Hayashi T, Nii M, Shimanuki S, Morozumi T, Awata T. 2005. Two quantitative trait loci on Sus scrofa chromosome 1 and 7 affecting the number of vertebrae. Journal of Animal Science 83, 2247–2254. Mikawa S, Morozumi T, Shimanuki S, Hayashi T, Uenishi H, Domukai M, et al. 2007. Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1). Genome Research 17, 586–593. Mikawa S, Sato S, Nii M, Morozumi T, Yoshioka G, Imaeda N, et al. 2011. Identification of a second gene associated with variation in vertebral number in domestic pigs. BMC Genetics 12, 5. Pérez-Enciso M, Misztal I. 2004. Qxpak: a versatile mixed model application for genetical genomics and QTL analyses. Bioinformatics 20, 2792–2798. Ren DR, Ren J, Ruan GF, Guo YM, Wu LH, Yang GC, et al. 2012. Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a White Duroc 3 Chinese Erhualian intercross resource population. Animal Genetics 43, 545–551. Rohrer GA, Keele JW. 1998. Identification of quantitative trait loci affecting carcass composition in swine. II. Muscling and wholesale product yield traits. Journal of Animal Science 76, 2255–2262. Sato S, Oyamada Y, Atsuji K, Nade T, Sato S, Kobayashi E, et al. 2003. Quantitative trait loci analysis for growth and carcass traits in a Meishan×Duroc F2 resource population. Journal of Animal Science 81, 2938–2949. Sellier P. 1998. Meat and carcass traits. In: Rothschild MF, Rubinsky A (eds), The Genetics of the Pig, pp. 463–510. CAB International, New York. Suzuki K, Kadowaki H, Shibata T, Uchida H, Nishida A. 2005. Selection for daily gain, loin-eye area, backfat thickness and intramuscular fat based on desired gains over seven generations of Duroc pigs. Livestock Production Science 97, 193–202. Suzuki K, Nishida S, Ujiie S. 1991. Correlated responses of carcass traits to selection for meat production performance in Landrace pigs. The Japanese Journal of Swine Science 28, 248–254. Uemoto Y, Nagamine Y, Kobayashi E, Sato S, Tayama T, Suda Y, et al. 2008. Quantitative trait loci analysis on Sus scrofa chromosome 7 for meat production, meat quality, and carcass traits within a Duroc purebred population. Journal of Animal Science 86, 2833–2839. Wada Y, Akita T, Awata T, Furukawa T, Sugai N, Inage Y, et al. 2000. Quantitative trait loci (QTL) analysis in a Meishan × Gottingen cross population. Animal Genetics 31, 376–384.
© 2014 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 125–131
doi: 10.1111/asj.12260
ORIGINAL ARTICLE Effect of VRTN gene polymorphisms on Duroc pig production and carcass traits, and their genetic relationships Hikaru NAKANO,1,2 Shuji SATO,2 Yoshinobu UEMOTO,2 Takashi KIKUCHI,2 Tomoya SHIBATA,3 Hiroshi KADOWAKI,3 Eiji KOBAYASHI4 and Keiichi SUZUKI1 1
Graduate School of Agricultural Science, Tohoku University, Sendai, 2National Livestock Breeding Center, Nishigo, 3Miyagi Prefecture Animal Industry Experiment Station, Oosaki, and 4National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, Japan
ABSTRACT The thoracic vertebral number is associated with body length and carcass traits, and represents one of the most important traits in the pig industry. Recent studies have shown that vertnin (VRTN) gene is associated with variations in the vertebral number in commercial European pigs. However, the genetic relationships and effect of this VRTN gene in pig production and carcass traits remain uncertain. Therefore, we investigated the genetic relationships among traits such as vertebral numbers, carcass weight and length-related traits, and meat production traits, and the effect of VRTN gene polymorphisms on these traits in a Duroc purebred population selected for its meat production traits. Highly positive genetic correlations were obtained between the thoracic vertebral numbers and length-related traits (0.56 to 0.84), whereas low correlations were obtained with production traits and carcass weight (−0.16 to 0.05). VRTN gene polymorphisms indicated that the number of thoracic vertebrae and length-related traits were significantly associated with the VRTN genotype, but had no significant effect on production traits and carcass weight. The results indicate that VRTN gene may be used as an effective selection marker to obtain pigs with high thoracic vertebral numbers and length-related traits, without adversely affecting meat production traits.
