A first complete catalog of highly expressed genes in eight chicken tissues reveals uncharacterized gene families specific for the chicken testis.

Based on next-generation sequencing, we established a repertoire of differentially overexpressed genes (DoEGs) in eight adult chicken tissues: the testis, brain, lung, liver, kidney, muscle, heart, and intestine. With 4,499 DoEGs, the testis had the highest number and proportion of DoEGs compared with the seven somatic tissues. The testis DoEG set included the highest proportion of long noncoding RNAs (lncRNAs; 1,851, representing 32% of the lncRNA genes in the whole genome) and the highest proportion of protein-coding genes (2,648, representing 14.7% of the protein-coding genes in the whole genome). The main significantly enriched Gene Ontology terms related to the protein-coding genes were "reproductive process," "tubulin binding," and "microtubule cytoskeleton." Using real-time quantitative reverse transcription-polymerase chain reaction, we confirmed the overexpression of genes that encode proteins already described in chicken sperm [such as calcium binding tyrosine phosphorylation regulated (CABYR), spermatogenesis associated 18 (SPATA18), and CDK5 regulatory subunit associated protein (CDK5RAP2)] but whose testis origin had not been previously confirmed. Moreover, we demonstrated the overexpression of vertebrate orthologs of testis genes not yet described in the adult chicken testis [such as NIMA related kinase 2 (NEK2), adenylate kinase 7 (AK7), and CCNE2]. Using clustering according to primary sequence homology, we found that 1,737 of the 2,648 (67%) testis protein-coding genes were unique genes. This proportion was significantly higher than the somatic tissues except muscle. We clustered the other 911 testis protein-coding genes into 495 families, from which 47 had all paralogs overexpressed in the testis. Among these 47 testis-specific families, eight contained uncharacterized duplicated paralogs without orthologs in other metazoans except birds: these families are thus specific for chickens/birds.NEW & NOTEWORTHY Comparative next-generation sequencing analysis of eight chicken tissues showed that the testis has highest proportion of long noncoding RNA and protein-coding genes of the whole genome. We identified new genes in the chicken testis, including orthologs of known mammalian testicular genes. We also identified 47 gene families in which all the members were overexpressed, if not exclusive, in the testis. Eight families, organized in duplication clusters, were unknown, without orthologs in metazoans except birds, and are thus specific for chickens/birds.