The PIT1 gene polymorphisms were associated with chicken growth traits

Background With crucial roles on the differentiation of anterior pituitary and the regulation of the prolactin (PRL), growth hormone (GH) and thyroid-stimulating hormone-β (TSH-β) genes, the chicken PIT1 gene is regarded as a key candidate gene for production traits. In this study, five reported polymorphisms (MR1-MR5) of the PIT1 gene were genotyped in a full sib F2 resource population to evaluate their effects on growth, carcass and fatty traits in chickens. Results Marker-trait association analyses showed that, MR1 was significantly associated with shank diameters (SD) at 84 days (P < 0.05), hatch weight (HW) and shank length (SL) at 84 days (P < 0.01), MR2 was significantly associated with BW at 28, 42 days and average daily gain (ADG) at 0–4 weeks (P < 0.05), and MR3 was significantly associated with ADG at 4–8 weeks (P < 0.05). MR4 was associated with SL at 63, 77, 84 days and BW at 84 days (P < 0.05), as well as SD at 77 days (P < 0.01). Significant association was also found of MR5 with BW at 21, 35 days and SD at 63 days (P < 0.05), BW at 28 days and ADG at 0–4 weeks (P < 0.01). Both T allele of MR4 and C allele of MR5 were advantageous for chicken growth. The PIT1 haplotypes were significantly associated with HW (P = 0.0252), BW at 28 days (P = 0.0390) and SD at 56 days (P = 0.0400). No significant association of single SNP and haplotypes with chicken carcass and fatty traits was found (P > 0.05). Conclusion Our study found that polymorphisms of PIT1 gene and their haplotypes were associated with chicken growth traits and not with carcass and fatty traits.

Until now, PIT1 cDNA has been identified in a variety of species, and previous studies showed that the PIT1 gene comprised 6 exons in mammals and 7 exons in birds and fishes, seen as differences in precursor length [12][13][14]. The chicken PIT1 cDNA has firstly been isolated and sequenced by Tanaka et al. (1999) [15], and its three isoforms of PIT1*, PIT1β* and PIT1ω* induced by alternative splicing have also been isolated and found to comprise 335, 363, and 327 amino acids, respectively [16]. The alternative splicing of PIT1 gene has also been reported in other species [17,18]. According to the chicken genome sequences released in May of 2006 [19], the chicken PIT1 gene is located at chromosome 1 (GGA1) and spans over 14 kb in length.
Due to its crucial regulatory function and a variety of bioactivities, PIT1 has been regarded as a key candidate gene for production performance. There are indications that variations of PIT1 gene are related to growth, carcass and fatty traits in pig [20][21][22][23][24], growth and carcass traits in cattle [25,26]. In chickens, although a total of 23 single nucleotide polymorphism (SNP) and a 57 bp indel have been lately identified in 2400 bp discrete region of PIT1 gene, their genetic effects on chicken production traits remain unclear [27]. Recently, it has been shown that a non-synonymous SNP at POU domain (A → T, Asn229Ile) is significantly associated with body weight at 8 wk [28].
In this study, five reported polymorphisms of the chicken PIT1 gene were genotyped in a full sib F 2 resource population to evaluate their genetic association with chicken growth, carcass and fatty traits were also observed.

Chicken populations
A full sib F 2 resource population as described by Lei et al. (2005) was used in this study [29]. The cross of 9 White Recessive Rock (WRR) males and 9 Chinese Xinghua (X) females and the reciprocal cross of 6 WRR females and 6 X males produced 17 F 1 families and 454 F 2 full-sib individuals. F 2 chickens were raised in floor pens and fed with commercial corn-soybean-based diets that met all NRC requirements. All birds from three generations were genotyped to evaluate the effects of PIT1 variations on chicken production performance.  [29].

Markers and primers
Five reported polymorphisms of the chicken PIT1 gene that could be easily genotyped by either PCR-RFLP or simple PCR were selected as markers to evaluate their effects on chicken production traits. These polymorphisms were a 57 bp indel (MR1) [27] and 4 SNP (MR2-MR5). Four primer pairs of PR1-PR4 were designed and synthesized to amplify specific fragments covering MR1-MR5 (Table 1). The detailed information for these polymorphisms is presented in Table 1.

