The draft genome of the Temminck's tragopan (Tragopan temminckii) with evolutionary implications

doi:10.21203/rs.3.rs-3240668/v1

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The draft genome of the Temminck's tragopan (Tragopan temminckii) with evolutionary implications

https://doi.org/10.21203/rs.3.rs-3240668/v1

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published 07 Dec, 2023

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Background

High-quality genome data of birds play a significant role in systematic studying their origin and adaptive evolution. The Temminck's tragopan (Tragopan temminckii) (Galliformes, Phasianidae), a larger pheasant, is one of the most abundant and widely distributed species of the genus Tragopan, and was defined as class II of the list of national key protected wild animals in China. The limited genomic data restricted evolutionary trait studies of this taxa.

Results

The whole genome of the Temminck's tragopan was sequenced using Illumina and PacBio platform, and then de novo assembled and annotated. The genome size was 1.06 Gb, with a contig N50 of 4.17 Mb. A total of 117.22 Mb (11.00%) repeat sequences were identified. 16,414 genes were predicted using three

methods

, with 16,099 (98.08%) annotated as functional genes based on five databases. In addition, comparative genome analyses were conducted across 12 Galliformes species. The

results

indicated that T. temminckii formed a basal position of the clade including Lophura nycthemera, Phasianus colchicus, Chrysolophus pictus, Syrmaticus mikado, Perdix hodgsoniae, and Meleagris gallopavo, with a corresponding divergence time of 31.43 million years ago (MYA). Expanded gene families associated with immune response and energy metabolism were identified. Genes and pathways associated with plumage color and feather development, immune response, and energy metabolism were found in positively selected genes (PSGs).

Conclusions

A genome draft of the Temminck's tragopan was reported, genome feature and comparative genome analysis were described, and genes and pathways related to plumage color and feather development, immune response, and energy metabolism were identified. The genomic data of the Temminck's tragopan considerably contribute to the genome evolution and phylogeny of the genus Tragopan and the whole Galliformes species underlying ecological adaptation strategies.

Temminck's tragopan

genome feature

comparative genomics

positive selection

Birds are the most species-rich monophyletic group of land vertebrates [1], containing generally smaller genome sizes ranging from 1 Gb to 2.1 Gb [2]. Compared with other vertebrates, avian genomes exhibited characteristics of relatively small, conserved synteny, and few repetitive elements, which made them suitable species used for evolutionary research [3]. The third-generation sequencing made it possible to provide reliable genome assembly, and currently, PacBio and Oxford Nanopore are the most popular technologies to generate long reads [4]. The advance in sequencing technologies have been facilitated genome data obtaining of avian species. The genome sequences of birds, together with the collection of their morphological, physiological, ecological, and behavioral traits, could provide information on studies of evolution, ecology, population genetics, neurobiology, development, and conservation [5]. For example, obtained avian genomic data were significant for understanding the origin and evolution of traits, and increasing genome sequences were focused on the evolution of powered flight, body size variation, beak morphology, plumage coloration, high-elevation colonization, migration, and vocalization [3], as well as aspects of phylogenetic evolution and adaption [6, 7], transposable elements (TEs) [1], vocal learning [8], and genome size evolutionary dynamics [2], etc.

The genus Tragopan belongs to Galliformes, Phasianidae, and consists of five species including T. melanocephalus, T. satyra, T. blythii, T. temminckii, and T. caboti [9]. The Temminck's tragopan (T. temminckii), a larger pheasant, is one of the most abundant, and widely distributed species of the genus Tragopan [10], and was defined as class II of the list of national key protected wild animals in China [11]. It is mainly distributed in southwest areas of China [12], including provinces of Sichuan, Yunnan, Xizang, and Chongqing, etc, and in abroad of India, Myanmar, and Vietnam [13, 14]. This species had a suitable altitude for survival of 1000–3500 m [12]. The Temminck's tragopan is an omnivorous bird, mainly with plant-based food [15]. For example, previous studies found that this species feeds chiefly on herbs and ferns in spring and winter, while the food is mature fruits in summer and autumn [16].

Some studies related to the Temminck's tragopan were mainly concentrated on behavioral and ecological investigations, such as the activity rhythm [17–19], niche [20], diet [10, 16], flocking behavior [21], habitat [22, 23], and breed [24]. In addition, previous research have shown that the karyotype of T. temminckii was 2n = 80, with seven pairs of macrochromosomes and 33 pairs of microchromosomes [25]. For phylogenetic status, Tragopan had a closer relationship with the clade containing Lophophorus and Tetraophasis, which was supported by different data types, such as mitochondrial genomes [26–28], mitochondrial and nuclear DNA sequence data [29], and ultraconserved elements (UCEs) [30], then the clade of Tragopan/Lophophorus/Tetraophasis clustered with the clade containing the genus Chrysolophus, Phasianus, Lophura, Crossoptilon, Syrmaticus, Perdix, Pucrasia, Bonasa and Meleagris [26–28]. To date, limited information about the underlying adaptation of the Temminck's tragopan was known, and consequently, comprehensive genetic analyses based on large-scale genomic data are essential to understand their ecological and evolutionary adaptations.

