Identification of leptin gene polymorphisms associated with carcass traits and fatty acid composition in Japanese Black cattle
Abstract
Previous studies have indicated that some leptin gene polymorphisms were associated with economically important traits in cattle breeds. However, polymorphisms in the leptin gene have not been reported thus far in Japanese Black cattle. Here, we aimed to identify the leptin gene polymorphisms which are associated with carcass traits and fatty acid composition in Japanese Black cattle. We sequenced the full-length coding sequence of leptin gene for eight Japanese Black cattle. Sequence comparison revealed eight single nucleotide polymorphisms (SNPs). Three of these were predicted to cause amino acid substitutions: Y7F, R25C and A80V. Then, we genotyped these SNPs in two populations (JB1 with 560 animals and JB2 with 450 animals) and investigated the effects on the traits. Y7F in JB1 and A80V in JB2 were excluded from statistical analysis because the minor allele frequencies were low (< 0.1). Association analysis revealed that Y7F had a significant effect on the dressed carcass weight in JB2; R25C had a significant effect on C18:0 and C14:1 in JB1 and JB2, respectively; and A80V had a significant effect on C16:0, C16:1, C18:1, monounsaturated fatty acid and saturated fatty acid in JB1. The results suggested that these SNPs could be used as an effective marker for the improvement of Japanese Black cattle.
Introduction
Japanese Black cattle is the main beef cattle breed in Japan and is appreciated worldwide for the quality of its beef, including its high marbling and good flavor (Wheeler et al. 2004). Some effective DNA markers for the improvement of this breed have been previously identified. For example, Sukegawa et al. (2010) reported an association between a single nucleotide polymorphism (SNP) in the EDG1 gene and beef marbling score, and Hayakawa et al. (2015) identified a FASN gene polymorphism in the promoter region noted to be associated with fatty acid composition. However, because beef quality traits are regulated by polygenes, additional DNA markers in other genes are required for a more effective marker-assisted selection in this breed.
Leptin, an adipocyte-derived circulating protein, is a central factor for decreasing food intake and virtually stopping body weight gain (Chen et al. 1996). Fantuzzi and Faggioni (2000) reported that leptin alters the balance of T cell-derived cytokines and acts as an inhibitor of apoptosis in T lymphocytes. Ingvertsen and Boisclair (2001) concluded that leptin has a direct effect on not only food intake and energy homeostasis but also immunity. Furthermore, leptin regulates the expression of some genes and the activation of enzymes (Siegrist-Kaiser et al. 1997; Minokoshi et al. 2002). Thus, polymorphisms in the leptin gene coding sequence may affect some important traits in Japanese Black cattle.
Previous studies have identified polymorphisms in the leptin gene coding sequence (CDS) and have reported that non-synonymous substitutions in the leptin gene are associated with many economically important traits in other breeds, including carcass traits (Corva et al. 2009; Tian et al. 2013; Silva et al. 2014), milk production traits (Buchanan et al. 2003; Chebel et al. 2008; Glantz et al. 2012) and fertility traits (Giblin et al. 2010). However, polymorphisms in the leptin gene have not been reported thus far in Japanese Black cattle. Therefore, we aimed to identify the leptin gene polymorphisms associated with carcass traits and fatty acid composition in Japanese Black cattle.
Materials and Methods
Animals
We used two Japanese Black cattle populations (JB1 and JB2) to evaluate the association of leptin genotypes with carcass traits and fatty acid composition. JB1 comprised the commercial cattle population produced in Miyazaki Prefecture, Japan from April 2006 to October 2007 and included 560 cattle (506 steers and 54 heifers), whereas JB2 comprised the cattle population fattened by the Wagyu Registry Association across Japan for field progeny testing from 2002 to 2011 and included 450 cattle (288 steers and 162 heifers). On fattening farms, a group of animals (usually a few to several individuals) are managed together in a pen. They have restricted access to roughage and ad libitum access to concentrate, which consists of corn, barley, and wheat bran. The average age at slaughter in JB1 and JB2 was 29.10 ± 1.62 and 28.54 ± 1.34 months, respectively.
