Polymorphisms of Pit-1 gene and its association with growth traits in chicken T. K. Bhattacharya,1 R. N. Chatterjee, and M. Priyanka Project Directorate on Poultry, Rajendranagar, Hyderabad, 500030 India ABSTRACT The Pit-1 gene is involved in regulation of muscle growth through controlling the expression of growth hormone, prolactin, and transforming growth factor-β genes in chicken. The objectives of the study were to explore polymorphisms of the Pit-1 gene and to estimate the effect of these polymorphisms on growth traits in PB-1 and control (broiler strain) and IWI (layer strain) chickens. Single-stranded conformation polymorphism followed by sequencing was performed to reveal polymorphisms of the gene. In total, 10 haplotypes were found across the lines. The mRNA ex-
pression of Pit-1 varied among haplogroups and had a significant effect on BW and growth rates. The haplogroups showed a significant effect on BW in wk 7 in PB-1 chickens. In control chickens there was a significant effect at d 1 and in wk 2 and 7, and in IWI strains, there was a significant effect at d 1 and wk 6 and 7. The significant association of haplogroups and growth rate was found between 0 and 2 wk in control and between 0 and 2 and 6 and 7 wk in IWI strains. It was concluded that the Pit-1 gene is polymorphic and has a significant effect on growth traits in chickens.
Key words: expression, growth, Pit-1, polymorphism 2012 Poultry Science 91:1057–1064 http://dx.doi.org/10.3382/ps.2011-01990
INTRODUCTION Growth is regulated by several genes, of which pituitary specific transcription factor-1 (Pit-1) is the most important, as it acts as a transcription factor for growth hormone, prolactin, and transforming growth factor-β genes that play the most pivotal role in controlling growth in chickens (Bodner et al., 1988; Cohen et al., 1996; Miyai et al., 2005). The Pit-1 gene is also involved in the development of the anterior pituitary gland (Li et al., 1990), silencing adrenarche (Taha et al., 2005), and inducing differentiation of hepatic progenitor cells into prolactin-producing cells (Lee et al., 2005). This gene is auto-regulated during expression, and the presence of this protein was reported in lactotrophs, somatotrophs, and thyrotrophs (Simmons et al., 1990). The Pit-1 cDNA has been identified in a variety of species. Chicken cDNA sequence was explored by Tanaka et al. (1999). In the chicken, this gene is located on chromosome 1 (Chicken Genome Browser Gateway). Twenty-three single-nucleotide polymorphism (SNP) have been reported in the 2,400-bp discrete region of the Pit-1 gene in chickens, but their genetic effects on production and immune competence traits remains unclear (Nie et al., 2005). A nonsynonymous SNP (AAC > ATC) has been reported to be associated with BW at 8 wk of age in chicken (Jiang et al., 2004). Keeping ©2012 Poultry Science Association Inc. Received November 3, 2011. Accepted January 10, 2012. 1 Corresponding author:
[email protected]
these facts in view, the objectives of our study were designed to identify genetic polymorphisms of the Pit-1 gene, to explore the expression profile of the gene during the juvenile stage, and to estimate the association of the polymorphisms with growth traits in chicken.
