Polymorphism in the promoter region of ACACB gene and its association with egg fat content in white leghorn layer chicken

Introduction

Chicken eggs and meat are relatively inexpensive and accessible sources of protein for human consumption. However, traditional within-population selection has been less effective, particularly when multiple traits with unfavorable genetic correlations are involved. The use of genetic markers has been shown to enhance selection efficiency, potentially increasing the genetic gain for growth rate in chicken populations by up to 15% compared to conventional family-based selection (Pongpisantham, 1994) [14] . Molecular markers exhibit distinct genetic characteristics and methodological advantages, making them more effective and suitable for genetic analysis than traditional markers. Various types of molecular markers, including RFLP, microsatellites, and single-strand conformation polymorphism (SSCP), have been widely used in the past to study complex genetic traits (Badola et al., 2004; Bhattacharya et al., 2011; Bhattacharya et al., 2015b; Bhattacharya et al., 2016) [2,6,5,7] . Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), developed by Orita et al. (1989) [13] , is a rapid, simple, and reproducible technique widely used for detecting DNA mutations, including deletions, insertions, and substitutions (Kakavas et al., 2008) [10] . SSCP identifies single-base sequence variations by detecting differences in the electrophoretic mobility of one or both single-stranded DNA fragments in a non-denaturing polyacrylamide gel (Gruszczynska et al., 2005) [9] . Early applications of the technique relied on radioactive labeling, which restricted its broader use. To improve simplicity and efficiency, alternative staining methods such as ethidium bromide (Yap and McGee, 1992) [22] and silver staining (Ainsworth et al., 1991) [1] were later introduced. High fat content in chicken meat poses potential health risks to humans (Milićević et al., 2014) [12] . In laying hens, excessive fat deposition can adversely affect regulating this pathway. As the ACACB gene is involved in fatty acid metabolism in chickens, it may influence egg quality. The enzyme encoded by ACACB produces malonyl-CoA, which inhibits carnitine/palmitoyl-transferase 1 (CPT1) and restricts the transport of fatty acyl groups into the mitochondria via the carnitine shuttle system for βoxidation (McGarry et al., 1977; Ruderman et al., 1999; Wakil and Abu-Elheiga, 2009) [11,16,20] . Consequently, this results in increased glucose utilization, decreased fatty acid oxidation, and enhanced synthesis of fatty acids and triglycerides (TG). Understanding genetic variation in the chicken ACACB gene may aid in identifying variants associated with egg fat content. In particular, characterization of variation in the regulatory region of this gene could support future breeding strategies aimed at improving egg quality in chickens. Therefore, the present study was undertaken to detect polymorphisms in the promoter region of the ACACB gene and to evaluate their association with egg fat content in IWI and IWK lines of White Leghorn layer chickens.

Materials and Methods

Experimental birds

The present study was conducted on 500 birds, belonging to IWI (250 birds) and IWK (250 birds) lines of the White Leghorn breed. The IWI and IWK lines are improved egg producing lines laying 298 and 276eggs annually (DPR Annual Report 2018-19). Both the lines are maintained at the ICAR-Directorate of Poultry Research (ICAR-DPR), Hyderabad, India.

Management

Day-old chicks from both lines were vent-sexed, wingbanded, and reared in an open-sided house on deep litter until 16 weeks of age. They were fed a layer chick starter diet containing 2, 600 kcal/kg metabolizable energy (ME) and 18% crude protein (CP) up to 8 weeks, followed by a grower diet with 2, 500 kcal/kg ME and 16% CP from 9 to 16 weeks. After 16 weeks, the birds were transferred to individual cages to enable recording of individual production. From the onset of egg production until the end of the experiment, they were provided a layer diet containing 2, 600 kcal/kg ME and 16% CP. A photoperiod of 16 hours, including natural daylight, was maintained during the laying period. All birds were maintained under uniform management conditions with ad libitum access to water. On the day of hatch, chicks were vaccinated against Marek's disease, and subsequently immunized against major diseases such as Ranikhet disease, Infectious Bursal Disease, and fowl pox following a standard vaccination schedule. During the summer season, cooling was provided by sprinkling water on the roof, and adequate lighting arrangements were ensured in the shed. Additionally, all routine management practices, including strict bio-security measures, were implemented in and around the sheds to provide a conducive environment for optimal performance of the birds.

Sample collection

Blood samples were collected from the wing vein, and genomic DNA was extracted following the standard protocol described by Sambrook and Russell (2001) [17] . Eggs were collected daily to record egg fat content.

Estimation

Whole eggs were dried at 60 o C temperature at the BOD incubator. The total fat (%) of an egg was estimated using Soxhlet extraction method following standard protocol.

PCR

The 5′ upstream region of the transcription start site of the ACACB gene, spanning 534 bp, was divided into two nonoverlapping fragments: ACACB-S1 (301 bp) and ACACB-S2 (233 bp). Primers were designed based on the chicken ACACB gene sequence (Accession No. NC_006102) to amplify these fragments (Table 1). Polymerase chain reaction (PCR) amplification was performed in 0.2 ml PCR tubes with a reaction mixture containing 1 µl of 10× DreamTaq buffer, 0.5 µl of 2.5 mM dNTP mix, 1 µl each of forward and reverse primers (20 ng/µl), 0.2 µl of Taq DNA polymerase (5 U/µl), approximately 100 ng of genomic DNA as template, and nuclease-free water to make up a final volume of 10 µl. The PCR cycling conditions included an initial denaturation at 95 °C for 10 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 64 °C for the S1 fragment and 66 °C for the S2 fragment for 30 seconds, and extension at 72 °C for 30 seconds, with a final extension at 72 °C for 10 minutes.