Sexually dimorphic plumage coloration is common in birds, with plumage in males brighter than females, which phenomenon is associated with environmental constraints on sexual selection or intraspecific competition between males and females [31]. The Temminck's tragopan contains a very gorgeous feather color in the male, consequently, candidate genes with a significant role in its plumage color are essential to be explored using genome data. Plumage color, a highly polygenic trait, can be affected by multiple coding genes, regulatory genes, and gene-gene epistasis interactions [32]. To date, several genes associated with the plumage color in birds have been reported, such as MC1R, MITF, ASIP, TYRP1, and BCO2 [31, 33, 34]. Some candidate genes, such as MITF, EDNRB2, TBC1D22A, EDA, SLC45A2, and GOLGB1, have been identified to be related to avian plumage color based on genome-wide analyses [35, 36]. Among them, the MC1R gene, a melanogenesis-related gene with polymorphisms, has been found in several birds [33, 37, 38], and was identified to play a potential role in the coloration of melanin and carotenoids in the sexual dimorphism plumages [31]. For example, an association between the genetic variation of MC1R and plumage color was found in pigeons [33].

In this study, a de novo assembled genome data of the Temminck's tragopan was obtained combined with PacBio long reads and Illumina short reads, then comparative genomic analyses were carried out with the other 11 available Galliformes species. With genome-scale insights, the genomic characteristic, phylogeny, and evolution of the Temminck's tragopan were investigated, then candidate genes and signaling pathways associated with plumage color and feather development, immune response, and energy metabolism were explored. The assembled genome resource of the Temminck's tragopan will benefit researchers on their genetic mechanisms and ecological aspects.

Genome assembly and characteristic

In total, 93.71 Gb raw data of Illumina platform were generated, with a depth of 88.41× (Table S1), while 3,271,105 reads of PacBio platform were obtained, with a total base of 29,732,708,638 bp. The genome size of the Temminck's tragopan was 1.06 Gb, and the GC content was 42.09%. The contig N50 was 4.17 Mb, with the largest contig of 16.6 Mb. In total, the gene annotation predicted 16,414 genes across the genome. The average length of genes was 19,220.68 bp, with an average length of 161.49 bp in exons and 1,716.97 bp in introns, respectively. In addition, non-coding RNAs predicted results identified 200 miRNAs belonging to 95 families, 129 rRNAs of four families, and 289 tRNAs of 23 families, respectively. A total of 283 pseudogenes were also found.

BUSCO analysis evaluated the genome assembly completeness and showed that 4,438 complete BUSCOs (90.30%) were identified, including 4,368 single-copy (88.87%), and 70 duplicated BUSCOs (1.42%) (Fig. 1a). 241 BUSCOs (4.90%) were fragmented, and 236 BUSCOs (4.80%) were missing (Fig. 1a). Besides, 427 CEGs (93.23%) were identified in the CEGMA database, and 224 out of 248 highly conserved CEGs were found accounting for 90.32%. Furthermore, 99.01–99.89% of Illumina clean reads from five libraries were mapped to the assembly, with 91.20–96.09% properly mapped. These results suggested that assembled genome sequences of the Temminck's tragopan were relatively complete and high-quality genome data.

Repeats percentage was 11.00% with a total length of 117.22 Mb in the Temminck's tragopan. Long interspersed nuclear elements (LINEs) accounted for most repeats, occupying about 8.11%, while the proportion of short interspersed nuclear elements (SINEs) was 0.02% (Table S2). In addition, the total length of 1,685,298 bp was classified as simple sequence repeats (SSRs), with a proportion of 0.16% (Table S2).

Gene function

Overall, a total of 16,099 (98.08%) genes were functionally annotated based on five databases. Collectively, 9,260 (56.42%) out of 16,414 total gene sequences were functionally annotated to the GO database, as well as 9,963 genes (60.70%) in the KEGG, 11,642 genes (70.93%) in the KOG, 16,040 genes (97.72%) in the TrEMBL, and 16,074 genes (97.93%) in the NR, respectively. The distributions of GO terms within three main GO domains including the cellular component (CC), molecular function (MF), and biological process (BP) are presented in Fig. 2a. The cell part (5,813 genes, GO:0005623) and cell (5,804 genes, GO:0044464) were the main GO classes found in the CC section. The binding (5,232 genes, GO:0005488) and catalytic activity (3,166 genes, GO:0003824) were the major GO terms in the MF section, while the cellular process (5,145 genes, GO:0009987) and single-organism process (4,477 genes, GO:0044699) were the top two dominant GO classes in the BP section. These GO term numbers in the Temminck's tragopan were similar to other birds [39].

KEGG functional annotation results suggested that these processes were found in six sections, including metabolism (1,434 genes), human diseases (323 genes), environmental information processing (1,448 genes), cellular processes (1,189 genes), genetic information processing (1,132 genes), and organismal systems (757 genes), with corresponding lipid metabolism (336 genes), infectious diseases: viral (235 genes), signal transduction (956 genes), transport and catabolism (505 genes), folding, sorting and degradation (427 genes), and endocrine system (414 genes) the highest term, respectively (Fig. 2b).