MATERIALS AND METHODS Experimental Birds and Husbandry Practices The study was conducted in 2 broiler lines [422 birds of PB-1 and 214 birds of control broiler (CB)] and a layer line (462 birds of IWI) of chickens maintained under a similar environment at the Institute farm, Rajendranagar, Hyderabad, India. The PB-1 strain is a synthetic color broiler line selected initially for 6 wk BW and later on for 5 wk BW for the last 18 generations. The overall BW of PB-1 at 5 wk of age was 924.3 ± 0.12 g (Anonymous, 2009). This line is used as a male line for commercial broiler production. The CB line is a synthetic color broiler line that is a randombred pedigree over the last 9 generations. The BW of the CB line at 5 wk of age was 625.5 ± 0.13 g (Anonymous, 2010). The IWI line is a selected line for egg production and egg weight developed by selecting over 12 generations. The birds were kept in the brooder house until the age of 6 wk and then shifted to the grower house. All birds were reared on a deep-litter system in the same shed under intensive management of farming, providing the same management regimen with ad libitum feeding
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Table 1. Primer sequences used for amplification of fragments of the Pit-1 gene Primer
Direction
Sequence (5′–3′)
Fragment
PITE1 PITE1 PITE2 PITE2 PITE2A PITE2A PITE3 PITE3 PITE4 PITE4 PITE5 PITE5
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
AGA TTG CCA GAT GGT TAT GTC CTG CTG ACA CGT TTC CCG GAC TAC ACT ACT CTG TGC GCC TTA ACT TGA GAC CTG GCA TCA AGC CTG CAA CTC ACT TTG CAG TGT GCT GAC A AGC CTG ACC CCT TGC CT CCA GCT TAA TTC TCC GCA G GTT ATA TAC AAA CCA ATG TTG CTC CCA CTT GTT CTG CTT C CTT CAT ACA ATG AAA AAG TTG GCG TTA CCG GCA CTC GTG GTG CT
87 bp (partial promoter) + 114 bp (exon-1)
201
66 bp (exon-2) + 110 bp (intron 2)
160
Exon-2a
116
Exon-3
244
Exon-4
165
Exon-5
275
and watering up to 5 wk of age, and after that, feed restriction was followed. All birds were hatched at the same time and housed all along in the shed at the age of 16 wk. The PB-1 and CB chicks up to 3 wk of age were fed 2,800 kcal of ME, 21% CP, 1.1% calcium, 0.45% phosphorus, 1.1% lysine, 0.46% methionine, 1.11% arginine, 0.2% tryptophan, 0.7% threonine, 15,000 IU of vitamin A, 4,000 ICU of vitamin D3, 4 mg of vitamin K, 100 mg of manganese, 100 mg of zinc, 110 mg of copper, 2.5 mg of iodine, and 0.5 mg of selenium. The PB-1 and CB chicks from 3 to 6 wk, and IWI chicks up to 6 wk, were fed 2,600 kcal of ME, 16% CP, 1% calcium, 0.45% phosphorus, 0.8% lysine, 0.36% methionine, 0.78% arginine, 0.14% tryptophan, 0.51% threonine, 15,000 IU of vitamin A, 4,000 ICU of vitamin D3, 4 mg of vitamin K, 100 mg of manganese, 100 mg of zinc, 110 mg of copper, 2.5 mg of iodine, and 0.5 mg of selenium. During the brooding stage, proper heating was provided via bulb and the temperature was 32°Cfor wk 1 with a weekly gradual decrease of −15°Cfrom wk 1 to 5. A proper vaccination schedule, such as Marek’s disease vaccine at d 1, New Castle disease vaccine on d 7, infectious bursal disease vaccine on d 14 and 24, and New Castle disease vaccine on d 28, was applied to all birds. From 1 to 6 wk, 0.03 to 0.09 m2 of space for female chicks and 0.09 m2 of space for male chicks were provided in the deep-litter system. Cooling facilities during the summer season through water sprinkling on the roof and proper lighting were provided in the shed so that birds have a congenial environment for performing their optimum potential.
DNA and RNA Extraction Blood samples were collected from 422 birds of PB1, 214 birds of CB, and 462 birds of IWI chicken lines, and genomic DNA was isolated from blood cells following the standard protocol (Sambrook and Russell, 2001). Fifteen birds of each haplotype group of the Pit1 gene were slaughtered to collect brain tissues near the pituitary gland, following the slaughter protocol as approved by the ethical committee of Project Directorate on Poultry, Hyderabad, India. Total RNA was
Size (bp)
isolated from these tissues with Trizol reagent (Invitrogen, Carlsbad, CA).
