Mol Biol Rep DOI 10.1007/s11033-014-3391-3

Analysis of sequence variability in the pig CART gene and association of polymorphism with fatness traits in a F2 population Xiaoping Zhu • Delin Mo • Chong Wang Xiaohong Liu • Jiaqi Li • Fei Ling • Yaosheng Chen



Received: 1 April 2013 / Accepted: 2 May 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract CART (cocaine- and amphetamine-regulated transcript) peptides are neuromodulators that are involved in appetite control and energy homeostasis. It can inhibit food intake and reduce body weight that have received much attention, but a direct and comprehensive relationship with pigs differing in fatness which could be applied to breeding well has not been established. This study aims to search for polymorphism within the porcine CART gene and evaluate the effect of specific genotypes with regards to an association with fatness traits in a F2 population consisting of 230 individuals. Screening of 2264 bp DNA fragment covering the entire CART gene revealed 29 mutations and four indels (insertion or a deletion), in which four unlinked SNPs (single-nucleotide polymorphisms) could be digested by enzymes and subsequently genotyped in two purebreds and a F2 population. Landrace (lean-type), one of purebreds, presented significantly higher CART expression level than Lantang (obese-type) in most tissues studied. Association analysis revealed that three SNPs (T415C, C640T and C847T) displayed significantly association (p \ 0.05) with fatness traits. Additionally, they are in almost complete linkage disequilibrium. Western Xiaoping Zhu and Delin Mo have contributed equally to this work. X. Zhu  C. Wang  J. Li  F. Ling College of Animal Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China X. Zhu Foshan University, Foshan 528000, People’s Republic of China D. Mo  X. Liu  Y. Chen (&) State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China e-mail: [email protected]

blotting experiments on these three SNPs loci revealed difference in CART expression among individuals with different genotypes, and the individuals with lower average live backfat thickness (BFAW) expressed CART protein at a bit higher level than others. Our study screened and mapped the genetic variations in the porcine CART gene, and confirmed three functional SNPs which are promising molecular markers for pig production traits. Keywords CART gene  Gene expression  SNPs  Association analysis  Fatness trait

Introduction Fatness traits are important factors that influence porcine carcass quality such as backfat thickness, leaf fat weight, lean mass ratio and intramuscular fat content (IMF). And they are affected by a number of genes owing to their function and structure variation [1]. Functions as one of candidate genes, cocaine- and amphetamine-regulated transcript (CART) gene codes for a neuropeptide system with a number of biological roles such as inhibiting food intake and increasing lipid substrate utilization [2, 3], and it is also involved in atherogenesis [4], modulation of the hypothalamo-pituitary-adrenal (HPA) axis and cardiovascular regulation [5]. After injecting CART recombinant protein into mice brain, appetite was inhibited and content of body adipose was reduced, which proved that CART was an endogenous inhibitor of food intake and can reduce fat deposition [2]. However, CART mRNA expression showed no difference between diet-induced obese (DIO) mice after obesity reversal and DIO mice without obesity reversal [6]. CART peptides are abundant but discretely distributed in central and peripheral nervous system, such as

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amygdale, thalamic nuclei, arcuate nucleus, paraventricular nucleus, hypophysis and adrenal gland [3, 7]. And CART mRNA expression was regulated by leptin, because it decreased the arcuate nucleus by disrupting leptin signaling whereas increased by peripheral administration of leptin [2]. And the leptin-CART pathway originating in the arcuate nucleus of a high fat diet could facilitate the regulation of lipid metabolism in order to control body fat [8].Although earlier studies have failed to find an association between 30 -untranslated region polymorphisms of CART gene and obesity or weight gain [9–11], there are still more and more research about association between genetic polymorphisms and obesity or obesity-related phenotypes as considering his important physiological role in food intake and fat deposition. Several researches focus the promoter sequence polymorphisms of CART gene, and have found some possible associations with obesity in Caucasian population or Japanese population [12, 13]. Recently, a significant association of CART with meat content in carcass, abdominal fat weight, and backfat thickness was found by analyzing microsatellite polymorphism located in intron 2 in a Large White population and a Landrace population [1]. These previous literature all have suggested that restricting polymorphism regions or detection methods analysis just not adequately describe all the common sequence variabilities or the functional mutations when all of the common variations are used to infer associations. Thus, in the present study, entire CART gene region that includes the three exons, as well as the two introns were screened by sequencing for SNPs using 17 pig breeds as the samples. And this study’s investigation of the genetic contribution of CART to fatness traits was performed in a Landrace 9 Lantang F2 population consisting of 230 individuals.

