黑龙江省马铃薯晚疫病菌的交配型和multi-locus基因型分析

2015~2017年在黑龙江省哈尔滨市、绥化市和齐齐哈尔市各马铃薯主产区共采集分离了126株马铃薯晚疫病菌菌株,测定交配型、mtDNA单倍型、SSR基因型并进行multi-locus基因型分析。结果显示,126株菌株中发现了A1、A2、自育型3种交配型,分别占分离菌株总数的88.1%、6.3%和5.6%。126株菌株中共鉴定出Ⅰa和Ⅱa两种mt DNA单倍型,分别占分离菌株总数的17.5%和82.5%。126株菌株中共鉴定出7种SSR基因型,分别为F-01、F-02、F-03、F-05、F-06、D-03和G-02,其中F-01基因型(77.8%)为优势基因型。根据马铃薯晚疫病菌的mt DNA单倍型和SSR基因型,共划分了9种multi-locus基因型,其中multi-locus基因型A(65.1%)是黑龙江省发现的优势基因型。2015~2017年黑龙江省马铃薯晚疫病菌群体结构逐年复杂,绥化市马铃薯晚疫病菌群体结构最为复杂。

mrMLM v4.0.2: An R Platform for Multi-locus Genome-wide Association Studies

Previous studies have reported that some important loci are missed in single-locus genome-wide association studies(GWAS),especially because of the large phenotypic error in field experiments.To solve this issue,multi-locus GWAS methods have been recommended.However,only a few software packages for multi-locus GWAS are available.Therefore,we developed an R software named mr MLM v4.0.2.This software integrates mr MLM,FASTmr MLM,FASTmr EMMA,p LARm EB,p KWm EB,and ISIS EM-BLASSO methods developed by our lab.There are four components in mr MLM v4.0.2,including dataset input,parameter setting,software running,and result output.The fread function in data.table is used to quickly read datasets,especially big datasets,and the do Parallel package is used to conduct parallel computation using multiple CPUs.In addition,the graphical user interface software mr MLM.GUI v4.0.2,built upon Shiny,is also available.To confirm the correctness of the aforementioned programs,all the methods in mr MLM v4.0.2 and three widely-used methods were used to analyze real and simulated datasets.The results confirm the superior performance of mr MLM v4.0.2 to other methods currently available.False positive rates are effectively controlled,albeit with a less stringent significance threshold.mr MLM v4.0.2 is publicly available at Bio Code(https://bigd.big.ac.cn/biocode/tools/BT007077)or R(https://cran.r-project.org/web/packages/mr MLM.GUI/index.html)as an open-source software.

Multi-locus association study of schizophrenia susceptibility genes with a posterior probability method

Schizophrenia is a serious neuropsychiatric illness affecting about 1% of the world’s population. It is considered a complex inheritance disorder. A number of genes are involved in combination in the etiology of the disorder. Evidence implicates the altered dopaminergic trans- mission in schizophrenia. In the present study, in order to identify susceptibility genes for schizophrenia in dopaminergic metabolism, we analyzed 59 single nucleotide polymorphisms (SNPs) in 24 genes of the dopaminergic pathway among 82 unrelated patients with schizophre- nia and 108 matched normal controls. Considering that traditional single-locus association stud- ies ignore the multigenic nature of complex diseases and do not take into account possible in- teractions between susceptibility genes, we proposed a multi-locus analysis method, using the posterior probability of morbidity as a measure of absolute disease risk for a multi-locus genotype combination, and developed an algorithm based on perturbation and average to detect the sus- ceptibility multi-locus genotype combinations, as well as to repress noise and avoid false positive results at our best. A three-locus SNP genotype combination involved in the interactions of COMT and ALDH3B1 genes was detected to be significantly susceptible to schizophrenia.

Multi-locus identification of Psilocybe cubensis by high-resolution melting(HRM)

Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin,psilocin and ibotenic acid,etc.The mushrooms containing hallucinogenic components are various,widely distributed and lack of standard to define,which made a great challenge to identification.Traditional identification methods,such as morphology and toxicology analysis,showed shortcomings in old or processed samples,while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA.In this paper,four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method,and the target markers include largest subunit of RNA polymerase II(marked as PC-R1),psilocybin-related phosphotransferase gene(marked as PC-PT),glyceraldehyde 3-phosphate dehydrogenase(marked as PC-3)and translation EF1α(marked as PC-EF).Real-time PCR with high-resolution melting(HRM)assay were used for the differentiation of the fragments amplified by these primer sets,which were tested for specificity,reproducibility,sensitivity,mixture analysis and multiplex PCR.It was shown that the melting temperatures of PC-R1,PC-PT,PC-3 and PC-EF of P.cubensis were(87.93±0.12)℃,(82.21±0.14)℃,(79.72±0.12)℃ and(80.11±0.19)℃ in our kinds of independent experiments.Significant HRM characteristic can be shown with a low concentration of 62.5pg/µL DNA sample,and P.cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa.In summary,the method of HRM analysis can quickly and specifically distinguish P.cubensis from other species,which could be utilized for forensic science,medical diagnosis and drug trafficking cases.