PCR The primers used for amplification of all 6 exons of the Pit-1 gene were designed from the chicken Pit-1 sequence (GenBank accession no. AF029892) using DNASTAR software (Lasergene Inc., Madison, WI; Table 1). The PCR reaction was set up with 50 μg of DNA template, 10 ng of each primer, 1.5 mM MgCl2, 100 μM of each dNTP, 1× assay buffer, and 0.25 U of Taq DNA polymerase (MBI Fermentas, Hanover, MD). The PCR was performed with initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 59°C (for exons 1 and 2) or 58°C (for exon 3), and extension at 72°C for 30 s, with a final extension at 72°C for 10 min.
Single-Stranded Conformation Polymorphism A 12% native PAGE (50:1, acrylamide and bis-acrylamide) with 5% glycerol was prepared to resolve the single-stranded conformation polymorphism (SSCP) pattern, as suggested by Vohra et al. (2006). Furthermore, 3 μL of PCR product was mixed with 15 μL of formamide dye [95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 0.5 M ethylene diamine tetra-acetic acid] was denatured at 95°C for 5 min, followed by snap-cooling on ice for 15 min. Then, the product was loaded into the gel and electrophoresis was performed at 4°C for 12 h at 200 V, followed by staining with silver nitrate to visualize banding patterns.
Sequencing Three PCR products amplified from each genotypic group pertaining to each of 6 fragments of the Pit-1 gene, derived with HotStar HiFidelity DNA polymerase (MBI Fermentas) were sequenced with the fragmentspecific primers from both ends by the automated dyeterminator cycle sequencing method in an ABI PRIZM 377 DNA sequencer (Perkin-Elmer, Waltham, MA).
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POLYMORPHISMS OF Pit-1
Haplotypes Haplotypes were constructed by combining SSCP patterns of all the fragments of the Pit-1 gene of each individual. Haplotypes in the diploid state of an individual reveal the haplotype combinations of that animal. Haplotype sequences were analyzed with DNASTAR software. Frequencies of haplotype and its combinations were calculated by the gene counting method.
Quantitative PCR The quantitative (q)PCR was performed for the Pit-1 gene along with the GAPDH gene as an internal control with the cDNA templates using a thermal cycler Stratagene Mx3000P machine with platinum SYBR green qPCR UDG supermix (Invitrogen). A pair of primers, namely PIT1Q forward: 5′-TGAGCATGCCCTGAGTGC-3′ and PIT1Q reverse: 5′-CCTGCTTGAAGTCTGAAGC-3′, was designed from the chicken cDNA sequences of the Pit-1 gene (GenBank accession no. AF029892) with DNASTAR software for qPCR study. The 104-bp fragment of the gene was amplified at 57°C annealing temperature. A fragment of 119 bp of the GAPDH gene was amplified at 57°C using a pair of primers, namely, QGAPDH forward: 5′-CTGCCGTCCTCTCTGGC-3′ and QGAPDH reverse: 5′-GACAGTGCCCTTGAAGTGT-3′, designed from the chicken GAPDH sequence (accession no. AF047874) with DNASTAR software. Reactions were prepared in duplicate with a final volume of 25 μL containing 12.5 μL of platinum SYBR green qPCR supermix, 0.5 μL of ROX reference dye, 0.2 μM of each primer, and 2 μL of cDNA. The qPCR conditions were initial denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 1 min, and extension at 72°C for 30 s. Following amplification, a dissociation melting curve analysis was conducted with programming the PCR machine from 55 to 95°C to detect possible nonspecific products. Fluorescence threshold was determined by default method at 32.5% with Stratagene software (Stratagene, La Jolla, CA) for Mx3000P real-time PCR machine. The threshold cycle (Ct) values of each sample was noted, and average Ct values of each sample generated in duplicate qPCR reactions were used in calculating the fold change (Fold change = 2−ΔΔCt) of gene expression at different ages of birds.
Traits Body weights at 1 d old and wk 2, 4, 6, and 7 of age were measured in all birds. Growth rates of each bird were estimated between 0 and 2, 2 to 4, 4 to 6, and 6 to 7 wk.