Table 1 Primers employed in these experiments for sequencing and quantitative analysis

Screening and mutations map of CART gene A total of 35 animals representing 17 pig breeds including Landrace, Duroc and 15 Chinese indigenous unrelated breeds were used to screen mutations in this study. The 15 Chinese indigenous pig breeds are Wuzhishan, Yushan Black, Lantang, Longlin, Putian, Luchuan, Dahuabai, Huaizhu, Tunchang, Bamaxiang, Dongshan, Guilin, Lingao, Wuyi Black and Erhualian. Based on the two porcine CART genomic sequences (GenBank accession number: EF581838, DQ235471), four primer pairs were designed to amplify DNA fragments of 474–1037 bp that span entire CART gene comprising three exons and two introns (Table 1). Template DNA was extracted from each individual and the DNA fragments were separately amplified by polymerase chain reaction (PCR). To control the quality of mutations identification, each PCR products were sequenced on both strands. Then the segmented data were automatically assembled using DNAstar software (http:// www.dnastar.com/). And the mutations were identified through manual checking and verification among the aligned sequences. Quantitative real-time PCR Fourteen tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, bladder, backfat, cerebrum, cerebellum, pituitary and hypothalamus) were collected from three individuals of breed, and used for CART expression level comparison between Landrace and Lantang pigs (150 days old, males). In all trials the pigs had ad libitum access to standard growing diet and water. These tissues were harvested, mixed, frozen in liquid nitrogen, and stored at -80 °C.

Name

Locus

Primer sequence

Tm (°C)

C-50

50 Flanking

F: 50 -GTCCCTGTTCTCCGCACTC-30

60

474

Intron 1

R: 50 -TCTGGGGAAAGGGGTAGG-30

Exon 1

F: 50 -CCGAGCCCTGGACATCTAC-30

60

495

62

1037

54

519

60

186

58

125

C-1 C-2 C-3 CART b-actin

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Materials and methods

0

0

Exon 2

R: 5 -ATCGGGATACGTTTACTCTTGA-3

Exon 2

F: 50 -GATTGAAGCGCTGCAGGAAGTC-30

Exon 3

R: 50 -CAGGAGGAAGGAATTGCAGGAG-30

Exon 3

F: 50 -TGCGAAAAGGAGCTAGAATC-30

Exon 3

R: 50 -TAGGGCAATTATGAAAAACACTT-30

Exon 1,2

F: 50 -TGGTAACCGAATGCTGATG-30

Exon 3

R: 50 -CGCATGTAATCTGGGATGA-30 F: 50 -CCACGAAACTACCTTCAACTC-30 R: 50 -TGATCTCCTTCTGCATCCTGT-30

Product length (bp)

