| The Asian small-clawed otter(Aonyx cinereus)is a small mammal with the characteristics of aquatic adaptation in the Mustelidae family,which is distributed throughout South and Southeast Asia.The otter undergone adaptive radiation in the mid to late Miocene and gradually diverged into multiple lineages of otters.The otter has undergone aquatic adaptive changes in the shape of its body and limb morphological structure.For example,it has a high density of hair,and the bristles form a protective and waterproof outer layer of structure.The dense wavy underhairs trap the air layer,which provides isolated environment and most of the thermal insulation.Otters have shortened limbs and use dorso-ventral undulation and pelvic oscillation to generate swimming propulsion.However,the molecular mechanisms of its evolution are still unknown.Therefore,this study explored the evolution of aquatic adaptation characteristics in Asian small-clawed otter from the perspective of genomics and comparative genomics,the hair development-related a-keratins(KRT)and keratin-associated protein(KRTAP)gene families and limb development-related homologous genes(Hox)family were selected as the research object to investigate the evolutionary patterns of genes related to hair development and limb development in Asian small-clawed otter and the pattern characteristics related to the aquatic adaptation of otter.The main contents of this study are as follows:(1)Whole genome sequencing and high-quality genome mapping of the Asian small-clawed otter.The genome of Asian small-clawed otter was sequenced for the first time by third-generation Nanopore sequencing and Hi-C-assisted assembly.The size of the Asian small-clawed otter genome was 2.42 Gb with a contig N50 of 122.83 Mb,and it was anchored to 19 chromosomes.BUSCO analysis showed 96.15%.Annotation of the Asian small-clawed otter genome revealed the presence of 20,726 genes with an average gene length of 45,285 bp.We obtained high-quality assembled and annotated genome of the Asian small-clawed otter in comparison with other closely related species.(2)Analysis of genomic comparison and evolutionary characteristics of Asian small-clawed otter.The Asian small-clawed otter is most closely related to Eurasian otter with an estimated divergence time of～5.22 Mya.The 209 gene families that were expanded in Asian small-clawed otter were mainly related to immune signaling pathways,fatty acid metabolism and olfactory transmission.In addition,selection pressure analysis showed that 179 genes in Asian small-clawed otter were positively selected,which were mainly related to immunity,lipid metabolism,signal transduction and angiogenesis.These selected genes may be related to transition between different habitats and adaptation to the environment in the long-term evolution of Asian small-clawed otter.(3)Evolutionary analysis of the keratin gene family in the Asian small-clawed otter.Taking the KRT genes of 23 species of Carnivora as the research objects,the high-quality Asian small-clawed otter genome obtained in this study were used to screen and identify keratin gene family,and other carnivore keratin genes were screened and identified through online database.The results showed that Asian smallclawed otter had 59 keratin genes,no pseudogenes or incomplete genes.The number of KRT genes in 23 carnivores varied from 35 to 59,and the otter group had the highest number of KRT genes and the proportion of pseudogenes was lowest.The KRT gene family is subjected to strong purifying selection in carnivores,and KRT20 and KRT9 genes have a relaxation of selective pressure in carnivora species.The branch model analysis with the otter group as the foreground branch showed that 10 KRT genes had undergone rapid evolution in otter group,and 12 KRT genes had significant positive selection sites.The positive selection of these genes in otters may be related to the adaptive evolution that occurred in otter to adapt to the aquatic environment and to maintain the core body temperature within a narrow range.(4)Evolutionary analysis of the keratin-associated protein gene family in the Asian small-clawed otter.The KRTAP genes of 17 species of Carnivora was used for this study.The KRTAP gene family was screened and identified using the high-quality Asian small-clawed otter genome and online database and the Eurasian otter genome data sequenced by our lab.The results showed that the number of KRTAP genes ranged from 80(spotted hyena)to 125(Asian small-clawed otter),with a higher number of KRTAP genes identified in otters.Branch model selection pressure analysis using otter group and species with aquatic adaptation characteristics of Carnivora as foreground branch,respectively,showed that KRTAP10 and KRTAP9 genes undergone rapid evolution in otters and species with aquatic adaptation in Carnivora.The analysis using branch-site model with otters and species with aquatic adaptation characteristics as foreground branch,which showed that positive selection signals were detected in 5 KRTAP genes.The expansion of KRTAP gene family in otter and the genes of positive selection signals detected by selection pressure analysis may be related to the adaptive evolution of otter hair density and hair morphological characteristics.(5)Evolutionary analysis of the Hox gene family in the Asian small-clawed otter.The Hox genes of 22 species of Carnivora was used for this study.The Hox gene family was screened and identified using the high-quality Asian small-clawed otter genome and online database and the Eurasian otter genome data sequenced by our lab.The results showed that 39 genes were identified in all species except for the meerkat HoxDll gene.Site model results in selection pressure analysis showed that HoxB9,HoxC10 and HoxDll genes had significant positive selection signals.The free ratio model showed that the Hox genes were subjected to strong purifying selection in Carnivora.Branch-site model analysis showed that 7 Hox genes were detected positive selection signals of otters.These selected and rapidly evolving genes may be associated with traits on adaptive evolution such as shorter limbs and swing-style swimming of otters.In this study,we analyzed the evolution of gene families related to hair and limb development in Asian small-clawed otter and found that expansion and rapid evolution of gene families related to hair density and morphological structure,as well as rapid evolution of some Hox genes related to limb development occurred in otter.In this study,we obtained a valuable genomic resource for Asian small-clawed otter,and provide a new perspective on its conservation.At the same time,the amphibious characteristics of Asian small-clawed otter provide a unique perspective on the evolution of mammals from terrestrial to completely aquatic. |
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