Genome-wide association with transcriptomics reveals a shade-tolerance gene network in soybean

Shade tolerance is essential for soybeans in inter/relay cropping systems.A genome-wide association study(GWAS)integrated with transcriptome sequencing was performed to identify genes and construct a genetic network governing the trait in a set of recombinant inbred lines derived from two soybean parents with contrasting shade tolerance.An improved GWAS procedure,restricted two-stage multi-locus genome-wide association study based on gene/allele sequence markers(GASM-RTM-GWAS),identified 140 genes and their alleles associated with shade-tolerance index(STI),146 with relative pith cell length(RCL),and nine with both.Annotation of these genes by biological categories allowed the construction of a protein–protein interaction network by 187 genes,of which half were differentially expressed under shading and non-shading conditions as well as at different growth stages.From the identified genes,three ones jointly identified for both traits by both GWAS and transcriptome and two genes with maximum links were chosen as beginners for entrance into the network.Altogether,both STI and RCL gene systems worked for shade-tolerance with genes interacted each other,this confirmed that shadetolerance is regulated by more than single group of interacted genes,involving multiple biological functions as a gene network.

Disease burden,antimicrobial resistance and molecular characterization of invasive group B Streptococcus among non-pregnant adults in Malaysia:A protocol study

and pili genes are also investigated.Methods:This multicentre,prospective,observational study is conducted in seven major tertiary hospitals in Malaysia among non-pregnant adults.Simultaneously,a retrospective study is conducted in the selected hospitals with similar approaches.GBS isolates are subjected to phenotyping,serotyping by multiplex PCR,antimicrobial susceptibility testing and PCR-detection of GBS virulence and pilus genes.Seven housekeeping genes are amplified and sequenced for multi-locus sequence typing.Discussion:Findings from the study may contribute to the management of clinical practice to diagnose and prevent GBS related diseases in a timely manner.Prudent use of antibiotics is encouraged by monitoring antimicrobial resistance.

Antimicrobial resistance and molecular typing of Salmonella in the chicken production chain in Hubei Province, China

Salmonella is a significant foodborne zoonotic pathogen that endangers both human and animal health.The goal of this research is to gain a preliminary understanding of Salmonella contamination and antimicrobial resistance in the chicken production chain in Hubei Province,China.1149 animal and environmental samples were collected from chicken farms,slaughterhouses,and retail markets in six cities across Hubei Province in China from 2019 to 2020,yielding Salmonella isolation rates of 4.68%(28/598),12.21%(47/385),and 9.64%(16/166),respectively.Seventeen distinct serotypes were detected among 53 non-clonal Salmonella strains,of which Meleagridis(26.42%,14/53)was the dominant serotype.Almost half of the strains(49.06%,26/53)were multi-drug resistant(MDR).Whole-genome sequencing(WGS)showed that 10 resistance genes(tetA,bla_(TEM),parC,qnrs1,floR,aac(6'-ly,aph(6)-ld,aph(3")-b,aac(6')-laa and sul2)and 7 categories of virulence genes were present in all three links in 22 non-clonal dominant serotype strains.It was shown that Salmonella in the chicken production chain in Hubei Province had a high resist-ance rate to Tetracycline(TET,73.58%),Ofloxacin(OFL,69.81%),Florfenicol(FFC,60.38%)and Ampicillin(AMP,39.62%)which was consistent with the widespread use of these drugs in the husbandry industry in China.Salmonella ST types determined by MLST and serotypes determined by WGS had a one-to-one correlation.Minimum spanning tree analysis revealed that there was cross contamination of Salmonella in farms and slaughterhouses,slaughterhouses and markets,animal samples and environmental samples.This work provides useful information for the prevention and control of contamination and antimicrobial resistance of Salmonella in the chicken production chain,as well as demonstrating the dependable role of WGS in Salmonella molecular typing.