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Total RNA was extracted from the tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and RNA quality was assessed with 1.2 % agarose gel electrophoresis and the ND-1000 Spectrophotometer (NanoDrop Technologies, USA), and the RNA was discarded if the 260/280 ratio was not between 1.8 and 2.1. Then, the RNA was reversetranscribed (RT) into cDNA using M-MLV reverse transcriptase (Promega Corp., Madison, WI). b-actin gene, expressed consistently in all samples, was used as an internal control for normalization. Negative control contained all components except templates. Relative quantification was performed using standard curves generated for b-actin and CART gene from a tenfold serial dilution of positive plasmid containing target fragment. In this assay, the efficiency of b-actin and CART gene primers (Table 1) were 1.969 and 1.984, respectively. The results were analyzed using the 2-DDCt method. The cycling conditions consisted of an initial single cycle of 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s, then a single cycle of 95 °C for 10 s, 65 °C for 15 s. The reaction was performed with SYBR Premix Ex Taq (TaKaRa, Biotechnology Co. Ltd) using LightCycler 480 System (Roche, Basel, Switzerland). PCR–RFLP analysis and genotype identification Four highly polymorphic mutations, which could be digested by restriction enzymes (Table 2), were chosen for further genotyping the above-mentioned two purebreds and a F2 population. The two pure pig breeds including Landrace (a typical lean-type pig breed, N = 31) and Lantang pig (one of Chinese obese-type pig breeds, N = 34) were used to analyze genetic variability of CART gene. The F2 population (N = 230), developed by self-pollinating the F1 hybrid between Landrace and Lantang, was used to perform association analysis with fatness traits. Each SNP was detected using PCR-restriction fragment length polymorphism (PCR–RFLP) method [14]. The PCR reaction contained 100 ng of genomic DNA, 0.4 lM of each primer, 10 ll 2 9 PCR Reaction Mix, and 0.2 U Taq polymerase Table 2 Details of four pairs of primers for genetic analysis

(Taq Mix Kit, Dongsheng Biotech Co., Ltd.) in a 20 ll reaction solution. The amplification conditions were 3 min at 95 °C, 32 cycles of 30 s at 94 °C, 30 s at 55–62 °C and 30 s at 72 °C, followed by a 5 min final extension at 72 °C. And the digested products were eletrophoresed on 1.5 % agarose gels for genotyping. Statistical analysis for linkage disequilibrium and association studies Fatness relevant traits including longissimus muscle area (LMA), IMF, leaf fat weight (LFW), lean meat percent (LMP), live backfat thickness between 6th and 7th ribs (BFA), live backfat thickness at last rib (BFB), rump fat depths measured at the intersection of the line from the high bone (third sacral vertebrae) with a line from the inside of the pin bone (BFC), average live backfat thickness (BFAW), carcass backfat thickness between 6th and 7th ribs (CBFA), carcass backfat thickness at last rib (CBFB), carcass rump fat depths (CBFC), mean of carcass backfat thickness (CBFM) were detected. The square of the correlation coefficient between two loci (r2) was used as an Linkage Disequilibrium (LD) measure and was carried out according to the procedure [15]. The associations between genotypes of SNPs and carcass traits were carried out using least-squares method (GLM procedure, SAS version 8.2, SAS Institute, Inc.), and the detail linear regression model list as following: yijkl ¼ l þ si þ bj þ gk þ ri CovW þ eijkl where yijkl is phenotypic value of the target trait, l is the least-square means, si is the i-th sex (i = 1 for male or 2 for female), bj is the effect of j-th bach (j = 1–4), gk is the effect of k-th genotype (k = AA, AB and BB), ri is the regression coefficient of covariate, CovW is Covariate of live weight before slaughter, eijkl is random error. Western blotting Total protein from the hypothalamus was lysed in buffer (40 mM Tris buffer (pH 8.0), 150 mM NaCl, 1.0 %Triton

Mutant site

Locus

Primers

Tm (°C)

Restriction enzyme

Digest (°C)

