Sex determination(SD)involves mechanisms that determine whether an individual will develop into a male,female,or in rare cases,hermaphrodite.Crustaceans harbor extremely diverse SD systems,including hermaphroditism,en...Sex determination(SD)involves mechanisms that determine whether an individual will develop into a male,female,or in rare cases,hermaphrodite.Crustaceans harbor extremely diverse SD systems,including hermaphroditism,environmental sex determination(ESD),genetic sex determination(GSD),and cytoplasmic sex determination(e.g.,Wolbachia controlled SD systems).Such diversity lays the groundwork for researching the evolution of SD in crustaceans,i.e.,transitions among different SD systems.However,most previous research has focused on understanding the mechanism of SD within a sin-gle lineage or species,overlooking the transition across different SD systems.To help bridge this gap,we summarize the understanding of SD in various clades of crustaceans,and discuss how different SD systems might evolve from one another.Furthermore,we review the genetic basis for transitions between different SD systems(i.e.,Dmrt genes)and propose the microcrustacean Daphnia(clade Branchiopoda)as a model to study the transition from ESD to GSD.展开更多
High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of ...High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.展开更多
Paramecium,a group of ciliates with a long evolutionary history,plays essential roles in freshwater ecosystems and has been model for genetic,cellular,and evolutionary studies for over a century.Despite the valuable c...Paramecium,a group of ciliates with a long evolutionary history,plays essential roles in freshwater ecosystems and has been model for genetic,cellular,and evolutionary studies for over a century.Despite the valuable contributions of genomic resources such as ParameciumDB,genomic data are still mostly limited to species in and near the P.aurelia group.This study addresses this gap by HiFi sequencing,assembling,and annotating the macronuclear genomes of five rare Paramecium species:P.calkinsi,P.duboscqui,P.nephridiatum,P.putrinum,and P.woodruffi.These genomes enable a comprehensive exploration of genomic diversity,genome evolution,and phylogenomic relationships within the genus Paramecium.The genome sizes range from 47.78 to 113.16 Mb,reflecting unexpected variation in genomic content,and genic features differ from those of other reported Paramecium genomes,such as larger intron sizes and higher GC content.Nonetheless,the de novo assemblies indicate that macronuclear genomes of all Paramecium are highly streamlined,with~77%being protein-coding gene regions.Based on gene-duplication depths,synonymous mutations in paralogs,and phylogenomic relationships,we discovered that the five species experienced at least three whole-genome duplication(WGD)events,independent of those previously found in the P.aurelia complex.Using all available WGD data for Paramecium,we further explore the paralog dynamics after WGD events by modeling.This study contributed to a more comprehensive and deeper understanding of genome architecture and evolution in Paramecium.展开更多
Antibiotic-resistant bacteria severely threaten human health.Besides spontaneous mutations generated by endogenous factors,the resistance might also originate from mutations induced by certain antibiotics,such as the ...Antibiotic-resistant bacteria severely threaten human health.Besides spontaneous mutations generated by endogenous factors,the resistance might also originate from mutations induced by certain antibiotics,such as the fluoroquinolones.Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems.However,the relative contributions of these molecular mechanisms remain unclear,hindering understanding of the generation of resistant pathogens.Here,using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains,as well as those of the strains deficient for the mismatch repair(MMR)and the oxidative damage repair pathways,we find that the SOS response is the major mutagenesis contributor in mutation elevation,responsible for~30–50%of the total base-pair substitution(BPS)mutation-rate elevation upon treatment with sublethal levels of norfloxacin(0~50 ng/mL).We further estimate the significance of the effects on other mutational features of these mechanisms(i.e.,transversions,structural variations,and mutation spectrum)in E.coli using linear models.The SOS response plays a positive role in all three mutational features(mutation rates of BPSs,transversions,structural variations)and affects the mutational spectrum.The repair systems significantly reduce the BPS mutation rate and the transversion rate,regardless of whether antibiotics are present,while significantly increasing the structural variation rate in E.coli.Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.展开更多