Phylogeny and divergence time

Family classification results showed that a total of 21,803 gene families were identified, with 5,846 gene families commonly sharing among 12 sampled Galliformes species (Fig. S1a). The number of species-specific gene families ranged from one in Chrysolophus pictus to 302 in Pavo cristatus, with eight in T. temminckii (Fig. S1a). Further analyses showed that 8,807 gene families were commonly shared in closely related five species, including Perdix hodgsoniae, M. gallopavo, T. temminckii, P. cristatus, and G. gallus (Fig. 1b).

The resulting phylogenetic tree contained relatively higher bootstraps, with values of 100 in all nodes except for 99 of the node formed by Lophura nycthemera and Phasianus colchicus (Fig. S1b). The reconstructing result recovered the monophyly of Phasianidae, and phylogenetic relationships of ((((((((((L. nycthemera + P. colchicus) + C. pictus) + Syrmaticus mikado) + P. hodgsoniae) + M. gallopavo) + T. temminckii) + P. cristatus) + (C. japonica + G. gallus)) + C. virginianus) + Numida meleagris) was supported (Fig. S1b). The phylogenetic analysis supported that the sampled Phasianidae taxa were divided into two major groups, with group I consisting of two species including one partridge (C. japonica) and one pheasant (G. gallus), group II including two partridges (P. hodgsoniae and M. gallopavo) and six pheasants, respectively (Fig. S1b). T. temminckii formed a basal position of the clade containing L. nycthemera, P. colchicus, C. pictus, S. mikado, P. hodgsoniae, and M. gallopavo.

The divergence time between Phasianidae and Odontophoridae was 43.96 million years ago (MYA) (95% highest posterior density (HPD) = 38.43–50.19 MYA) (Fig. S1c). The divergence times of genera within Phasianidae ranged from 11.65 MYA between L. nycthemera and P. colchicus to 38.16 MYA between the clade of C. japonica/G. gallus and other sampled Phasianidae species (Fig. S1c), corresponding to Miocene and Eocene. T. temminckii diverged from the recent common ancestor approximately 31.43 MYA with 95% HPD = 28.17–36.46 MYA (Fig. S1c), corresponding to Oligocene.

The Temminck's tragopan genome showed high synteny with the chicken genome, with contig00419 and contig00124 in the Temminck's tragopan fused into chromosome 4 in chicken, and contig00295 and contig02393 fused into chromosome 5, respectively (Fig. S2).

Expansion and contraction

Comparative genomic investigation among 12 sampled Galliformes indicated that 27 expanded gene families and 14 contracted gene families were identified in the Temminck's tragopan (Fig. 1c). As functional identities presented in GO terms, the cell part (40 genes) and cell (40 genes) were the main GO terms found in the CC section, the binding (33 genes) was the major GO term in the MF section, and the cellular process (19 genes), biological regulation (19 genes, GO:0065007) and response to stimulus (18 genes) were three dominant GO classes in the BP section, respectively (Fig. S3a). Among GO functional enrichment results, several significantly expanded gene families were involved in the immune response including defense response to virus (GO:0051607) and immune response (GO:0006955) in the BP section, and energy metabolism containing fatty acid biosynthetic process (GO:0006633) in the BP section, and oxidoreductase activity (GO:0016491), GTPase activity (GO:0003924) and GTP binding (GO:0005525) in the MF section.

In addition, in the functional KEGG enrichment, the expanded gene families were identified with the immune response involving drug metabolism - other enzymes (ko00983), regulation of autophagy (ko04140), RIG-I-like receptor signaling pathway (ko04622), MAPK signaling pathway (ko04010), Toll-like receptor signaling pathway (ko04620), Jak-STAT signaling pathway (ko04630), and cytokine-cytokine receptor interaction (ko04060). These immune-related pathways have been also found in previous studies based on avian genomes, such as the MAPK signaling pathway [39], which may be related to reply to the environment, and could enhance their adaptations. These unique expansions of GO and KEGG enrichment in the Temminck's tragopan might be important for the ability of immune response and energy metabolism for their ecological environmental adaptation.

PSG analysis

The results indicated that a total of 2,022 genes were found under positive selection in the Temminck's tragopan. As functional enrichment analysis presented in the GO section Fig. S3b), the cell part (751 genes) and cell (749 genes) were the main GO terms in the CP section, the binding (705 genes) was the major GO classes in the MF section, and the cellular process (670 genes) were the top dominant of GO terms in the BP section, respectively. The top 10 significant GO terms were shown in Fig. S4, which revealed that the integral component of Golgi membrane (GO:0030173), ATP-dependent helicase activity (GO:0008026), and binding of sperm to zona pellucida (GO:0007339) were the most significant GO terms in a corresponding CC, MF, and BP section, respectively (Fig. S4a-c). While the phosphatidylinositol signaling system (ko04070) was the most significant KEGG term (Fig. S4d).