T415C

intron 1

F: 50 -CCGAGCCCTGGACATCTAC-30

60

HaeIII

37

R: 50 -TTCGGGATACGTTTACTCTTGA-30 C640T

exon 2

F: 50 -GATTGAAGCGCTGCAGGAAGTC-30 R: 50 -CAGGAGGAAGGAATTGCAGGAG-30

62

TaqI

65

C847T

intron 2

F: 50 -GATTGAAGCGCTGCAGGAAGT-30

59

Hin6I

37

55

HinfI

37

0

R: 5 -CCGGAAGTCAGGAAGAAAATTG-3 T1531C

exon 3

F: 50 -GGATCAGTCACGTGTGG-30

0

R: 50 -TGGAGTTCTGGGAACAAG-30

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X-100, 1 mM PMSF, 1 mM EDTA, 5 mM NaF, and 10 mM Na3VO4). 30 lg protein separated on a 12 % SDSPAGE gel, and electronically transferred to 0.45 lm PVDF membrane (Roche) using a semi-dry transblot apparatus (Bio-Rad Laboratories, Inc). The membrane was blocked for 2 h in Tris Buffered Saline with Tween (TBST) containing 5 % (w/v) skimmed milk, and incubated with primary antibody for 1 h. The primary antibody for CART was Anti-human CART Antibody (diluted 1:500, AF163, R&D Systems, Inc.). The primary antibody for GAPDH (diluted 1:1000, sc-59540, Santa Cruz Biotechnology) was mouse anti-GAPDH. The relative densities of the protein bands were visualized using a Kodak Imaging Station 4000 MM PRO (Carestream Molecular Imaging).

equivalent of short tandem repeat (STR) polymorphism (CA)2(CG)n(CA)n reported by Stachowiak et al. [1], was also discovered. However, not consistent with the report completely, the STR locus has 5 alleles: (CA)2(CG)1(CA)9, (CA)2(CG)3(CA)11, (CA)2(CG)1(CA)8, (CA)2(CG)3(CA)10, and (CA)12 (Fig. 1). And some alleles of this STR locus showed linkage relationship with adjacent mutations and formed several kinks of haplotype. Besides 1067STR, there are three indels including 217id (GCCTCCCTACCCC[-), G1257- (G[-), C1626-(C[-), and that were found located in intron 1, intron 2 and 30 UTR respectively. Interestingly, there is a variant CC778TT (CC[TT) which is a kind of rare genetic variation. Meanwhile, the rate of C?T transitions was the highest among these mutations, which is due to the fact that cytosine is subject to a high rate of hydrolytic deamination [16].

Results The polymorphisms of porcine CART gene

Comparison of differential expression between Landrace and Lantang pigs

To investigate variation within porcine CART gene, four amplicons covering a genetic region of 2264 bp were sequenced separately in a total of 35 subjects (5 lean and 30 obese individuals). We identified a total of 29 mutations and four Indels (insertion or a deletion) within the complete genomic sequence of the porcine CART gene (2264 bp) including the 50 untranslated region (UTR), the coding region, and complete 30 UTR (436 bp). As shown from the Fig. 1, all of exons and introns have polymorphisms except exon 1, in which two introns and 30 UTR shared the most mutations and all of indels. Notably, 1067STR, the

In order to investigate the association between SNPs and fatness traits in Landrace 9 Lantang F2 population, we firstly identified the differential expression of CART gene in the two purebreds. Then fourteen tissues were employed to measure the CART gene expression in lean-type pig breed (Landrace) and obese -type pig breed (Lantang pig). The quantitative real-time PCR results showed that CART mRNA expression of Landrace is significantly higher than that of Lantang pigs in all of tested tissues (p \ 0.05) (Fig. 2). It suggested that the F2 population originated from Landrace and Lantang could be used for association

Fig. 1 Mutations map of porcine CART gene. Mutations are reported assigning ?1 to the A of the ATG start codon. The mutations underlined are selected for association analysis in this study. The STR reported in the literature is given in bold, and its detail information is indicated in the box. Black boxes represent the untranslated regions (UTRs) of porcine CART gene while the pink boxes refer to the coding region. (Color figure online)

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Fig. 2 Differential expression profiles between two pig breeds. Porcine CART mRNA expression levels in 14 tissues were means and standard error, assessed by qPCR, normalized against b-actin housekeeping gene. Error bars represent the standard error (n = 3). Results were averaged from three independent assays. Differential expression was calculated between same tissues from different purebreds

