Gene characterization and phylogenetic analysis of four mitochondrial genomes in Caenogastropoda

Caenogastropoda is a highly diverse group,containing~60%of all existing gastropods.Species in this subclass predominantly inhabit marine environments and have a high ecological and economic value.Owing to the increase in relevant phylogenetic studies,our understanding of between species relatedness in Caenogastropoda has improved.However,the biodiversity,taxonomic status,and phylogenetic relationships of this group remain unclear.In the present study,we performed next-generation sequencing of four complete mitochondrial genomes from three families(Buccinidae,Columbellidae,and Cypraeidae)and the four mitogenomes were classical circular structures,with a length of 16177 bp in Volutharpa ampullacea,16244 bp in Mitrella albuginosa,16926bp in Mauritia arabica asiatica and 15422 bp in Erronea errones.Base composition analysis indicated that whole sequences were biased toward A and T.Then compared them with 171 complete mitochondrial genomes of Caenogastropoda.The phylogenetic relationship of Caenogastropoda derived from Maximum Likelihood(ML)and Bayesian Inference(BI)trees constructed based on CDS sequences was consistent with the results of traditional morphological analysis,with all three families showing close relationships.This study supported Caenogastropoda at the molecular level as a separate clade of Mollusca.According to our divergence time estimations,Caenogastropoda was formed during the middle Triassic period(~247.2–237 Ma).Our novel mitochondrial genomes provide evidence for the speciation of Caenogastropoda in addition to elucidating the mitochondrial genomic evolution of this subclass.

Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

Identification and Phylogenetic Analysis of an Orf Virus Isolated from an Outbreak in Boer Goat in Shanxi Province

To identify and analyze the Orf virus in Shanxi Province, China, an Orf virus strain was successfully isolated from crust materials of boer goat with clinical sore mouth symptom from a goat farm of Shanxi Province by passaging in lamb testis （LT）. The Orf virus was identified by enzyme linked immunosorbent assay （ELISA） test, recurrent infection test, transmission electron microscopy, and PCR. The nucleotide and amino acid sequences of two genes of the Orf virus were analyzed. The results showed that under the electron microscopy the virus had a presence of typical parapoxvirus virions and there were many eosinophilic intracytoplasmic inclusions observed by hematoxylin-eosin （H＆E） stain. In ELISA test, optical density （OD） readings of the sample showed a positive result, and the rabbits infected with the virus showed a typically Orf virus-infected appearance. All these findings proved that the sample was an Orf virus. The phylogenetic studies of Orf B2L and Orf F1L genes showed that the virus clustered in different branches and were closer to the Orf virus Nantou （DQ904351） and the OV-SA00 isolates （AY386264）. Furthermore, the above results may provide some insight into the genotype of the etiological agent responsible for the Orf outbreak in Shanxi Province, and could also provide a comparative view of the B2L and F1L genes of parapoxvirus.

Characterization and phylogenetic analysis of a phenanthrene-degrading strain isolated from oil-contaminated soil

Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp. based on analysis of 16S rDNA sequence,cellular fatty acid composition and physiological-chemical tests. The salicylate hydroxylase and catechol 2,3-dioxygenase(C23O) were detected in cell-free lysates, suggesting a pathway for phenanthrene catabolism via salicylate and catechol. Alignment showed that both of the C23O and GST genes of the strain EVA17 had high similarity with homologues of strains from genus Sphingomonas. The phylogenetic analysis based on 16S rDNA and C23O gene sequence indicated that EVA17 should be classified into genus Sphingomonas, although the two phylogenetic trees were slightly different from each other. The results of co-amplification and sequence determination indicated that GST gene should be located upstream of the C23O gene.

Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)

Mitogen activated protein kinases （MAPK） cascades based on protein phosphorylation play an important role in plant growth and development. In this study, we have identified 15 putative members of the wheat MAPK gene （TaMPK） family through an in silico search of wheat expressed sequence tags （EST） databases based on the presence of amino acid sequence of Arabidopsis and rice MAPKs. Phylogenetic analyses of MAPKs from wheat, rice and Arabidopsis genomes have classified them into seven subgroups （A, B, C, D, E, F, and G）. Using the available EST information as a source of expression data, the MAPK family genes from Triticum aestivum were detected in diverse tissues. Further expression analysis of the MAPKs in NCBI EST database revealed that their transcripts were most abundant in callus （20%）, followed by leaf （12%） and inflorescence （12%）. Most MAPK family genes showed some tissue specificity.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Characterization of the Complete Mitochondrial Genome of the Hybrid Epinephelus moara♀ × Epinephelus lanceolatus♂,and Phylogenetic Analysis in Subfamily Epinephelinae

This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 r RNA genes, 22 t RNA genes, a light-strand replication origin and a control region. Additionally, phylogenetic analysis based on the nucleotide sequences of 13 conserved protein-coding genes using the maximum likelihood method indicated that the mitochondrial genome is maternally inherited. This study presents genomic data for studying phylogenetic relationships and breeding of hybrid Epinephelinae.

Phylogenetic analysis and arsenate reduction effect of the arsenic-reducing bacteria enriched from contaminated soils at an abandoned smelter site

Microbial reduction of As(V)(i.e.,arsenate)plays an important role in arsenic(As)mobilization in aqueous environment.In this study,we investigated As(V)reduction characteristics of the bacteria enriched from the arsenic-contaminated soil at an abandoned smelter site.It was found that As(V)was completely reduced to As(III)(i.e.,arsenite)in 21 h.After 3-d incubation,a yellow solid was precipitated and the concentration of As(III)decreased sharply.After 150 h incubation,ca.65%of soluble arsenic was removed fro...

Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

The genetic variation and phylogenetic relationships of Citrus tristeza virus （CTV） isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions （p23, p20, p13, p18, p25, p27, POL, HEL and k17） with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3′ proximal region was relatively conserved, and the 5′ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.

Comparative mitochondrial genome analysis of Sesarmidae and its phylogenetic implications

Here,we sequenced the complete mitogenome of Parasesarma eumolpe(Brachyura:Grapsoidea:Sesarmidae)for the first time.The characteristics of this newly sequenced mitogenome were described and compared with other Sesarmidae species.The 15646-bp mitogenome contains 13 protein-coding genes(PCGs),two ribosomal RNA genes(r RNAs),22 transfer RNA genes(t RNAs),and an A-T rich region.All of the PCGs are initiated by the start codon ATN and terminated by the standard TAN codon or an incomplete T.The pairwise Ka/Ks ratio analysis shows that all 13 PCGs are under purifying selection,whereas the ATP8 gene is an outlier,with pairwise comparison values ranging from neutral selection(0.000)to positive selection(1.039).The gene arrangement of P.eumolpe compared with ancestral Decapoda shows the translocation of two t RNAs(t RNA-His and t RNA-Gln),which is identical to other Sesarmidae species.Phylogenetic analyses show that all Sesarmidae species are placed into one group,and the polyphyly of Eriphioidea,Ocypodoidea,and Grapsoidea is well supported.The relationship between gaps in the QIM region and the phylogeny of Sesarmidae is analyzed.It is obvious that both the G5(the gap between Q and I)and G6(the gap between I and M)decrease progressively with the evolution process.These results will help to better understand the genomic evolution within Sesarmidae and provide insights into the phylogeny of Brachyura.

Biodiversity and phylogenetic analysis of the gut microbiome of Euphausia superba Dana from the Rose Sea of the Antarctic Ocean

