Quantitative PCR analysis showed that FATP4 mRNA was expressed in all eight MB chicken tissues. At 84 days, the mRNA levels of the chicken FATP4 gene were extremely high in the small intestine, followed by the leg muscle weight (LMW), brain, abdominal fat weight (AW), breast muscle weight (BMW), liver, subcutaneous fat (SFT) and heart (Table 1). The relative expression level of FATP4 in small intestine at 84 days was significantly higher (p < 0.01) than other all tissues and no significant expression differences among other tissues.

To clarify whether the FATP4 mRNA expression had an age-related expression pattern, we investigated the ontogenic expression of this gene in eight MB chicken tissues. As shown in Figure 1, FATP4 mRNA expression levels showed no difference in leg muscle and subcutaneous fat at various days of age (p > 0.05). The FATP4 mRNA in breast muscle, contrastingly, had the highest expression on 0 day and the lowest expression on 14 days (p < 0.01). In abdominal fat, the expression level of FATP4 mRNA on 84 days was significantly higher than the expression on 28 days, 42 days and 70 days by 79.3%, 85.8% and 86.9%, respectively. In liver tissue, the level of FATP4 mRNA showed no difference between 0 day and each of the three growth points on 28 days, 42 days and 84 days. However, the expression of FATP4 mRNA on 56 days was significantly higher than the expression level on 0 day by 91.7% (p < 0.01) (Figure 1). In heart tissue, the FATP4 mRNA levels presented a unimodal distribution pattern with increasing age peaked on 42 days, but there were no significant differences at various ages (p > 0.05). In brain tissue, the FATP4 mRNA expression exhibited a “rise-decline-rise-decline” developmental change, and its expression on 56 days was significantly higher than at other times (p < 0.01). Finally, in small intestine, FATP4 was expressed at 56 days with a low level then increased to a peak at 70 days and down at 84 days. In addition, although the expression level of FATP4 mRNA on 70 days was higher than the expression on 56 days and 84 days by 48.9% and 29.8%, respectively, there were no differences at various days of age (p > 0.05).

The direct sequencing of these fragments in representative samples showing different bands on SSCP gels (see Materials and Methods section) yielded a total of nine genetic variants. We named these polymorphisms relative to their respective positions on sequences of chicken genomic chromosome 17 (reverse strand; GenBank accession number NC_006104.2, GI: 118135643), and we deposited the SNPs in GenBank under dbSNP numbers ss345102396-ss345102405. The locations of the nine SNPs are as follows: one in intron 7 (g.5608272C>G, synonymous), one in intron 6 (g.5608695 G>A, synonymous), one in exon 9 (g.5607545T>C, synonymous), one in intron 10 (g.5606740C>T, synonymous), one in exon 13 (g.5606005A>G, synonymous), two in exon 14 (g.5605454C>T, synonymous; g.5605481G>A, synonymous), and the remaining two (which are also the only ones causing amino acid changes) in exon 6 (g.5608814G>A, p.A443T; g.5608778C>T, p.R455*).

We further estimated the likelihood that the two missense variants could cause a putatively functional impact on the FATP4 protein by using the cSNP analysis tool in PANTHER. The subPSEC score was −2.18124, and the p_deleterious value was 0.30603 for variant p.A443T. According to these results, the functional effect of variant p.A443T is unlikely to be harmful, but it may somewhat change normal gene functions. Variant p.R455* was found to be a nonsense mutation by analysis with PANTHER, so it likely alters normal gene function. Therefore, to confirm our hypothesis, we focused on these two variants with putatively functional roles in S01 and S05 chicken populations.

Table 2 presents the association results of g.5608778C>T (p.R455*) and g.5608814G>A (p.A443T) genotypes with chicken carcass traits in two different populations. With the exception of g.5608778C>T in S01 populations, the allele frequencies of both g.5608778C>T and g.5608814G>A were not deviated from Hardy-Weinberg equilibrium (HWE) in two populations. In the S01 population, the g.5608778C>T SNP was significantly associated with BMW and with LMW (p < 0.05). All other traits were not significantly associated with this SNP (p > 0.05). In the S05 population, g.5608778C>T was significantly associated with all carcass traits except for AW. Genotype CC was associated with lower values of BW (p = 0.0003), CW (p = 0.0004), SEW (p = 0.0015), EW (p = 0.0006), zBMW (p = 0.0156) and LMW (p = 0.0047). Similarly, genotypes of g.5608814G>A in exon 6 were also associated with some chicken carcass traits in different populations (Table 2). In the S01 population, chickens with the GG genotype had the highest BW (p = 0.0229), CW (p = 0.0147) and EW (p = 0.0272), while the other traits were not significantly associated with this SNP. In the S05 population, the g.5608814G>A SNP was significantly associated with BW, CW, SEW and EW (p < 0.05), whereas it was negatively associated (p > 0.05) with BMW, LMW and AW. In addition, we found that the two SNPs were significantly associated with BW, CW, EW and SEW when both S01 and S05 were included in the analysis (data not shown).

