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. 2024 Jun 19;103(9):103963. doi: 10.1016/j.psj.2024.103963

Further screening of SNP loci of eggshell translucency related genes and evaluation of genetic effects

Geng-Yun Liu ⁎, Xiang-Yu Chen ⁎,†, Xue-Lu Liu ⁎, Rong-Yan Zhou ⁎, Xiao-Yu Zhao ‡, Li-Jun Xu †, Zhong-Hua Ning §, De-He Wang ⁎,1
PMCID: PMC11519685  PMID: 39013295

Abstract

Eggshell translucency is a widespread issue in the field of egg quality. Previous research has established that the heritability of eggshell translucency is relatively low or moderate. Scientists have also successfully identified SNP loci related to eggshell translucency on different chromosomes by using gene chips and single-variant GWAS. However, the specific impact of single or multiple genes on the trait of eggshell translucency remains unknown. In an effort to investigate this, we examined 170 SNPs associated with eggshell translucency obtained by our research group. We selected 966 half-sibling laying hens from 2 generations in 3 pure lines: Dwarf Layer-White, Rhode Island Red-White Strain, and Rhode Island Red. Eggs were collected from each hen over a period of 5 consecutive days, and eggshell translucency was measured using a grading method in which the hens were divided into 2 groups: an opaque group and a translucent group. We collected blood samples from the laying hens and extracted DNA. Time of flight mass spectrometry (TOF-MS) was used for genotyping to identify SNP loci that influence the trait of eggshell translucency. The results of our analysis revealed that using TOF-MS in 3 chicken strains, we were able to eliminate loci with low gene polymorphism, genetic effect contribution less than 1%, and deviation from Hardy-Weinberg equilibrium. Ultimately, 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were identified on chromosomes 1, 5, and 19. Additionally, nine candidate genes (DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A) were found to be associated with these SNPs. No linkage disequilibrium relationship was observed between the 2 pairs of SNP loci on chromosomes 1 and 19. Based on previous studies on the formation mechanism of eggshell translucency, we hypothesize that NUCB2, FTL, and ACACA genes may be affecting the eggshell structure through different mechanisms, such as increase the water permeability or make thin of eggshell membrane, which promote moisture or part of other egg contents and ultimately lead to the formation of eggshell translucency. These findings validate and identify five SNP loci that regulate the translucency trait, and provide molecular markers for breeding non-translucent populations. Furthermore, this study serves as a reference for further investigation of the genetic regulatory mechanisms underlying eggshell translucency.

Key words: chicken, eggshell translucency, genotyping, SNP loci, candidate gene

INTRODUCTION

Eggshell translucency refers to watermark-like spots on the surface of eggshells, with diameters ranging from 300 to 800 μm. These spots are caused by the transfer of moisture or part of other egg contents from the eggshell membrane to accumulation in the eggshell (Tyler and Geake, 1964; Solomon, 1991; Wang et al., 2019). When light penetrates the eggshell, these spots become translucent in appearance (Holst et al., 1932). Relevant data that the proportion of translucent eggs has exceeded 30%, with the most severely affected groups reaching over 70%. It not only affects the appearance of the eggs but also renders the egg contents susceptible to salmonella infection, posing a food safety hazard (Chousalkar et al., 2010; Xiong et al., 2022). Additionally, the poor appearance of translucent eggs is typically priced lower or refused by consumers, which causes significant revenue loss for egg producers. Studies have demonstrated that eggshell translucency is a moderately or weakly heritable trait, with heritability estimates ranging from 0.20 to 0.30. Consequently, there is a high probability of this trait being passed on to offspring (Wang et al., 2017; Liu et al., 2023).

In the early stages of molecular marker research, microsatellites were widely used in livestock breeding owing to their ease of development and genotyping (Selkoe and Toonen, 2006). However, with the advancements in sequencing technology, single nucleotide polymorphisms (SNPs) are rapidly replacing microsatellites as the main methods of molecular markers due to their higher abundance of genetic variation within the genome (Ferreira et al., 2018; Mengulluoglu et al., 2019). Genotyping arrays comprising of whole genome-wide SNPs have proven to be very valuable tools in genomic analysis (Matukumalli et al., 2009; Ramos et al., 2009). Noteworthy examples include the Illumina 60K SNP arrays (Groenen et al, 2011) and Axiom 600K whole genome array (Kranis et al, 2013), which have been successfully applied in chicken research.