Functional annotation results of PSGs in the Temminck's tragopan identified pathways and genes related to plumage color and feather development, immune response, and energy metabolism. The contents related to plumage color and feather development were identified, including melanocyte differentiation (GO:0030318), hair follicle morphogenesis (GO:0031069), hair follicle development (GO:0001942), keratinocyte differentiation (GO:0030216) in the BP section in GO terms, and MAPK signaling pathway, Notch signaling pathway and melanogenesis pathway in the KEGG (Table S3). Nine PSGs including WNT10A, DVL1, ADCY5, MAPK1, CBP, tcf7l1, GNAO1, CAMK2G and EP300 were functional candidate genes involved in the melanogenesis pathway (ko04916). 12 candidate genes associated with plumage coloration were identified, such as SLC45A2 (Table 1). The 362 position of amino acid Q in the SLC45A2 gene was under significant positive selection effects (prob = 0.958), which may show a significant role in adaptive evolution.

Table 1

Candidate genes associated with plumage coloration in PSGs in the Temminck's tragopan.
Gene ID	Gene
EVMG011321.1	PDGFRA
EVMG001445.1	SLC6A6
EVMG001495.1	CHCHD6
EVMG001503.1	RPN1
EVMG005723.1	PSMD6
EVMG004808.1	BTK
EVMG013095.1	Cnot6l
EVMG011466.1	SMAD6
EVMG000561.1	MAPKAPK2
EVMG007178.1	PBRM1
EVMG008392.1	TTBK2
EVMG011707.1	SLC45A2

As functional identities presented, several immune-related contents were found in the Temminck's tragopan, such as fibroblast growth factor binding (GO:0017134) in the MF section, and defense response to virus (GO:0051607) in the BP section of the GO result, and mTOR signaling pathway (ko04150) in the KEGG (Table S3). 30 immune-related KEGG pathways was found, such as MAPK, Jak-STAT, and Toll-like receptor signaling pathway (Table S3). Among them, 39 PSGs involved in the MAPK signaling pathway (ko04010) were identified, while 17 PSGs (e.g., MTOR, EGFR, PIK3R2, MAPK1, and CAMK2G) were identified as involved in the ErbB signaling pathway.

Genes related to energy metabolism were also identified in the Temminck's tragopan, such as mitochondrion (GO:0005739) in the CC section, ATPase activity (GO:0016887) in the MF section, and metabolic process (GO:0008152) in the BP section of GO terms, respectively, as well as oxidative phosphorylation (ko00190) in the KEGG (Table S3).

Genome feature

The genome size of the Temminck's tragopan (1.06 Gb) was similar to other Galliformes species reflected by the previous studies, ranging between 0.93 Gb of Coturnix japonica to 1.25 Gb of Colinus virginianus [7], as well as other birds such as 1.07 Gb of Malurus cyaneus in Passeriformes [40]. The GC content of the Temminck's tragopan (42.09%) was within the ranges of other Galliformes species from 40.95% of Odontophorus gujanensis to 42.66% of C. virginianus [7], and was also similar to that of other avian genomes, such as 41.9% of Acrocephalus scirpaceus [41]. In total, the predicted gene number of the Temminck's tragopan (16,414) was within ranges of total gene numbers of other Galliformes species, from 15,429 of Alectura lathami to 17,883 of Gallus gallus reported for the previous genome assembly [7]. The pseudogene numbers of the Temminck's tragopan (283) were slightly higher than that of Larus relictus (221) [42]. The proportion of complete BUSCOs of the Temminck's tragopan (90.30%) was similar to that of other Galliformes species ranging from 89.6% of A. lathami to 94.9% of G. gallus [7].

Repeat sequence

Repeats proportions of the Temminck's tragopan (11.00%) was within the ranges of other Galliformes species from 8.68% of A. lathami to 15.12% of Meleagris gallopavo [7], and also similar with that of some other birds, such as 10.94% in A. scirpaceus genome [41]. Compared to other vertebrates, avian genomes are characterized by a low proportion of repetitive DNA, with species belonging to Piciformes showing relatively higher repeats [43]. TEs, a source of endogenous small RNAs in animals and plants [44], are low-complexity elements, make up to two-thirds of the human genome, and play an essential role in genome architecture and regulation associated with both normal function and disease states [45]. Based on sequence analyses of more than 75 species from all major avian taxa, flying birds have smaller genomes yet more TEs compared with flightless birds [1]. Among TEs, LINEs and SINEs belonged to Class I with representing by non-LTR retrotransposons. LINEs proportion of the Temminck's tragopan (8.11%) was larger than some other avian genomes, such as ~ 6.79% of four manakins [36]. The highest LINEs were also found in other avian genomes [7, 36, 39, 40], such as M. cyaneus [40]. A previous study showed that LINEs of the chicken genome were longest on the Z chromosome, and their length was negatively correlated with the local GC content [46]. The proportion of SINEs of the Temminck's tragopan (0.02%) was lower than that in several birds, such as 0.09% in A. scirpaceus genome [41].

Divergence time

The divergence time between Phasianidae and Odontophoridae (43.96 MYA) was similar to that of previous studies based on a combination of mitochondrial and nuclear data, such as 42.8 MYA [47] and 53.6 MYA [29], UCEs (39.9 MYA) [30], and genome data (46.46 MYA) [39]. Divergence times of genera within Phasianidae (11.65 MYA-38.16 MYA) were consistent with the previous study [48]. The divergence time of Tragopan divided with other closely related Phasianidae species (31.43 MYA) was similar to previous studies [28, 29], however, was earlier than that of the results of a combination of mitochondrial and nuclear data, and UCEs [30, 47].