To further define the haplotype structures of the FATP4 gene, haplotype blocks were analyzed using the Haploview program. Two most common LD measures, D′ and, were used to determine the extent of LD in the FATP4 gene. The D′ values for combinations of these two SNPs were 0.45 and r² values were 0.12, which indicated that these two polymorphisms were linked.

Therefore, to discern the potential effect of haplotypes on carcass traits, the g.5608778C>T and g.5608814G>A haplotypes were analyzed in the S05 population. Four possible haplotypes were discerned: haplotype 1 = C-G with a frequency of 31.3%, haplotype 2 = C-A with a frequency of 24.6%, haplotype 3 = T-G with a frequency of 26.8%, and haplotype 4 = T-A with a frequency of 17.3%. In the S01 population, only haplotype 1 was well represented (data not shown) with the other haplotype frequencies equaling less than 10%; therefore, this population was not fully informative for a haplotype association analysis. Table 3 shows the estimated regression coefficient of the haplotype substitution effect for the carcass traits. For the S05 population, haplotype 3 (T-G) was the most favorable haplotype due to its association with higher BW, CW, EW, SEW, BMW and LMW (p < 0.05). On the contrary, animals with haplotype 2 (C-A) showed lower BW, CW, EW and SEW (p < 0.05). Haplotype 1 (C-G) showed lower BMW and LMW (p = 0.0421 and p = 0.0461, respectively), but had no correlation with other carcass traits. Haplotype 4 (T-A) did not correlate with any carcass traits (p > 0.05), possibly due to the small number of chickens with this haplotype.

The Mountainous Black-boned chicken (MB) is a well-known bird with spotty feathers, black or yellow skin and favorable meat quality natively bred in Sichuan Province, China. MB chickens were used in the expression study, and all chickens were raised on an experimental farm for poultry breeding at the Sichuan Agricultural University (Ya’an, China) under the same conditions. For the ontogeny studies, different tissues including heart, liver, brain, leg muscle, breast muscle, abdominal fat, subcutaneous fat and the small intestine from male chicken were collected at 0 day, 14 days, 28 days, 42 days, 56 days, 70 days and 84 days of age (five samples of each age). Fresh tissues were identified, excised, then immediately frozen in liquid nitrogen and stored at −80 °C until mRNA isolation took place. For quantification of the overall tissue expression profile of chicken FATP4 mRNA, five adult male chicken at growth point 84 days were used, and the above eight tissues were collected and frozen in the same way.

Two groups of chicken (S01 and S05) were selected for association analysis between DNA markers and carcass traits. S01 and S05 belong to Erlang Mountainous chickens. The first group was made up by 128 S01 chickens (65 females and 63 males) with yellow partridge plumage, blue shanks and white skin. These chickens had a favorable quality of meat and grew at a fast rate. The second group of chickens was made of 136 S05 chickens (69 females and 67 males) with big bodily forms and a high quantity of meat. Eggs of the two breeds were hatched in different incubators under the same conditions on 15 March 2008; the chickens were kept in single-breed pens and were slaughtered on 15 June 2008. During the growth period, all birds had free access to food and water ad libitum under the same temperature and lighting conditions. From birth to 3 weeks of age, chickens were fed, ad libitum, a starting diet containing 2.90 megacalories (Mcal) of metabolizable energy (ME)/kg of body weight and 20.5 g of crude protein (CP)/kg of body weight. Birds were fed a grower diet (3.00 Mcal of ME/kg and 18.5 g of CP/kg) from 4 to 6 weeks of age and were transferred to growing pens at 7 weeks. Before the chickens were slaughtered, a blood sample was collected and stored at −20 °C for each one.

The two groups of chicken used for the association study were measured for performance-tested traits at the Sichuan Dahen Poultry Breeding Company. At the age of 90 days, live weight (BW) was measured on chickens restricted from feed for 12 h. After slaughter on that same day, the carcass traits were measured, including carcass weight (CW), eviscerated weight (EW), semi-eviscerated weight (SEW), breast muscle weight (BMW), leg muscle weight (LMW) and abdominal fat weight (AW). CW was measured of chilled carcasses removed of feathers. SEW was measured of carcasses removed of the trachea, esophagus, gastrointestinal tract, spleen, pancreas, and gonad. EW was measured of the semi-eviscerated carcasses after additional removal of the head, claws, heart, liver, gizzard, glandular stomach and abdominal fat.

Total RNA was extracted from chicken tissues using the TRIzol reagent (Invitrogen) and was treated with RNase-free DNase I using the RNase-free DNase Set (TakaRa Biotechnology Co. Ltd., Dalian, China). For each sample, about 1 μg of total RNA was reverse transcribed using the ImProm-II Reverse Transcription System (TakaRa Biotechnology Co. Ltd., Dalian, China). The reaction was performed in a volume of 10 μL containing 5× PrimerScript Buffer, 10 mM of each dNTPs, 40 U/μL RNase Inhibitor and 2.5 μM oligo-dT Primer. The reverse transcription was maintained at 30 °C for 10 min, and then at 45 °C for 25 min. The cDNA product was stored at −20 °C.