Single strand conformation polymorphism (SSCP)

The amplicons of both fragments were denatured at 95 °C for 5 minutes, followed by rapid cooling on ice for 15 minutes. The samples were then separated on a 12% polyacrylamide gel prepared using 9.6 ml Acrylamide: Bisacrylamide (49:1), 28.3 ml 1× Tris-borate-EDTA (TBE) buffer, 1.5 ml glycerol, 100 µl of 10% ammonium persulfate (APS), and 75 µl tetramethylethylenediamine (TEMED) to detect genetic variants. The PCR products were loaded onto the gel, and electrophoresis was carried out at 200 V for 12 hours. Following electrophoresis, the gel was stained with silver nitrate, as described by Vohra et al. (2006) [19] , to visualize the banding patterns and determine genotypes. Different fragment size combinations were scored as distinct genotypes.

Haplotypes

The SSCP variants observed in the ACACB-S1 and ACACB-S2 fragments were recorded for each individual. The ACACB-S1 fragment exhibited three distinct patterns, while the ACACB-S2 fragment showed six different patterns. The combination of patterns from both fragments in each bird was used to infer its haplotype. In total, 12 haplotypes (designated h1 to h12) were identified based on these combinations. The frequencies of haplotypes and haplogroups were estimated using the direct counting method (Bhattacharya et al., 2013). In diploid individuals, the pair of haplotypes constituted the haplogroup.

Statistical analysis

Allelic and genotypic frequencies for each fragment were estimated using the POPGENE software (Yeh et al., 1997) [23] . A Univariate General Linear Model was applied to assess the association between traits across the two lines using the Statistical Package for Social Sciences (SPSS) version 17.0. Haplogroups with fewer than three observations were excluded from the analysis.

Results

Polymorphism in ACACB gene promoter

The ACACB-S1 (301 bp) fragment exhibited six distinct banding patterns in both IWI and IWK lines, while the ACACB-S2 (233 bp) fragment showed three different patterns in both populations. The combination of patterns from these two fragments resulted in the identification of twelve haplotypes (h1 to h12) and eighteen haplogroups, namely h1h1, h1h2, h2h2, h3h3, h3h4, h4h4, h5h5, h5h6, h6h6, h7h7, h7h8, h8h8, h9h9, h9h10, h10h10, h11h11, h11h12, and h12h12.

Association of haplogroups with total egg fat content

In IWI lines within group there is a significant (p≤0.05) effect on total egg fat. Whereas In IWK lines within group there is a non-significant (p≤0.05) effect on total egg fat was observed. The haplogroups of ACACB gene promoter had non-significant (p≤0.05) effect on total egg fat between the group of IWI and IWK lines with the exception of h10h10 and h11h11. In general h11h11 haplogroup birds was showed lowest total egg fat and h6h6 haplogroup birds was showed highest total egg fat. In IWI and IWK lines the haplogroups were associated with the total egg fat (%), which was the most important indicators depicting the egg quality. While performing the association, the haplogroups which had the less number were excluded. In the least square means for total egg fat in h10h10 haplogroup birds was the highest (25.94±0.95) and in the h11h11 haplogroup it was found to be the lowest (20.35±1.12) total egg fat. In case of IWK, the h6h6 haplogroup birds were the highest (24.24±1.01) and in the h2h2 haplogroup it was found to be the lowest (22.06±0.70) total egg fat (Table 2)

Discussion

The ACACB gene has been reported to be associated with conditions such as nephropathy, obesity, diabetes, and endstage renal disease (Tang et al., 2010; Riancho et al., 2011; Zain et al., 2017) [18,15,24] . Polymorphisms in the promoter region may influence gene function, thereby affecting its association with various traits in chickens (Cui et al., 2006; Zhu and Jiang, 2014) [8,25] . For the present polymorphism analysis, two genetic lines of White Leghorn layer chickens, IWI and IWK, were selected. The IWI line has been developed for egg production, whereas the IWK line has been selected for both egg production and egg weight.Both the fragments in the present study revealed polymorphism based on SSCP analysis. The 301 bp fragment revealed higher polymorphism compared to 233 bp fragment. Based on analysis of both the fragments, 12 haplotypes were identified. These 12 haplotypes formed 18 haplogroups in both the populations studied. Polymorphism in the promoter region of genes in chicken is common phenomenon for several genes. Promoter polymorphism is also observed in matrix metalloproteinase 9 gene (Zhu and Jiang 2014) [25] , prolactin gene (Cui et al., 2006; Bhattacharya et al., 2015a) [8, 4] etc. in chicken. Generally the variation is associated with difference in function. The total egg fat% is significantly higher within IWI line, when they have h10h10 haplogroup. The same haplogroup also showed higher total fat in IWI line compared to IWK line. But the h11h11 haplogroup is associated with higher total fat% in IWK line. Since ACACB gene plays a key role in fatty acid synthesis and fatty acid oxidation, it would be interesting how this particular haplogroup is influencing the total fat% in IWI line.

Conclusion

From the results obtained in this study, it can be concluded that the promoter region of ACACB gene is polymorphic and certain haplogroups are associated with egg fat percentage. It would be interesting to further validate the specific mutations to understand their precise role in the egg fat percentage in the two lines, which would help in marker assisted selection.

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