Effect of PMEL17 Plumage Colour Gene Diversity on Production Performance of Indigenous Chicken Variety of Bangladesh

The genetics of feather colour have been well characterized in the chicken, with several genes (for example, PMEL17, MC1R and ASIP). Of these genes, the gene PMEL17 affects plumage colour in the chicken with polymorphisms within this gene have been associated with a variety of different colours (Kerje et al., 2004). The variation in plumage colouration ranges from the dominant white to dun and smoky, depending on the alleles present in the PMEL17 gene (Kerje et al., 2004). Several studies have seen on phenotypes besides colouration to the PMEL17 gene, for example, social behaviour (Keeling et al., 2004). However, no study found whether production traits are affected by these alleles.
 
The most commonly distinct chicken varieties of Bangladesh are the Hilly, Naked Neck, Assel and Full-feathered. The plumage colours present in full-feathered chicken are red, white, black, black with red stripes, white with red stripes and brownish (Faruque et al., 2010). Generally, these chickens having single comb with yellowish shank colour (Faruque et al., 2010). The yearly egg production of a white indigenous chicken is 90 eggs (Khan et al., 2017), that are higher than other chicken varieties.
 
Genetic characterizations of native chickens have been performed by several researchers (Islam and Nishibori, 2012; Nedup et al., 2012), however, no study have observed elsewhere plumage colouration and its potential role in production traits. Therefore, the present study was conducted to know the effect of PMEL17, plumage colour gene diversity on production performance of indigenous chicken variety of Bangladesh.

The research work was conducted following the animal ethics rule and the ethical committee of Chattogram Veterinary and Animal Sciences University (CVASU) (Memo no. -CVASU/Dir (R&E) EC/2015/799, Date 05/07/2017).
 
Study area, animal and production traits in relation to colour
 
The field study was conducted in the three locations of Patia upazila (sub-district) of Chittagong district and the molecular study was done in the lab of Poultry Research and Training Centre (PRTC) at CVASU of Bangladesh. The research was conducted under the Department of Genetics and Animal Breeding at Chittogram Veterinary and Animal Sciences University, Khulshi, Chattogram-4225, Bangladesh from March 2017 to May 2018. A total of 240 households (80 household from each location and having at least 3 chickens) from the Badamtal (location 1), Kusumpura (location 2) and Kolagaon (location 3) areas of Potia upazila (sub district) of Chattogram district was directly surveyed by the researchers and field assistant. All the locations had a similar ambient temperature, humidity, bird management protocols and feed used. Colour was recorded and divided into three different colour morph classes (Black and white, brownish and spotted), whilst comb type (predominantly single) and body shape (round and cylindrical) were also selected. Production traits, consisted of mature live weight (g), clutch size (number of days spent laying) and egg weight (g) were recorded. Mature live weight (g) was taken with top loading balance at the chicken’s age about 40 to 42 weeks of age of the chicken.
 
External and internal egg characteristics
 
Fifty eggs from each variety of chicken from each location were collected. Eggs were cleansed, measured and weighed using digital electronic balance (Model: DWA-224, Dawer Scales India Private Limited) and recorded. Egg-width and egg-length were measured using Vernier slide calipers (Mitutoyo Corp, Japan). An egg shape index was calculated according to Yakuba et al., (2008) as:  
 Eggs shell surface area was determined according to Carter (1975) as,  
The thickness of the egg shell was determined using a micrometer screw gauge (Mitotoyo Corp, Japan). The accuracy of shell thickness dimensions was ensured by measuring shell samples at the broad end, the mid-portion and the narrow end of each egg. Each egg was broken and the yolk and the albumen were weighed and separated. The albumen and yolk height were determined using a spherometer (RAC Expots, Haryana, India); the albumen height was measured in the middle of the thick albumen equidistant from the outer edge of the albumen and the yolk using Vernier slide calipers (Mitutoyo Corp, Japan). Both albumen and yolk ratios of each egg were determined as: In order to correct for the difference in egg weight, the albumen height was converted into Haugh units (Haugh 1937), Haugh unit = 100 log (H + 7.6- 1.7W^0.37), where, H = Observed height of the albumen in millimetres and W = Weight of egg in grams.
 
Molecular analysis of PMEL17
 
Molecular characterisation of the PMEL17 gene was performed in three colour morphs: black and white single round (variety 1), brownish single round (variety 2) and spotted single round (variety 3) chickens. Blood samples were collected from 60 laying hens (variety 1, n=20, variety 2, n=10 and variety 3, n=30). DNA was extracted from the whole blood samples using the FavorPrep^TM blood genomic DNA extraction mini kit (FAVORGEN biotech corporation, Taiwan) and stored at -20°C. Exon 6 of the PMEL17 gene was then amplified using the following PCR primers (forward primer: GTGGATGTGACACAGCTGGA-3´, reverse primer: R-5´-GGAGCATCACCACCTGA-3´), with a resulting product size of 542bp. PCR products were cleaned using 2μl of ExoSAP-IT (enzyme: ExoASP-IT) per sample.
 
Polymerase chain reactions (PCR) and agarose gel electrophoresis
 
A total of 25µl (12.5µl mastermix, 2.5µl each primers (forward and reverse), buffer 5µl and 2.5µl DNA template) PCR mix was prepared (FavorPrep^TM). The PCR amplification was conducted in a MJ PTC-200 per litre. Thermal cycler or a Bio-Red C 1000 thermal cycler with an initial denaturation at 95°C for 10 minutes, followed by 50 cycles of denaturation at 95°C for 30s, annealing for 30s at the 65°C, a primary extension at 72°C for 2 minutes, and a final extension at 72°C for 10 minutes. The PCR products were electrophoresed on 2.5% agarose gel (Lonza USA) at 90 V for 1.5 to 2h and stained with ethidium bromide and their sizes were estimated using a 100-bp DNA ladder. The amplified PCR band pattern was visualized by on a UV trans-illuminator and photographed in a computer.
 