The high density gene chip containing 600K SNP has been utilized to identify 4 SNPs that are significantly associated with the trait of eggshell translucency, as well as 170 suggested significant SNPs (Wang et al., 2017). In a separate study by Qu et al. in 2021, nine SNPs were found to be significantly associated with the occurrence of translucent eggs in chicken through genome-wide association analysis (GWAS). The differential SNPs may be situated in functional regions such as UTR and exons, or non-functional regions such as introns and intergenic regions, due to genetic linkage, as reported in previous studies by Selkoe and Toonen (2006), Matukumalli et al. (2009), Ramos et al. (2009), and Liu et al. (2021). Therefore, these SNP loci merely serve as potential molecular markers for the screening of the eggshell translucency trait, and further investigations are necessary to uncover the specific functional genes involved.

In this study, a total of 966 individuals from 3 different strains and 2 generations of laying hens were genotyped using TOF-MS in 3 chicken strains. The objective was to validate the SNP loci affecting eggshell translucency and investigate the genetic effects associated with this trait. The obtained results not only offered molecular markers for the breeding of non-translucent populations but also served as a valuable reference for the deeper understanding of the genetic regulatory mechanisms underlying eggshell translucency.

MATERIALS AND METHODS

Animals and Data Collection

In the present study, the same populations of laying hens as previously described by Liu et al., (2023) were utilized. A total of 966 hens were selected from the 6th and 7th generations of 3 strains of Dwarf Layer-White (DWL-White), Rhode Island Red-White Strain (RIR-White), and Rhode Island Red (RIR) laying hens, consisting of 198 hens from the 6th generation and 768 hens from the 7th generation. Each strain comprised of 322 hens. All 3 strains were sourced from Hebei Rongde Poultry Breeding Co., Ltd. (Hengshui, China). The chicks were reared in a temperature and light-controlled coop for 42 d and subsequently transferred to individual cages in an underground facility at 16 wk, where they were subjected to identical environmental conditions. Throughout the experiment, the hens were provided with ad libitum access to the same feed and water, and maintained on 16 h: 8 h light/dark cycle (Temperature: 22–25°C; Humidity: 55–65%).

Eggshell translucency was assessed at the age of 40 wk. In order to ensure a sufficient sample size, eggs were collected from each hen over a five-day period, with 3 qualifying eggs per hen being selected. These selected eggs were then stored for five days under relatively stable temperature and humidity conditions (Temperature: 17–20°C; Humidity: 40–50%). Subsequently, candling of the eggs was conducted using a light emitting diode (LED) cold light source (HLK, Tongfa Corp., DeZhou), and the eggs were categorized into 4 grading levels. All the aforementioned procedures and standards were carried out in accordance with the protocols described by Wang et al. (2019). Laying hens with different grades of translucent eggs were classified as individuals with corresponding levels of eggshell translucency and level 4 of translucent egg is the most serious case (Ray and Roberts, 2013). Based on the measurement results, the hens were divided into distinct groups. The opaque group consisted of hens with level 1 and 2 translucent eggs, while the translucent group comprised hens with level 3 and 4 translucent eggs.

Selection of SNPs

The present study screened out 170 SNP loci, which were reported to have suggested significant effects on the trait of eggshell translucency by Wang (2017), and were subsequently divided into 3 batches for genotyping. After 2 rounds of SNPs genotyping screening, a subset of 40 SNP loci were ultimately selected for the third genotyping assay.