DNA polymorphism identification
To detect DNA polymorphisms, we sequenced the full-length CDS of the bovine leptin gene using genomic DNA from eight Japanese Black cattle, which were selected from a JB2 population based on the pedigree information. Primer sets for the PCR amplification and sequencing analysis were designed based on GenBank sequences (NC_007302.5) using OLIGO 7.41 (Table 1). Following purification of the PCR product using ExoSAP-IT® (Affymetrix, Inc., Santan Clara, CA, USA), we performed standard double-stranded DNA cycle sequencing with approximately 20 ng of amplified product using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan) on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems).
| Primer | Sequence | Product size (bp) | Annealing temperature |
|---|---|---|---|
| Sequencing exon 2, genotyping c.20A > T | F 5′-TTTCTTGATTCCGCCGCACCTCT-3′ | 721 | 63 |
| R 5′-GCTCAGTTACCAGGCAGGAAGAA-3′ | |||
| Sequencing exon 3 | F 5′-CAGAAAGATAGGAGCCCAGGAGA-3′ | 974 | 62 |
| R 5′-GCTTCCATCGTATGTTGTGTGGG-3′ | |||
| Genotyping c.73C > T | F 5′-GGGGAGTGCCTTTCATTACTGTC-3′ | 495 | 62 |
| R 5′-GGAGGAGAGGAGCTGTCTTTATG-3′ | |||
| Genotyping c.239C > T | F 5′-CACCTCTACGCTCTAGGGAAAG-3′ | 221 | 65 |
| R 5′-AGGCAGACTGGTGAGGATCTGTTGGTTGAT-3′ |
- Exon 1 in bovine leptin gene is a non-coding exon.
Genotyping
We applied PCR-RFLP (restriction fragment length polymorphism) to genotype three nucleotide substitutions (Y7F, R25C and A80V) in the bovine leptin gene. Three PCR primer sets were designed for DNA fragments flanking these polymorphisms (Table 1). PCR was performed with 20 ng of genomic DNA and TaKaRa Ex Taq™ polymerase (TaKaRa, Kyoto, Japan) using the GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) with the following thermo-cycling protocol: initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 63°C (Y7F) or 62°C (R25C and A80V) for 30 s, and 72°C for 1 min (Y7F and R25C) or 30 s (A80V), with a final extension step of 72°C for 7 min. The fragments flanking Y7F, R25C and A80V were digestible with BspDI, AciI and BclI, respectively. We performed the digestions in 20 μL reaction mixture, with approximately 5 µg of PCR products and 2.5 units of each restriction enzyme. The digested PCR products were confirmed by agarose gel electrophoresis of the undigested products and sequenced homozygous and heterozygous samples.
Traits
The carcass weight (kg), rib eye area (cm2), rib thickness (cm), subcutaneous fat thickness (cm), yield estimate (%) and beef marbling standard were measured by official graders of the Japan Meat Grading Association. Cervical muscles were obtained for genomic DNA extraction and genotyping; and adipose tissues were collected from the perirenal fat (JB1) and intramuscular fat of the Longissimus muscle (JB2) to measure fatty acid composition. All samples were stored at −20°C prior to DNA extraction or fatty acid purification. The extraction and methylation of fatty acid were performed following the methods described by Folch et al. (1957) and O'Keefe et al. (1968), respectively. We analyzed the fatty acid methyl esters C14:0, C14:1, C16:0, C16:1, C18:0, C18:1 and C18:2 and calculated the percentage composition of each (Table 2).
