Abstract：Blue eggshell is an important economic trait in the poultry, as it presents the thicker eggshell and plays more important role in filtering solar radiation and damage resistance than the brown eggshell and the white eggshell. Now the genetic basis underlying blue eggshell in the chicken (Gus gallus) has been uncovered. However, its genetic mechanism remains unknown in ducks (Anas platyrhynchos domesticus). In this study, to comprehensively analyze the transcriptome basis of the blue eggshell in ducks, homozygous blue-shelled and homozygous white-shelled Shaoxing ducks were bred by using the test cross, and transcriptome sequencing of uterus from the 2 genotypes was performed using the Illumina HiSeq 2500 platform with 3 biological replicates per genotypes. After removing sequencing adaptors and the low-quality reads, we totally obtained 288 008 582 clean reads, and 71.62% of them were successfully mapped to the duck genome. 93.04% of the mapped reads were uniquely aligned to the duck genome. These mapped reads were assembled into 64 587 transcripts, including 19 589 novel ones. Based on these RNA-seq data 5 known alternative splicing event, including Skipped exons (SE), Retained intron, Alternative 5' splicing site (A5SS), Alternative 3' splicing site (A3SS) and Mutually exclusive exon (MEX) were also analyzed, and 20 302 alternative splicing events were identified. Among these alternative splicing event, SE was found to be the major type, with the times of 8388, followed by 4891 A5SS and 3649 A3SS, and MEX was the rarest events, happening 603 times. In this study, reads per kilobase per million of mapped reads (RPKM) was calculated for gene expression. Based on the RPKM value, 21 829 genes were expressed (RPKM≥1), and most of these genes were expressed between 1 to 100 RPKM. Further differential expression analysis between the 2 genotypes showed that 1 768 genes exhibited differential expression (P＜0.05,｜fold change｜≥1.5), including 700 up-regulated and 1 068 down-regulated in homozygous blue-shelled ducks when compared with the white-shelled individuals. Among these differentially expressed genes we found that several genes were related with the main pigments (Protoporphyrin-IX, biliverdin, and biliverdin zinc chelate) of the eggshell, such as biliverdin reductase A (BLVRA), uroporphyrinogen Ⅲ synthase (UROS), hemoglobin subunit alpha-1 (HBA1), heme binding protein 1 (HEBP1), and so on. To verify the gene expression data from the RNA-seq, Real-time PCR was carried out for 4 randomly selected genes, including ferrochelatase (FECH), heme oxygenase 1 (HMOX1)、RasGEF domain family member 1B (RASGEF1B) and polycystic kidney disease 2 (PKD2). Results showed that all of the 4 genes exhibited consistent expression pattern in Real-time PCR with that of RNA-seq. Further Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these differentially expression genes took part in numerous functions and pathways, including functions associated with heme and bile metabolism, such as heme binding, tetrapyrrole binding, primary bile acid biosynthesis and bile secretion. This study provides the first transcriptome basis of eggshell color in ducks, and the differentially expressed genes identified in this study will provide valuable resources for the further genetic analysis of the eggshell color in ducks.
