Development and Characterization of Microsatellite Markers for Harpadon nehereus Based on High-Throughput Sequencing and Cross-Species Amplification in Three Myctophiformes Fishes

Harpadon nehereus is a widespread economical fish found in the coastal seas of China and has important ecological value in the marine ecosystem.H_(o)wever,its germplasm resources have been seriously degraded due to natural factors and anthropogenic activities.In this study,high-throughput sequencing was applied to search for microsatellite loci in H.nehereus transcriptome to provide references for its resource conservation and utilization.Polymorphic loci were developed by non-denaturing polyacrylamide gel electrophoresis,and their cross-species amplified ability was detected in three related species.A total of 5652 microsatellites were identified from 16974320 unigenes.Among the primer pairs designed for 100 SSRs for PCR amplification,80%were successfully amplified,and 26 loci were polymorphic with a high number of alleles from 3 to 11 each.The expected(H_(e))and observed(H_(o))heterozygosities were 0.355–0.885 and 0.375–0.958,respectively.Most of the loci were highly polymorphic(polymorphism information content:0.316–0.852;mean:0.713),and these markers can be applied in the population genetic diversity research of H.nehereus.H_(o)wever,the transferability of these primers was low,probably because of the close relation of the collected species.In follow-up work,simple sequence repeats will be excavated with genome-based technologies,and related species will be gathered to address the present inadequacies.

A panel of polymorphic microsatellites in the Blue Eared Pheasant (Crossoptilon auritum) developed by cross-species amplification

Polymorphic microsatellites are among the versatile genetic markers in molecular ecology studies. In contrast to de novo isolation of microsatellites from target species, cross-species amplification is a costeffective approach for a fast development of microsatellite markers from closely related taxa. In our study, we crossamplified a panel of polymorphic microsatellite markers for the Blue Eared Pheasant (Crossoptilon auritum), a species endemic to China of considerable conservation concern. We obtained 11 polymorphic microsatellite markers selected from 112 candidate loci, originally isolated from other Galliforme species. This panel of makers has shown moderate to high levels of polymorphism and include a Z-chromosomal linkage locus. We carried out preliminary analyses of parentage among captive individuals with a known pedigree using this new panel of microsatellites. Our results suggest that the high utility of these markers may be powerful tools for studies in conservation genetics of eared-pheasants and other endangered Galliforme species.

Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

AIM： To demonstrate the feasibility of using woodchuck samples on human microarrays, to provide insight into pathways involving positron emission tomography （PET） imaging tracers and to identify genes that could be potential molecular imaging targets for woodchuck hepatocellular carcinoma. METHODS： Labeled cRNA from woodchuck tissue samples were hybridized to Affymetrix U133 plus 2.0 GeneChips. Ten genes were selected for validation using quantitative RT-PCR and literature review was made. RESULTS： Testis enhanced gene transcript （BAX Inhibitor 1）, alpha-fetoprotein, isocitrate dehydrogenase 3 （NAD＋） beta, acetyI-CoA synthetase 2, carnitine palmitoyltransferase 2, and N-myc2 were up-regulated and spermidine/spermine N1-acetyltransferase was down-regulated in the woodchuck HCC. We also found previously published results supporting 8 of the 10 most up-regulated genes and all 10 of the 10 most downregulated genes. CONCLUSION： Many of our microarray results were validated using RT-PCR or literature search. Hence, we believe that woodchuck HCC and non-cancerous liver samples can be used on human microarrays to yield meaningful results.

Development and characterization of novel polymorphic microsatellite markers for the Korean freshwater snail Semisulcospira coreana and cross-species amplification using next-generation sequencing

Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.

High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (<i>Epinephelus bruneus</i>) and cross-species amplifications for Epinephelinae species

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

Hepatitis B virus lineages in mammalian hosts:Potential for bidirectional cross-species transmission

The hepatitis B virus(HBV)is a cosmopolitan infectious agent currently affecting over 350 million people worldwide,presently accounting for more than two billion infections.In addition to man,other hepatitis virus strains infect species of several mammalian families of the Primates,Rodentia and Chiroptera orders,in addition to birds.The mounting evidence of HBV infection in African,Asian and neotropical primates draws attention to the potential crossspecies,zoonotic transmission of these viruses to man.Moreover,recent evidence also suggests the humans may also function as a source of viral infection to other mammals,particularly to domestic animals like poultry and swine.In this review,we list all evidence of HBV and HBVlike infection of nonhuman mammals and discuss their potential roles as donors or recipients of these viruses to humans and to other closely-related species.