Key words: genetic parameter, pig, thoracic vertebral number, VRTN gene.
INTRODUCTION Carcass traits, such as carcass and loin lengths, are economically important traits in pig breeding programs, with these traits being associated with the number of vertebrae. King and Roberts (1960) showed that carcass length increases by an average of about 0.8 cm per additional vertebra. In general, wild pigs have 19 thoracic and lumber vertebrae and European breeds have 20 to 23, with the number of dorsal vertebrae varying considerably (King & Roberts 1960). An increase in vertebral number is expected to improve carcass traits, with these traits potentially being genetically correlated with other economically important traits, such as production traits. However, detailed studies of the genetic relationships among the number of vertebrae, carcass traits and production traits have yet to be reported. Recently, several studies have reported the detection of two quantitative trait loci (QTLs) for vertebral number on SSC1 and SSC7 in crossbred pig popula© 2014 Japanese Society of Animal Science
tions (Rohrer & Keele 1998; Wada et al. 2000; Sato et al. 2003; Mikawa et al. 2005; Ren et al. 2012; Fan et al. 2013) and on SSC7 in a Duroc purebred population (Uemoto et al. 2008). One is a nuclear receptor Germ Cell Nuclear Factor (NR6A1) gene on SSC1 (Mikawa et al. 2007), while the other is a vertnin (VRTN) gene on SSC7 (Mikawa et al. 2011). The NR6A1 gene influences the number of both dorsal and lumbar vertebrae; however, genetic variation caused by NR6A1 gene polymorphisms was not detected in commercial European breeds. The haplotype of VRTN gene polymorphism takes part in the number of thoracic vertebra, and the heterozygote of this VRTN gene genotype was detected in the commercial European
Correspondence: Keiichi Suzuki, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 981-8555, Japan. (Email: [email protected]) Received 30 January 2014; accepted for publication 17 April 2014.
126 H. NAKANO et al.
Table 1 Descriptive statistics of selection traits and carcass traits
Selection traits Daily gain Loin eye muscle area Backfat thickness Intramuscular fat content Carcass traits Carcass weight Carcass length Back loin length 1 Back loin length 2 Loin length Carcass width Thoracic vertebrae number Lumber vertebrae number Total vertebrae number
Abbreviation
n
Unit
Mean
SD
DG LEA BF IMF
1642 1639 1642 545
g/day cm2 cm %
873.6 37.0 2.4 4.2
109.3 4.1 0.4 1.5
77.1 90.5 75.0 65.3 48.9 35.4 14.9 6.0 20.9
2.7 2.6 2.1 2.2 2.1 1.4 0.6 0.3 0.6
CWT CL BL1 BL2 LL CW TVN LVN TotalVN
population (Fan et al. 2013; Hirose et al. 2013a). In addition, Fan et al. (2013) showed that g.19034A>C and g.20311_20312ins291 in the haplotype are the most likely causal mutations in the VRTN gene. The discovery of the genes responsible for the numbers of vertebrae would facilitate the improvement of carcass and production traits that are related to the number of vertebrae, by using a simple and unambiguous selection process with a DNA marker. Recently, Hirose et al. (2013a) demonstrated the significant effect of VRTN gene polymorphisms on intramuscular fat content (IMF) and body length in a Duroc pig population. However, a subsequent report by Hirose et al. (2013b) indicated that there was no significant association between VRTN gene polymorphisms and IMF in the same population. Therefore, further study is required to evaluate the effect of VRTN gene polymorphisms on production traits and other economically important traits related to the number of vertebrae in different pig populations. This study aimed to estimate the genetic relationship among the number of vertebrae, production traits and carcass traits, and to evaluate the effect of VRTN gene polymorphisms on these traits by using a Duroc breed selected for meat production and meat quality traits.