Plumage color and feather development

Traits, such as plumage coloration, usually have lineage specificity, are commonly related to selection pressure, and reflect the adaptive characteristics of species [3]. Major changes in key genes or at the genomic level may drive plumage color differentiation on a larger evolutionary scale [3]. The plumage color of chicken is a complex trait controlled by several genes [49]. Research related to plumage color has been widely studied in birds based on different data types, such as transcriptomes and genomes, and identified several significant signaling pathways and genes [3, 36, 37, 50, 51]. For example, manakins are a family of small suboscine passerine birds characterized by a variety of plumage colors, their genome evolutionary features were analyzed to explore the possible genetic mechanism of color diversities, and four plumage color-related genes (TBC1D22A, EDA, SLC45A2 and GOLGB1) under positive selection were found [36].

For related signaling pathways on plumage coloration, several confirmed pathways involved in pigmentation, such as the BMP signaling pathway, and pathways of cAMP, SCF-KIT, Notch, ERK, CREB/MITF/tyrosinase, Wnt/β-catenin and MAPK, and genes, such as MC1R, TYR, SLC24A5, and DCT, played a role in melanin proportion synthesis [37]. Some signaling pathways and genes were commonly found in previous investigations of birds, such as the TYR gene in chicken [52], the crested ibis [53], and ducks [54]. The melanogenesis pathway found in PSGs of the Temminck's tragopan was also been identified in other birds [32, 37, 38, 55–57], such as the chicken based on transcriptome [57] and genomic SNP data [32]. The WNT10A gene was identified in melanogenesis pathway in PSGs of the Temminck's tragopan, and a previous study showed that miR-193b might participate in the adjustment of coat color in the skin tissue of Cashmere goats by targeting WNT10A and GNAI2 [58]. Some other genes were also found enriched in the melanogenesis pathway in previous studies. For example, integrative analysis of transcriptomic and metabolomic data of chickens, 25 differentially expressed genes (DEGs) (such as TYRP1, PDZK1, and DCT) and 11 transcription factors (such as FOXA1 and SOX10) in the melanogenesis pathways were identified, and the dual physiological functions of the PDZK1 gene were revealed [55]. Based on transcriptomics and proteomics analyses, a total of eight DEGs, such as ASIP and WNT9A, and nine differentially expressed proteins (DEPs), such as DVL3 and CAMK2A, in chicken to be involved in the melanogenesis pathway was identified [37]. In addition, genes, such as MAPK1, the main member of MAPKs, were identified enriched in the MAPK signaling pathway in PSGs of the Temminck's tragopan. By comparing transcription and protein levels between the red and black skin of Plectropomus leopardus, ERK1/2 (corresponding to the MAPK1 gene) was interfered with after RNAi, and the local skin of the tail would turn black [59].

For related genes on plumage coloration, more than 200 genes associated with pigmentation have been found in mammals, and genes associated with plumage color mutations were increasingly identified in birds [51]. Multiple genes related to plumage color have been found in birds, such as TBC1D22A, EDA, SLC45A2, and GOLGB1 [36], EDNRB2 [51], GRM5, RAB38 and NOTCH2 [60], GPNMB, PMEL, TYRP1, GPR143, OCA2, SOX10, KRT75 and TYR [61], LATS1, MAPKAPK2, PBRM1 and TTBK2 [62], MC1R, TYR and ASIP [63]. For example, 18 candidate genes (such as CKMT1B and MIR1772) and 89 putative gene-gene combinations responsible for plumage color variation in chickens were distinguished [32]. The down-regulation of TYRP1, DCT, PMEL, MLANA, and HPGDS may result in reduced eumelanin and increased pheomelanin synthesis in yellow plumage in chicken [64]. The association between plumage color and genetic variation of MC1R, TYR, and ASIP in chicken was indicated [63].

Several candidate genes have been identified with color differentiation, such as SOX10 and MITF. For example, the light yellow phenotype in chicken is caused by a 7.6 kb non-coding deletion near the SOX10 gene [49], while this mutation has also been reported to lead to dark brown plumage in chickens with an 8.3 kb deletion upstream of the SOX10 gene [65]. The MITF gene seems to be primarily associated with loss of pigmentation and patterning [54], and a stop codon caused by a 2-bp deletion in exon 11 of this gene was responsible for the silver plumage color in Japanese quail [66]. In addition, the ASIP, MC1R, TYRP1, and BCO2 genes were found to play a potential role in the coloration of melanin and carotenoids in bird sexual dimorphism plumages [31] Based on the genome-wide association study, several candidate genes in chicken, such as TYR for pure white, GOLGB1, OSBPL3, PCDH15 and REEP3 for yellow, CAMKK1 and IFT22 for black, and CDKN2A for barred plumage in adults were found [52]. The white color feathers in chickens were due to the recessive insertion mutation of TYR, while the formation of black area size and color depth may be caused by the expression levels of GPNMB, PMEL, TYRP1, GPR143, OCA2, SOX10, SLC45A2, KRT75, and TYR [61]. MITF and EDNRB2 genes were functional candidate genes that contributed to white and black plumage due to their indirect involvement in the melanogenesis pathway in ducks based on genome-wide analyses [56], and the EDNRB2 gene was responsible for tyrosinase-independent recessive white and mottled plumage colors in chickens [51]. The MC1R gene was also associated with the extended black color in chickens [67].