Characterization of 14 anonymous nuclear loci in Pinus thunbergii and their cross-species transferability

We characterized 14 anonymous nuclear loci from Pinus thunbergii Parl., an important pine species native to Japan. One hundred and twenty-six single nucleotide polymorphisms （SNPs） were identified from these loci, giving a frequency of 1 SNP per 51 bp. Nucleotide di- versity （0） ranged from 1.06 × 10^-3 to 11.87 × 10^-3, with all average of 4.99 × 10^-3. Only one locus （mK45） deviated significantly from the Hardy-Weinberg equilibrium. Thirteen of 14 loci were applicable in other pine species. These loci will be useful for nucleotide variation studies and will provide material for SNP-based marker development in P. thun- bergii and related species.

Identification,development,and application of cross-species intron-spanning markers in lentil(Lens culinaris Medik.)

Lentil(Lens culinaris Medik) is one of the most important food legumes in the world. The use in lentil of molecular marker-assisted breeding is limited, owing to the low availability of polymorphic markers. In the present study, we developed a set of polymorphic intron-spanning markers(ISMs) using a cross-species mapping approach. In this approach, putative unique transcripts(PUTs) of L. culinaris were mapped onto the Medicago truncatula genome, exploiting its closeness with the lentil genome. Spliced alignment of the PUTs resulted in a total of 25,717 alignments, allowing the development of 1703 ISMs. From these, a subset of 105 ISMs were synthesized and validated with a 51% amplification success rate in 32 lentil genotypes. Of these ISMs, 40(74%) were polymorphic and generated 2–11 alleles per locus in a genetically diverse panel of 32 lentil genotypes including wild species.This set of polymorphic ISMs along with their functional annotation data will be useful in lentil breeding.

Safety,immunogenicity,and cross-species protection of a plasmid DNA encoding Plasmodium falciparum SERA5 polypeptide,microbial epitopes and chemokine genes in mice and olive baboons

Incorporation of biomolecular epitopes to malarial antigens should be explored in the development of straintranscending malarial vaccines.The present study sought to determine safety,immunogenicity and cross-species efficacy of Plasmodium falciparum serine repeat antigen 5 polypeptide co-expressed with epitopes of BacilleCalmette Guerin（BCG）,tetanus toxoid（TT） and a chemokine gene.Olive baboons and BALB/c mice were randomly assigned into vaccine and control groups.The vaccine group animals were primed and boosted twice with pIRES plasmids encoding the SERA5 ＋ BCG ＋ TT alone,or with either CCL5 or CCL20 and the control group with pIRES plasmid vector backbone.Mice and baboons were challenged with P.berghei ANKA and P.knowlesi H strain parasites,respectively.Safety was determined by observing for injection sites reactogenicities,hematology and clinical chemistry.Parasitaemia and survivorship profiles were used to determine cross-species efficacy,and T cell phenotypes,Th1-,Th2-type,T-regulatory immune responses and antibody responses were assessed to determine vaccine immunogenicity.The pSeBCGTT plasmid DNA vaccines were safe and induced Thl-,Th2-type,and Tregulatory responses vaccinated animals showed enhanced CD4~＋（P〈0.01）,CD 8~＋ T cells（P〈 0.001） activation and IgG anti-SE36 antibodies responses（P〈 0.001） at week 4 and 8 post vaccination compared to the control group.Vaccinated mice had a 31.45-68.69%cumulative parasite load reduction and 60%suppression in baboons（P〈0.05）and enhanced survivorship（P〈 0.001） with no clinical signs of malaria compared to the control group.The results showed that the vaccines were safe,immunogenic and conferred partial cross-species protection.