METHODS Animal and phenotype measurement The Duroc purebred population used in the this experiment was selected over seven generations for daily gain (DG) of 30 to 105 kg body weight (BW), loin eye muscle area (LEA), backfat thickness (BF) at 105 kg BW measured using ultrasound technology, and IMF. Details of the selection and measurement methods are described by Suzuki et al. (2005). DG, LEA and BF were measured in all pigs (1639 to 1642 pigs), while IMF was measured in slaughtered sibtested pigs (545 pigs). These slaughtered sib-tested © 2014 Japanese Society of Animal Science
545 545 545 545 545 545 545 545 545
kg cm cm cm cm cm
Duroc pigs were also measured for carcass weight (CWT), carcass length (CL), carcass width (CW), back loin length 1 (BL1), back loin length 2 (BL2), loin length (LL), the number of thoracic and lumbar vertebrae (TVN and LVN, respectively), and the number of total vertebrae (TotalVN) as carcass traits. CL was measured from the first cervical spine to the anterior extremity of the pubic bone; BL1, BL2 and LL are the measurements from the leading edge of the first thoracic vertebra to the anterior extremity of the pubic bone, from the leading edge of the first thoracic vertebra to the posterior border of the last lumbar vertebra, and from the leading edge of the fifth thoracic to the leading edge of the last lumbar vertebra, respectively. TotalVN is the total number of thoracic and lumber vertebrae. The descriptive statistics of these traits are shown in Table 1.
Genetic parameter estimates First, the genetic parameters of selection and carcass traits were estimated by the following multiple trait animal model: Yijkl = μ i + G ij + Sik + a il + eijkl ,
where Yijkl = observation for trait i, μi = common constant for trait i and Gij = fixed effect of selection generation j (7 classes) for trait i. The effect of selection on the generations included the genetic effect of selection and the environmental effect on each generation. Sik = fixed effect of sex k (three classes) for trait i, ail = random additive genetic effect of animal l for trait i and eijkl = random residual effect for trait i. Pedigree information was obtained for 1642 animals across seven generations, along with production trait data from 152 ancestors born before the fourth generation (totalling 1794 animals) being included in this analysis. In total, 547 pigs (394 barrows and 153 gilts) were selected as sib-tested pigs, which were then Animal Science Journal (2015) 86, 125–131
GENETIC ASSOCIATIONS OF VRTN IN PIGS 127
Table 2 Number of male and female pigs by generation of selection
Male
Female
Total
Generation
All
Sibtested
All
Sibtested
All
Sibtested
1 2 3 4 5 6 7 Total
91 109 119 118 100 101 135 773
44 59 69 68 52 51 51 394
109 149 120 122 114 121 134 869
18 30 14 16 24 21 30 153
200 258 239 240 214 222 269 1642
62 89 83 84 76 72 81 547
slaughtered to measure meat quality and carcass traits. The number of pigs in each generation is shown in Table 2. The ASREML 3.0 program (Gilmour et al. 2009) was used to estimate the (co)variance components and their respective standard errors.
