This paper probes into the molecular genetic mechanism of the formation of species, subspecies and variety in evolving progression, and brings forward 5 criteria of an ideal strategy in species identification: stating...This paper probes into the molecular genetic mechanism of the formation of species, subspecies and variety in evolving progression, and brings forward 5 criteria of an ideal strategy in species identification: stating the specific characteristics at species, subspecies and variety level without any interference of too high polymorphism at individual or population level; keys should be distributed as 0 or 1, e. g. yes or no; satisfying repeatability and simple operation; high veracity and reliability; adaptability to widely various specimen. Respectively, this paper reviews two strategies focusing on detecting the fragment length polymorphism and base replacement and lays out some detail methods under above strategies. It demonstrates that it is not possible to solve all species problems by pursuing identification with only a single gene or DNA fragment. Only based on thorough consideration of all strategies, a method or combined several methods could bring satisfying reliability. For advanced focuses, it requires not only development and optimization of methods under above strategies, but also new originality of creative strategies.展开更多
Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The current review show...Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The current review shows common target genes and screening criteria suitable for species identification, and described various DNA-based molecular biology methods about species identification. Additionally, it discusses the future development of species identification combined with real-time PCR and sequencing technologies.展开更多
DNA barcoding is an increasingly prevalent molecular biological technology which uses a short and conserved DNA fragment to facilitate rapid and accurate species identification. Kalidium species are distributed in sal...DNA barcoding is an increasingly prevalent molecular biological technology which uses a short and conserved DNA fragment to facilitate rapid and accurate species identification. Kalidium species are distributed in saline soil habitat throughout Southeast Europe and Northwest Asia, and used mainly as forage grass in China. The discrimination of Kalidium species was based only on morphology-based identification systems and limited to recognized species. Here, we tested four DNA candidate loci, one nuclear locus(ITS, internal transcribed spacer) and three plastid loci(rbc L, mat K and ycf1b), to select potential DNA barcodes for identifying different Kalidium species. Results showed that the best DNA barcode was ITS locus, which displayed the highest species discrimination rate(100%), followed by mat K(33.3%), ycf1b(16.7%), and rbc L(16.7%). Meanwhile, four loci clearly identified the variant species, Kalidium cuspidatum(Ung.-Sternb.) Grub.var. sinicum A. J. Li, as a single species in Kalidium.展开更多
Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania specie...Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.展开更多
Human and animal hairs have been used in forensic investigations for over a century.Hair is stable under adverse natural conditions;hence,it is often recovered at the crime scene,and it is necessary to determine wheth...Human and animal hairs have been used in forensic investigations for over a century.Hair is stable under adverse natural conditions;hence,it is often recovered at the crime scene,and it is necessary to determine whether the hair is of human or animal origin.Morphological and genetic characteristics are useful to differentiate human hair from animal hair.In the present study,we analyzed the distinguishing characteristics of hair of various species.In addition,we explore species identification by cytochrome c oxidase I mitochondrial gene analysis.We confirm that both the microscopic and molecular analyses of hairs are useful in forensic investigations.展开更多
Roscoea is an alpine or subalpine genus from the pan-tropical family Zingiberaceae,which consists of two disjunct groups in geography,namely the"Chinese"clade and the"Himalayan"clade.Despite extens...Roscoea is an alpine or subalpine genus from the pan-tropical family Zingiberaceae,which consists of two disjunct groups in geography,namely the"Chinese"clade and the"Himalayan"clade.Despite extensive research on the genus,Roscoea species remain poorly defined and relationships between these species are not well resolved.In this study,we used plastid genomes of nine species and one variety to resolve phylogenetic relationships within the"Chinese"clade of Roscoea and as DNA super barcodes for species discrimination.We found that Roscoea plastid genomes ranged in length from 163,063 to 163,796 bp,and encoded 113 genes,including 79 protein-coding genes,30 tRNA genes,four rRNA genes.In addition,expansion and contraction of the IR regions showed obvious infraspecifc conservatism and interspecific differentiation.Plastid phylogenomics revealed that species belonging to the"Chinese"clade of Roscoea can be divided into four distinct subclades.Furthermore,our analysis supported the independence of R.cautleoides var.pubescens,the recovery of Roscoea pubescens Z.Y.Zhu,and a close relationship between R.humeana and R.cautloides.When we used the plastid genome as a super barcode,we found that it possessed strong discriminatory power(90%)with high support values.Intergenic regions provided similar resolution,which was much better than that of protein-coding regions,hypervariable regions,and DNA universal barcodes.However,plastid genomes could not completely resolve Roscoea phylogeny or definitively discriminate species.These limitations are likely related to the complex history of Roscoea speciation,poorly defined species within the genus,and the maternal inheritance of plastid genomes.展开更多