Cross-species transmission and host range genes in poxviruses

The persistent epidemic of human mpox, caused by mpox virus (MPXV), raises concerns about the future spread ofMPXV and other poxviruses. MPXV is a typical zoonotic virus which can infect human and cause smallpox-likesymptoms. MPXV belongs to the Poxviridae family, which has a relatively broad host range from arthropods tovertebrates. Cross-species transmission of poxviruses among different hosts has been frequently reported andresulted in numerous epidemics. Poxviruses have a complex linear double-strand DNA genome that encodeshundreds of proteins. Genes related to the host range of poxvirus are called host range genes (HRGs). This reviewbriefly introduces the taxonomy, phylogeny and hosts of poxviruses, and then comprehensively summarizes thecurrent knowledge about the cross-species transmission of poxviruses. In particular, the HRGs of poxvirus aredescribed and their impacts on viral host range are discussed in depth. We hope that this review will provide acomprehensive perspective about the current progress of researches on cross-species transmission and HRGvariation of poxviruses, serving as a valuable reference for academic studies and disease control in the future.

Pathogenicity and risk of cross-species transmission of Aerococcus viridans isolated from Tian-e-zhou National Reserve for Lipotes vexillifer in Shishou,China

During the survey of potential pathogens in the Tian-e-zhou National Reserve for Lipotes vexillifer in Shishou,China,isolate SS20210504 was isolated from the diseased prey fish-Pelteobagrus fulvidraco and was identified as Aerococcus viridans through 16S rRNA gene,morphological observation,and physiological and biochemical analysis.Artificial infection trials with fish through different methods were conducted.Results indicated that the isolate of SS2021504 caused mortality in fish with the LD50 of 1.0×106.2 CFU/mL,1.0×107.9 CFU/mL and 1.0×106.8 CFU/mL with the infection method of intraperitoneal injection,co-immersion,and scratch immersion,respectively.Challenged fish exhibited hemorrhages signs similar to naturally diseased fish.Histology analysis showed that the liver and kidney were the principal target organs of isolate SS2021504 in fish,and obvious vacuolation and karyopyknosis occurred in the above organs.To evaluate the pathogenicity of isolate SS20210504 in mice,healthy mice and immunosuppressed mice were further experimentally infected with bacteria,respectively.No death was observed either in healthy mice or in immunocompromised mice.However,the immunocompromised mice inoculated with bacteria showed obvious clinical symptoms and severe damage to multiple organs.The inflammatory signaling pathways were significantly activated with upregulated levels of Myd88,TNFα,AP-1,IL-1β,IL-6,and IFN-γgene in the spleen from the immunocompromised mice after infected with isolate SS20210504.These findings imply that isolate SS20210504 has the potential risk to YFPs and residents.Continued surveillance of A.viridans and other pathogens in the TZO reserve is suggested to prevent outbreaks in prey fish,YFPs,and residents as well.

Cross-species single-cell transcriptomic analysis of animal gastric antrum reveals intense porcine mucosal immunity

As an important part of the stomach,gastric antrum secretes gastrin which can regulate acid secretion and gastric emptying.Although most cell types in the gastric antrum are identified,the comparison of cell composition and gene expression in the gastric antrum among different species are not explored.In this study,we collected antrum epithelial tissues from human,pig,rat and mouse for scRNA-seq and compared cell types and gene expression among species.In pig antral epithelium,we identified a novel cell cluster,which is marked by high expression of AQP5,F3,CLCA1 and RRAD.We also discovered that the porcine antral epithelium has stronger immune function than the other species.Further analysis revealed that this may be due to the insufficient function of porcine immune cells.Together,our results replenish the information of multiple species of gastric antral epithelium at the single cell level and provide resources for understanding the homeostasis maintenance and regeneration of gastric antrum epithelium.