Statistical Analysis The haplotype distribution was tested for HardyWeinberg equilibrium by chi-squared and likelihood ratio tests. The association of haplotype combinations
and traits were explored following least-square maximum likelihood method of the LSML90 package (Harvey, 1991), where haplotype and breed were used as fixed effects and sire as a random effect in the following model: Yijklm = μ + Si + Bj + HPLk + Bj × HPLk + eijkl, where μ is the overall mean, Si = ith sire effect, Bj = jth breed effect, HPLk = kth haplotype effect, Bj × HPLk is the interaction effect between jth line and kth haplotypes, and eijkl = random error with normal independent distribution (0, σ2e). The effect of Pit-1 expression on growth traits were also estimated by linear regression analysis.
RESULTS Polymorphism All of the exons of the Pit-1 gene, except exon 5, were found as polymorphic in 3 lines of chickens. In the PB-1 strain, 4 haplotypes were observed, of which h6 and h2 haplotypes showed the highest (0.32) and lowest (0.17) frequencies, respectively. The frequencies of h1 and h8 haplotypes in this line were 0.31 and 0.19, respectively. However, a total of 3 haplotype combinations (h1h2, h1h6, and h6h8) were found in this strain, where h6h8 was the most predominant and h1h6 was the least frequent haplogroup. A total of 9 haplotypes was observed in the CB strain, of which h1 had the highest (0.45) frequency. The lowest frequency (0.02) was found in h2, h3, and h7 haplotypes, while frequencies of h4, h5, h6, h8, and h9 were 0.07, 0.03, 0.32, 0.03, and 0.04, respectively. In this strain, 7 haplotype combinations were observed, of which the most predominant one was h1h6 (0.54). In the IWI line, 8 haplotypes were present, in which h1 and h2 had the highest (0.39) and lowest (0.02) frequencies, respectively, whereas frequencies of h4, h5, h6, h8, h9, and h10 were 0.13, 0.05, 0.23, 0.05, 0.06, and 0.06, respectively. In total, 7 haplotype combinations were observed in this line, where h1h6 combination had the highest (0.32) and h4h8 and h5h10 had the lowest (0.04) frequencies. The haplotype distribution was tested for Hardy-Weinberg equilibrium with both chi-squared and likelihood ratio tests. Both of the tests revealed that all lines followed the Hardy-Weinberg equilibrium. Furthermore, the sequences of haplotypes were submitted to the NCBI GenBank, and the accession numbers were obtained as JN613435, JN613436, JN613437, JN613438, JN613439, JN613440, JN613441, JN613442, JN613443, and JN613444 for h1, h2, h3, h4, h5, h6, h7, h8, h9, and h10 haplotypes, respectively.
Nucleotide Variabilities of Pit-1 Haplotypes Among Pit-1 haplotypes, nucleotide changes were found both in partial promoter (1–88 bp from the 5′
g
a
g
g
g
g
g
a
a
g
h1
h2
h3
h4
h5
h6
h7
h8
h9
h10
c
t
t
c
c
c
c
c
t
c
65
a
t
t
a
a
a
a
a
t
a
105 a K t I a K a K a K a K a K t I t I a K
143 t N a K t N t N t N t N t N a K a K t N
156 c P t S c P c P c P c P c P t S t S c P
160 a H t L a H a H a H a H a H t L t L a H
164 a H t L a H a H a H a H a H t L t L a H
170 g K c N g K g K g K g K g K c N c N g K
195
g
a
a
g
g
g
g
g
a
g
204 c H c H c H a N a N a N a N c H a N a N
459
t
c
t
t
t
c
c
t
c
c
668
c
g
c
c
c
g
g
c
g
g
669
g
a
g
g
g
a
a
g
a
a
670
Position in the gene2
c D c D a R c D c D a R a R a R c D a R
671
c
a
c
c
c
a
a
c
a
a
672
a
t
a
a
a
t
t
a
t
t
673 g M g M t H g M g M t H t H t H g M t H
674
g
a
g
g
g
a
a
g
a
a
676
t D t D a G t D t D a G a G a G t D a G
677
c S c S t F c S c S t F t F t F c S t F
679
c
t
c
c
c
t
t
c
t
t
683
g
a
a
g
a
g
a
g
a
a
825
t L t P c L t P c L t P c L t L t P c L
835
2Position
letters are nucleotides and uppercase letters in bold italics are amino acids. 1–87 in the haplotypes is partial promoter. Positions 88–205, 206–271, 482–597, 598–841, 842–1006, and 1007–1281 in the haplotypes are exon 1, exon 2, exon 2a, exon 3, exon 4, and exon 5, respectively. Position 272–381 in the haplotypes is partial intron 2.