Genotyping of VRTN gene polymorphisms Genomic DNA was collected from 759 Duroc pigs. Of these pigs, 314 were selected as candidate pigs to contribute to the next generation, with DG, LEA and BF being measured. The other 445 were sib-tested pigs, from which IMF and carcass traits were measured. Genomic DNA was isolated from ear tissue or blood samples using a phenol-chloroform method. Mikawa et al. (2011) reported that nine polymorphic sites have been clearly divided between the Q haplotype of a European type, resulting in the VRTN polymorphism of a gene increasing the number of vertebrae and the Wt haplotype of a wild type. Fan et al. (2013) showed that out of the nine polymorphic sites, g.19034A>C and g.20311_20312ins291 were the most likely causal mutations in the VRTN gene. Therefore, the insertion (Q) or deletion (Wt) of g.20311_20312ins291 in the intron region was genotyped. The primer array used for PCR was designed following Mikawa et al. (2011). PCR was performed in a total volume of 15 μL containing 20 ng genomic DNA, 6.25 pmol of each primer, 0.2 mmol/L deoxynucleoside triphosphate (dNTP), 10 mmol/L Tris–HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2 and 0.375 U Taq DNA polymerase (Takara, Kyoto, Japan). The PCR conditions were as follows: denaturation at 94°C for 10 min, 30 cycles of amplification at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final extension step at 72°C for 5 min. The PCR product sizes were measured using a 3130 DNA Sequencer (Applied Biosystems Japan, Tokyo, Japan) and analyzed with GENESCAN and GENOTYPER software (Applied Biosystems, Tokyo, Japan). The allele of Q (insertion: 411bp) and Wt (deletion: 120 bp) of g.20311_20312ins291 was determined. Animal Science Journal (2015) 86, 125–131
A mixed-inheritance animal model was used to evaluate the effects of genotypes on the traits in this population. The snp_ad option of Qxpak software (Pérez-Enciso & Misztal 2004) was used for VRTN gene polymorphism. The assumed model for the phenotypic data of each trait was as follows: y ijk = sex i + generation j + Ca a + Cd d + u k + eijk ,
where yijk is the ijk-th observation for these traits, sexi is the fixed effect of sex i (three classes), generationj is the fixed effect of generation year j (seven classes), uk is the infinitesimal genetic effect of animal k, which is distributed as N ( Aσ 2u ), where A is the numerator relationship matrix and eijk is the residual effect. The additive effect (a) is a covariate coefficient with Ca having values of −1, 0 and 1, and the dominance effect (d) is a covariate coefficient with Cd, with values of 0, 1 and 0 for genotypes Wt/Wt, Wt/Q and Q/Q, respectively. Pedigrees of base population animals were traced back to the parental population to create the numerator relationship matrix. A total of 1004 animals were used in this study. Likelihood ratio test statistics (LRT) were calculated by excluding the additive and dominance effects from the model. Nominal P-values were obtained by assuming a chi-square distribution (d.f. = 2) of the LRT. To account for multiple testing, we considered the false discovery rate (FDR), and calculated q-values by the R procedure (http://www.rproject.org/) using the method of Benjamini and Hochberg (1995). The FDR procedure was run for 13 tests (selection and carcass traits). The proportion of additive genetic variance accounted for by this genotypic effect was calculated as: 2pq {a + d (q − p )} Va
2
Variance percentage =
