Relative Expression Profile of AA Genotype of BMP4 Gene in Broiler and Layer Chicken

Bone morphogenetic protein 4 (BMP4) gene is primarily involved in regulation of bone development and is considered as positive regulator for osteogenesis. The objectives of the study were to explore the polymorphism of BMP4 gene and to determine the expression profile of the predominant genotype observed in broiler (81%) and layer (95%). PCR-SSCP revealed differential pattern with AA genotype being most frequent over AB and AC genotypes. The fast-growing birds displayed higher magnitudes of growth traits over their counter layers. The mRNA expression level was relatively higher in broiler at day 42 and in layer on day 1 and lower on day 28 of both the lines. Significant difference (p≤0.05) in gene expression was observed during different stages of growth in layer line only. It can be concluded that the BMP4 gene was polymorphic and the mRNA expression varied distinctly over different developmental stages of juvenile period in broiler and layer chicken.

Bone morphogenetic protein 4 (BMP), a member of transforming growth factor-β superfamily found predominantly in mesenchymal cells, soft bone cells, periosteum, bone marrow cavity and the muscle cells in the nearby regions of bone fracture site (Nakase et al., 1994). BMP4 are secretory, hydrophobic and acidic glycoprotein with mature polypeptide encompassing 114 amino acids and signal peptide region (1 st 19 amino acids) at N-terminal site with 4 possible glycosylation sites (144 th , 208 th , 347 th and 362 nd ). They form a close cluster with BMP2, a positive regulator for osteogenesis. They are one of the key players in osteogenesis where they act as stimulating factor for soft bone ossification (Yaoita et al., 2000) at early stages and inhibits osteoclastogenesis process. Various studies using gene knock-in-knock-out technology disclosed the functional diversity of BMP4 gene i.e. in the processes of craniofacial morphogenesis, limb morphogenesis, chondrogenesis and osteogenesis. Their variable effect irrespective of tissue and stage of development was well documented in chicken (Miyazaki et al., 2003;Onagbesan et al., 2003;Shimizu et al., 2004;Weber et al., 2008 andPerry et al., 2009). However, a detail comprehension in the expression at various stages of development and the associated-based study of expression is lagging in chicken. Thus, an attempt was made to explore the polymorphism of BMP4 gene and to study the expression pattern of most predominant genotype at different stages of growth period in broiler and layer chickens.

Experimental birds
The birds of control broiler and layer chicken population maintained at ICAR-Directorate of Poultry Research, Hyderabad was used for the study where all managemental and biosecurity measures with respect to watering, feeding, health, vaccination etc. were taken besides summer management.

Sample collection
Bone marrow tissues of 40 birds (in total) of broiler and layer lines of 1, 14, 28 and 42 days of age (i.e. 10 birds/ day/line) was collected after the approval of IAEC, DPR, Hyderabad in 1.5 ml sterile polypropylene tube (formerly treated with DEPC) and preserved until processing at -80ºC for the purpose of gene expression study. Congruently, blood from broiler (286) and layer (270) birds was collected in to an anticoagulant added sterile polypropylene tube (2.7% EDTA i.e. 60-70 µl/ 1ml of blood) and stored at -20ºC till succeeding for DNA isolation.

Genomic DNA and RNA isolation process
Phenol-chloroform method (Sambrook and Russell, 2001) was used to extract genomic DNA from blood samples of each bird under study. The purity was checked in terms of quality and quantity and the ones with good DNA were subjected to further procedures. Trizol (Qiagen, Invitrogen) method of RNA isolation was done from the bird's bone marrow tissue and stored at -80°C.

cDNA synthesis
Isolated RNA samples of each bird of different age group of both the lines were converted in to cDNA by using a high capacity cDNA Reverse transcription kit (ABI, USA). The conversion mix prepared as per the manufacturer's protocol, blended with RNA sample (10 µl) and placed in thermocycler (Mastercycler, Eppendorf) for reverse transcription @ 25ºC/10 min (denaturation), 37ºC/120 min (annealing), 85ºC/5 min (extension) and held at 4ºC. The resulted cDNA's were kept at -20ºC till use for expression studies.

Single stranded conformation polymorphism (SSCP) and Statistical analysis
The populations of broiler and layer birds of different age groups were screened for the presence of polymorphism by using single stranded conformation polymorphism (SSCP). Native PAGE was prepared with the ingredients such as 49:1 Acrylamide:Bisacrylamide (9.6 ml), 1X TBE buffer (28.3 ml), Glycerol (1.5 ml), 10% APS (100 µl ) and TEMED (75 µl). Individual samples in formamide dye were denatured by means of heating and were not allowed for renaturation by means of immediate snap cooling. The chilled products were then electrophoresed @ 195 V (8 hrs) in vertical gel electrophoresis, silver stained and observed for differential pattern. The samples that showed variation in expression pattern was sequenced by using automated dye-terminator cycler in ABI PRIZM 377 DNA sequencer (Perkin-Elmer), ScieGenom lab. The association study of genotype with expression was performed using Univariate General Linear Model of SPSS 16.0 with the model: Y ijk = μ + G i + A j + S k + e ijk where, Y ijk = dependent variable; μ = overall population mean; G i = fixed effect of genotype; A j = fixed effect of age; S k = is the fixed effect of sex and e ijk = random error with NID (0, σ 2 ). Significant differences were calculated between least-squares means of genotype by Duncan's method (5% LS).
So obtained Ct (comparative threshold) values of BMP4 (target) and reference (GAPDH) genes were quantified using the formula: ΔCt = maen Ct of target gene -mean Ct value of endogenous control