Genotyping

Chicken genomic DNA was extracted from blood samples using a specialized blood DNA extraction kit (Tiangen Biotech, Beijing, China). The quality of the DNA was evaluated using 1.5% agarose gel electrophoresis, and the concentration was determined using a spectrophotometer (Nano Drop ND-1000, Thermo Scientific, Wilmington, DE). The resulting DNA concentrations ranged from 20 to 50 ng/μL. SNP loci gene sequences were obtained from the Ensembl database (http://asia.ensembl.org). For each SNP, 2 PCR primers and one extension primer were designed. Subsequently, genotyping of 966 chickens was performed using MALDI-TOF-MS on the MassARRAY iPLEX Platform (Sequenom, San Diego, CA).

Statistical Analysis

SNPs with a genotype detection rate of less than 95% and a minor allele frequency of less than 5% in all individuals of the 3 strains were also excluded. Using the Chi-square test to examine the SNP allele frequency (IBM SPSS Statistics version 20, Armonk, NY) and the Hardy-Weinberg equilibrium (R version 3.6.0). The loci effects of SNPs were calculated using variance component estimation that a general stochastic linear model in SPSS software. The Haploview program (Version 4.2; Broad Institute of MIT and Harvard, Cambridge, MA) was used to determine the linkage disequilibrium (LD) among multiple SNPs in a gene. Pairwise LD between SNP was assessed using the Lewontin D' statistic and squared correlation coefficient r2. The D' > 0.8 and r2 >1/3 means sufficiently strong LD (Ardlie et al., 2002; Grunau et al., 2006).

Ethics Statement

All experimental procedures were conducted in strict adherence to the guidelines outlined in the “Guide for the Care and Use of Laboratory Animals” provided by the Institutional Animal Care and Use Committee of Hebei Agricultural University, China.

RESULTS AND DISCUSSION

In this study, a total of 40 SNPs were included. After performing genotype quality control analyses, one SNP with a minimal minor allele frequency and two SNPs with a low genotype detection rate were excluded. The remaining 37 SNPs exhibited polymorphic characteristics, with a minor allele frequency exceeding 5% and a genotype detection rate exceeding 95%.

Table 1 presents the Chi-square test results of SNPs allele frequencies in the 3 chicken strains. In RIR, there were highly significant differences between group opaque and translucent that rs15050262, rs315151141, rs315487058, and rs317580829 (P < 0.01). In RIR-White, there were highly significant differences between group opaque and translucent that rs14123691, rs316121113, rs317580829, and rs317703750 (P < 0.01).

Table 1.

The Chi-square test of allele frequency at SNP loci in opaque and translucent groups of DWL-White, RIR and RIR-White Strain.

SNP Loci Variation type P/DWL-White P/RIR P/RIR-White
Affx-50362536 A>G 0.725 0.197 0.808
Affx-50362599 A>G 0.830 0.053 0.812
Affx-51384820 T>C 0.320 0.335 0.467
Affx-51435050 A>C 0.083 0.684 0.080
rs13575798 A>T 0.949 0.787 0.448
rs14123691 A>G 0.831 0.637 0.001
rs15050262 C>T 0.411 0.001 1.000
rs15050550 A>G 0.729 0.655 0.131
rs15112231 T>C 0.734 0.432 1.000
rs15150251 A>T 0.260 1.000 1.000
rs15515730 A>G 0.852 0.572 0.729
rs15906182 C>T 0.452 0.525 0.161
rs312295391 A>G 0.515 0.948 0.338
rs312943734 T>C 0.183 0.578 1.000
rs312973678 C>G 0.533 0.698 1.000
rs313041752 A>G 0.737 0.685 0.325
rs313232800 T>C 1.000 0.704 0.250
rs314255298 A>G 0.563 0.482 0.379
rs314914762 A>G 1.000 0.304 0.232
rs314940570 T>G 0.580 0.486 0.735
rs315144816 A>G 0.589 0.531 0.579
rs315151141 A>G 0.257 0.017 0.602
rs315487058 T>G 0.850 0.001 1.000
rs315487879 T>C 0.862 0.936 0.482
rs315532707 T>G 0.653 0.922 0.977
rs315664306 T>C 0.979 0.555 0.710
rs316121113 T>C 0.731 1.000 0.005
rs316163751 T>C 0.299 0.955 0.674
rs316987262 T>C 0.537 0.593 0.919
rs317149140 C>G 0.461 0.526 0.980
rs317389181 T>G 0.121 0.138 0.702
rs317580829 A>G 0.622 0.001 0.001
rs317703750 T>C 0.501 0.818 0.001
rs317723964 A>G 0.801 0.477 0.781
rs317809001 A>C 0.806 0.637 0.653
rs317875370 A>G 0.557 0.781 0.378
rs318104288 A>G 0.653 0.939 0.558
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There were significant differences in SNP loci between the opaque and translucent groups of different strains (P<0.05). Conversely, P>0.05 indicated no significant difference.