| JB1 | JB2 | |||
|---|---|---|---|---|
| Mean | SD | Mean | SD | |
| Carcass traits | ||||
| Dressed carcass weight (kg) | 432.09 ± 3.94 | 444.17 ± 3.64 | ||
| Rib-eye area (cm2) | 56.28 ± 0.64 | 55.77 ± 0.64 | ||
| Rib thickness (cm) | 7.60 ± 0.07 | 7.73 ± 0.06 | ||
| Subcutaneous fat thickness (cm) | 2.46 ± 0.06 | 2.87 ± 0.06 | ||
| Yield estimate (%) | 74.22 ± 0.11 | 73.69 ± 0.11 | ||
| Beef marbling standard | 6.17 ± 0.17 | 6.42 ± 0.17 | ||
| Fatty acid composition (%) | ||||
| C14:0 | 2.48 ± 0.05 | 2.63 ± 0.04 | ||
| C14:1 | 0.87 ± 0.03 | 0.87 ± 0.02 | ||
| C16:0 | 23.69 ± 0.22 | 26.77 ± 0.15 | ||
| C16:1 | 2.72 ± 0.05 | 3.91 ± 0.06 | ||
| C18:0 | 19.65 ± 0.25 | 11.45 ± 0.12 | ||
| C18:1 | 48.59 ± 0.38 | 51.98 ± 0.21 | ||
| C18:2 | 1.99 ± 0.04 | 2.33 ± 0.05 | ||
| SFA | 45.83 ± 0.39 | 40.85 ± 0.22 | ||
| MUFA | 52.18 ± 0.39 | 56.75 ± 0.22 | ||
- MUFA, monounsaturated fatty acid; SD, standard deviation; SFA, saturated fatty acid.
- JB1: the commercial cattle population produced in Miyazaki Prefecture.
- JB2: the cattle population fattened across Japan for field progeny testing from 2002 to 2011.
Statistical analysis
The effects of the polymorphisms on each trait were statistically tested by analysis of variance (ANOVA) using a model that accounted for age at slaughter, sire, sex and genotype without interactions. Factors that were found to be significant were then further tested using Tukey's honestly significant difference (HSD) test.
Results
Polymorphism identification
We sequenced the CDS of the leptin gene from the genomic DNA of eight Japanese Black cattle. Sequence comparison revealed eight SNPs (20A/T, 73 T/C, 150C/G, 239C/T, 396C/T, 399 T/C, 411 T/C and 495C/T, with the translation initiation site assigned as +1) in the CDS. Three of these were predicted to cause amino acid substitutions: tyrosine to phenylalanine at 20A/T (Y7F; rs29004487), arginine to cysteine at 73 T/C (R25C; rs29004488) and alanine to valine at 239C/T (A80V; rs29004508).
Genotyping
We genotyped three polymorphisms (Y7F, R25C and A80V) in two Japanese Black cattle populations (JB1 and JB2) (Table 3). In Y7F, the allele A frequency was 0.98 and 0.89 in JB1 and JB2, respectively. In R25C, the allele C frequencies were 0.84 and 0.70 in JB1 and JB2, respectively. In A80V, the allele C frequency was 0.87 and 0.96 in JB1 and JB2, respectively. Y7F in JB1 and A80V in JB2 were excluded from statistical analysis because the minor allele frequencies (MAFs) of these SNPs were <0.1 in each group. Homozygous cattle for minor alleles of Y7F in JB2, R25C in JB1 and A80V in JB1 were also excluded because of the small sample sizes.
| Population | JB1 | JB2 | ||
|---|---|---|---|---|
| Y7F | Genotype frequency (n) | AA | 0.97 (118) | 0.79 (356) |
| AT | 0.03 (4) | 0.19 (86) | ||
| TT | 0.00 (0) | 0.02 (7) | ||
| Allele frequency | A | 0.98 | 0.89 | |
| T | 0.02 | 0.11 | ||
| R25C | Genotype frequency (n) | CC | 0.70 (387) | 0.48 (212) |
| CT | 0.28 (158) | 0.44 (197) | ||
| TT | 0.02 (11) | 0.08 (37) | ||
| Allele frequency | C | 0.84 | 0.70 | |
| T | 0.16 | 0.30 | ||
| A80V | Genotype frequency (n) | CC | 0.76 (424) | 0.96 (172) |
| CT | 0.23 (129) | 0.04 (8) | ||
| TT | 0.01 (7) | 0.00 (0) | ||
| Allele frequency | C | 0.87 | 0.96 | |
| T | 0.13 | 0.04 | ||
- JB1: the commercial cattle population produced in Miyazaki Prefecture.
- JB2: the cattle population fattened across Japan for field progeny testing from 2002 to 2011.