Candidate genes related to plumage coloration found in PSGs of the Temminck's tragopan have been also found in the other birds, such as MAPKAPK2 [62], SLC6A6 and SMAD6 [56]. The PDGFRA gene was identified as associated with the coat color of mammals, such as cattle and goats. For example, genes (e.g., PDGFRA and MITF) were promising candidates for black and teat color in Holstein cattle [68]. According to previous studies, the PDGFRA gene was related to a proportion of black [68], SLC6A6, CHCHD6, RPN1, and PSMD6 genes were associated with white plumage, BTK, Cnot6l, and SMAD6 genes were correlated with grey plumage [56], MAPKAPK2, PBRM1, and TTBK2 genes were associated with melanogenesis [62], and the SLC45A2 gene played an important role in vesicle sorting in the melanocytes [50], respectively. Among them, the SLC45A2 gene encoded a transporter protein, which mediated melanin synthesis, and has been also reported as related to plumage color in birds [36, 50, 69]. Besides, the SLC45A2 gene played a significant role in vesicle sorting in the melanocytes, and two independent missense mutations (Tyr277Cys and Leu347Met) of this gene were associated with the silver plumage color in chicken [50]. Furthermore, the SLC45A2 gene was also found to be positively selected in Machaeropterus deliciosus, and may explain its unique reddish-brown body plumage among other studied manakins [36]. The identified protein-coding and cis-regulatory mutations in TYRP1, SOX10, and SLC45A2 underlay classical color phenotypes of pigeons [70].

Immune response

The immune system was an interactive network of lymphoid organs, cells, humoral factors, and cytokines [71]. Multiple immune-related pathways have been also found in previous studies based on avian genomes, such as toll-like receptors (TLRs) [72, 73] and the MAPK signaling pathway [39], which play a significant role in their immune responses against infections. Among them, TLRs, one family of functional genes, are present in nearly all multicellular organisms [74], and play a crucial role in the recognition of pathogens and activation of the immune system [73]. TLRs can be a possible thread linking hypoxia and venous thromboembolism (VTE) by recognition of damage-associated molecular patterns (DAMPs) generated by hypoxia [75]. Based on the reference genome and whole-genome resequencing population data, the results showed that the TLR gene diversity was low in Thinornis novaeseelandiae, and formed two distinct captive and wild genetic clusters [73]. The MAPK signaling pathway identified in PSGs in the Temminck's tragopan was similar to previous genome data also including several related genes under positively selected in this pathway [39, 76, 77]. In addition, ErbB signaling pathway has been found significantly enriched in ducks based on genome-wide analyses, in which the candidate gene (ABL1) was enriched [78]. The ErbB and Wnt signaling pathway were also identified in genome data in the Chinese monal [77]. Consequently, the positive signals of genes and pathways identified in the Temminck's tragopan may indicate adaptation to the ecological environment.

In this study, the assembled and annotated genome of the Temminck's tragopan (T. temminckii) was obtained by utilizing long-read PacBio and short-read Illumina sequencing. The genome characteristics, such as the genome size and repeat sequence, were reported, and comparative genomic studies were analyzed among sampled 12 Galliformes species. The results indicated that the genome size of the Temminck's tragopan was 1.06 Gb, with 11.00% repeat sequences. T. temminckii formed a basal position of the clade containing L. nycthemera, P. colchicus, C. pictus, S. mikado, P. hodgsoniae, and M. gallopavo, with a corresponding divergence time of 31.43 MYA. Multiple genes and pathways related to plumage color and feather development, immune response, and energy metabolism were identified in the Temminck's tragopan, such as the SLC45A2 gene, melanocyte differentiation, MAPK signaling pathway, and lipid metabolic process, and which may be significant with their ecological adaptation. This genome will serve as an important resource to increase our knowledge of genome data in Galliformes birds, and will provide a deeper insight into their genomic characteristics and significance for avian evolution.

Sample collection and sequencing

The muscle sample of male T. temminckii was collected from a captive breeder of Lantian, Xi'an, Shaanxi Province, China in 2017, and preserved in the College of Life Sciences of Shaanxi Normal University with the voucher of LGJZ01. The genomic DNA was extracted using the CTAB method, while total RNA was extracted with TRIzol reagent following the recommended protocol provided by the manufacturer. DNA and RNA quality was checked using a combination of a NanoDrop 2000, Qubit 2.0, and Agilent 2100. DNA qualified with a DNA integrity number (DIN) and RNA with RNA integrity number (RIN) scores larger than 8.0, and OD260/280 ranged between 1.8 to 2.2 were used for the library preparation and construction. Short reads were sequenced on the Illumina NovaSeq 6000 platform, employing five small libraries (three of 270 bp and two of 350 bp), while long reads were obtained on the PacBio Sequel platform. The standard protocol of library preparation and sequencing method were consistent with previous studies [39, 42].