without association with traits [19], and its T allele frequency in Landrace (0.39) was consistent with our result (0.37). Other three SNPs were identified and analyzed for the first time. It showed that every genotypes frequency of these four SNPs displayed differences between Landrace and Lantang pigs. Especially, the obese-type Lantang pigs were nearly homozygous at C640T and T1531C sites, whereas more heterozygotes appeared at these sites in leantype Landrace (Table 3). Besides that, in Lantang pigs, T allele appeared with a relatively higher frequency than Landrace at the T415C and T1531C sites, but was absent at C640T site (Table 3). These results indicated that the two purebreds exhibit significant difference not only in genotype frequencies, but also in allele frequency for CART gene. Compared with the purebreds, the F2 generation presents high in the homozygous genotype which was found weaker in the parental generation (Table 3, 4). Thus it could be used for association analysis between genotype and phenotype well. Linkage disequilibrium and association analysis between the SNPs and porcine fatness traits

analysis with fatness traits. In addition, this assay further confirmed that lean animals had higher expression in almost all tissues. And the significantly lower expression in Lantang pigs may contribute to the development of obesity [17], which is consistent with the fact that the fat deposition capacity of obese pig significantly higher than that of lean pigs [18]. Consistent with previous literatures, central nervous systems (CNS) exhibit higher expression level than others except cerebrum. Genotype frequency of selected SNPs in purebreds and F2 population A total of 33 substitutions were identified in our study. Considering about linkage disequilibrium among several loci, four SNPs were selected and subsequently genotyped the two purebreds and a F2 population. In the two purebreds, the four non-linkage SNPs included T415C, C640T, C847T and C1524T (the number between the two nucleotides represents the SNP location on the genomic sequence). In which, SNP T415C has been found in 1999 Table 3 Genotype and allele frequency of four SNPs in Landrace and Lantang pigs

Related to the production performance, live weight before slaughter was used as a covariant to adjust other traits in the least squares method. After multiple comparisons among different genotypes values, it revealed: (1) At T415C site, TT genotype was significantly associated with leaf fat weight, LMA, average living backfat, living backfat B, carcass backfat B and C, intramuscular fat, and lean meat rate (p \ 0.05); (2) At C640T site, the statistical significant difference were mainly presented between genotype CC and CT in leaf fat weight, living backfat A and B, average living backfat and average carcass backfat (p \ 0.05); (3) At C847T site, CC genotype was significantly associated with lean meat rate, average living backfat, living backfat B and C, carcass backfat B and C (Table 4); (4) However, no evidence of association or linkage between genotypes and fatness traits was detected on T1531C site (data not shown). The Linkage Disequilibrium measured between the three SNPs loci (T415C, C640T and C847T) exhibited

Landrace

Lantang pig

CC

CT

TT

C

T415C

0.16 (5)

0.42 (13)

0.42 (13)

C640T

0.58 (18)

0.43 (13)

0 (0)

C847T

0.35 (11)

0.42 (13)

0.23 (7)

T1531C

0.06 (2)

0.39 (12)

0.55 (17)

T

CC

CT

TT

0.37

0.63

0.03 (1)

0.44 (15)

0.53 (18)

0.79

0.21

1 (34)

0.56

0.44

0.53 (18)

0.26

0.74

0 (0)

0 (0) 0.29 (10) 0 (0)

0(0)

C

T

0.25

0.75

1

0

0.18 (6)

0.68

0.3

100 (34)

0

1

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Mol Biol Rep Table 4 SNPs genotypes value and their association with fatness traits in a F2 population

SNP T415C Genotype (mean ± SE) TT (83)

CT (124)

CC (23)

TT–CT

CC–TT

CT–CC

LFW

1.28 ± 0.06

1.42 ± 0.06

1.50 ± 0.10

0.045*

0.0368*

0.4215

IMF

2.44 ± 0.17

2.66 ± 0.17

3.17 ± 0.27

0.199

0.0166*

0.0609

LMA

29.02 ± 0.65

27.31 ± 0.56

27.63 ± 1.14

0.0166*

0.2612

0.7821

LMP

0.48 ± 0.01

0.46 ± 0.01

0.46 ± 0.01

0.0087**

0.0157*

0.9902

BFB

2.43 ± 0.08

2.61 ± 0.07

2.74 ± 0.12

0.0289*

0.0239*

0.31

BFAW

2.66 ± 0.08

2.79 ± 0.08

2.94 ± 0.12

0.0872

0.0369*

0.2541

CBFB

2.51 ± 0.10

2.78 ± 0.09

2.63 ± 0.18

0.0174*

0.5684

0.3909

CBFC

2.62 ± 0.11

2.87 ± 0.09

2.96 ± 0.19

0.0364*

0.0992

0.6317

Traits

SNP C640T genotype (mean ± SE)