Differentially expressed genes identified by cross-species microarray in the blind cavefish Astyanax

Changes in gene expression were examined by microarray analysis during development of the eyed surface dwelling(surface fish)and blind cave-dwelling(cavefish)forms of the teleost Astyanax mexicanus De Filippi,1853.The cross-species microarray used surface and cavefish RNA hybridized to a DNA chip prepared from a closely related species,the zebrafish Danio rerio Hamilton,1822.We identified a total of 67 differentially ex-pressed probe sets at three days post-fertilization:six upregulated and 61 downregulated in cavefish relative to surface fish.Many of these genes function either in eye development and/or maintenance,or in programmed cell death.The upregulated probe set showing the highest mean fold change was similar to the human ubiquitin specific protease 53 gene.The downregulated probe sets showing some of the highest fold changes corresponded to genes with roles in eye development,including those encoding gamma crystallins,the guanine nucleotide binding pro-teins Gnat1 and Gant2,a BarH-like homeodomain transcription factor,and rhodopsin.Downregulation of gam-ma-crystallin and rhodopsin was confirmed by in situ hybridization and immunostaining with specific antibodies.Additional downregulated genes encode molecules that inhibit or activate programmed cell death.The results suggest that cross-species microarray can be used for identifying differentially expressed genes in cavefish,that many of these genes might be involved in eye degeneration via apoptotic processes,and that more genes are downregulated than upregulated in cavefish,consistent with the predominance of morphological losses over gains during regressive evolution.

Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus

Recently,increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus(IAV)infections.Notably,some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans,posing a threat to public health.Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32(ANP32)is the most important one among them.However,role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown.In the present study,a total of 10 feline ANP32(feANP32)splice variants(including 5 feANP32A,3 feANP32B,and 2 feANP32E)were obtained from domestic cats by RT-PCR.Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants,but all feANP32 proteins were primarily localized to cell nucleus.Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated.The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains,though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains.In addition,the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro.Collectively,our study systematically investigated the support activity of feANP32 on IAV,providing a clue for further exploring the mechanism of susceptibility of cats to IAV.

Learning Sequential and Structural Dependencies Between Nucleotides for RNA N6-Methyladenosine Site Identification

N6-methyladenosine(m6A)is an important RNA methylation modification involved in regulating diverse biological processes across multiple species.Hence,the identification of m6A modification sites provides valuable insight into the biological mechanisms of complex diseases at the post-transcriptional level.Although a variety of identification algorithms have been proposed recently,most of them capture the features of m6A modification sites by focusing on the sequential dependencies of nucleotides at different positions in RNA sequences,while ignoring the structural dependencies of nucleotides in their threedimensional structures.To overcome this issue,we propose a cross-species end-to-end deep learning model,namely CR-NSSD,which conduct a cross-domain representation learning process integrating nucleotide structural and sequential dependencies for RNA m6A site identification.Specifically,CR-NSSD first obtains the pre-coded representations of RNA sequences by incorporating the position information into single-nucleotide states with chaos game representation theory.It then constructs a crossdomain reconstruction encoder to learn the sequential and structural dependencies between nucleotides.By minimizing the reconstruction and binary cross-entropy losses,CR-NSSD is trained to complete the task of m6A site identification.Extensive experiments have demonstrated the promising performance of CR-NSSD by comparing it with several state-of-the-art m6A identification algorithms.Moreover,the results of cross-species prediction indicate that the integration of sequential and structural dependencies allows CR-NSSD to capture general features of m6A modification sites among different species,thus improving the accuracy of cross-species identification.

EST-derived microsatellites in P seudosciaena crocea and their applicability to related species

Simple sequence repeat （SSR） markers were obtained for the large yellow croaker Pseudosciaena crocea using 1 205 expressed sequences tags （ESTs） from the NCBI database.Primers for 48 ESTSSR loci were designed and screened with 30 P.crocea specimens captured from Guanjingyang sea area in Fujian Province of China.Sixteen of the loci were polymorphic,which were amplified with 3 to 11 alleles per locus and the mean of 6.13.The observed and expected heterozygosity per locus ranged from 0.091 to 0.844 （mean 0.544） and from 0.118 to 0.892 （mean 0.644）,respectively.Polymorphic information content （PIC） ranged from 0.115 to 0.866 （mean 0.593）.The results for cross-species amplification of the 16 large yellow croaker EST-SSRs on P.polyactis,C.niveatus,C.lucidus,A.argentatus and J.belengeri revealed that 14,12,11,7 and 6 loci were successfully amplified with 1 to 10 alleles with an average of 4.5 per locus,respectively,which are suitable for population genetics studies of these species and useful for phylogenetic relationship analysis among these species.Overall,this study provides a set of type I markers for population genetics studies and genome mapping for large yellow croaker and its closely related species.