ΔΔCt = ΔCt
Expression of target Expression of target gene at different ages gene at day 1

Statistical analysis
The expression data was analyzed with ANOVA following univariate GLM with Duncan's as post-hoc test (between different age groups) using SPSS 16.0 version. The data were being presented in mean ± SE with LSD at 5% level of significance (p<0.05). The statistical model for the present study was:

Y ijk = µ+ G i + A j + S k +e ijk
Where, Y ijk is expression of each individual at each age group in the population, µ is the overall mean, G i is the fixed effect of genotype, A j is the fixed effect of age, S k is the fixed effect of sex and e ijk is the random error distributed normally with mean 0 and common variance σ 2 (NID-0, σ 2 ).

RESULTS AND DISCUSSION
A 536 bp 5' upstream region (partial promoter) of BMP4 gene was amplified using gene specific primers and was subjected to SSCP to determine polymorphism in the fragment (Fig. 1).

Fig. 1: AGE showing PCR amplified product from genomic DNA of BMP4 gene promoter
The study revealed the presence of polymorphism in the fragments of both broiler and layer chicken lines ( Fig.  2) with AA genotype being predominant one having frequency of >80% over other two genotypes (AB and AC with combined frequency of <13%). The mean weights of growth and conformation traits were tabulated in table 1 where the broiler birds recorded significantly higher (p≤0.05) magnitude of body weights and conformation traits such as shank length and breast angle at 6 weeks of age. The occurrence of polymorphisms in the promoter region may influence the binding of transcription factors with promoters and ultimately interfere the gene expression, which may thereby impact the bone development and skeletal integrity in chicken.
The bone marrow tissues of the predominant AA genotype at respective age groups (day 1, 14, 28 and 42) were subjected to expression profiling. In the present study, the amplified products of BMP4 and GAPDH genes through real time PCR was run in 0.8% agarose gel and the respective bands have been detected in Fig. 3. The mean Cq values were recorded separately and the relative standard curve was made by taking cDNA on X-axis (independent variable; in log) and quantification cycle on Y-axis (dependent variable) where a coefficient of determination (R 2 ) of 0.9583 and 0.9316 was observed in respective BMP4 and GAPDH genes. Correspondingly, the relative expression of BMP4 gene was analyzed up to day 42 in broiler and layer chicken lines wherein the 40-ΔCt value directly corresponds to the gene expression i.e. the higher the 40-ΔCt value, the higher will be the expression of gene and vice-versa.
A non-significant effect of sex was observed in both the lines on gene expression. The highest and lowest BMP4 gene expression was found with the magnitude of 42.29 ± 0.71 on day 42 (D42) and 39.03 ± 0.70 on day 28 (D28) in AA genotype of broiler and 39.84 ± 0.4 on D42 and 38.01 ± 0.5 on D28 in AA genotype of layer lines, respectively (Table 2). In other words, the broiler population exerted a slightly higher mRNA expression than their counter parts implicating the role of BMP4 expression in regulating higher growth in the broiler line. The significant differences (p≤0.05) in gene expression were observed during different ages of growth (D1, D14, D28 and D42) in the layer line only. Zhen et al. (2006) revealed the significant effect of genotypes on gene expression for Hsp70 gene in chicken. However, the expression trend was found to be decreased in both the fast (broiler) and slow growing  (Kingsley, 1994;Winnier et al., 1995;Chang et al., 2002;Bandyopadhyay et al., 2006;Shu et al., 2011). Tissues where BMP4 is expressed were osteoblastic cell, hippocampus, brain, astrocyte, oligodendrocyte and ependymal cells of spinal cord in rat (Harris et al., 1994;Fan et al., 2003;Mikawa et al., 2006;Miyagi et al., 2012), branchial arches, retinal pigment epithelium and embryo in chick (Francis-West et al., 1994;Zhang et al., 2013;Judge et al., 2016), alveolar bone, hair follicle and digit in mouse (Zhang et al., 2002;Han et al., 2003;Ou et al., 2015), embryo (MartÍnez-Barberá et al., 1997) in zebra fish and bone marrow tissue in chicken.
Their over expression displayed ectopic bone formation (Alden et al., 1999;Jane et al., 2002;Kubota et al., 2002) and under expression caused osteogenic impairment characterized by spontaneous bone fractures. In this study, we observed differential growth performance of broiler and layer birds, which could be attributed to the breed differences and effect of genotypes of candidate genes related to controlling growth in chicken. The expression of BMP4 gene in AA genotyped birds of broiler was found to be relatively higher than that of layer birds indicating its role in regulating growth and skeletal conformation traits.

CONCLUSION
The polymorphism was observed in 5'upstream region of BMP4 gene in chicken in which the predominant AA genotype showed differential expression between breeds and among different age groups with mRNA expression being higher at D42 in broiler and at D1 in layer line.