The FATP4 mRNA expression levels in different tissues collected at different developmental stages of male MB chicken were quantified by the SYBR Green I assay on an IQ5 real-time PCR thermal cycle instrument (Bio-Rad, USA). Chicken mRNA sequences of the β-actin gene (GenBank accession number NM_205518) and SLC27A4 (accession number XM_415504) were retrieved from GenBank. Primers were designed using Oligo 6.0 (Table 1). Reactions were performed in triplicate in a volume of 12.5 μL containing 10 mM Tris-HCl (pH 8.3), 50 mM KCL, 3.5 mM MgCL₂, 50 U/mL Takara ExTaq™ R-PCR (Takara, Biotechnology Co. Ltd., Dalian, China), 0.2 μM of each specific primer and 0.25 μL/300 diluted SYBR Green I (Takara, Biotechnology Co. Ltd., Dalian, China). The cycling condition consisted of an initial denaturation cycle for 5 min at 95 °C, then 35 cycles of 30 s at 95 °C, 40 s at 61 °C, and 45 s at 72 °C, ending with 8 min at 72 °C. Gene expression levels were quantified relatively to the expression of β-actin using the comparative 2^−ΔΔCT method [33]. Then, data of the mRNA expressions were subjected to ANOVA and/or t-test to determine the difference between tissues/growth points using SAS software 6.12 (SAS Institute Inc.). The p-value for significance was set at p ≤ 0.01.

Genomic DNA was extracted from whole blood using the standard phenol/chloroform method. According to the UCSC Genome Browser information for the chicken genome, May 2006 Assembly [34] that was retrieved by searching for the Gallus gallus FATP4 sequence (GenBank accession number XM_415504), the chicken FATP4 gene contains 15 exons. Fifteen pairs of primers were designed to amplify these exons (and partially cover some introns) (Table 4). Primer synthesis was completed by Shanghai Yingjun Biotechnology Co. Ltd. (Shanghai, China). PCR reactions were performed using the Gene Amp PCR System 9700 (Bio-Rad, USA) thermal cycler in a final volume of 10 μL reaction containing 10–100 ng genomic DNA, 10 pmol of each primer and 5 μL 2× Master mix (including Mg²⁺, dNTPs, Taq DNA polymerase; Beijing TIAN WEI Biology Technique Corporation, Beijing, China). The amplification condition included an initial step of denaturation for 5 min at 94 °C; 35 cycles of 45 s at 94 °C, 35 s at 55 °C (or other apt annealing temperature as shown in Table 1), and 45 s at 72 °C; then ended with a final extension step for 7 min at 72 °C. Genetic variants in the FATP4 genomic sequence were analyzed by using the PCR-SSCP (single strand conformation polymorphism) method. Briefly, after a denaturation at 99 °C for 10 min, 3 μL of PCR product was rapidly cooled on ice and then loaded on 12% acrylamide/bisacrylamide (39:1) gels. Electrophoresis was performed at 120–150 V for 13–15 h in 1× TBE buffer, and gels were silver-stained. Three DNA samples showing different patterns on SSCP gel were further amplified and purified, and then sequenced by Shanghai Yingjun Biology Technique Corporation (Shanghai, China).

We performed in silico functional predictions for FATP4 gene nonsynonymous variations using PANTHER (Protein analysis through evolutionary relationships) [35], which estimates the likelihood of a particular amino acid substitution to cause a functional impact on a protein. It calculates the substitution position-specific evolutionary conservation (subPSEC) score and the probability of a deleterious effect on protein function caused by a given variant (p_deleterious). PANTHER subPSEC scores are continuous values from 0 (neutral) to about −10 (most likely to be deleterious), and a subPSEC score of −3 corresponds to a p_deleterious of 0.5 [36].

Association analyses were carried out independently for the S01 and S05 chickens. The general linear model (GLM) procedure of SAS 6.12 (Statistical Analysis Systems Institute Inc., Cary, NC) was used to test associations between the genotyped markers and carcass traits. The model is as follows:

where Y_ijk is the trait measured in animal k, S_i is the effect of the ith sex category, G_j is the effect of the genotype j for SNP in the FATP4 gene, Sire_k is the effect of the sire k and e_ijk is the residual random effect associated with animal k.

In addition, the identified SNPs (g.5608778C>T and g.5608814G>A) in this gene were tested for Hardy-Weinberg equilibrium and the linkage disequilibrium (LD) between SNPs was tested by the Haploview [37]. Two parameters, including D′ and r², were used to characterize LD patterns within the FATP4 gene. The value of D′ > 0.33 and r² > 0.1 were applied as the criterion for meaningful LD [36]. Haplotypes between the g.5608778C>T and g.5608814G>A were constructed using the PHASE program v. 2.0 [38]. Haplotypes were analyzed using the model applied for the single marker association test with consideration for animals having 0, 1, or 2 copies of the haplotype in question. The PROC REG procedure of SAS (version 8.02, SAS Institute Inc.) was used to perform the analysis. Significant associations were declared when p < 0.05.