Loci Effect Analysis and HWE Test

In order to further investigate the impact of SNP loci on eggshell translucency, estimates of variance components were analyzed within 3 strains, the contribution to the genetic variances (CGV) of each SNP loci was calculated. Furthermore, the allele frequencies were examined for Hardy-Weinberg equilibrium in the 3 chicken strains using R. Table 2 presents the variation type, chromosome, physical location, contribution to genetic variances, and results of the HWE equilibrium test for 37 SNP loci. Out of those, ten SNP loci (Affx-50362599, Affx-51435050, rs15050262, rs15150251, rs312943734, rs315144816, rs315487058, rs316121113, rs317389181, and rs317580829), with a CGV higher than 1%, were identified. The SNP loci with the highest CGV was rs315487058 at 4.30%. Moreover, the allele frequencies of Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181 were found to be in Hardy-Weinberg equilibrium across the 3 strains.

Table 2.

Analysis of genetic effects and Hardy-Weinberg equilibrium tests for SNP loci related to eggshell translucency.

SNP Loci Variation type Chromosome Physical location Loci effect HWE Test
Affx-50362536 A>G 1 44335419 0.00% 0.001
Affx-50362599 A>G 1 44373423 1.92% 0.281
Affx-51384820 A>G 4 19384095 0.32% 0.002
Affx-51435050 A>C 4 50213513 1.92% 0.005
rs13575798 A>G 19 7971429 0.32% 0.428
rs14123691 T>C 19 8621768 0.79% 0.016
rs15050262 A>G 19 8178375 1.56% 0.333
rs15050550 A>G 19 8703728 0.32% 0.001
rs15112231 A>C 2 66785467 0.16% 0.023
rs15150251 C>T 3 121924887 1.71% 0.003
rs15515730 T>C 1 176337652 0.48% 0.594
rs15906182 T>C 8 7724782 0.48% 0.001
rs312295391 A>G 8 6113990 0.32% 0.080
rs312943734 A>G 19 8568577 1.25% 0.829
rs312973678 C>G 22 1459833 0.48% 0.000
rs313041752 T>G 19 8152430 0.79% 0.241
rs313232800 T>G 19 8161530 0.16% 0.881
rs314255298 A>G 4 4069423 0.00% 0.000
rs314914762 A>T 1 2981793 0.00% 0.016
rs314940570 T>G 14 9278184 0.79% 0.002
rs315144816 A>C 8 6039973 1.10% 0.001
rs315151141 T>C 19 7875559 0.96% 0.321
rs315487058 A>G Z 63366191 4.30% 0.001
rs315487879 A>G 19 8308269 0.48% 0.001
rs315532707 T>C 19 8057448 0.79% 0.004
rs315664306 T>G 1 6095270 0.48% 0.000
rs316121113 T>C 1 91718015 1.25% 0.628
rs316163751 T>C 4 5259298 0.48% 0.014
rs316987262 T>C 2 121984356 0.48% 0.298
rs317149140 A>G 1 34372133 0.32% 0.001
rs317389181 T>C 5 11925413 1.44% 0.483
rs317580829 A>G 19 8905818 1.25% 0.002
rs317703750 C>G 13 10333115 0.48% 0.517
rs317723964 T>C 19 8279964 0.64% 0.068
rs317809001 T>C 6 6250394 0.80% 0.038
rs317875370 A>G 14 182144 0.00% 0.385
rs318104288 A>G 3 98986619 0.48% 0.163
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LD Analysis and Candidate Genes