Gene sequencing, scoring, alignment and detection of mutation
 
A 5μl aliquot of a post-PCR reaction product was mixed with 2μl of ExoSAP-IT (enzyme: ExoASP-IT). This combined 7μl reaction volume was incubated at 37°C for 15 minutes to degrade the remaining primers and nucleotides. Finally, the ExoSAP-IT enzymatic reaction mixed sample was inactivated by incubation at 80°C for 15 minutes. The purified PCR products were Sanger-sequenced with a big dye terminator v3.1 sequencing kit and a 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences were thereafter determined on both strands of PCR amplification products at the Macrogen sequencing facility (Macrogen Inc., Seoul, Korea) using an ABI PRISM 3730xl Analyzer (96 capillary type). Eight best sequences from each chicken variety of the PMEL17 gene, including DNA, were taken and from the NCBI information gene bank and using the tool BLAST on the website http://ncbi.nlm.nih.gov, similar sequences, their similarity score and possible mutations were investigated using MEGA6 software package (Tamura et al., 2013).
 
Statistical analysis
 
The least square means of the different recorded parameters on the basis of the chicken variety and location using PROC GLM and PROC MIXED of SAS (2008) followed by randomized block design (RBD). Mean differences were compared using least significant difference (lsd) test at the 5% level of significance.

Production traits in relation to colour morphs
 
The different production traits of the indigenous chicken varieties and locations are presented in Table 1. The spotted single round variety chicken was found to be best for egg production (number/ clutch in days, 15.3±0.9 days) and also have the heaviest live weight compared to other variety chickens (Table 1).
 

 
The clutch size of the different indigenous chicken variety was higher than the Nigerian native chicken (Daikwo et al., 2011). On the other hand, a similar live weight of the different varieties of indigenous chicken was observed by Khan et al., (2017). The variation of clutch size and live weight of this study might be due to differences between regions, genetics and feeding of chickens. Similar factors were described by other researchers elsewhere (Grobbelaar et al., 2010; Khan et al., 2017: Sarma et al., 2018).
 
External and internal egg characteristics in relation to colour
 
The highest egg weight was observed for the spotted single round and the lowest was for the same colour with a cylindrical body type (Table 2). In addition to varying by colour morph, a significant (P<0.05) effect of location on egg weight was also observed. Similar values of egg weight of different genotypes of chicken were reported by several researchers (for example, Khan et al., 2017; Sarma et al., 2018). The variation in egg weight might be due to the differences in genetics, feeding and management of the chicken and these factors was also reported by other researchers (Khan et al., 2004; 2017).
 

 
No effect of colour morph on egg shell thickness was observed. Iqeobi et al., (2004) reported a higher average shell thickness value in normal plumage genotype than the current study. An effect of colour morph was found on shape index and the highest values was observed in the spotted single round colour morph than others (Table 2). The shape index was similar with Khan et al., (2004) and variation of shape index may be due to variation of the breed and management. The surface area of different varieties of chicken eggs did not differ between location, but differed (P <0.05) among varieties in a location (Table 2).
 
An effect of colour morph was found for albumin and yolk weight, but not albumin and yolk ratio (Table 3). The Haugh units varied significantly between colour morphs, but not between locations (Table 3). The albumen and yolk weight, haugh unit observed in the current study agreed with those reported by Nonga et al., (2012) and Yakubu et al., (2008) and this variation may be due to variety differences of chicken.
 

 
Molecular analysis of PMEL17
 
The scoring of similarity and matching rate of three different varieties of chickens was compared for the sequenced plumage colour gene (PMEL17) of Gallus gallus are presented in Table 4. The sequences revealed a 99% homology (NCBI accession no: AY 636126.1, AY636129.1, respectively) with the sequence of Gallus gallus, these were concord with Kerje et al., (2004) as well as the sequence of the domestic chicken (Kerje et al., 2004; Kuliawa et al., 2009).
 

 
Of the sequences, no polymorphisms were found in the spotted colour genotype, but polymorphisms were identified at 64bp (C to T) and 91bp (G to A) in the black-white and brown genotypes, respectively (Fig 1a, b). The nucleotide changes for the black-white morph is predicted from a protein-coding changes. Therefore, the sequence analysis showed that the dominant white and the black alleles were exclusively associated with an insertion and deletion of amino acids in the gene, PMEL17 transmembrane region. Vaez et al., (2008) also found the mutation was together with the recessive silver polymorphism in the mouse, the only PMEL17 gene. The present results show that the different colour morphs have different production characteristics.
 

It can be concluded that the spotted single round variety chickens were found to be best for egg production, live weight and external and internal quality of eggs than the other studied variety of chickens. Mutation of plumage colour genes, PMEL17 was detected in white-black and brownish single round variety chickens and appeared the variability of the production traits. The spotted variety can be used in the traditional systems in rural areas as dual purpose chicken production.

The authors have expressed their sincere gratitude to the Swedish Research Council FORMAS, Stockholm (Project 2017-00946) and the Swedish Research Council (Vetenskapsrådet, VR; project 2015-05919) and Chittagong Veterinary and Animal Sciences University (Grant No. CVASU/CASR (HR)/MS-5/2013/113 (27), 16/10/2017) for providing funds for implementing this research. The authors are immensely grateful to the farmers for helping them during experimental work.

The authors declare no conflict of interest with the funding body.