1Lowercase
58
Haplotype
Table 2. Nucleotide and amino acid changes among haplotypes in chicken1
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POLYMORPHISMS OF Pit-1
Figure 1. Haplotype-wise expression profile of the Pit-1 gene in PB-1 (A), control broiler (B), and IWI (C) chicken lines. Different letters indicate significance at P < 0.05.
end) as well as in the coding region. In the partial promoter, nucleotide substitution was observed at the 58th and 65th positions of the fragment. In the coding region, nucleotide substitutions were found in 22 positions. The nucleotide changes in the 105th, 204th, 668th, 669th, 670th, 672nd, 673rd, 676th, 683rd, and 825th positions showed synonymous types of mutations, whereas the remaining 12 changes revealed nonsynonymous types of mutations (Table 2).
the CB strain, haplotypes had a significant effect (P < 0.05) on Pit-1 expression, where h1h2, h1h3, h1h4, and h1h6 haplogroups had nonsignificant differences of expression and h1h9, h6h7, and h6h8 haplogroups revealed relatively higher expression than other haplogroups. The highest and lowest expressions of the Pit-1 gene were found in h1h9 and h1h6 haplotype combinations, respectively. The h1h9 birds had 1,801-fold higher expression than that of the h1h6 group (Table 3). In the IWI strain, haplotype had a significant effect on Pit-1 expression, in which h5h10 and h1h4 haplogroups were the highest and lowest expression groups, respectively. The birds of h5h10 haplogroup showed 5,752-fold higher expression than that of the h1h4 haplogroup. The regression analysis revealed that Pit-1 expression showed a significant effect (P = 0.077 with R2 = 0.925) on BW at 7 wk of age in the PB-1 line, whereas other growth traits were not found to be significantly affected by the gene expression. In the CB line, Pit-1 expression
mRNA Expression The expression of Pit-1 was the highest in the forebrain of birds with h6h8 haplotype combination, and the lowest expression of Pit-1 was found in h1h2 haplogroup in the PB-1 strain (Figure 1). The expression pattern differed significantly (P < 0.05) among haplogroups. The h6h8 haplogroups had approximately 12fold higher expression than that of the h1h2 group. In
Table 3. Fold change of Pit-1 expression in different haplogroups of PB-1, CB, and IWI lines of chickens1 PB-1 h1h2 h1h6 h6h8 CB
h1h2 h1h3 h1h4 h1h5 h1h6 h1h9 h6h7 h6h8 IWI
h1h2 h1h4 h1h5 h1h6 h1h9 h4h8 h5h10 h6h8 h6h10 1CB
h1h2
h1h6
h6h8
—
5.24 —
h1h2
h1h3
11.79 2.25 — h1h4
—
3.1 —
0.7 0.2 —
h1h5
12.7 4.0 18.0 —
h1h6
0.2 0.07 0.3 0.01 —
h1h2
h1h4
h1h5
h1h6
h1h9
—
0.02 —
1.6 59.3 —
6.1 213.7 3.6 —
23.2 814.6 13.7 3.8 —
= control broiler.
