Genomic analysis reveals variant association with high altitude adaptation in native chickens

Native chickens are endangered genetic resources that are kept by farmers for different purposes. Native chickens distributed in a wide range of altitudes, have developed adaptive mechanisms to deal with hypoxia. For the first time, we report variants associated with high-altitude adaptation in Iranian native chickens by whole genome sequencing of lowland and highland chickens. We found that these adaptive variants are involved in DNA repair, organs development, immune response and histone binding. Amazingly, signature selection analysis demonstrated that differential variants are adaptive in response to hypoxia and are not due to other evolutionary pressures. Cellular component analysis of variants showed that mitochondrion is the most important organelle for hypoxia adaptation. A total of 50 variants was detected in mtDNA for highland and lowland chickens. High-altitude associated with variant discovery highlighted the importance of COX3, a gene involved in cell respiration, in hypoxia adaptation. The results of study suggest that MIR6644-2 is involved in hypoxia and high-altitude adaptations by regulation of embryo development. Finally, 3877 novel SNVs including the mtDNA ones, were submitted to EBI (PRJEB24944). Whole-genome sequencing and variant discovery of native chickens provided novel insights about adaptation mechanisms and highlights the importance of valuable genomic variants in chickens.

as the most importance biological pathway.
In males, the results of molecular function analyses showed that damaged DNA binding protein (MSH3) was shared mostly between highland and lowland chickens and it involves in DNA repair process. Midbody structure (CEP55) and cell proliferation (ROS1) had the most frequency in cellular component and biological process analysis. More results of gene ontology analysis are shown in supplementary Tables S6 and S7 online.