De novo genome assembly and assessment

The short- and long-reads were used to assemble the genome. Firstly, low-quality sequences and short-length reads of the PacBio raw data were filtered out. LoRDEC v. 0.7 [79] was employed to correct long-read PacBio sequencing data using Illumina data. Subsequently, the short-read Illumina sequencing data were assembled preliminarily with platanus v. 1.2.4 [80]. Finally, the dbg2olc [81] was run for mixed assemblies with corrected PacBio data and Illumina assemble results.

Three methods were used to assess the quality of the genome assembly, including Benchmarking Universal Single-Copy Orthologs (BUSCO) evaluation, Core Eukaryotic Genes Mapping Approach (CEGMA) analysis, and reads remapping. Among them, BUSCO v. 2.0 [82] was employed to estimate the completeness of the de novo assembled genome draft by searching for 4,915 universal avian single-copy orthologs (aves_odb09), running under the genome mode to compute the proportion of complete, fragmented, and missing genes across the dataset. CEGMA v. 2.5 [83] was also used, with 458 CEGs. To evaluate the mapping rate of reads, the Illumina short reads were realigned to genome assembly.

Genome annotation

To identify repeat regions of the assembled genome, the software LTR-FINDER v. 1.05 [84], MITE-Hunter [85], RepeatScout v. 1.05 [86], and PILER-DF v. 2.4 [87] were used to construct a repeat library based on the structure-based and de novo predictions. PASTEClassifier [88] was employed to classify this repeat library. Then the repeat library was combined with the Repbase [89] database to build a final library. Finally, RepeatMasker v. 4.0.6 [90] was employed to annotate the repeat sequences based on the constructing repeat library.

The assembled genome was annotated through three methods, including de novo, homologous, and RNA-seq gene annotation. For de novo gene prediction, Augustus v. 2.4 [91], Genscan [92], GlimmerHMM v. 3.0.4 [93], GeneID v. 1.4 [94], and SNAP v. 2006-07-28 [95] were used. For homologous gene annotation, GeMoMa v. 1.3.1 [96] was employed, with referring protein sequences of the following five species: chicken, wild turkey, zebra finch, collared flycatcher, and great tit. For the RNA-seq method, Hisat v. 2.0.4 [97] and StringTie v. 1.2.3 [98] were used for assembly based on transcripts. TransDecoder v. 2.0 (available online: https://transdecoder.github.io/) and GeneMarkS-T v. 5.1 [99] were employed for gene prediction. PASA v. 2.0.2 [100] was used to predict unigene sequences with non-reference assembly based on transcriptome data. Finally, the predicted results were integrated to generate a consensus gene set by EVidenceModeler (EVM) v. 1.1.1 [101] pipeline, and PASA v. 2.0.2 [100]was run to modify.

For non-coding RNAs prediction, Infenal 1.1 [102] was run to determine rRNAs and microRNAs according to the Rfam [103] and miRBase [104] databases, while tRNAscan-SE v. 1.3.1 [105] was used to identify tRNAs. For pseudogene prediction, through GenBlastA v. 1.0.4 alignment [106], homologous gene sequences were searched on the genome after shielding the true gene loci. Then, GeneWise v. 2.4.1 [107] was run to search for immature stop codons and frameshift mutation.

The functions of predicted genes in the assembled genome were identified based on the best matches across five databases using the threshold of -evalue 1e-5: Gene Ontology (GO) [108], Kyoto Encyclopedia of Genes and Genomes (KEGG) [109], eukaryotic ortholog groups (KOG) [110], Translation of the EMBL database (TrEMBL) [111], and Non-Redundant Protein Sequence database (NR).

Phylogenetic reconstruction

To reconstruct the phylogenetic history of the Temminck's tragopan, 11 other closely related Galliformes species with relatively higher assembled qualities were selected, including one Numididae species, one Odontophoridae species, and nine Phasianidae species, with detailed information listed in Table S4. The Numididae species (N. meleagris) was used as the outgroup. Orthofinder v. 2.4 [112] was employed to classify protein sequences. In total, 4,907 one-to-one orthologous genes were used for phylogenetic tree construction in IQ-TREE v. 1.6.11 [113]. Genes were firstly aligned using MAFFT v. 7.205 [114]. Gblocks v. 0.91b [115] was employed to remove the regions with poorly aligned scores or large difference areas, and then the alignments were concatenated to form one supergene. Subsequently, the best substitution model (JTT + F + I + G4) was selected using ModelFinder [116], and the maximum likelihood (ML) method was used to reconstruct the phylogenetic tree with the bootstrap set to 1000.

Divergence time estimation

The divergence time estimation of Galliformes species was based on the ML phylogenetic tree above. Divergence times were inferred by MCMCTree in the PAML v. 4.9i [117], with three calibration points retrieved from the TimeTree database [118] used for calibrating, including 6.83–13.4 MYA between L. nycthemera and C. pictus, 27.5–37.5 MYA between L. nycthemera and M. gallopavo, and 41–52 MYA between N. meleagris and P. hodgsoniae. The parameters of gradient and Hessian were used. Using the ML method, the correlated molecular clock and JC69 model were run to estimate divergence times. Two repeated calculations were performed to observe the consistency (value = 1). The parameters were set as burnin of 5 000 000, sampfreq of 30, and nsample of 10 000 000.