h1h9
386.7 124.0 548 30.4 1,801 —
h6h7
h4h8
0.06 2.3 0.03 0.01 0.002 —
h6h8
25.1 8.0 35.6 1.9 116.9 0.07 —
28.0 9.0 39.8 2.2 130.6 0.07 1.1 — h6h8
h5h10
164.2 5,752.6 97.0 26.9 7.0 2,486 —
0.85 29.8 0.5 0.1 0.03 12.9 0.005 —
h6h10 1.9 68.1 1.1 0.3 0.08 29.4 0.01 2.2 —
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287 ± 36ab — 294 ± 21ab 261 ± 30a 313 ± 18b 281 ± 23a 386 ± 35c 258 ± 37a — 332 ± 29c 274 ± 21a a–cColumn-wise
1f
different superscripts indicate significance. = frequency; BW1, BW2, BW6, and BW7 are the BW (g) at 1 d old, and 2, 6, and 7 wk of age, respectively. CB = control broiler.
36.2 ± 1.0b — 33.7 ± 0.5a 34.8 ± 0.8ab 33.0 ± 0.4a 35.1 ± 0.7b 33.0 ± 1.2a 32.7 ± 1.2a — 35.9 ± 1.0b 36.1 ± 0.8b 0.05 — 0.21 0.09 0.32 0.11 0.04 0.04 — 0.05 0.09 ± 61bc ± 56ab ± 39c ± 51c ± 20b ± 43b — — 816 ± 61a 839 ± 52a — 961 882 992 1,013 925 928
± 5.4b ± 6.1a ± 4.1a ± 5.0a ± 2.1b ± 5.0a — — 101 ± 6.3a 93 ± 5.4a — 120 107 101 106 111 94
± 1.0b ± 1.1a ± 0.9a ± 1.4b ± 0.5b ± 1.1b — — 30.7 ± 1.0a 34.5 ± 1.2b — 0.05 0.04 0.13 0.06 0.54 0.08 — — 0.04 0.06 — 1,376 ± 38a — — — 1,532 ± 61b — — — — 1,541 ± 41b — 0.35 — — — 0.26 — — — — 0.39 — h1h2 h1h3 h1h4 h1h5 h1h6 h1h9 h4h8 h5h10 h6h7 h6h8 h6h10
f BW7 (P = 0.11) f Haplogroup
PB-1
Table 4. Haplotype-wise BW in chicken1
The Pit-1 gene acts as a transcription factor for expression of growth hormone, prolactin, and transforming growth factor-β gene in chickens. We determined a total of 10 haplotypes in different lines of chickens, and their frequencies were found to be varied among themselves in each line. In the PB-1 strain, 4 haplotypes were found, whereas a total of 9 and 8 haplotypes were found in the CB and IWI strains, respectively. In the PB-1 line, selection for BW at 5 wk of age was employed for the last 18 generations, which resulted in a reduction of variation for growth traits. It is known that Pit-1 is the prime factor for regulating growth in chickens through controlling expression of growth hormone, prolactin, and transforming growth factor-β genes, which are the most important genes involved in muscular growth during the posthatch period. Selection for growth traits reduced variability in growth traits indirectly indicates low variability in the Pit-1 gene that is expressed in terms of haplotypes. As a result, a fewer number of haplotypes was observed in the PB-1 line. On the other hand, in the CB and IWI lines, selection was not applied for growth traits, and hence, variability of growth traits was a bit high, with
BW1 (P = 0.006)
DISCUSSION
BW2 (P = 0.14)
CB
BW7 (P = 0.16)
f
BW1 (P = 0.01)
The Pit-1 haplogroups showed significant association with BW at 7 wk of age in the PB-1 strain of broiler chicken (Table 4). The h6h8 haplogroup revealed 12% higher BW than the h1h2 group. In the CB strain, haplogroups revealed a significant effect on BW at 1 d old and 2 and 7 wk of age. The h1h5 haplogroup showed 20% higher BW than that of the h1h4 group at 1 d of age. At wk 2, the h1h2 haplogroup showed 29% higher BW than that of the h6h8 haplogroup, whereas at 7 wk of age, h1h5 group revealed 24% higher BW than that of the h6h7 group. In the IWI strain, haplogroups showed a significant effect on BW at 1 d old and wk 6 and 7 of age. At 1 d of age, chicks with h1h2 haplogroup had 11% higher BW than those of the h5h10 haplogroup. At 6 and 7 wk of age, the h4h8 haplogroup showed 49 and 55% higher BW than that of the h5h10 haplogroup, which was the lowest-yielding group. The growth rate between 0 and 2 wk was highest in the h1h2 haplogroup of the CB strain, which was about 52% higher than that of the h6h8 haplogroup (Table 5). In the IWI strain, h4h8 haplogroup revealed 63 and 85% higher growth rates over the h510 haplogroup, respectively, during 0 to 2 and 6 to 7 wk of age.