Genetic Diversity, Antimicrobial Resistance, and Virulence Genes of Aeromonas Isolates from Clinical Patients, Tap Water Systems, and Food

Objective This study aimed to evaluate the genetic diversity,virulence,and antimicrobial resistance of Aeromonas isolates from clinical patients,tap water systems,and food.Methods Ninety Aeromonas isolates were obtained from Ma’anshan,Anhui province,China,and subjected to multi-locus sequence typing(MLST)with six housekeeping genes.Their taxonomy was investigated using concatenated gyr B-cpn60 sequences,while their resistance to 12 antibiotics was evaluated.Ten putative virulence factors and several resistance genes were identified by PCR and sequencing.Results The 90 Aeromonas isolates were divided into 84 sequence types,80 of which were novel,indicating high genetic diversity.The Aeromonas isolates were classified into eight different species.PCR assays identified virulence genes in the isolates,with the enterotoxin and hemolysin genes act,aer A,alt,and ast found in 47(52.2%),13(14.4%),22(24.4%),and 12(13.3%)of the isolates,respectively.The majority of the isolates(≥90%)were susceptible to aztreonam,imipenem,cefepime,chloramphenicol,gentamicin,tetracycline,and ciprofloxacin.However,several resistance genes were detected in the isolates,as well as a new mcr-3 variant.Conclusions Sequence type,virulence properties,and antibiotic resistance vary in Aeromonas isolates from clinical patients,tap water systems,and food.

Culturable plant pathogenic fungi associated with sugarcane in southern China

Sugarcane(Saccharum officinarum)is one of the oldest crops cultivated by mankind.Numerous fungal taxa have been reported from this host,although most were not identified beyond genus level.In this study,we explored 31 sampling sites in Guangxi and Guangdong Provinces(China),and collected 370 diseased samples from leaves and roots of sugarcane,from which 762 strains were isolated.Our preliminary analysis based on internal transcribed spacer(ITS)sequences suggested that these isolates belonged to 143 species in 51 genera,but we could not assign 129 strains.Bipolaris,Chaetomium,Curvularia,Phoma and Nigrospora represented the top five most common genera identified,while 27 rare genera comprised only one identified species.In this study,we chose above mentioned common genera for in-depth morphological observations and multi-locus analyses,in order to identify these strains to species level.In this paper,we described one new genus,32 new species,and reported 19 new records for China and the asexual morph of Chaetomium olivaceum.Hitherto,this is the most comprehensive study with molecular identification and illustration of fungi associated with sugarcane,which greatly improves our understanding of culturable mycota associated with this host.

Taxonomic revisionofthegenus Lactarius(Russulales,Basidiomycota)in Korea

The genus Lactarius Pers.(Russulales)is a cosmopolitan group of Basidiomycota that forms ectomycorrhizal relationships primarily with both deciduous and coniferous trees.Although the genus has been well-studied in Europe and North America,only fragmentary researches have been carried out on Asian species.In particular,the distribution of Lactarius species in South Korea is poorly understood due to insufficient morphological descriptions and a lack of DNA sequence data.In addition,the misuse of European and North American names has added to confusion regarding the taxonomy of Asian Lactarius species.In this study,the diversity of Lactarius in South Korea was evaluated by employing both morphological and phylogenetic approaches.A multi-locus phylogenetic analysis of 729 Lactarius specimens collected between 1960 and 2017 was performed using the internal transcribed spacer(ITS)region,partial nuclear ribosomal large subunit(nrLSU),partial second largest subunit of RNA polymerase II(rpb2),and minichromosome maintenance complex component 7(mcm7).49 Lactarius species were identified in three Lactarius subgenera:L.subg.Russularia(17 spp.),L.subg.Lactarius(22 spp.),and L.subg.Plinthogalus(10 spp.).Among them,28 Lactarius species were identified as new to science,while just 17 were previously described Lactarius species.Four of the taxa remain un-named due to paucity of materials.A key to Korean Lactarius species,molecular phylogenies,a summary of diversity,and detailed description are provided.

Lichenicolous fungi of the genus Abrothallus (Dothideomycetes: Abrothallales ordo nov.) are sister to the predominantly aquatic Janhulales

This study provides new insights on the phylogenetic position of the lichenicolous fungal genus Abrothallus based on six molecular markers(nuSSU,nuLSU,mtSSU,RPB1,RPB2 and TEF-α).In a broad-scale analysis,we detected high support for inclusion of the genus within Dothideomycetes.A further analysis provided support for Abrothallus as a member of the subclass Pleosporomycetidae as a sister group of Jahnulales,an order of aquatic Dothideomycetes.Given the exclusive characters of this group of apotheciate fungi within the Dothidiomycetes,a new monotypic order Abrothallales is here introduced together with the new family Abrothallaceae.In a multi-locus analysis(based on the six loci indicated above plus ITS)restricted to 12 putative Abrothallus species,two clearly separated clades were observed:one comprising species growing on lichens of the families Parmeliaceae and Ramalinaceae,and the second including species that live on lichens of the order Peltigerales and the family Cladoniaceae.

Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics

Early efforts to classify Mortierellaceae were based on macro-and micromorphology,but sequencing and phylogenetic studies with ribosomal DNA(rDNA)markers have demonstrated conflicting taxonomic groupings and polyphyletic genera.Although some taxonomic confusion in the family has been clarified,rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae.In this study,we applied two parallel approaches to resolve the Mortierel-laceae phylogeny:low coverage genome(LCG)sequencing and high-throughput,multiplexed targeted amplicon sequenc-ing to generate sequence data for multi-gene phylogenetics.We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset.Resolving the Mortierellaceae phylogeny into monophyletic genera resulted in 13 genera,7 of which are newly proposed.Low-coverage genome sequencing proved to be a relatively cost-effective means of generating a high-confidence phylogeny.The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach,but has limitations too.We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity and evolution.

Biphasic taxonomic approaches for generic relatedness and phylogenetic relationships of Teichosporaceae

The placement of the dothideomycetous family Teichosporaceae has been controversial.Recent phylogenetic investigations have used a taxonomic lumping approach with the Floricolaceae and its genera have been synonymized under the earlier fam-ily name,Teichosporaceae.Intergeneric relationships were therefore obscure and proper generic delimitation was needed in upcoming studies.We here taxonomically revised the family Teichosporaceae based on both morphological and phylogenetic evidence.Teichosporaceae species have immersed or semi-immersed,erumpent to superficial,ostiolate ascomata,cellular or trabeculate pseudoparaphyses,cylindrical to oblong or sub-clavate asci and ellipsoid to oblong or fusiform,cylindric-fusiform or oblong to elliptical,ovoid to clavate,symmetric or asymmetric,initially hyaline or pale brown to dark brown or yellowish brown,1-3-septate or muriform ascospores.Asexual morphs are coelomycetous.Type or representative speci-mens of Teichosporaceae were loaned and fresh specimens were collected from China and Thailand.Maximum likelihood and Bayesian analyses of a combined ITS,LSU,SSU,tef1-αand rpb2 dataset were performed to clarify the phylogenetic affinities of taxa and examine monophyly of newly proposed genera.One new species(Floricola festucae),one new host record(Ramusculicola thailandica)and four new combinations(Aurantiascoma nephelii,A.quercus,Magnibotryascoma acaciae,M.melanommoides)are introduced.The broad genus concept of Teichospora is dismissed based on morphological dissimilarities and the monophyletic status of the proposed genera.We accept Asymmetrispora,Aurantiascoma,Floricola,Magnibotryascoma,Misturatosphaeria,Pseudoaurantiascoma,Pseudomisturatosphaeria,Ramusculicola and Teichospora as distinct genera in the Teichosporaceae.All recognized genera are phenotypically characterized and phylogenetically well-supported.The phylogenetic placements of three genera(Chaetomastia,Loculohypoxylon and Sinodidymella),which do not have molecular data cannot be conclusively clarified at present,but are still placed in Teichosporaceae for future studies.

Surveillance and examination of microbial contamination in ice cream in China

This study investigated the microbial contamination of ice cream in China.A total of 2887 ice cream samples were collected from different regions of China.Contamination by the aerobic plate count(APC),coliforms,and three foodborne pathogens,Listeria monocytogenes(L.monocytogenes),Staphylococcus aureus(S.aureus),and Salmonella spp.,was detected in the samples.L.monocytogenes isolates were further analyzed for antibiotic susceptibility and multi-locus sequence typing(MLST).The results showed that APC was more than 10^(5) colony forming units(CFU)/g in 6.10%(176/2887)and coliform was more than 10^(2) CFU/g in 15.69%(453/2887)of all samples.The positive rates of S.aureus,L.monocytogenes,and Salmonella spp.were 0.66%,0.62%,and 0.10%,respectively.Among these,S.aureus contamination was more than 102 CFU/g in two samples,and L.monocytogenes in the positive sample was in the range of 0.3-240 most probable number(MPN)/g.with a median of 4.3 MPN/g.The hygienic status of the packaged samples was much better than that of the bulk samples(P<0.05).Catering samples were more frequently and heavily contaminated than samples from retail and wholesale outlets(P<0.05).No significant differences were observed in samples bought from urban and rural areas(P>0.05).For 18 L.monocytogenes strains isolated from ice cream,the resistance rate of nine antibiotics was 5.56%(1/18).By MLST,the L.monocytogenes strains were classified into nine sequence types(STs),of which ST8 was the most common(six isolates).These results indicate that a potential health risk to the public may be caused by ice cream,particularly in susceptiblepopulations.