The SNP loci associated with eggshell translucency and their corresponding candidate genes are presented in Table 3. Notably, these SNPs were situated on chromosomes 1, 5, and 19, which aligns with the chromosomes previously reported by Qu (2019) in relation to the translucent trait. Nine candidate genes were identified to influence translucency, namely Decorin (DCN), B-cell translocation gene anti-proliferation factor 1 (BTG1), Zinc Finger Protein 92 (ZFP92), POU Class 2 Homeobox 1 (POU2F1), Nucleobindin 2 (NUCB2), Ferritin Light Chain (FTL), Gametogenetin Binding Protein 2 (GGNBP2), Acetyl-CoA Carboxylase Alpha (ACACA), and Transcriptional Adapter 2 Alpha (TADA2A). In order to further assess the linkage of single SNP loci on the same chromosome, we utilized the Haploview program to analyze LD blocks on chromosomes 1 and 19 in 3 different strains. The measurements of pairwise LD can be found in Table 4. It was observed that rs15050262 and rs312943734 on chromosome 19 exhibited a D' value of 0.378 and an r2 value of 0.131 across all 3 strains, while no data regarding the linkage between Affx-50362599 and rs316121113 on chromosome 1 were available. Based on the aforementioned criteria, it can be concluded that no evidence of LD was detected among the 2 pairs of SNP loci.

Table 3.

SNP loci and candidate genes associated with eggshell translucency.

SNP Loci Chromosome Physical location Variation type Candidate gene
Affx-50362599 1 44373423 A>G DCN, BTG1
rs316121113 1 91718015 T>C ZFP92, POU2F1
rs317389181 5 11925413 T>C NUCB2, FTL
rs15050262 19 8178375 C>T GGNBP2
rs312943734 19 8568577 T>C ACACA, TADA2A
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Table 4.

Pairwise SNPs linkage disequilibrium measures for between Affx-50362599, rs316121113 and rs15050262, rs312943734 in 3 laying chicken strains.

SNP1 SNP2 D' r2
Affx-50362599 rs316121113 - -
rs15050262 rs312943734 0.378 0.131
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In previous reports, the main physiological processes and functions of DCN, TADA2A, ZFP92, and GGNBP2 genes in laying hens have not been examined by scientists. While DCN and TADA2A are primarily implicated in regulation in mammals, their roles in poultry have not been explored. Such as extracellular signaling, development or regulation of other physiological processes were involved by DCN (Karamanos et al., 2018; Mochizuki et al., 2020), which can promote the corneal development of human and mouse eyes (Rodahl et al., 2006; Hill et al., 2018). TADA2A is involved in histone H3/H4 acetylation, which enhances nucleosome affinity with histone H1 and promotes chromosome remodeling (Lee et al., 2021; Qian et al., 2005). Moreover, TADA2A plays a crucial role in its acetyltransferase activity and the repair of UV-induced DNA damage in humans (Huang et al., 2012). In contrast, ZFP92 and GGNBP2 primarily participate in the expression process of human sperm. Variations in the ZFP92 gene have been associated with non-obstructive azoospermia (NOA) in humans (Zhuang and Liu, 2017), and ZFP92 has been shown to be absent in female ovaries (Ramos, 2022). GGNBP2 significantly influences the morphology and function of Sox9-positive Sertoli cells during the spermatogenesis process and is closely linked to sperm production (Ohbayashi et al., 2001; Chen et al., 2017).

BTG1 primarily regulates the initial differentiation of chondrocytes and tendon cells, which has been demonstrated by Lorda-Diez et al. (2016). On the other hand, POU2F1 is found to be highly expressed in various types of tumors, including osteosarcoma (Jeong et al., 2014), gastric cancer (Xie et al., 2016), and head and neck squamous cell carcinoma (Sharpe et al., 2014). It has been shown that POU2F1 enhances the activity of cancer cells by activating the expression of specific genes, as evidenced by studies conducted by Li et al. (2021) and Wang et al. (2021). Moreover, both BTG1 and POU2F1 play crucial roles in chick development. BTG1 exhibits high expression during spinal development (Kamaid and Giraldez, 2008), while POU2F1 regulates the response of chicks to stress during late development by binding to CpG at the distal end of the HSP70 promoter through methylation (Kisliouk et al., 2017).