33.6 32.3 30.1 36.2 34.2 33.9
IWI
Association with Haplotypes
BW6 (P = 0.13)
BW7 (P = 0.08)
had a significant effect (P = 0.046 and = 512) on BW at 2 wk of age and growth rate (P = 0.017 and R2 = 0.644) between 0 to 2 wk, whereas other traits were not significantly affected by the gene expression. In the IWI line, Pit-1 expression revealed significant association with BW at 6 (P = 0.108 and R2 = 0.32) and 7 (P = 0.105 and R2 = 0.33) wk of age.
414 ± 41b — 359 ± 21a 338 ± 34a 379 ± 18a 361 ± 23a 501 ± 42c 323 ± 51a — 398 ± 49ab 351 ± 28a
R2
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POLYMORPHISMS OF Pit-1 Table 5. Haplotype-wise growth rates in
chicken1
CB Haplogroup h1h2 h1h3 h1h4 h1h5 h1h6 h1h9 h4h8 h5h10 h6h7 h6h8 h6h10
IWI
gr0–2 (P = 0.14)
gr0–2 (P = 0.02)
gr6–7 (P = 0.02)
± 0.4b ± 0.5b ± 0.2b ± 0.3b ± 0.1b ± 0.3a — — 5.0 ± 0.4ab 4.0 ± 0.4a —
3.2 ± 0.3a — 3.6 ± 0.3a 3.7 ± 0.1a 4.2 ± 0.2b 3.1 ± 0.2a 4.9 ± 0.3b 3.0 ± 0.4a — 3.3 ± 0.4a 3.4 ± 0.4a
8.7 ± 0.8c — 5.1 ± 0.7a 6.0 ± 0.5b 5.1 ± 0.3a 5.7 ± 0.9ab 8.9 ± 1.0c 4.8 ± 1.3a — 6.1 ± 1.1b 5.5 ± 0.9a
6.1 5.3 5.5 5.2 5.4 4.1
a–cColumn-wise 1gr0–2,
tively.
different superscripts indicate significance. gr2–4, and gr6–7 are the growth rates (g/d) between 1 d old to 2 wk, 2 to 4 wk, and 6 to 7 wk, respec-
existence of a large number of haplotypes in the lines. Nie et al. (2008) revealed the presence of 13 haplotypes in the Pit-1 gene of White Recessive Rock × Chinese Xinghua chickens. In our study, the h10 haplotype was only found in the IWI strain, whereas h3 and h7 were absent in this line. Likewise, the h6 haplotype was predominant in the PB-1 line, whereas h1 was predominant in both the CB and IWI lines. The PB-1 line had relatively low polymorphism at the Pit-1 gene, whereas in the CB line, it was highly polymorphic. This haplotype distribution is the characteristic of the strain, which confirms the specificity of gene pool in chickens. Like microsatellite markers, SNP markers in terms of haplotype constitution can be used for genetic divergent study as had been highly polymorphic. Nie et al. (2008) also depicted that out of 13 haplotypes, h1 and h2 haplotypes contributed approximately 87.4% of the total haplotypes while 5 haplotypes were the rare haplotypes with less than 1% frequency. The nucleotide composition varied among haplotypes. Some of the nucleotide changes determined nonsynonymous types of mutation depicting changes of amino acids in the haplotype composition. In addition to the structural differences, haplotypes showed differential expression patterns in different chicken lines. The mRNA expression of Pit-1 haplotypes differed significantly within the lines, which indicates the role of haplotype compositions on the effect of mRNA expression. The effect of mRNA expression was also analyzed on growth traits through linear regression analysis, which revealed the significant association of the Pit-1 gene expression with BW and growth rates in PB-1, CB, and IWI strains. The regression effect of expression on growth traits, although it has not possessed high precision, delineates the trend of effect of gene expression on growth traits. But, for obtaining higher precision on haplotype-growth relationship, the GLM approach may be followed with several factors affecting the dependent trait; here, it is growth traits. In our study, we adopted the GLM approach to delineate the effect of haplogroups on growth traits. The