Marker-trait association analyses
Marker-trait association analyses were performed with SAS GLM procedure (SAS Institute, 1996) and the genetic effects were analyzed using the following mixed model: where Y is a trait observation, µ is the overall population mean, G is the fixed effect of genotype, D is the random effect of dam, H is the fixed effect of hatch, S is the fixed effect of sex (male or female), and e is the residual random error. With the above model, association of each of MR1-MR5 and their haplotypes with the 57 production traits was performed to evaluate its genetic effect on chicken growth, body composition and fat deposition.

Association of single SNP with chicken production traits
Results showed that MR1 was significantly associated with SD at 84 days (P < 0.05) and highly significantly associated with HW and SL at 84 days (P < 0.01), and MR2 was significantly associated with BW at 28, 42 days and ADG at 0-4 weeks (P < 0.05). MR3 was significantly associated with ADG at 4-8 weeks (P < 0.05). Moreover, MR4 was significantly associated with SL at 63, 77, 84 days and BW at 84 days (P < 0.05) and highly significantly associated with SD at 77 days (P < 0.01), and T rather than C was advantageous for chicken growth (Table 2). MR5 was significantly associated with BW at 21, 35 days, and SD at 63 days (P < 0.05) and highly significantly associated with BW at 28 days and ADG at 0-4 weeks (P < 0.01), and C allele was advantageous for chicken growth ( Table 3).
None of these polymorphisms was significantly associated with any of chicken carcass and fatty traits (P > 0.05).  lotypes were significantly associated with growth traits of HW (P = 0.0252), BW at 28 days (P = 0.0390) and SD at 56 days (P = 0.0400). Among ten diplotypes, H2H4 had much higher value of HW (mean = 30.2), BW at 28 days (mean = 352.6) and SD at 56 days (mean = 9.24) compared with other ones. Nevertheless, the PIT1 haplotypes were not significantly associated with any of chicken carcass and fatty traits (P > 0.05).

Association of PIT1 haplotypes with chicken production traits
It was concluded that polymorphisms of PIT1 gene were associated with chicken growth traits, but not with carcass and fatty traits.

Discussion and Conclusion
In this study, polymorphisms of the PIT1 gene were related to chicken growth traits. Until now, associations of the PIT1 gene with growth traits were reported in human [31], pig [19][20][21][22][23] and cattle [25,26]. In chicken, a non-synonymous SNP (Asn299Ile) in exon 6 of the PIT1 gene was significantly associated with body weight at 8 wk, and its allele frequencies differed significantly between meat-type and lay-type chickens [28]. Another SNP in exon 6 (MR5) was associated with ADG at 0-4 weeks, BW at 21, 28, 35 days and SD at 63 days as indicated by this study. Furthermore, three adjacent SNP in intron 5 (MR2-MR4) were associated with ADG at 0-4 and 4-8 weeks, BW at 28, 42 and 84 days, SL at 63, 77, and 84 days, as well as SD at 77 days. Until now, some QTL for body weight were identified in GGA1 [32][33][34][35], however, only one reported QTL covered the 96 Mb region (total chromosomal size of 201 Mb) where the chicken PIT1 gene located [36]. It seemed that these SNP may be in linkage disequilibrium with causative mutation(s), which situated in this region and played crucial roles on chicken growth.
It was interesting that the PIT1 gene polymorphisms were associated with none of chicken carcass and fatty traits. In previous studies, variations of the PIT1 gene were related to carcass and fatty traits in pig [20][21][22]24], carcass traits in cattle [25]. However, no association was found of five polymorphisms in the PIT1 gene with 26 carcass and fatty traits in this study. In addition, haplotype analysis also provided similar results. This was surprising because some carcass traits were correlated with growth traits to some extent, and therefore it still required further study for confirmation.
It was further indicated that polymorphisms of the PIT1 gene affected chicken growth at different stages. MR2, MR3 and MR5 seemed to have higher effects on chicken early growth, as they were associated with ADG at 0-4 and 4-8 weeks, BW at 21, 28, 35 and 42 days, and SD at 63 days, respectively. Otherwise, MR1 and MR4 seemed to have higher effects on chicken growth in middle stage, as they were associated with SL at 63, 77 and 84 days, SD at 77 and 84 days, and BW at 84 days. As far as different genotypes were compared, both T allele of MR4 and C allele of MR5 were advantageous for chicken growth.
It was concluded that polymorphisms of PIT1 gene and their haplotypes were associated with chicken growth traits.

Authors' contributions
QN analyzed the data and drafted the manuscript. MF, XL and MZ participated in the data analyses. Both of ZL, GW, WB, CL and WZ carried out the genotyping studies. QN and XZ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.