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Full Research Article
The Analysis of Reproduction-related Genes Provides Insights into the Adaptive Evolution of Fecundity Traits in Yangtze Finless Porpoise
First Online 02-12-2021|
Methods: Orthofinder software was employed to search homologous genes based on protein sequence. CAFÉ software was used to obtain the expansion and contraction gene families of YFP. Then, GO terms and pathway enrichment analyses were performed using TBtools software and Swissprot database. PAML package was used to calculate Ka/Ks (i.e., ω). Evolution rate changes in the positive selected genes were examined using the GU99 process in Diverge (v3.0) program.
Result: In YFP, 501 rapid expansion gene families GO enrichment results showed that the reproductive activities related pathways were mainly three significant enrichment process, participation of germline stem cells maintain androgen receptors signaling pathways regulating and male reproductive tract stem cell population to maintain. The most significant GO terms of 220 rapidly contraction gene families associated with reproductive activities mainly consisted of biological processes which were involved in positive regulation of estrogen secretion, mating and estrogen metabolic process. GDF9 and BMP15 genes exhibited purifying selection. However, significant signs of positive selection were detected in FSHR and FSHβ genes, but only FSHβ showed specific changes in the YFP lineage.
The cetacean oxidative stress-related gene families, such as the peroxiredoxin (PRDX) family, glutathion peroxidase (GPx) and the OGT gene family, had been found to have expanded and it was an adaptive mode for Cetacean tolerance to hypoxia stress. But, neuronal differentiation-related gene families showed contraction, suggesting that they might be associate with smaller brains of porpoises (Sun, 2018). However, there have been no reports on the rapid evolution gene families related to reproductive activity in aquatic mammals such as YFP. And more and more studies have found that many hormones, cytokines and adhesion factors are linked with ovulation through endocrine activities and play a very important role in follicular development and ovulation process, such as: Luteotropic hormone LH), follicle stimulating hormone (FSHβ) (Chu et al., 2012; Miyano et al., 2014) and follicle stimulating hormone receptors (FSHR) (Lazaros et al., 2012), growth and differentiation factors-9 (Growth different-I ation factor-9, GDF-9), BMP 15 (Bone morphogenetic protein-15, BMP-15) (Otsuka et al., 2014; Migaud et al., 2013). Therefore, in order to analysis of the adaptive evolution of fecundity traits in YFP, the present study was performed to rapidly evolving gene families of YFP. At the same time, positive selection test and protein functional differentiation test of GDF9, BMP15, FSHβ and FSHR genes related to the hypothalamo-pituitary-gonadal axis system were carried out to find the specific mutation sites of YFP, thus providing new insights into the adaptive evolution of fecundity traits in order to facilitate current and future breeding protection.
MATERIALS AND METHODS
The assembled genome sets are available from NCBI, Bos taurus (GCF_002263795.1), Felis catus (GCF_000181335.3), Cavia porcellus (GCF_000151735.1), Heterocephalus glaber (GCF_000247695.1), Chinchilla lanigera (GCF_000276665.1), Castor canadensis (GCF_001984765.1), Jaculus jaculus (GCF_000280705.1), Nannospalax galili (GCF_000622305.1), Camelus bactrianus (GCF_000767855.1), Ovis aries (GCF_002742125.1), Sus scrofa (GCF_000003025.6), Homo sapiens (GCF_000001405.39), Mus musculus (GCF_00000 1635.27), Lipotes vexillifer (GCF_000442215.1), Phocoena sinus (GCF_008692025.1), Neophocaena asiaeorientalis (GCF_003031525.1).
Orthologous family identification
To define a set of conserved genes for cross-taxa comparison, we employed Orthofinder (v2.3.3) to search homologous genes of 16 species based on protein sequence (Emms et al., 2015; 2019). Toolbox for Biologists (TBtools, v1.096) software was used to obtain whole-genome representative CDS sequences from the genome and then representative protein sequences were extracted for subsequent analysis according to the corresponding relationship between the CDS sequence number and the protein sequence number (Chen et al., 2020). To identify expanding and contracting gene ortholog groups across the phylogeny. The species tree was obtained from the Orthofinder (v2.3.3) (Emms et al., 2017; 2018) and the time of differentiation was obtained from the website Timetree (Kumar et al., 2017). We estimated the gene numbers on internal branches using a random birth and death process model implemented in the software CAFÉ (v3.0), a tool for the statistical analysis of the evolution of the size of gene families (De et al., 2006).
GO enrichment and statistical analyses
After the expansion and contraction gene families of the target species were obtained by CAFÉ v3.0 software, the expansion and contraction gene sets were further obtained according to the results of Orthofinderv 2.3.3 software. Then, GO terms and pathway enrichment analyses were performed using TBtools and Swissprot with the default parameters. And the significantly enriched clusters associated with reproductive activities were reported (Benjamini-Hochberg corrected q<.05). Visualization of results were carried out using the R package ‘ggplot2’ (Hadley et al., 2021).
Positive selection test
PAML package were used to calculate Ka (nonsynonymous substitution rate of nonsynonymous sites), Ks (synonymous substitution rate of synonymous sites) and Ka/Ks (i.e., ω) between the homologous GDF9, BMP15, FSHβ and FSHR genes of 16 species (Yang, 2007). In addition, the ω value between paternal gene and retrogene pair was used to estimate functional constraints with ω < 1 representing the purifying selection, ω = 1 for the neutral selection and ω > 1 for the positive selection (Yang, 2007). Sequence alignment was performed using MEGA X software (Kumar et al., 2018).