haplotype combinations of the Pit-1 gene showed a significant (P < 0.05) effect on different growth traits. The haplotypic trend for BW at 7 wk of age in PB-1 chickens was h6h8/h1h6 > h1h2. The haplotypic trend in the CB strain for BW at 1 d old and 2 and 7 wk old were h1h2/h1h5/h1h6/h1h9/h6h8 > h1h3/h1h4/h6h7, h1h2/h1h6 > h1h3/h1h4/h1h5/h1h9/h6h7, and h1h4/ h1h5 > h1h6/h1h9 > h6h7/h6h8, respectively. In the IWI strain, the trend of haplotypes for 1 d old and 6 and 7 wk old were h6h10/h6h8/h1h9/h1h2 > h1h4/ h1h6/h4h8/h5h10, h4h8/h6h8 > h1h6 > h1h5/h1h9/ h5h10/h6h10 and h4h8 > h1h2 > h1h4/h1h5/h1h6/ h5h10/h6h10, respectively. In the case of growth rate, the haplotypic trend for 0 to 2 wk in the CB strain was h1h2/h1h3/h1h4/h1h5/h1h6 > h1h9/h6h8. The trends in the IWI strain for growth rate between 0 and 2 and 6 to 7 wk were h4h8/h1h6 > h1h2/h1h4/h1h5/ h1h9/h5h10/h6h8/h6h10 and h4h8/h1h2 > h1h5/ h6h8 > h1h4/h1h6/h5h10/h6h10, respectively. Thus, haplotype combination revealed their association with growth traits at different ages. In the past, associations of the Pit-1 gene with growth traits were reported in humans (Aarskog et al., 1997), pigs (Song et al., 2005), cattle (Xue et al., 2006), and chickens (Jiang et al., 2004). In chickens, a nonsynonymous SNP (Asn299Ile) in exon 6 of the Pit-1 gene was significantly associated with BW at 8 wk of age (Jiang et al., 2004). Another SNP in exon 6 was associated with average daily gain between 0 and 4 wk and BW in wk 3, 4, and 5. Three more adjacent SNP in intron 5 were associated with average daily gain between 0 and 4 and 4 to 8 wk, BW at wk 4, 6, and 12, shank length at wk 8, 11, and 12, and shank diameter at wk 11 (Jiang et al., 2004). McElroy et al. (2006) reported a QTL for BW in GGA1 covering a 96-Mb region, where the chicken Pit-1 gene was located. Study revealed that SNP could be in the linkage disequilibrium with causative mutations, which was situated in this region and played crucial roles in chicken growth (Sewalem et al., 2002). The gene expression also confirms the significant association between haplotype combinations and growth traits. Haplotypes with high-
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er BW may be favored both for broiler and layer chickens. In broilers, high growth rate is desired to attain an early slaughter age, whereas in layers, higher BW produces birds that reach puberty early, making them more productive during the egg-laying cycle. Thus, certain haplotypes of the Pit-1 gene possessing favorable associations with growth traits may be given priority over other haplotypes while selecting elite birds for regenerating the next generation. This event will not only enhance the frequency of superior haplotypes in the farm but also enable augmentation of performance in the desired direction. In conclusion, it may be stated that the Pit-1 gene was highly polymorphic and has significant association with growth traits in chickens.
ACKNOWLEDGMENTS Authors are thankful to the Department of Biotechnology (Govt. of India) for providing financial support to carry out the research work.
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