Effect of leptin gene polymorphisms on traits
We investigated the effects of three polymorphisms in the leptin gene on carcass traits and fatty acid composition using ANOVA (Table 4). Y7F had a significant effect on the dressed carcass weight in JB2; R25C had a significant effect on the C18:0 and C14:1 contents in JB1 and JB2, respectively; and A80V had a significant effect on the C16:0, C16:1, C18:1, saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) contents in JB1.
| Y7F | R25C | A80V | ||
|---|---|---|---|---|
| JB2 | JB1 | JB2 | JB1 | |
| Carcass traits | ||||
| Dressed carcass weight (kg) | * | ns | ns | ns |
| Rib-eye area (cm2) | ns | ns | ns | ns |
| Rib thickness (cm) | ns | ns | ns | ns |
| Subcutaneous fat thickness (cm) | ns | ns | ns | ns |
| Yield estimate (%) | ns | ns | ns | ns |
| Beef marbling standard | ns | ns | ns | ns |
| Fatty acid composition (%) | ||||
| C14:0 | ns | ns | ns | ns |
| C14:1 | ns | ns | ** | ns |
| C16:0 | ns | ns | ns | ** |
| C16:1 | ns | ns | ns | * |
| C18:0 | ns | * | ns | ns |
| C18:1 | ns | ns | ns | ** |
| C18:2 | ns | ns | ns | ns |
| SFA | ns | ns | ns | ** |
| MUFA | ns | ns | ns | ** |
- * P < 0.05
- ** P < 0.01, ns non-significant
- SFA, saturated fatty acid; MUFA, monounsaturated fatty acid
- JB1: the commercial cattle population produced in Miyazaki Prefecture.
- JB2: the cattle population fattened across Japan for field progeny testing from 2002 to 2011.
Tukey–Kramer HSD test was conducted to investigate the effects of Y7F, R25C and A80V on traits in JB1 and JB2 in more detail. Table 5 presents the mean values for each trait and the significant differences among genotypes in JB1 and JB2. The AA type in Y7F exhibited a significantly higher dressed carcass weight than the AT type in JB2. R25C had a significant effect on the C14:1 content in JB2, with an additive effect among genotypes being observed; animals with the C allele exhibited a higher percentage of C14:1 than those with the T allele. In addition, the CC type in R25C contained a significantly lower percentage of C18:0 than the CT type in JB1. The CC type in A80V had a significantly greater effect on C16:0 and SFA but a smaller effect on C16:1, C18:1 and MUFA than the CT type.
| Population | SNP | Trait | Genotype | Mean | SD |
|---|---|---|---|---|---|
| JB1 | R25C | C18:0 (%) | CC | 19.27B ± 0.29 | |
| CT | 20.14A ± 0.34 | ||||
| A80V | C16:0 (%) | CC | 24.02A ± 0.25 | ||
| CT | 22.70B ± 0.36 | ||||
| C16:1 (%) | CC | 2.66B ± 0.06 | |||
| CT | 2.84A ± 0.09 | ||||
| C18:1 (%) | CC | 48.34B ± 0.39 | |||
| CT | 49.78A ± 0.57 | ||||
| SFA (%) | CC | 46.16A ± 0.40 | |||
| CT | 44.49B ± 0.59 | ||||
| MUFA (%) | CC | 51.87B ± 0.40 | |||
| CT | 53.48A ± 0.59 | ||||
| JB2 | Y7F | Dressed carcass weight (kg) | AA | 444.41A ± 2.70 | |
| AT | 431.17B ± 5.19 | ||||
| R25C | C14:1 (%) | CC | 0.92A ± 0.02 | ||
| CT | 0.86AB ± 0.02 | ||||
| TT | 0.80B ±0.05 | ||||
- NSP, single nucleotide polymorphism; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid
- A,BMeans with different superscripts within the same trait differ significantly at P < 0.05 (Tukey's HSD analysis).
- JB1: the commercial cattle population produced in Miyazaki Prefecture.
- JB2: the cattle population fattened across Japan for field progeny testing from 2002 to 2011.
Discussion
Previous studies have demonstrated an association between leptin gene polymorphisms and economically important traits in cattle. However, this is the first study to investigate leptin gene polymorphisms in Japanese Black cattle.