Whole-genome duplication(WGD)events are widespread across eukaryotes and have played a significant role in moulding the genetic architectures of diverse organisms.In the present study,the newly sequenced genome of a g...Whole-genome duplication(WGD)events are widespread across eukaryotes and have played a significant role in moulding the genetic architectures of diverse organisms.In the present study,the newly sequenced genome of a giant ciliated protist,Stentor roeselii,provides an opportunity for the analysis of the collinearity and retention of reciprocal best-hit genes between two Stentor species.As a main result,we have unveiled a previously undetected ancient WGD event shaping the genome of its congener,Stentor coeruleus,a model protist used in cytological and evolutionary studies.Genomes of two congeners,S.coeruleus and S.roeselii,are compared and analyzed,revealing that:(i)the former exhibits a much higher retention rate of colinear-gene pairs(28%)than does S.roeselii,and in S.coeruleus,75%of genes that have a RBH hit in S.roeselii,have paralogs with high amino-acid identity,consistent with a WGD event in the lineage leading to S.coeruleus;(ii)the S.roeselii genome possesses extremely short intergenic regions,implying that the lengths of intergenic regions are under strong selection;(iii)the unique characteristics of introns may have been shaped in the common ancestor of heterotrichs;(iv)gene families that play a role in activities of multiple protein kinases and voltage-gated ion channels expanded rapidly in the ancestor of both taxa,possibly relating to the remarkable regenerative ability in Stentor.This study offers new insights into the evolutionary dynamics of ciliate genomes,with implications for understanding of the processes underlying the evolution of genomic complexity.展开更多
Many plant disease resistance(R)genes function specifically in reaction to the presence of cognate effectors from a pathogen.Xanthomonas oryzae pathovar oryzae(Xoo)uses transcription activator-like effectors(TALes)to ...Many plant disease resistance(R)genes function specifically in reaction to the presence of cognate effectors from a pathogen.Xanthomonas oryzae pathovar oryzae(Xoo)uses transcription activator-like effectors(TALes)to target specific rice genes for expression,thereby promoting host susceptibility to bacterial blight.Here,we report the molecular characterization of Xa7,the cognate R gene to the TALes AvrXa7 and PthXo3,which target the rice major susceptibility gene SWEET14.Xa7 was mapped to a unique 74-kb region.Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element(EBE)in its promoter and encoded a 113-amino-acid peptide of unknown function.Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains.Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana.The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility.Ectopic expression of Xa7 triggers cell death in N.benthamiana.Xa7 is prevalent in indica rice accessions from 3000 rice genomes.Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.展开更多
Mutation is a primary source of genetic variation that is used to power evolution.Many studies,however,have shown that most mutations are deleterious and,as a result,extremely low mutation rates might be benefcial for...Mutation is a primary source of genetic variation that is used to power evolution.Many studies,however,have shown that most mutations are deleterious and,as a result,extremely low mutation rates might be benefcial for survival.Using a mutation accumulation experiment,an unbiased method for mutation study,we found an extremely low base-substitution mutation rate of 5.94×10^(-11) per nucleotide site per cell division(95%Poisson confdence intervals:4.65×10^(-11),7.48×10^(-11))and indel mutation rate of 8.25×10^(-12) per site per cell division(95%confdence intervals:3.96×10^(-12),1.52×10^(-11))in the bacterium Photorhabdus luminescens ATCC29999.The mutations are strongly A/T-biased with a mutation bias of 10.28 in the A/T direction.It has been hypothesized that the ability for selection to lower mutation rates is inversely proportional to the efective population size(drift-barrier hypothesis)and we found that the efective population size of this bacterium is signifcantly greater than most other bacteria.This fnding further decreases the lower-bounds of bacterial mutation rates and provides evidence that extreme levels of replication fdelity can evolve within organisms that maintain large efective population sizes.展开更多
基金This work was financially supported by NIH grant R35-GM122566-01 to M.L.NIH Enabling Discovery through GEnomics(EDGE)grant IOS-1922914 to M.L.and Andrew Zelhof(Indiana University).