In the investigation of the underlying mechanisms of translucent egg formation, scientists have reached a consensus that water is the primary cause of the direct translucency of eggs (Holst et al., 1932; Tyler and Geake, 1964; Tyler and Standen, 1969). Based on previous studies (Wang, 2017) and our own experiments, we propose that the susceptibility of the eggshell membrane to breakage is the fundamental reason for the formation of translucent eggs. The trait of eggshell translucency is a complex characteristic that is produced within the eggshell and may be influenced by either a single differential gene or the interaction of multiple differential genes. Nie (2013) and Jiang (2015) reported a decrease in the expression levels of CaBP mRNA and Ca2+-ATPase mRNA in laying layers with translucent eggs. Additionally, Foo et al. (2010) found that NUCB2 could regulate the maintenance of Ca2+ levels in the body, serving as a universal sensor involved in Ca2+ signal transduction, and that the NUCB2 protein possesses a Ca2+-dependent mosaic structure (Skorupska et al., 2020). Therefore, we propose that NUCB2 may affect mRNA expression by influencing the Ca2+ signaling pathway and subsequently impacting the ultrastructure of the eggshell, thereby facilitating water accumulation within the eggshell and leading to eggshell translucency. Previous investigations demonstrated that the protein content and total amino acid levels of the eggshell membrane in the translucent group were significantly lower than those in the opaque group, and the maximum longitudinal tensile force of the eggshell membrane was reduced (Wang, 2017; Zhang et al., 2019). Ovotransferrin, a matrix protein present in the eggshell membrane, is primarily responsible for the transport and metabolism of Fe2+ (Gautron et al., 2001). Conversely, the light chain ferritin encoded by the FTL gene predominantly binds and stores Fe2+ within cells (Cadenas et al., 2019). Additionally, the structure of chicken ferritin differs from that of mammals, consisting solely of H-chain subunits that enhance the protein's functionality (Levi et al., 1988; Levi et al., 1989). Thus, we hypothesize that FTL may affect the structure of the eggshell membrane by influencing the expression of ovotransferrin, leading to increased permeability and water penetration of the eggshell membrane, consequently resulting in eggshell translucency. He (2021) reported significant differences in the glycerophospholipid metabolism and linoleic acid metabolism pathways, among other 13 metabolic pathways, between translucent and opaque eggs, with the ACACA gene playing a central regulatory role in fat synthesis (Takai et al., 1988; Wang et al., 2005). Furthermore, this gene is involved in the liver glycolipid metabolism process in laying hens (Liu et al., 2019). Therefore, it is speculated that the lipid metabolism in hens laying translucent eggs is inhibited, leading to decreased expression of genes involved in eggshell film synthesis or protein composition, resulting in a thinner eggshell film and the subsequent formation of eggshell translucency.

CONCLUSIONS

In this study, genotyping was employed to conduct a further screening of 170 single nucleotide polymorphisms (SNPs) loci that have the potential to influence the trait of eggshell translucency, which have been investigated by our research group. Loci exhibiting low gene polymorphism, genetic effects contributing less than 1%, and deviations from the Hardy-Weinberg equilibrium were eliminated using triple SNP flight mass spectroscopy. Ultimately, a total of 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were screened. These SNPs were associated with 9 candidate genes, namely DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A. Building upon previous investigations into the underlying mechanisms of eggshell translucency, it is postulated that NUCB2, FTL, and ACACA genes may influence the characteristics of eggshell translucency.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS

The work was supported by the Modern agricultural industry technology system of layer industry innovation team in Hebei (HBCT2024240204), Natural Science Foundation for The Excellent Youth of Hebei Province, China (C2023204186), the National Natural Science Foundation of China (31902141), Key Research and Development Project of Hebei Modern Breeding Science and Technology Target Project (20326631D).

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