where p and q were the frequencies of alleles Q and Wt at the VRTN polymorphism, and Va was the additive genetic variance of the trait obtained from an animal model analysis that did not take this genotypic effect into account (Falconer & Mackay 1996). In this study, some individuals had a missing genotype, with the allele frequencies in this population being estimated using the Sequential Oligogenic Linkage Analysis Routines package (SOLAR; Almasy & Blangero 1998), which accounted for pedigree structure.
RESULTS AND DISCUSSION Descriptive statistics and genetic parameter estimates Table 3 shows the genetic parameter estimates for the selection and carcass traits. High heritability was estimated for CL, BL1, BL2 and LL, with values of 0.56, 0.65, 0.63 and 0.70, respectively. In addition, the heritability estimate for TVN and TotalVN was also high © 2014 Japanese Society of Animal Science
128 H. NAKANO et al.
Table 3 Genetic parameter estimates on the selection traits and carcass traits
Trait†
DG
LEA
BF
IMF
CWT
CL
BL1
BL2
LL
CW
TVN
LVN
TotalVN
DG
0.51 (0.05) −0.08 (0.03) 0.28 (0.03) 0.06 (0.05) 0.01 (0.05) −0.23 (0.05) −0.16 (0.05) −0.16 (0.05) −0.11 (0.05) −0.16 (0.05) 0.04 (0.05) −0.07 (0.05) −0.01 (0.05)
−0.08 (0.10) 0.50 (0.06) −0.29 (0.03) −0.23 (0.05) 0.11 (0.05) 0.08 (0.05) 0.03 (0.05) 0.05 (0.05) 0.04 (0.05) 0.03 (0.05) −0.05 (0.05) 0.03 (0.05) −0.03 (0.05)
0.35 (0.08) −0.43 (0.08) 0.72 (0.04) 0.18 (0.05) 0.21 (0.05) −0.43 (0.04) −0.42 (0.04) −0.41 (0.04) −0.36 (0.04) 0.10 (0.05) −0.06 (0.05) −0.08 (0.05) −0.11 (0.05)
0.19 (0.15) −0.27 (0.15) 0.14 (0.14) 0.44 (0.11) 0.12 (0.05) 0.00 (0.05) −0.03 (0.05) −0.02 (0.05) −0.05 (0.05) 0.04 (0.05) 0.08 (0.05) 0.00 (0.05) 0.07 (0.06)
0.11 (0.19) −0.03 (0.20) 0.39 (0.19) 0.29 (0.25) 0.24 (0.10) 0.11 (0.05) 0.06 (0.05) 0.07 (0.05) 0.05 (0.05) 0.32 (0.04) −0.06 (0.05) 0.04 (0.05) −0.04 (0.05)
−0.07 (0.14) 0.07 (0.15) −0.53 (0.10) 0.33 (0.19) −0.21 (0.25) 0.56 (0.12) 0.86 (0.01) 0.82 (0.02) 0.71 (0.03) 0.03 (0.05) 0.25 (0.05) 0.12 (0.05) 0.32 (0.05)
0.01 (0.14) 0.10 (0.14) −0.53 (0.10) 0.18 (0.19) −0.30 (0.23) 0.93 (0.04) 0.65 (0.12) 0.88 (0.01) 0.79 (0.02) −0.01 (0.05) 0.40 (0.05) 0.11 (0.05) 0.47 (0.04)
−0.01 (0.13) 0.14 (0.14) −0.51 (0.10) 0.13 (0.19) −0.22 (0.23) 0.97 (0.03) 0.93 (0.04) 0.63 (0.12) 0.88 (0.01) 0.02 (0.05) 0.45 (0.04) 0.23 (0.04) 0.59 (0.04)
−0.05 (0.13) 0.10 (0.14) −0.42 (0.10) 0.11 (0.20) −0.18 (0.24) 0.91 (0.05) 0.95 (0.03) 0.96 (0.02) 0.70 (0.12) 0.11 (0.20) 0.55 (0.04) 0.25 (0.04) 0.70 (0.03)
−0.02 (0.16) −0.31 (0.16) 0.13 (0.16) 0.27 (0.22) 0.30 (0.25) 0.04 (0.21) 0.07 (0.20) 0.03 (0.20) 0.06 (0.05) 0.37 (0.11) 0.01 (0.05) 0.05 (0.05) 0.03 (0.05)
−0.03 (0.13) 0.05 (0.13) −0.16 (0.12) −0.07 (0.18) −0.12 (0.22) 0.56 (0.14) 0.67 (0.11) 0.70 (0.10) 0.84 (0.07) 0.26 (0.19) 0.77 (0.12) −0.30 (0.05) 0.85 (0.01)
−0.28 (0.25) 0.23 (0.26) −0.14 (0.23) 0.34 (0.39) 0.39 (0.40) 0.54 (0.35) 0.38 (0.33) 0.41 (0.30) 0.31 (0.29) −0.27 (0.33) 0.19 (0.33) 0.13 (0.09) 0.24 (0.05)
−0.08 (0.12) 0.11 (0.13) −0.19 (0.11) −0.09 (0.17) −0.04 (0.22) 0.62 (0.12) 0.70 (0.10) 0.75 (0.08) 0.86 (0.05) 0.13 (0.19) 0.98 (0.02) 0.37 (0.27) 0.91 (0.12)
LEA BF IMF CWT CL BL1 BL2 LL CW TVN LVN TotalVN
†Heritability on the diagonal, upper diagonal is genetic correlation, lower diagonal is phenotypic correlation. Standard errors are shown in prarentheses. BF, backfat thickness; BL1, back loin length 1; BL2, back loin length 2; CL, carcass length; CW, carcass width; CWT, carcass weight; DG, daily gain; IMF, intramuscular fat content; LEA, loin eye muscle area; LL, loin length; LVN, lumber vertebrae number; TotalVN, total vertebrae number; TVN, thoracic vertebrae number.