Comparative transcriptomic analysis of rat versus mouse cerebral cortex after traumatic brain injury

The heterogeneity of traumatic brain injury(TBI)-induced secondary injury has greatly hampered the development of effective treatments for TBI patients.Targeting common processes across species may be an innovative strategy to combat debilitating TBI.In the present study, a cross-species transcriptome comparison was performed for the first time to determine the fundamental processes of secondary brain injury in Sprague-Dawley rat and C57/BL6 mouse models of TBI, caused by acute controlled cortical impact.The RNA sequencing data from the mouse model of TBI were downloaded from the Gene Expression Omnibus(ID: GSE79441) at the National Center for Biotechnology Information.For the rat data, peri-injury cerebral cortex samples were collected for transcriptomic analysis 24 hours after TBI.Differentially expressed gene-based functional analysis revealed that common features between the two species were mainly involved in the regulation and activation of the innate immune response, including complement cascades as well as Toll-like and nucleotide oligomerization domain-like receptor pathways.These findings were further corroborated by gene set enrichment analysis.Moreover, transcription factor analysis revealed that the families of signal transducers and activators of transcription(STAT), basic leucine zipper(BZIP), Rel homology domain(RHD), and interferon regulatory factor(IRF) transcription factors play vital regulatory roles in the pathophysiological processes of TBI, and are also largely associated with inflammation.These findings suggest that targeting the common innate immune response might be a promising therapeutic approach for TBI.The animal experimental procedures were approved by the Beijing Neurosurgical Institute Animal Care and Use Committee(approval No.201802001) on June 6, 2018.

Seroprevalence and evolutionary dynamics of genotype 4 hepatitis E virus in Shandong Province,China

AIM: To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus (HEV) and assess the ancestor of HEVs in China&#x02019;s Shandong Province.

Species Diversity and Elevational Distribution of Amphibians in the Xianxialing and Wuyishan Mountain Ranges,Southeastern China

The species diversity and altitudinal distribution of amphibians along an eleva tional gradient of 200-1600 m in the Xianxialing and Wuyishan Mountain Ranges in Southeastern China were investiga ted through time-constra ined visual surveys along 32 transect lines in 9 survey areas,in which the ha bitat types were also recorded.A total of 27 amphibian species belonging to 19 genera,7 families,and 2 orders were found.The species diversity of the amphibians plateaued at low elevation,and the altitudinal boundary of their distribution was at 800 m.Their species compositions were dissimilar in the two mountain ranges probably beca use the annual average temperature and annual rainfall were different in both areas.The eleva tional Rapoport's rule demonstrated that the species range size of the amphibians expanded as the elevation increased in both mountain ranges.The results of the cross species method supported the rule only when the influence of the low-frequency occurrence proba bility of an investigated species was excluded,whereas those of the Steven's method strongly corroborated the rule rega rdless of the incidental occurrence or absence of the species.

Isolation and Characterization of 17 Microsatellite DNA Loci for Odorrana margaretae(Anura: Ranidae)

We isolated and characterized 17 microsatellite DNA loci for the odorous frog Odorrana margaretae from its transcriptome sequence data. These loci were screened with 24 individuals from Mt. Emei. All loci were polymorphic, with the number of alleles ranging from 2 to 8. The observed and expected heterozygosity, polymorphism information content, ranged from 0.04 to 1, 0.04 to 0.81, and 0.040 to 0.763, respectively. All loci were in linkage equilibrium and six loci were significantly deviated from Hardy-Weinberg equilibrium after sequential Bonferroni corrections. Cross- species amplification test was conducted for ten odorous frog species, and 12 loci were amplifiable in most species. With the high cross-species amplification rates, thesc markers will provide useful molecular tools for conservation genetic and phylogeographic studies on the genus Odorrana and Bamburana.