Genome-wide Association Studies And Measurement Of Eggshell Ultrastructure And Crystal Structure Of Chicken Eggs

Eggshell provides a relatively independent and stable internal environment,as well as the source of calcium for embryonic growth and development.Improved quality eggshell could not only lower the risk in food safety,but also reduce the economic losses from the damage of eggshell,and increase the economic efficiency.On the other hand,the formation process of eggshell is also a classic biomineralization model.In this study,we constructed the F2 population derived from the cross of White Leghorn and Dongxiang chickens and measured eggshell ultrastructure and crystal structure.Genome-wide association studies were conducted to reveal the genetic basis of eggshell ultrastructure and crystal structure.927 hens from F2 population were selected for SNP genotyping.Eggshells were collected one per hen at 66 weeks to measure eggshell thickness(EST),effective layer thickness(ET),mammillary layer thickness(MT),mammillary density(MD)under the scanning electron microscope.Linear mixed model was used for genome-wide association analysis.We found that the coefficients of variances of EST,ET,MT and MD were 12.17%,14.83%,20.44%and 23.83%,and their SNP-based heritabilities were 0.39,0.36,0.17 and 0.19 respectively.719,784,1 and 10 genome-wide significant SNPs were associated with EST,ET,MT and MD separately.Those SNPs were located in the region from 59.4 Mb to 68.5 Mb in GGA1 for ET and EST,20.6 Mb to 21.4 Mb in GGA6 for MD and around 4.9 Mb in GGA9 for MT.Four candidate genes,ABCC9,KCNJ8,ITPR2,WNK1 were considered relating to ET and EST for their regulation roles in uterus ions transport system.MT was determined by both of physical space around mammillae affected by mammillary density and physiological process of calcium carbonate deposition regulated by the protein interactions of ITM2C-RIT2-Calmodulin-ITPR.MD might be regulated by the modification of KNDC1 to fiber core proteins at nucleation sites directly or indirectly.The crystal parameters were determined using a Rigaku R-AXIS SPIDERX X-ray diffraction.The integral intensity of the first nine major diffraction peaks,total integral intensity(TA)and crystal orientation degree(OD)were obtained.Genome-wide association studies were performed using a mixed linear model.The results showed that TA had a positive correlation with the ultrastructure and common quality traits of eggshells.There was a negative correlation between TA and OD,indicating that the larger the crystals,the lower the degree of orientation,the more prone to random distribution.The coefficients of variances of TA and OD were 9.4%and 23.73%,and their SNP-based heritabilities were 0.23 and 0.06.621 SNPs located in 55.7?69.3 Mb in GGA1 were significantly associated with TA.According to their mRNA and protein expression in uterine tissue cells and their function,we suggested that DERA gene may play an important role in the regulation of eggshell crystal growth.There might be three possible pathways:a)DERA provides energy for the synthesis of matrix protein and ions transport in the uterine tissue;b)DERA reduces the stress of high Ca2+ concentration to cells to maintain cell activity,and provides Ca2+ stably for the growth of eggshell crystals;c)DERA affects the growth of eggshell crystals through its regulation on uterine fluid ATP concentration.