(0.77 and 0.91, respectively). In contrast, the heritability estimate for LVN was very low (0.13). The value for TVN is in good agreement with the estimates of Fredeen and Newman (1962), who reported heritability estimates of 0.73 and 0.59 for thoracic number and 0.60 and 0.59 for vertebrae number by offspring on mid-parent regression and full-sib correlation, respectively. Generally, medium values of heritability estimates for CL are reported to be 0.56–0.57 (Sellier 1998). Furthermore, Borchers et al. (2004) estimated a heritability of 0.51 for thoracic number and 0.62 for vertebrae number. Holl et al. (2008) reported a high heritability estimate of 0.88 for thoracic vertebrae and a low heritability estimate of 0.26 for lumber vertebrae. The genetic correlation values of TVN with CL, BL1, BL2 and LL were 0.56, 0.67, 0.70 and 0.84, respectively. LL was particularly highly correlated with TVN. The higher genetic correlation of LL with TVN than BL1 and BL2 was because they were dependent on the shape of the dorsal line. In the carcass sites from the first to fourth thoracic vertebra and from the last lumbar vertebra to the pubic bone, the dorsal lines were curved. The curved dorsal lines affected body length, and the differences in the lengths of the sites among pigs caused variances in BL1 and BL2. On the © 2014 Japanese Society of Animal Science
other hand, the dorsal line in the LL site was straight, and the difference of LL among pigs was very small, if all pigs had the same TVN. Therefore, TVN difference directly affected LL, and the genetic correlation of LL with TVN was the highest in length-related traits. Although the genetic and phenotypic correlations between BF and length-related traits (CL, BL1, BL2 and LL) were moderately negative (−0.53 to −0.42 and −0.43 to −0.36, respectively), the correlations with TVN were very low (−0.16 and −0.06, respectively). The genetic and phenotypic correlations between CWT and length-related traits (CL, BL1, BL2 and LL) were very low (−0.30 to −0.18 and 0.05 to 0.11, respectively), and also had low correlations with TVN (−0.12 and −0.06, respectively). The genetic correlation between BF and CWT was moderately positive (0.39). In summary, the genetic correlations between length-related traits and TVN were highly positive, whereas those with BF were moderately negative. However, the genetic correlation between TVN and BF was almost zero. In addition, the genetic correlation of CWT with length-related traits and TVN was very low. The results indicate that the increase in the thoracic segment number influences length-related traits, and subsequently influences fat accumulation. However, the promoting increases in thoracic segment number Animal Science Journal (2015) 86, 125–131
GENETIC ASSOCIATIONS OF VRTN IN PIGS 129
Table 4 Number of pigs in each thoracic vertebrae number (TVN) by vertnin (VRTN) genotype
VRTN genotype
TVN
Total
13 14 15 16
Total
Q/Q
Q/Wt
Wt/Wt
0 5 46 50 101
0 21 202 9 232
1 90 19 2 112
1 116 267 61 445
had no direct genetic relationship with fat accumulation and weight gain traits.
VRTN gene frequency and its relationship with the number of thoracic vertebrae Table 4 shows the number of pigs with different TVN in relation to VRTN genotype. In the Q/Q genotype, a total of 50, 46, 5 and 0 individuals had 16, 15, 14 and 13 thoracic vertebrae, respectively. In the Wt/Wt genotype, a total of 2, 19, 90 and 1 individuals had 16, 15, 14 and 13 thoracic vertebrae, respectively. A pig with the Wt/Wt genotype had 13 thoracic vertebrae. The proportion of each TVN showed an asymmetric ratio between the Q/Q and Wt/Wt genotype of the VRTN gene. This asymmetric diversity may be caused by the following process. Genes that influence the thoracic segment number act as continuous variants, even though the phenotype of the number of thoracic vertebra is a threshold characteristic. The expression of the wild type (Wt type) of the VRTN gene is of very short duration compared to the mutant type (Q type) in heterozygous embryos (Mikawa et al. 2011). The difference of gene expression influences the number of thoracic segments, leading to an increase in TVN, because the Q type increases VRTN gene expression in the domesticated pig. The genotypic and allelic frequencies of VRTN gene polymorphisms are shown in Table 5. Pigs that had both genotype information and phenotypic value for the traits were used in this analysis. The allelic frequencies of Q and Wt alleles in this population were 0.51 and 0.49, respectively. Hirose et al. (2013a) reported that the allelic frequency of this gene was approximately 0.60 for Q and 0.40 for Wt in a purebred Duroc population. Fan et al. (2013) also reported similar Q allele frequencies (0.54, 0.71 and 0.66 for Duroc, Landrace and Large White, respectively). The results indicate that variant types of the VRTN gene are present in European breeds and that a difference in the allele frequency causes a variety of major TVN types in pig populations. Table 5 also presents the average number of TVN and the number of pigs in each TVN in each selected generation. The average number of pigs with each Animal Science Journal (2015) 86, 125–131
TVN changed with selection for DG, LEA, BF and IMF from the first generation to the seventh generation (15.2, 15.0, 15.0, 15.0, 14.9, 14.5 and 14.7, respectively). In addition, the number of pigs with TVN 14 increased (from four to 29 pigs), with the number of pigs with TVN 16 decreased (from 11 to four pigs). The genotypic and allelic frequencies of VRTN changed from the first generation to the seventh generation, with only 6% of pigs in the seventh generation having the Q/Q genotype. In the Duroc population, selection was conducted for DG, LEA, BF and IMF based on the desired gains, and the aggregate breeding value was used as an index of selection. When the aggregate breeding value and inbreeding coefficient of the pigs showed the same values, pigs with less body length were selected considering the demand of pig farmers, because the purpose of the selection experiment was to ensure a pig population that is useful to the farmer. In such pig populations, the average body lengths of the first and seventh generations were 107.4 cm and 106.0 cm, respectively. Therefore, the selection for body length might have an effect on the genotypic frequencies of VRTN gene polymorphisms.