CC–TT

CT–TT

CC (147)

The number of detected samples was 230 individuals in F2 population, and the numbers in parentheses indicate the number of special genotype in the F2 population * (p \ 0.05), ** (p \ 0.01)

P value

CT (73)

P value TT (10)

CC–CT

LFW

1.33 ± 0.05

1.48 ± 0.06

1.44 ± 0.15

0.0156*

0.446

0.7687

BFA

2.77 ± 0.08

2.97 ± 0.10

2.91 ± 0.24

0.0422*

0.5679

0.7748 0.9157

BFB

2.50 ± 0.07

2.67 ± 0.08

2.65 ± 0.19

0.0461*

0.4593

BFAW

2.72 ± 0.07

2.84 ± 0.09

3.11 ± 0.18

0.1077

0.0275*

0.1495

CBFM

3.76 ± 0.26

4.41 ± 0.12

4.28 ± 0.09

0.0178*

0.0484*

0.2988

Traits

SNP T847 C Genotype (mean ± SE)

P value

CC (71)

CT (122)

TT (37)

CC–CT

CC–TT

CT–TT

LMP BFB

0.48 ± 0.01 2.43 ± 0.08

0.46 ± 0.01 2.59 ± 0.07

0.45 ± 0.01 2.69 ± 0.10

0.0466* 0.0625

0.0104* 0.0313*

0.1799 0.364

BFC

2.47 ± 0.09

2.65 ± 0.08

2.75 ± 0.11

0.0683

0.0352*

0.3753

BFAW

2.61 ± 0.08

2.80 ± 0.08

2.95 ± 0.11

0.0222*

0.0055**

0.1757

CBFB

2.45 ± 0.11

2.80 ± 0.09

2.72 ± 0.14

0.0039**

0.0983

0.6062

CBFC

2.56 ± 0.11

2.91 ± 0.09

2.92 ± 0.15

0.0343*

0.0053**

0.9174

different levels. The r2 between T415C and other two SNP loci is 0.0096 (C640T), 0.0090 (C847T), respectively. They are in almost complete linkage disequilibrium. But the r2 between T415C and C847T is 0.1939, which shows a greater LD for its relative close distance. Expression level of animals with different genotypes We sought to elucidate this possible correlation by comparing CART protein levels with genotypes and phenotype. In this study, Western blot analysis was used to measure CART protein in hypothalamus of individuals from the F2 population. The animals employed in this assay have different genotypes in their genome sequence, and that their phenotypes (BFAW) are also different. As a result, CART protein level was higher in NO.1 which had TT genotype at T415C, CC genotype at other two loci. Conversely, it had the lowest BFAW. Whereas the remaining two individuals with CT genotype at T415C had reduced levels of CART protein, and both of them were richer in BFAW (Fig. 3). In which, the animal with CC at C640T and CT at C847T seems has higher CART protein than the third animal which has CT at C640T and TT at C847T.

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Fig. 3 Effect of different genotypes on expression level of CART. Proteins were extracted from hypothalamus of three individuals (no. 1, 2, 3) possessing different genotypes in the three SNP loci. The protein was separated on a 12 % SDS-PAGE gel and immunoblotted with anti-human CART antibody (diluted 1:500). GAPDH was used as an internal control. The live backfat thickness (BFAW) of the three individuals was also listed. Protein quantification was performed by densitometry

Discussion This is the first extensive study about the porcine CART gene that includes looking for mutations throughout the