文摘Sex determination(SD)involves mechanisms that determine whether an individual will develop into a male,female,or in rare cases,hermaphrodite.Crustaceans harbor extremely diverse SD systems,including hermaphroditism,environmental sex determination(ESD),genetic sex determination(GSD),and cytoplasmic sex determination(e.g.,Wolbachia controlled SD systems).Such diversity lays the groundwork for researching the evolution of SD in crustaceans,i.e.,transitions among different SD systems.However,most previous research has focused on understanding the mechanism of SD within a sin-gle lineage or species,overlooking the transition across different SD systems.To help bridge this gap,we summarize the understanding of SD in various clades of crustaceans,and discuss how different SD systems might evolve from one another.Furthermore,we review the genetic basis for transitions between different SD systems(i.e.,Dmrt genes)and propose the microcrustacean Daphnia(clade Branchiopoda)as a model to study the transition from ESD to GSD.
基金supported by the National Natural Science Foundation of China(31961123002,31872228)the Fundamental Research Funds for the Central Universities of China(202041001)+1 种基金the Young Taishan Scholars Program of Shandong Province(tsqn201812024)the National Science Foundation(DEB-1927159).
文摘High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.
基金supported by the Laoshan Laboratory(LSKJ202203203)the National Natural Science Foundation of China(31961123002,32270435 and 32471688)+1 种基金the National Institutes of Health(R35-GM122566-01)the National Science Foundation(DBI-2119963,DEB-1927159 and 1911449).
文摘Paramecium,a group of ciliates with a long evolutionary history,plays essential roles in freshwater ecosystems and has been model for genetic,cellular,and evolutionary studies for over a century.Despite the valuable contributions of genomic resources such as ParameciumDB,genomic data are still mostly limited to species in and near the P.aurelia group.This study addresses this gap by HiFi sequencing,assembling,and annotating the macronuclear genomes of five rare Paramecium species:P.calkinsi,P.duboscqui,P.nephridiatum,P.putrinum,and P.woodruffi.These genomes enable a comprehensive exploration of genomic diversity,genome evolution,and phylogenomic relationships within the genus Paramecium.The genome sizes range from 47.78 to 113.16 Mb,reflecting unexpected variation in genomic content,and genic features differ from those of other reported Paramecium genomes,such as larger intron sizes and higher GC content.Nonetheless,the de novo assemblies indicate that macronuclear genomes of all Paramecium are highly streamlined,with~77%being protein-coding gene regions.Based on gene-duplication depths,synonymous mutations in paralogs,and phylogenomic relationships,we discovered that the five species experienced at least three whole-genome duplication(WGD)events,independent of those previously found in the P.aurelia complex.Using all available WGD data for Paramecium,we further explore the paralog dynamics after WGD events by modeling.This study contributed to a more comprehensive and deeper understanding of genome architecture and evolution in Paramecium.
基金supported by Laoshan Laboratory(LSKJ202203203)the National Natural Science Foundation of China(31961123002,32270435)+3 种基金the Fundamental Research Funds for the Central Universities(202161064)the Young Taishan Scholars Program of Shandong Province(tsqn201812024)the Natural Science Foundation of Shandong Province(ZR2023QC191)the National Institutes of Health award(R35-GM122566).
文摘Antibiotic-resistant bacteria severely threaten human health.Besides spontaneous mutations generated by endogenous factors,the resistance might also originate from mutations induced by certain antibiotics,such as the fluoroquinolones.Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems.However,the relative contributions of these molecular mechanisms remain unclear,hindering understanding of the generation of resistant pathogens.Here,using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains,as well as those of the strains deficient for the mismatch repair(MMR)and the oxidative damage repair pathways,we find that the SOS response is the major mutagenesis contributor in mutation elevation,responsible for~30–50%of the total base-pair substitution(BPS)mutation-rate elevation upon treatment with sublethal levels of norfloxacin(0~50 ng/mL).We further estimate the significance of the effects on other mutational features of these mechanisms(i.e.,transversions,structural variations,and mutation spectrum)in E.coli using linear models.The SOS response plays a positive role in all three mutational features(mutation rates of BPSs,transversions,structural variations)and affects the mutational spectrum.The repair systems significantly reduce the BPS mutation rate and the transversion rate,regardless of whether antibiotics are present,while significantly increasing the structural variation rate in E.coli.Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.
基金supported by the Science & Technology Innovation Project of Laoshan Laboratory(LSKJ202203203)the National Science Foundation of China (32270558)+4 种基金the National Institutes of Health Grant (R35-GM122566-01)the National Science Foundation (DEB-1927159, DBI-2119963)the Moore and Simons Foundations (735927)the Young Taishan Scholar Program of Shandong Provincethe Project of Outstanding Young Innovative Team in Higher Education Institutions of Shandong Province (2023KJ038).