Metabolites derived from marine symbiotic microorganisms have great potential as lead compounds for the discovery of novel marine drugs. Euphausia superba Dana, which lives in the Antarctic Ocean, is regarded as a new source of marine microbial natural products. However, no studies have examined the biodiversity of the symbiotic intestinal microbiome of E. superba. To address this issue, the species diversity and abundance of the gut microbiome of E. superba Dana from the Rose Sea of the Antarctic Ocean were analyzed by culture-independent high-throughput sequencing and pure culture methods. A comparison with gene databases revealed that the microbiome contained 61 known microbial species and a plethora of uncultivable microorganisms. Additionally, 7% of the species in the microbiome were currently unknown. The microbes belonged to 56 genera, eight of which, including Arthrobacter, Bacillus, Candidatus, Lactococcus, Lysinibacillus, Leuconostoc, Solibacillus, and Vibrio, were dominant, as were Vibrionaceae spp. Moreover, 81 microbial strains were isolated by the pure culture method, and they belonged to 36 genera, including Mobilicoccus, Rhodococcus, Arthrobacter, and Microbacterium. The results obtained by two different methods demonstrate the richness of the microbial biodiversity of the gut microbiome of E. superba, and it also suggests that they have good potential for the discovery of novel marine microbial species.

Isolation and Phylogenetic Analysis of Vibrio ichthyoenteri Strains from Hapalogenys nitens

[ Objective] This study aimed to isolate and identify Vibrio ichthyoenteri strains from diseased Hapalogenys nitens. [ Method] In January 2013, dis- eased H. nitens juveniles from a maricultural farm in Luoyuanwan Bay of Fuzhou City were selected to investigate the incidence, clinical symptoms and pathogenic characteristics. Liver, spleen, kidney and gill of five diseased H. nitens juveniles were collected for bacterial isolation and pure culture to conduct morphological, physiological and biochemical assays, 16S rRNA gene sequencing and phylogenetic analysis. The susceptibility of five strains to 30 kinds of antimicrobial drugs was detected. [ Result] The death of H. nitens juveniles was caused by bacterial infections. The isolated strains fzlyw201301071 and fzlyw201301072 belonged to the same genus as Vibrio ichthyoenteri. Drug susceptibihty test suggested that there were no significant differences in drug sensitivity and resistance among different strains. [ Conclusion] This study provided theoretical basis for the prevention and control of diseases in aquaculture animals.

PCR Based Detection and Phylogenetic Analysis of Fowl Adenovirus Strains Isolated from 2019 Epidemic from Punjab and Sindh, Pakistan

Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.

Identification and phylogenetic analysis of two canine coronavirus strains

Canine coronavirus(CCoV),a member of the genus Alphocoronavirus,is an enveloped,single-stranded positive-sense RNA virus that responsible for gastroenteritis in dogs.In this study,two CCoV isolates were successfully propagated from 53 CCoV-positive clinical specimens by serial passaging in A-72 cells.These two strains,CCoV JS1706 and CCoV JS1712,caused cytopathic effects in A-72 cells.The sizes of virus plaque formed by them differed in early passages.Electron microscopy revealed a large quantity of typical coronavirus particles with 80-120 nm in diameter in cell culture media and cytoplasm of infected cells,in which they appeared as inclusion bodies.RT-PCR analysis of 5 gene indicated that these two isolates were belonged to CCoV lla subtype.Homology of RdRp,S,M and N proteins between the two strains were 100,99.6,99.2 and 100.0%,respectively,whereas they were 99.4-100%,83.1-95.2%,88.5-99.2% and 91.9-99.7%identity compared to CCoV II reference strains.Phylogenetic analysis of RdRp,S,M and N protein showed that they were closely related to CCoV II strains.These two subtype lla isolates will be useful for evaluating the pathogenesis and evolution of CCoV and for developing diagnostic reagents and vaccines.

Complete Mitochondrial Genome Analysis of Daphniopsis tibetana(Branchiopoda:Diplostraca):New Insights into the Taxonomy of the Genus and Its Phylogenetic Implications for Branchiopoda