The effect of the VRTN gene on selection and carcass traits Additive genetic effects, dominance effects and the contribution ratio of VRTN gene to selection and carcass traits are shown in Table 6. Six carcass lengthrelated traits (CL, BL1, BL2, LL, TVN and TotalVN) were significantly related to the VRTN genotype. In particular, the contribution ratio of VRTN gene polymorphisms to TVN was very high (95.3%). Furthermore, this VRTN genotype had significant effects on carcass length-related traits. Although the contribution ratios of VRTN gene polymorphism to these traits exceeded 11.0%, this value was considerably lower compared to that of TVN. The contribution of this VRTN gene to genetic variance in carcass lengthrelated traits was low compared to that of TVN. This result indicates that carcass length-related traits are influenced by both the number of vertebrae and the length of each vertebra. CL includes the cervical, thoracic, lumbar and sacral vertebrae. BL1 includes the thoracic, lumbar and sacral vertebrae, and BL2 includes the thoracic and lumbar vertebrae, respectively. LL includes the fifth to last thoracic and lumbar vertebrae (Suzuki et al. 1991). Therefore, the economically important parts are BL2 and LL, with TVN being highly genetically correlated with BL2 and LL (0.70 and 0.84, respectively) in this population (Table 3). The contribution of the VRTN gene to BL2 and LL was comparatively high (24.7% and 35.3%, respectively). There were large differences in additive effect (1.00 and 1.17 cm) for BL2 and LL between Q and Wt alleles. The difference of additive effect in TVN between the Q and Wt alleles was 0.57. Large additive © 2014 Japanese Society of Animal Science
130 H. NAKANO et al.
Table 5 Average number of thoracic vertebrae number (TVN), number of pigs in each TVN, and genotypic and allelic frequencies of vertnin (VRTN) gene polymorphisms in each generation of selection
Generation
Mean
1 2 3 4 5 6 7 Mean
15.2 15.0 15.0 15.0 14.9 14.5 14.7 14.9
Number of pigs in each TVN
Genotypic frequency
Allelic frequency
13
14
15
16
Total
Wt/Wt
Wt/Q
Q/Q
Wt
Q
0 1 0 0 0 0 0 1.8
4 17 10 12 11 33 29 16.3
25 49 25 50 40 33 45 35.3
11 16 9 15 4 2 4 9.6
40 83 44 77 55 68 78 63.6
0.25 0.17 0.18 0.16 0.22 0.44 0.33 0.25
0.50 0.49 0.48 0.52 0.55 0.49 0.60 0.52
0.25 0.34 0.34 0.32 0.24 0.07 0.06 0.23
0.50 0.42 0.42 0.42 0.49 0.68 0.63 0.51
0.50 0.58 0.58 0.58 0.51 0.32 0.37 0.49
Table 6 Effects of the vertnin (VRTN) gene polymorphisms on selection and carcass traits
Trait
DG LEA BF IMF CWT CL BL1 BL2 LL CW TVN LVN TotalVN
Unit
g/day cm2 cm % kg cm cm cm cm cm
Descriptive statistics
LRT†
n
Mean
SD
759 756 759 445 445 445 445 445 445 445 445 445 445
874.9 37.0 2.5 4.3 76.9 90.4 74.9 65.2 48.9 35.3 14.9 6.0 20.9
98.3 4.2 0.4 1.5 2.7 2.6 2.1 2.2 2.1 1.4 0.6 0.3 0.6
1.4 4.7 1.7 3.5 1.8 23.0 43.7 57.5 77.1 2.9 233.8 2.0 2.0
P-value
0.50 0.10 0.44 0.18 0.41 1.0 × 10−5 3.3 × 10−10 3.3 × 10−13 1.8 × 10−17 0.24 1.7 × 10−51 0.37 2.7 × 10−49
q-value
0.50 0.19 0.48 0.29 0.48 2.2 × 10−5 8.5 × 10−10 1.1 × 10−12 7.9 × 10−17 0.35 2.2 × 10−50 0.48 1.8 × 10−48
Additive effect‡
Dominance effect‡
Mean
SE
Mean
SE
3.24 −0.45 −0.01 0.19 0.02 0.78 0.87 1.00 1.17 0.12 0.57 −0.03 0.53
4.57 0.22 0.02 0.10 0.18 0.17 0.13 0.14 0.14 0.10 0.03 0.02 0.03
−5.38 −0.09 −0.03 0.01 0.29 0.21 0.18 0.37 0.44 0.14 0.13 0.01 0.14
5.12 0.24 0.02 0.13 0.22 0.21 0.16 0.17 0.17 0.13 0.04 0.03 0.04
Variance§
0.002 0.015 0.000 0.022 0.000 0.112 0.213 0.247 0.353 0.008 0.953 0.015 0.856
