Study of Differential Genes Expression of Uterine tissue Related to EggShell Using Transcriptomics data

Introduction: Due to the high importance of egg shell quality in terms of hardness, extensive transportation with high volume of this product between different countries, the importance of shell quality in terms of protecting the internal content from extra-shell contamination and also one of the ways to improve traits, relying on The genetic structure of a trait is the application of genetic selection. Therefore, the aim this study is to use transcriptome data obtained from uterine tissue of two groups of laying hens with hard and weak eggshell, in each of the three stages of the calcification period (initiation, growth and termination). Gene expression profile was determined and differential expression of genes is analyzed so that different index genes can be expressed and their ontology can be studied to help the results. As a result, the genetic structure of the trait and the list of index genes have been regularly completed, and with the information obtained from the present study, the correct and practical genetic selection can be made in laying hen breeds.Materials and Methods: The aim of this study was to investigate the gene expression profile between two groups of chickens with hard and weak eggshells in three stages of calcification period (initiation, growth and termination), to identify significant index genes with different expression and analysis. Ontology and the paths involved are related to them. Therefore, in this study, transcripts (total mRNA sequences) of 18 samples of uterine tissue including three replicates in each stage of each group were prepared. The process of converting the initial format of readings was done using SRAtoolkit software, measuring the quality of readings with fastQC software, editing readings with Trimmomatic software and re-measuring the quality of readings after editing with fastQC software; Also, the alignment and mapping of the readings were done using Hisat2 software and format conversion and sorting of results was performed using Samtools software. Finally, the differential expression analysis of genes was performed using Cufflinks software package (Cufflink, Cuffmerge and Cuffdiff software) and Then, considering p-value <0.00025 and the criterion for comparing the value of differential gene expression, log2 (fold_change) less than -4 and greater than +4 were identified as the most significant genes with different and significant expression. Histogram of the results was performed using CummeRbund package in R software environment and ontological analysis was performed using DAVID and Ensembl databases.Results and Discussion: According to the results of gene expression profile, 28833 genes were identified on the transcript of the samples. Finally, by analyzing the differential expression of genes, 1338 genes at the initiation stage, 81 genes at the growth stage and 190 genes at the termination stage were identified with significantly different expressions, that Considering log2(fold_change) > +4 and log2(fold_change) < -4, 51 genes at the initiation stage, two genes at the growth stage and four genes at the termination stage had different and significant expression. The largest difference in gene expression between the two groups at the initiation stage, ENSGALG0000000044418 gene with log2 (fold_change) equal to 8/63 and at the growth stage, ENSGALG0000000049618 gene with log2 (fold_change) equal to 6/82 and at the termination stage, ENSGALG0000000049618 gene with log2 (fold_change) equal to 5/14 was observed. Ontological analysis of index genes showed that they are mainly involved in protein binding activities (ENSGAL000000444418, NMRAL1, SLITRK4, CHL1, ENSGAL00000009041, PHACTR3 and CORIN genes), DNA transcription regulation (LHX1, TFAP2A, RUNX3, ETV1, NR4A3 and RUNX1 genes), immune response (ENSGAL0000000043996, TMEM117, AvBD9, GCNT3, ENSGAL00000046947 and ENSGAL00000051617 genes), fat metabolism (PNPLA3, NR4A3, CD36, FFAR4 and DGKB genes) and calcium ion binding (ANXA10, CAPN8, CDH18 and DGKB genes), respectively.Conclusion: In the differential expression analysis of genes, considering log2 (fold_change) more than +4 and less than -4, in the initial stage, 51 genes, in the growth stage, two genes and in the termination stage, four genes were identified. The highest differences in gene expression between the two groups were observed in the initial stage, ENSGALG00000044418 and PDZK1IP1 genes, in the growth stage, ENSGALG00000049618, ENSGALG00000048945 and TMEM63C genes and in the termination stage, ENSGALG000000004400 and ENSG26 genes; The results of ontological analysis of index genes showed that they are mainly involved in protein binding, DNA transcription regulation, immune response, lipid metabolism and calcium ion binding, respectively.