Daphniopsis tibetana is widely distributed in Xinjiang,Qinghai,Tibet of China,as well as in Russia and India,which is the dominant zooplankton in many high-altitude(4000 m)salt lakes.D.tibetana can adapt to saline waters,whereas the other species of the superorder Cladocera can only inhabit in freshwater.However,the phylogenetic status of D.tibetana in Branchiopoda remains unclear primarily because of limited mitogenome.In this study,complete mitochondrial genome sequences of D.tibetana were sequenced and annotated for the first time to obtain a comprehensive understanding of its phylogenetic status.The complete mitogenome of D.tibetana is 16196 bp in length.It contains 37 genes,including two ribosomal RNAs(12S and 16S rRNAs)genes,22 transfer RNA(tRNA)genes,13 protein-coding genes,and one non-coding region.The overall base composition is 29.6%A,33.2%T,19.0%G,and 18.2%C with a high AT bias(62.8%).Except for trnS1(GCT),most tRNAs have a typical cloverleaf secondary structure.Phylogenetic analysis based on maximum likelihood and Bayesian inference generates identical phylogenetic topology and shows the phylogenetic status of D.tibetana,which reconfirm the distinction between the genera Daphniopsis and Daphnia.Meanwhile,the class Branchiopoda is clustered into three clades(Anostraca,Notostraca,and Diplostraca)with high support values.These results provide not only a comprehensive understanding of the characteristics of D.tibetana mitogenome and its phylogenetic position in Diplostraca,but also information for future research on the phylogeny of Branchiopoda.

Phylogenetic and Comparative Genomic Analysis of Lactobacillus fermentum Strains and the Key Genes Related to their Intestinal Anti-Inflammatory Effects

Emerging evidence shows that some Lactobacillus fermentum(L.fermentum)strains can contribute to the prevention and treatment of ulcerative colitis(UC).In this study,105 isolates of L.fermentum strains were separated from fecal samples of populations in different regions in China and their draft genomes were sequenced.Pan-genomic and phylogenetic characterizations of these strains and four model strains(L.fermentum 3872,CECT5716,IF03956,and VRI003)were performed.Phylogenetic analysis ind icated that there was no significant adaptive evolution between the genomes of L.fermentum strains and the geographical location,sex,ethnicity,and age of the hosts.Three L.fermentum strains(FWXBH115,FGDLZR121,and FXJCJ61)from different branches of the phylogenetic tree and strain type L.fermentum CECT5716 were selected and their anti-inflammatory and immune modulatory activities in a dextran sulphate sodium(DSS)-induced colitis mouse model were further investigated.Both L.fermentum FXJCJ61 and CECT5716 significantly alleviated UC by reducing all colitis-associated histological indices,maintaining mucosal integrity,and stimulating replenishment of short-chain fatty acids(SCFAs),while the other two strains failed to offer similar protection.The anti-inflammato ry mechanisms of L.fermentum FXJCJ61 and CECT5716 were related to the inhibition of nuclear factor kappa-B(NF-κB)signaling pathway activation and enhancement of interleukin 10(IL-10)production.Comparative genomic analysis of these strains identified candidate genes that may contribute to the anti-inflammatory effects of specific L.fermentum strains.

Molecular phylogenetic analysis of sulfate-reducing bacteria from deep sediment layers of the tropical West Pacific warm pool

The diversity of sulfate-reducing bacteria （SRB） from deep layers of deep-sea sediments [ more than 2 m bsf （below seafloor） ] of two sites （WO1 -3 and WPO1 -4） in a tropical West Pacific warm pool region was characterized by using molecular phylogenetic analysis. The results of culture-independent samples demonstrated that the dominant clones from both sites were related to Grampositive spore forming genus, Desulfotomaculum, which accounted for 36.8% of all the sequencing clones from Site WP01 - 3 and 62.8% from Site WP01 -4. However, the other SRB group which was generally reported to be predominant in the deep-sea sediments of other regions, δ- subclass of the proteobacteria was found to be in very low percentages. Therefore, it could be speculated that there existed a unique chemical environment in the deep-sea sediment of this warm pool region. When comparing the Desulfotomaculum sp. related sequences from both sites, it was revealed that though the Desulfotomaculum-like sequences from Site WP01 -3 were more diverse than those from Site WP01 -4, all these sequences from both sites showed high similarity and formed a new phylogenetically homogeneous cluster in the Desulfotomaculum genus which had never been reported before. Successful enrichment of SRB was only achieved from samples of Site WP01 -4 and the sequence analysis of culture-dependent samples further confirmed the dominance of Desulfotomaculum genus. But Desulfotomaculum-related sequences from culture-dependent and culture-independent samples belonged to two different clusters respectively. This difference showed the choice of cultivation to the microorganisms.