Discussion
Common variants analysis indicated that shared variants are involved in cell survival, protein modification and reproduction. Several candidate genes such as ROS1, PRSS3, BRCA2, TDRD9, POT1, CEP55 were detected for cell proliferation, digestion, chromosome organization, RNA processing, telomere capping and cytokinesis, respectively. In human, BRCA2 (breast cancer 2) is involved in chromosomal damage and DNA repair. The mutated BRCA2 gene leads to unrepair DNA and finally breast cancer 1 . Furthermore, we showed that the regulation of ERK (extracellular signal-regulated kinases) was shared between highland and lowland samples. ERK is involved in mitosis, meiosis and post meiotic function in different cells.
The results of gene ontology indicated that protein structure modification was shared between highland and lowland chickens. For example, protein depalmitoylation is a dynamic post translational modification. In this biological process, 16-carbons fatty acidis added to cysteine of protein and finally it is removed by acyl protein thioesterases (APTs). This modification leads to modulation of protein sorting, targeting and signaling 2 . Additionally, here we suggested LYPLAL1 gene as a candidate gene for this biological process.
LYPLAL1 gene (Lysophospholipase like 1) has depalmitoylating activity and able to hydrolyze only short chain substrates due to its shallow active site (Entrez gene: 127018). In addition, the results of molecular function analysis showed that palmitoyl-(protein) hydrolase activity function can be considered for protein modification as was described. CEP55 gene was reported for establishment of protein localization in cells.
Reproduction is one of the most important factors for species survival. Therefore, it seems logical that there should be common variants for reproduction between highland and lowland chickens. Our study identified TDRD9 gene for P granule. Vornina (2013) 3 , reported that P granule are conserved cytoplasmic organelles that are present in C.elegans and germ cells. In embryogenesis process, P granules are segregated asymmetrically into those blastomeres and finally the germ line is produced. There is correlation between P granules distribution and germ line developments. Consequently, P granules might have been associated with germ line specification and differentiation 4 . Also, in humans, TDRD9 (Tudor Domain Containing 9) has critical role in spermatogenesis and germ line integrity (UniProtKB: Q8NDG6).
Differential variants analysis showed that adaptive variants were classified to ion channels, PeBoW complex and histone binding.
Previous studies have noted that there is association between cell viability and hypoxia condition. Ions channels have critical role in regulating most of the biological process (e.g., T cell activation, apoptosis, glucose metabolism, pancreatic β cell insulin release and transport of nutrients) 5-6 . Therefore, cell function depends on ions and their performance directly. K + channels are blocked by hypoxia condition, thus, the K + efflux is reduced in intracellular. Finally, it causes increased Ca 2+ ion 5 . An increase in Ca 2+ leads to cell growth, proliferation and inhibition of cell apoptosis 7 . In this study, ATP13A4 gene (ATPase type13A4) was found to be associated with hypoxia adaptation. Vallipuram et al. (2010) 8 illustrated that ATP13A4 gene may be involved in calcium regulation. They showed that over-expression of ATP13A4 in COS-7 cells lead to increase in the intracellular calcium level. These findings further support the idea of Wang et al. (2015) 9 who reported that the genes involved in Ca 2+ signaling pathway can be considered as candidate genes for adaptation of high altitude chickens to hypoxia.
PeBoW complex is checkpoint response between nucleus and cell cycles in mature cells. It is located in nucleus and involved in ribosome biogenesis specially 60s ribosomal subunit and cell proliferation. In mammalian cells, it consists of three proteins Pes1 (pescadillo), Bop1 (block of proliferation) and WDR12 (WD-repeat protein) [10][11] . In human cells, PeBow complex is linked to another process such as mitosis. For example, inactivation of Pes1 and Bop1 leads to abnormal mitosis 12 . Furthermore, over expression of PeBoW complex proteins has been detected in cancers 13 . Pervious evidence suggests that the signal stress such as hypoxia and heat shock causes disruption of PeBoW complex function and finally cell cycle process 14 18 who showed that there was association between histone binding and high altitude conditions.
In this study, sex chromosomes were also analyzed. Several GO terms were reported for common and differential variants. Differential variants analysis showed that there were two GO terms as molecular function in females on chromosome Z (  21 showed that the amount of alanine is decreased in hypoxic cell compared with normoxic cells. In the other words, the decrease of alanine might be due to the conversion to pyruvate and finally ATP production. In males, common variants analysis indicated several GO terms were associated with Y-form DNA binding, loop DNA binding and damaged DNA binding (  The main goal of parameter optimizing was to decrease in false variants. In each step the threshold of parameters was increased and it leads to decrease in number of variation. Finally, parameters in step 3 were selected for variant detections.   Male  333262  280411  11049  16557  22755  2490  Male  419734  352375  13446  21422  29175  3316  Male  405642  349559  15680  15233  22726  2444  Female  248202  213114  10032  9704  13739  1613  Female  262688  222119  9511  12385  16643  2030 Variants of highland chickens were compared with reads of lowland chickens as a control tool in order to remove the common variation between lowland and highland samples. Then, a comparison was carried out within the male and female birds based on the frequency threshold optimization information (TableS4). Finally, differential variants are kept for further analysis including filtrations and gene ontology enrichment analysis. Five frequency thresholds were considered in order to be utilized in the frequency threshold optimization (0, 25, 50, 75 and 100). The percentage of samples that have variants is considered as the threshold frequency. Thus, frequency thresholds of 50% and 100% were determined in order to make comparisons within males and females groups. Consequently, a total of 97610 and 17024 variants were detected as differential variants between highland and lowland samples. Table S5: The results of common variant detection between highland and lowland chickens   Sex  Total variants New variants Coding variants Amino acid changes  Male  35090  9810  504  270  Female  18690  7025  937  246  Total  53780  16835  1441  516 In current study, common variants were collected between highland and lowland chickens and classification of them were performed based on available annotations. Novel variants: variants were reported for the first time.

Supplementary references
Coding variants: variants were located in coding regions. Amino acid changes: variations can change protein sequence.    In this study, differential variants between highland and lowland chicken, and mt-DNA variants were filtered based on known variant annotation. Consequently, many variants are discovered as novel variants for the first time. Finally, a total of 3877 SNVs variants are collected and are also submitted to EBI as novel variants with an accession number of PRJEB24944, for the first time Description Chromosome Counts type Novel differential variants between highland and lowland chickens -females Z 112 SNV Novel differential variants between highland and lowland chickens -males 276 Novel differential variants between highland and lowland chickens -females Somatic 591 Novel differential variants between highland and lowland chickens -males 2851 Novel mt-DNA variants, highland chickens-female Mt-DNA 8 Novel mt-DNA variants, highland chickens-male 21 Novel mt-DNA variants, lowland chickens-female 10 Novel mt-DNA variants, lowland chickens-male 8 Figure S1: The Summary of signature selection analysis of native chicken ecotypes based on FST calculation.