†Likelihood ratio test statistics. ‡Additive and dominance effects were genotypic values of (QQ-WtWt)/2 and QWt-(QQ+WtWt)/2, respectively. §The proportion of additive genetic accounted for by the VRTN gene polymorphism. BF, backfat thickness; BL1, back loin length 1; BL2, back loin length 2; CL, carcass length; CW, carcass width; CWT, carcass weight; DG, daily gain; IMF, intramuscular fat content; LEA, loin eye muscle area; LL, loin length; LVN, lumber vertebrae number; TotalVN, total vertebrae number; TVN, thoracic vertebrae number.
effects were noted for BL2 (1.00 cm) and LL (1.17 cm), which corresponded to the additive effect in TVN (0.57), because LVN was not affected by the VRTN gene. Because the dorsal line in a carcass site from the first to fourth thoracic vertebrae is curved and BL2 has a larger variance than LL, LL shows larger contribution and additive effect of VRTN gene than that by BL2. These results show that VRTN gene polymorphisms could be used as effective DNA markers for the improvement of LL. In contrast, VRTN gene polymorphism had no significant effect on genetic variance in CWT, CW and LVN. Mikawa et al. (2011) also reported that the VRTN gene was effective only in the thoracic vertebrae, and that its relationship to LVN was not statistically significant. The estimation of genetic correlation between TVN and CWT was very low, and this value is in good agreement with the results of the effect of the VRTN gene on CWT. Table 6 also shows the effect of VRTN gene polymorphisms on selection traits. In our population, © 2014 Japanese Society of Animal Science
there was no significant effect of VRTN gene polymorphisms on DG, LEA, BF and IMF. The results of DG and BF were the same as those described by Hirose et al. (2013a). In the present study, the estimate of genetic correlation between TVN and LEA was close to zero (0.05) (Table 3). Moreover, that of LEA and LL was also estimated to be close to zero (0.10). This finding indicates that LL might be improved by constantly maintaining the loin area. The Duroc population used in this study was selected by IMF, with the genotypic and allelic frequencies being changed by the selection process (Table 5). However, VRTN gene polymorphism had no significant effect on IMF. In addition, the genetic correlation between VRTN and IMF was very low (−0.07). Therefore, these results indicate that the VRTN gene is not associated with IMF in this population. Hirose et al. (2013a) reported that IMF content in the Longissimus muscle is significantly associated with the VRTN genotype in a Duroc population. In a subsequent analysis, Hirose et al. (2013b) reported no significant association between the VRTN Animal Science Journal (2015) 86, 125–131
GENETIC ASSOCIATIONS OF VRTN IN PIGS 131
gene and IMF in the same Duroc population. The biological explanation for the result obtained by Hirose et al. (2013a) is uncertain, with further study being needed by using other populations. In this study, we investigated the genetic relationships among the number of vertebrae, carcass traits and production traits, and the effect of VRTN gene polymorphisms on these traits. VRTN gene polymorphism greatly contributed to TVN and carcass lengthrelated traits, while exhibiting an extremely low relationship with other traits. In particular, the VRTN gene was not associated with selection traits. These results also indicate that economically important meat production and meat quality traits were not adversely affected when selection was conducted by using this VRTN gene polymorphism as an index for targeting an increase in the number of dorsal vertebrae. The results of this study indicate that the selection of the Q allele in the VRTN gene increased the number of dorsal vertebra, which, in turn, was effective in increasing the economically important trait of LL, without creating adverse effects on meat production traits.
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