Phylogenetic Analysis of mtDNA from the Ancient Human of Yuan Dynasty in Inner Mongolia in China

A study of the genetic structure of an ancient human excavated from the Yikeshu site of Yuanshangdu ancient city in Inner Mongolia and the relationships between the ancient population and the extant populations was carried out. Sequences of the control region and coding region of mtDNA from the ancient human were analyzed by using direct sequencing and restriction-fragment length polymorphism （RFLP） methods, Phylogenetic analysis and multidimensional scaling analysis were also performed on the mtDNA data of the ancient population and 12 extant populations. These results show that the ancient individuals of Yikeshu site can be assigned to D, G, B and Z haplogroups that are prevalent in Duars and Mongolians from Inner Mongolia. The ancient population is also closer to Duar and Mongolian populations in genetic distance than other compared populations. This study reveals that the ancient population from Yikeshu site in the Yuan Dynasty shares a common ancestor with Mongolic-speaking Daur and Mongolian tribes.

Isolation and Identification of Porcine Epidemic Diarrhea Virus(PEDV) HLJ Strain with IPEC-J2 Cells and Phylogenetic Analysis of Its S Gene

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV),and is characterized by vomiting,diarrhea and dehydration of suckling pigs from 80% to 100% morbidity and 50% to 90% mortality,and resulted in tremendous economic losses to swine industry.The PEDV mainly infects small intestine of pigs,resulting in vacuolar degeneration and necrosis of mucosal epithelium.The IPEC-J2 is a pig intestine epithelial cell line,which is similar to the intestinal environment of piglets,can be used to isolate and identify the PEDV field isolates.In this study,it appeared the PEDV typical postmortem changes and histopathological lesion of degeneration and destruction of small intestine in infected piglets,and IHC identified that the PEDV distributed in the mucosa and submucosa of small intestine mostly.Furthermore,the PEDV HLJ strain was successfully isolated and characterized in the IPEC-J2 cells,and indicated that the IPEC-J2 cell line was sensitive to isolate and adapt the PEDV field strain,and could be utilized to multiply the PEDV rapidly.The S gene analysis indicated that the PEDV HLJ strain was the prevailed virus,belonged to Group 1 with attenuated virulent DR13,SC1402 and J-S2/2015 strains isolated in South Korea and China from 2014 to 2015.This study had important theoretical and practical significances on analyzing genetic variation of the PEDV,understanding the pathogenic characteristics of the virus and developing new vaccines for the PED.

Isolation, Molecular and Phylogenetic Analysis of Porcine Encephalomyocarditis Virus Strain HLJ in China

Encephalomyocarditis virus(EMCV)is a positive single-stranded small RNA virus without envelope,which can infect a variety of mammals.Swines are the most susceptible animals,which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows.Diseases caused by EMCV have a wide range of effects on the global swine industry.In this study,a strain of EMCV was isolated from a swine aborted fetus in northeast China.It was identified by reverse transcriptase polymerase chain reaction(RT-PCR),electron microscopic observation and indirect immunofluorescence assay.The subsequent results showed that the virus titer of HLJ strain grew to 8.3 lgTCID50 on baby hamster kidney 21(BHK-21)cells.And HLJ strain caused the specific cytopathic effect(CPE)on BHK-21 cells and severe pathological changes in mice.Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5%-99.7%.Phylogenetic analysis showed that EMCV isolates fell into five clusters:lineageⅠ,Ⅱ,Ⅲ,ⅣandⅤ,based on the nucleotide sequences of the entire open reading frame(ORF)and VP1 gene.HLJ isolate was grouped into lineage I.The analyses of amino acid mutation sites of VP1 protein showed that the amino acids at positions 20 and 54 in VP1 junction were unique to HLJ strain.The isolation of HLJ strain enriched the epidemiological database of EMCV.