Abstract Background Although multiple chicken genomes have been assembled and annotated, the number of protein-coding genes in chicken genomes is still uncertain due to the low quality of these genome assemblies and limited resources used in gene annotations. Results To fill the gap, we annotated our four recently assembled high-quality genomes of four indigenous chickens with distinct traits using a combination of RNA-seq- and homology-based approach. Our annotated genes in the four chickens recovered 51 of the 274 “missing” genes in birds in general and 36 of the 174 “missing” genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in each chicken breed, we found a total of 1,420 new protein-coding genes in the four chicken genomes, which were missed in the reference chicken genome annotations. These newly annotated genes (NAGs) tend to have high G/C contents and be located in sub-telomeric regions of almost all assembled chromosomes and some unplaced contigs. The NAGs showed tissue-specific expression and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. We found that most of the NAGs also are encoded in previously assembled chicken genomes. The NAGs form functional modules with homology-supported genes that are involved in many important biological pathways. We also identified numerous unique genes in each indigenous chicken genome that might be related to the unique traits of each breed. Conclusion The ubiquitous presence of the NAGs in various chicken genomes indicate that they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in sub-telomeric regions with high G/C contents