We found three non-synonymous SNPs in the leptin gene (Y7F, R25C and A80V). These SNPs have previously been found in other breeds, with MAFs of 0.02 and 0.025 in Simmental (Orrù et al. 2011) and Holstein Friesian (Clempson et al. 2011) cattle, respectively, in Y7F; MAFs of 0.42, 0.34, 0.45 and 0.32 in Angus, Charolais, Hereford and Simmental cattle, respectively, in R25C (Buchanan et al. 2002); and a MAF of 0.33 in Jersey cow in A80V (Komisarek & Antkowiak 2007). The Japanese Black cattle investigated in the present study exhibited lower MAFs than these breeds, ranging from 0.02 to 0.30. This is possibly due to the strong selection for beef quality in Japanese Black cattle. In addition, Gutiérrez-Gil et al. (2010) reported a quantitative trail locus (QTL) that influenced fatty acid composition at 93.5–98.6 Mbp on BTA4 near the leptin gene. Therefore, given the low MAF and the detection of this QTL, the region around the leptin gene may be a possible candidate for improving beef quality.
Y7F was predicted to cause the substitution tyrosine with phenylalanine in leptin. However, this position is not so important in mature leptin because a signal peptide (1st to 21st amino acids) is cleaved off before leptin is excreted from the adipose tissue (Zhang et al. 1994; Buchanan et al. 2002). In the present study, we found an association between Y7F and dressed carcass weight in JB2, with the A allele (Y type) being detected as economically favorable because it increased dressed carcass weight. However, this association was not strong (0.01 < P < 0.05) because Y7F is not included in mature leptin and may be due to a linkage disequilibrium. In addition, the genotyping results of JB1 and JB2 suggested that the A allele frequency is very high in Japanese Black cattle as well as in Simmental and Holstein Friesian cattle (Clempson et al. 2011; Orrù et al. 2011). Therefore, it would be difficult for Y7F to be used as a selective DNA marker in these breeds.
R25C was predicted to cause the substitution arginine with cysteine in leptin. This substitution may affect the tertiary structure of leptin because it adds an unpaired cysteine (Liefers et al. 2003). In the present study, R25C was associated with the C18:0 and C14:1 contents in JB1 and JB2, respectively; and a previous study reported that R25C was associated with the C14:0, C16:0, C17:1 and C18:0 contents in Chinese Simmental cattle (Tian et al. 2013). This SNP has also been associated with many other traits, such as dressed carcass weight, rib eye area, loin thickness, milk yield, milk fat and protein content, somatic cell count linear scores, calving difficulty and length of gestation (Buchanan et al. 2003; Chebel et al. 2008; Giblin et al. 2010; Tian et al. 2013). Buchanan et al. (2002) demonstrated that the T allele in R25C was associated with fatter carcasses, whereas the C allele was associated with leaner carcasses in Angus, Charolais, Hereford and Simmental cattle, suggesting that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and thus could be the causative mutation for carcass fat content. Therefore, considering the effect that R25C has on the tertiary structure of leptin and its association with many carcass traits, it is possible that this polymorphism would directly affect fatty acid composition and thus could be used as an effective marker in Japanese Black cattle.
A80V was predicted to cause the substitution alanine with valine in leptin. In the present study, this SNP was significantly associated with the C16:0, C16:1, C18:1, SFA and MUFA contents in JB1. An association between A80V and fatty acid composition has also been reported in Chinese Simmental cattle (Orrù et al. 2011). In addition, Reicher et al. (2012) reported that the A80V mutation affects leptin functioning by decreasing its affinity toward the leptin receptor. These findings suggest that A80V is a prospective DNA marker for fatty acid composition in Japanese Black cattle. Furthermore, although the presence of the T allele in A80V had a positive effect on fatty acid composition, its frequency was low in our study populations (0.13 and 0.04 in JB1 and JB2, respectively), which matches the previous finding of Orrù et al. (2011), for Chinese Simmental cattle (0.30). Therefore, A80V may have a large effect on the improvement of fatty acid composition in Japanese Black cattle.
In conclusion, we found that three of the eight SNPs (Y7F, R25C and A80V) in the coding region of the leptin gene in Japanese Black cattle were non-synonymous and associated with carcass traits or fatty acid composition, and two of these (R25C and A80V) could be used as effective DNA markers for improving fatty acid composition in this breed.
Acknowledgments
This work was partially supported by JSPS KAKENHI Grant Number 16H05015. We also thank Wagyu Registry Association for sampling and their data collection.