文摘Whole-genome duplication(WGD)events are widespread across eukaryotes and have played a significant role in moulding the genetic architectures of diverse organisms.In the present study,the newly sequenced genome of a giant ciliated protist,Stentor roeselii,provides an opportunity for the analysis of the collinearity and retention of reciprocal best-hit genes between two Stentor species.As a main result,we have unveiled a previously undetected ancient WGD event shaping the genome of its congener,Stentor coeruleus,a model protist used in cytological and evolutionary studies.Genomes of two congeners,S.coeruleus and S.roeselii,are compared and analyzed,revealing that:(i)the former exhibits a much higher retention rate of colinear-gene pairs(28%)than does S.roeselii,and in S.coeruleus,75%of genes that have a RBH hit in S.roeselii,have paralogs with high amino-acid identity,consistent with a WGD event in the lineage leading to S.coeruleus;(ii)the S.roeselii genome possesses extremely short intergenic regions,implying that the lengths of intergenic regions are under strong selection;(iii)the unique characteristics of introns may have been shaped in the common ancestor of heterotrichs;(iv)gene families that play a role in activities of multiple protein kinases and voltage-gated ion channels expanded rapidly in the ancestor of both taxa,possibly relating to the remarkable regenerative ability in Stentor.This study offers new insights into the evolutionary dynamics of ciliate genomes,with implications for understanding of the processes underlying the evolution of genomic complexity.
基金supported by the United States Department of Agriculture National Institute of Agriculture and Food(2017-67013-26521 to B.Y.)the National Science Foundation(1238189 to F.F.W.,V.P.B.,and B.Y.,1741090 to F.F.W.)subawards to University of Missouri and University of Florida from the Heinrich Heine University Düsseldorf funded by the Bill&Melinda Gates Foundation[OPP1155704](B.Y.and F.F.W.).
文摘Many plant disease resistance(R)genes function specifically in reaction to the presence of cognate effectors from a pathogen.Xanthomonas oryzae pathovar oryzae(Xoo)uses transcription activator-like effectors(TALes)to target specific rice genes for expression,thereby promoting host susceptibility to bacterial blight.Here,we report the molecular characterization of Xa7,the cognate R gene to the TALes AvrXa7 and PthXo3,which target the rice major susceptibility gene SWEET14.Xa7 was mapped to a unique 74-kb region.Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element(EBE)in its promoter and encoded a 113-amino-acid peptide of unknown function.Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains.Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana.The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility.Ectopic expression of Xa7 triggers cell death in N.benthamiana.Xa7 is prevalent in indica rice accessions from 3000 rice genomes.Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.
基金This work is supported by the Young Taishan Scholars Program of Shandong Province(tsqn201812024)the Fundamental Research Funds for the Central Universities of China(201822020)to H.L.+1 种基金the Multidisciplinary University Research Initiative Award from the US Army Research Ofce(W911NF-09-1-0444 and W911NF-09-1-0411)National Institutes of Health award(R35-GM122566)to M.L.
文摘Mutation is a primary source of genetic variation that is used to power evolution.Many studies,however,have shown that most mutations are deleterious and,as a result,extremely low mutation rates might be benefcial for survival.Using a mutation accumulation experiment,an unbiased method for mutation study,we found an extremely low base-substitution mutation rate of 5.94×10^(-11) per nucleotide site per cell division(95%Poisson confdence intervals:4.65×10^(-11),7.48×10^(-11))and indel mutation rate of 8.25×10^(-12) per site per cell division(95%confdence intervals:3.96×10^(-12),1.52×10^(-11))in the bacterium Photorhabdus luminescens ATCC29999.The mutations are strongly A/T-biased with a mutation bias of 10.28 in the A/T direction.It has been hypothesized that the ability for selection to lower mutation rates is inversely proportional to the efective population size(drift-barrier hypothesis)and we found that the efective population size of this bacterium is signifcantly greater than most other bacteria.This fnding further decreases the lower-bounds of bacterial mutation rates and provides evidence that extreme levels of replication fdelity can evolve within organisms that maintain large efective population sizes.