Synteny analysis

The genome assembly of the Temminck's tragopan was aligned against the chicken genome assemble result using diamond v. 0.9.29.130 [119] to determine similar gene pairs with e value less than 1e − 5 and C score greater than 0.5. MCScanX [120] was used to estimate neighboring links on chromosomes of similar gene pairs. JCVI [121] was run to display large-scale synteny blocks.

Gene family expansion and contraction

The expansion and contraction of orthologous gene clusters were performed among 12 sampled Galliformes species to understand the evolutionary dynamics of genes. These species were analyzed using CAFE v. 4.2 [122] based on the phylogenetic tree with divergence times and gene family cluster results. A random birth and death process model was used, and both the family-wide P-Values and viterbi P-Values less than 0.05 were used to detect significantly expanded or contracted gene families of each lineage on the phylogenetic tree. The expansion of gene families of the Temminck's tragopan were annotated using PANTHER V. 15 [123]. Functional enrichment analyses were further performed on the expanded and contracted gene families using clusterProfile v3.14.0 [124].

Positive selection analysis

With the phylogenetic tree, one-to-one orthologous genes of five closely related Galliformes species including the Tibetan partridge, wild turkey, Reeves’s pheasant, Indian peafowl, and chicken were retrieved to identify potential positively selected genes (PSGs). MAFFT v. 7.205 [114] was used to align protein sequences of each gene family, and the corresponding coding sequence alignments were back-translated from protein alignments employing PAL2NAL [125]. The branch-site model of CodeML in PAML [117] was used to detect PSGs, with the Temminck's tragopan set as the foreground branch and other species performed as the background branch. Two models, model A, and null model, were run, and the likelihood ratio tests (LRTs) were then employed to compare these two models using the chi2 program. Genes with P values less than 0.05 was considered to be significant differences. Furthermore, the posterior probability (PP) was obtained using a Bayes Empirical Bayes (BEB) method, and genes with a PP value greater than 0.95 was defined as significant PSGs. Finally, functional enrichment analysis of identified PSGs was performed via clusterProfile v3.14.0 [124].

Author’s contributions

XL designed the research plan, analyzed the sequenced data, and wrote the manuscript. XW, CY and LL helped to analyze the sequenced data. XW and XY conducted the samples collection. YH conceived the research plan, and carried out the manuscript revision. All authors read and approved the content of this manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31601846), China Scholarship Council (202106875010), Fundamental Research Funds for the Central Universities, China (GK202304021 and GK202101003), and China Postdoctoral Science Foundation (2016M602760).

Data Availability Statement

The sequence reads of the Temminck's tragopan genome were deposited to the National Center for Biotechnology Information (NCBI) database with accession number of BioProject (PRJNA1000723), BioSample (SAMN36776254), and Sequence Read Archive (SRA) (SRXSRR25487939 and SRR25487940), respectively.

Ethics approval and consent to participate

We adhered to all local, national, and international regulations and conventions, and respect normal scientific ethical practices. The specimen used in this study was dead naturally, and no living animal experiments were conducted. The animal experiment program was approved by animal ethics committee of Shaanxi Normal University, and conforms to animal protection, animal welfare and ethical principles, as well as relevant regulations of national ethical welfare of experimental animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹College of Life Sciences, Shaanxi Normal University, Xi’an, China. ²School of Biological and Environmental Engineering, Xi’an University, China. ³Shaanxi Institute of Zoology, Xi’an, China.

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No competing interests reported.

Fig.S1.png
Fig. S1 Phylogenetic evolution of 12 sampled Galliformes species. a gene family cluster. b phylogenetic tree. c divergence time.
Fig.S2.png
Fig. S2 Genome synteny between the Temminck's tragopan and the chicken.
Fig.S3.png
Fig. S3 GO enrichment results in the Temminck's tragopan. a expanded gene families, b PSGs.
Fig.S4.png
Fig. S4 Top 10 significant GO and KEGG terms of PSGs in the Temminck's tragopan. a cellular component in the GO database. b molecular function in the GO database. c biological process of the GO database. d the KEGG database.
Supplementarytables.docx
Table S1 The sequencing data based on Illumina platform in the Temminck's tragopan. Table S2 Repeat sequences in the Temminck's tragopan. Table S3 Identified pathways and genes related with plumage color and feather development, immune response, and energy metabolism in PSGs in the Temminck's tragopan. Table S4 12 sampled Galliformes species used for comparative genomic analyses.

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The draft genome of the Temminck's tragopan (Tragopan temminckii) with evolutionary implications

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Journal Publication

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Abstract

Background

Results

methods

results

Conclusions

Figures

Background

Results

Genome assembly and characteristic

Gene function

Expansion and contraction

PSG analysis

Discussion

Genome feature

Repeat sequence

Divergence time

Plumage color and feather development

Immune response

Conclusions

Materials and methods

Sample collection and sequencing

De novo genome assembly and assessment

Genome annotation

Phylogenetic reconstruction

Divergence time estimation

Synteny analysis

Gene family expansion and contraction

Positive selection analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1