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gene and an analysis in association with fatness traits. Indeed, CART gene have been well characterized in both human and mouse, and have been identified several sites relating to obesity within 50 upstream region [13, 20]. For the coding sequence, Dominguez et al. have confirmed that CART peptide levels were altered by a mutation associated with obesity at codon 34 [21]. However, there are some other studies about the sequence variability and association with phenotype in swine. In 1999, porcine CART gene was first characterized, and the Allele 2 (the equivalent of allele C in T415C) was observed with a frequency of 0.39 in Landrace, 0.23 in Meishan [19], which were similar to that of our results (Table 3), respectively. As different pig breeds have vastly different allele frequencies [19], it suggested that this SNP might be a functional QTN (quantitative trait nucleotide). In 2009, Stachowiak et al. identified a STR polymorphism (CA)2(CG)n(CA)n which is the equivalent of 1067STR in our study. And this STR was revealed significant allelic additive effects on meat content and backfat thickness. However, its genotyping procedure was complex and inaccurate. From the biologist’s point of view, the genotyping procedure should be as simple and has a low-cost as possible, in order to generate a large amount of genotyping data often necessary [22]. Considered the SNPs to be studied involve restriction enzyme site, PCR–RFLP can be a genotyping procedure that is easy to set up and a highly sensitive method to detect SNPs [22, 23]. Therefore, 4 SNPs were chosen to cover looking for mutations in different parts of the gene. Fortunately, there are three SNPs (T415C, C640T and C847T) exhibited significantly association with fatness traits including backfat thickness, leaf fat weight, lean meat ratio, intramuscular fat content and longissimus muscle area (p \ 0.05). It further confirmed that the chromosome region harboring the CART gene is a promising quantitative trait locus (QTL) for pig production traits [1]. However, it could be argued that SNPs identified in sequence studies will have little value for enhancing understanding of complex trait formation unless they are identified in relevant tissues of the individuals [24]. In human, the Leu34Phe missense mutation in CART has been found with severe early-onset obesity, and this mutation has a biochemical effect and alters CART peptide levels in mouse pituitary tumor cells [21]. In the 50 upstream region of human CART, it was also found that one SNP T3608C may possibly contribute to the genetic risk for obesity in the Caucasian population by modulates nuclear protein binding [25]. These SNPs have genetic effect and alter CART peptide levels, and they greatly enriched our understanding of how genotype leads to phenotype. Furthermore, trait-associated SNPs are more likely than other SNPs to be expression quantitative trait loci (eQTLs), and will often affect phenotype by altering

the amount or timing of protein production [24]. In order to determine whether the three SNPs affect fatness traits by altering protein amount or not, we measured the CART expression level of animals with different genotype on the three SNP sites. From the western blot results, it seems that the individuals possessing different genotypes in the three SNP loci expressed different protein amount. And the animal with TT at T415C, CC at other two SNPs sites seems to produce a highest CART protein production which might result in the thinnest backfat. The NO.3, possessing TT at C847T, and CT at other two SNP loci showed the opposite result (Fig. 3). It suggested that T allele of T415C and C alleles of C640T and C847T all contribute to CART protein production. Additionally, the TT genotype of C640T was absent in both purebred, which was likely due to a universal rule that deleterious allele is necessarily rare and thus mostly present in heterozygotes [26]. It provided further evidence that these three polymorphisms were potential functional SNPs. Predictably, the three SNPs associated with fatness traits could be used as genetic markers for improvement of carcass traits in pig breeding in the future. Our study presented a possible modest contribution of variation in the porcine CART gene to the genetic susceptibility to fatness traits, which enriched our understanding of how genotype leads to phenotype. And further analysis of these three SNPs assigned to alter CART expression levels seems to be reasonable. Acknowledgments This research was supported by the National Natural Science Foundation of China (31272417), the earmarked fund for Modern Agro-industry Technology Research System (CARS-36), and Science and Technology Planning Project of Guangzhou (2008Z1-E121).

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Analysis of sequence variability in the pig CART gene and association of polymorphism with fatness traits in a F2 population.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuromodulators that are involved in appetite control and energy homeostasis. It can...
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