DE NOVO ASSEMBLIES OF GENOMES OF FOUR INDIGENOUS CHICKENS AND A WHITE EARED PHEASANT REVEAL GENETIC BASIS OF CHICKEN DOMESTICATION AND ALTITUDE ADAPTATION

High-quality assembly and annotation of the genome of a species are critical in understanding the genetic basis of almost all aspects of the biology of the species. Although many genome assembly pipelines have been developed, they are either difficult to use or their assemblies are too fragmental. Moreover, although gene annotation pipelines have been developed at large genome centers, they are either too complicated for individual labs to use or not available to public. In this dissertation project, we have proposed a user-friendly pipeline that can assemble genome at chromosome-level with high-quality using PacBio/Nanopore long reads, Illumina paired-end short reads and Hi-C paired-end short reads. We have also developed an accompanying gene annotation pipeline using a combination of homology-based and RNA-based approached. The pipeline achieves high accuracy in protein-coding gene and pseudogene annotations.
Moreover, although multiple chicken genomes have been assembled, high-quality indigenous chicken genomes are still lacking, hampering the understanding of chicken domestication and evolution. Using the pipelines, we assembled and annotated the genomes of four indigenous chickens with distinct morphological traits at the chromosome-level. Our results challenge two earlier conclusions regarding chicken domestication and evolution. First, we found a total of 1,420 new protein-coding genes in the four chickens and recovered 51 of the 274 “missing” genes in birds in general and 36 of the 174 “missing” genes in chickens in particular. Most of these new genes are also found in previously assembled GRCg6a and GRCg7b/w chicken genomes, and might play house-keeping roles. Counting these new genes, chicken genomes encode more genes than originally thought. Second, we identified a total of 2,015 non-processed pseudogenes in the seven genomes. Most pseudogenization mutations are fixed in their respective populations and preferentially occur at the two ends of genes. Purifying selection is relaxed on the pseudogenes, suggesting that they might lose their gene functions. Pseudogenization mutations segregate in the chickens as their phylogenetic tree does, which is based on more than 6,000 essential protein-coding genes. Thus, in contrast to the previous conclusion, loss-of-function mutations play a critical role in chicken domestication and evolution. Moreover, these assembled genomes are valuable resources for studying chicken domestication and evolution.
Furthermore, although many studies related to artificial selection signatures of commercial and indigenous chickens have been carried out, quite a small number of genes have been found to be under selection. To fill these gaps, we re-sequenced 85 individuals of five indigenous chicken breeds with distinct traits from Yunnan, a southwest province of China. By analyzing these indigenous chickens together with 116 individuals of commercial chickens (broilers and layers) and 35 individuals of red jungle fowl (RJF), we find a substantially large number of selective sweeps and affected genes for each chicken breed using a rigorous statistic model than previously reported. We confirm most of previously identified selective sweeps and affected genes. Meanwhile, the vast majority (~98.3%) of our identified selective sweeps overlap known chicken quantitative trait loci. Thus, our predictions are highly reliable. For each breed, we also identify candidate genes and selective sweeps that might be related to the unique traits of the chickens. Most of these genes do not contain nonsense mutations, we therefore quantified the expression levels of eight genes in relevant tissues using RT-qPCR and found that most of them showed differential expression compared to their counterparts.
Finally, eared pheasant species are closely related but inhabit at highly varying altitudes from northeast to southwest China. To understand genetic bases of closely related species to adapt to different altitudes, we sequenced a population of 10 white eared pheasants (WT) (Crossoptilon crossoptilon) inhibiting in 3,000~4,300 m altitude niches in Yunnan, China, and assembled the genome of an individual at chromosome-level with a contig N50 of 19.63 Mb, a scaffold N50 of 29.59 Mb, and a total length of 1.02 Gb. This assembly with only few gaps is of higher quality than a previous one of a brown eared pheasant (BR) (C. mantchuricum) individual living at 20~1000 m altitude in northeast China. Interestingly, the WT genome encodes more protein genes than the BR genome (16,315 VS. 15,003), while the later contains more pseudogenes than the former (1,519 VS. 1,976). The two genomes shared 14,178 genes and 1,040 pseudogenes with 2,137 and 825 unique genes, and 479 and 936 unique pseudogenes, respectively. The unique genes and unique pseudogenes of both species are mainly involved in biological pathways of cardiovascular, energy metabolic, neuronal and immune functions, which are known to be related to adaptation to high altitude. Moreover, we compared the selective sweeps in the genomes of WT, BR and an additional species blue eared pheasant (BL) (C. auritum) inhibiting at 1,500~3,000 m altitude in central west China, using re-sequencing data of 10 WT, 12 BL and 41 BR individuals, respectively. Interestingly, genes under selection in each species converge on the same pathways of the aforementioned four functional categories. These results suggest that these species adapted to highly varying altitudes by loss-of-function mutation and fine-tuning of genes in these common pathways. Our assembled WT genome and re-sequencing data can be valuable resources for studying the biology, evolution and developing conservation strategies of these endangered species.