Protein functional differentiation test
Evolution rate changes in the positive selected genes were examined using the GU99 process in Diverge V. 3.0 program to predict amino acid sites associated with functional differentiation (Gu et al., 2013). θ indicates the likelihood of functional differentiation between two sets of genes (Thompson et al., 2018). In fact, GDF9 and BMP15 genes exhibited purifying selection. For FSHR gene, no functional differentiation was found between the clusters of 16 species. Only FSHβ gene needs to be further expanded for protein functional differentiation test. For FSHβ gene, the above gene family analysis showed that YFP and Phocoena sinus had two homologous genes. By comparing these four sequences, it was found that they could be divided into two groups, which constituted one of the YFP and one of Phocoena sinus (XP_024620387.1 and XP_032497072.1) and the other two constituted another group (XP_024620395.1 and XP_032496507.1). Therefore, protein sequence sets A and B were respectively constructed by the two distinct sequences groups described above and another 32 common sequences downloaded from the FSHβ gene NCBI Orthologs, as shown in Table 1.
RESULTS AND DISCUSSION
In 16 species, orthogroups number of genes was 324580 and percentage of genes in orthogroups was 97.8%; number of orthogroups was 19498; number of species-specific orthogroups was 752; number of genes in species-specific orthogroups was 4002; mean orthogroup size was 16.3; median orthogroup size was 16; number of single-copy orthogroups was 8809. In YFP, 501 gene families showed rapidly expansion and 220 gene families showed rapidly contraction, which was consistent with the number of expanding and contracting gene families as shown in Fig 1. For YFP, 860 genes were obtained; 270 genes were lost; 18777 genes were no change; in general, YFP showed genes expansion.
Functional enrichment analysis of 1158 genes (genes of rapidly expansion 501 gene families) was executed. Only clusters associated with reproductive activities enrichment with p<0.05 were listed (Fig 2). The most significant GO terms mainly consisted of biological processes which were involved in germ-line stem cell population maintenance, regulation of androgen receptor signaling pathway and male germ-line stem cell population maintenance. This could be a genetic adaptation for YFP to remain fertile when it was old. According to the conversion of body length and age, the females of YFP in Poyang Lake may still be able to reproduce at the age of 18-19. YFP, meanwhile, had 220 gene families showed rapidly contraction.
Functional enrichment analysis of 726 genes (220 gene families of YFP rapidly contraction and the gene set was the corresponding Mus musculus genes) was executed. Only clusters associated with reproductive activities enrichment with p<0.05 were listed (Fig 3). The most significant GO terms mainly consisted of biological processes which were involved in positive regulation of estrogen secretion, mating and estrogen metabolic process. Ireland et al., 1984 believed that estrogen was mainly secreted by dominant follicles on the ovary (Ireland et al., 1984) and the conception rate of female animals might be affected by the concentration of estrogen secreted by dominant follicles (Kiewisz et al., 2011). This might be related to YFP’s polygyny mating system (Hao et al., 2006), the fact that no YFP gave birth to more than 1 baby per birth (Li, 2017) and the pregnancy rate of YFP in Poyang Lake was as high as 70% (Mei, 2013).
Positive selection genes and protein functional differentiation
According to the branch site model detection, GDF9 and BMP15 genes exhibited purifying selection. For FSHR gene, 11 positive selection sites with a posterior probability greater than 0.5 with experience were found. For FSHβ gene, YFP, Phocoena sinus and Bos taurus all had two homologous genes. Therefore, FSHβ genes could form 8 sequence combinations. No positive selection sites were found in one combination and positive selection sites were found in the other seven combinations. Only FSHβ gene needs to be further expanded for protein functional differentiation test, seeing Materials and Methods for detailed description. For B FSHβ gene set, no functional differentiation was found between the clusters. For A FSHβ gene set, there were 1-4 functional differentiation sites in cetaceans, as shown in Table 2. Further analysis revealed that the L at position 40 is a specific site for the YFP, Phocoena sinus and baiji, as shown in Fig 4-5. The FSHβ protein sequence of YFP and Phocoena sinus had fragment insertions, as shown in Fig 6. YFP, Phocoena sinus and Bos taurus all had two homologous genes for FSHβ, but only FSHβ protein sequence of YFP and Phocoena sinus had fragment insertions. Further analysis revealed that the L at position 40 was a specific site for the YFP, Phocoena sinus and baiji.
The relationship between genes of GDF9, BMP15, FSHβ, FSHR and ovulation number is the main object of research on multiple fertility traits in cattle and sheep. Similar to previous studies examining positive selection, the majority of genes investigated in this study were under purifying selection, which is not surprising given the evolutionary constraint on protein coding genes. However, signs of significant positive selection were detected in 2 genes (FSHR and FSHβ). This suggests that FSHR and FSHβ evolved fast along all YFP lineages examined and their positive selection might be associated with fecundity traits. The mutation rate of FSHR gene exon 10 was significantly different between twin and single bovine cattle (Lei et al., 2004). It was inferred that FSHR gene exon 10 was related with the major gene that controls the high prolificacy of goat and the individuals with FSHR gene mutation showed high lambing number, indicating that the high expression of FSHβ in the ovary promoted the high ovulation number, which was the result of the mutual regulation between FSHR and FSHβ gene (Ji, 2007).
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