Thirty-five new microsatellite loci from the sea cucumbers H olothurian scabra(Jaeger, 1833) and Apostichopus japonicas(Selenka, 1867) were screened and characterized using the method of magnetic bead enrichment. Of t...Thirty-five new microsatellite loci from the sea cucumbers H olothurian scabra(Jaeger, 1833) and Apostichopus japonicas(Selenka, 1867) were screened and characterized using the method of magnetic bead enrichment. Of the twenty-four polymorphic loci tested, eighteen were consistent with Hardy-Weinberg equilibrium after a modified false discovery rate(B-Y FDR) correction, whereas six showed statistically significant deviations(CHS2 and CHS11: P <0.014 790; FCS1, FCS6, FCS8 and FCS14: P <0.015 377). Furthermore, four species of plesiomorphous and related sea cucumbers(Holothurian scabra, Holothuria leucospilota, Stichopus horrens and Apostichopus japonicas) were tested for mutual cross-amplification using a total of ninety microsatellite loci. Although transferability and universality of all loci were generally low, the results of the cross-species study showed that the markers can be applied to identify individuals to species according to the presence or absence of specific microsatellite alleles. The microsatellite markers reported here will contribute to the study of genetic diversity, assisted breeding, and population conservation in sea cucumbers, as well as allow for the identification of individuals to closely related species.展开更多
The dried shellfish products with rich nutrients and low-calorie content are favorite food in China,especially in coastal areas.However,the species of dried shellfish products in the market are usually unknown,as the ...The dried shellfish products with rich nutrients and low-calorie content are favorite food in China,especially in coastal areas.However,the species of dried shellfish products in the market are usually unknown,as the taxonomic features were removed during the production process.This study described the application of DNA barcoding technique to the identification of 100 dried shellfish(scallop,squid,octopus and cuttlefish)products in markets.Samples were authenticated by comparing mitochondrial cytochrome oxidase subunit I(COI)gene and 16S ribosomal RNA(16S rRNA)gene sequences with public reference taxonomic databases.The results showed that all the 100 products can be identified at species level.Sixty four scallop adductor products were processed using the bay scallop,Argopecten irradians,and one was from Portuguese oyster,Crassostrea angulata.All the 27 squid,2 cuttlefish and 6 octopus products were produced by the Jumbo flying squid,Dosidicus gigas.The neighbour-joining tree is in agreement with the results of DNA barcoding analysis.The 64 scallop samples formed one A.irradians cluster,leaving Sca65 clustered with the reference oyster sequence C.angulata(MH997922).All the 35 cephalopod(squid,octopus and cuttlefish)samples formed a D.gigas cluster.This investigation revealed a low variety of dried shellfish products sold on the market,and highlighted the high rate of mislabeling and species substitution.Our present work provides a practical method for tracing and authenticating shellfish products.展开更多
Due to its unique advantages, molecular marker technology is widely applied in the research of forest tree species. This paper reviewed the application of molecular marker technology in tree species resource diversity...Due to its unique advantages, molecular marker technology is widely applied in the research of forest tree species. This paper reviewed the application of molecular marker technology in tree species resource diversity, germplasm identification, genetic map construction, gene mapping and marker-assisted selection (MAS) breeding. In addition, it elaborated the great significance of molecular marker technology to promote the sustainable development of forestry production in China.展开更多
Objective To select a more suitable DNA barcode to identify the species in Artemisia L. Methods ITS, ITS2, and three other major universal barcode candidates(mat K, rbc L, and psb A-trn H) were evaluated in the identi...Objective To select a more suitable DNA barcode to identify the species in Artemisia L. Methods ITS, ITS2, and three other major universal barcode candidates(mat K, rbc L, and psb A-trn H) were evaluated in the identification efficiency using a total of 1433 sequences downloaded from Gen Bank representing 343 species in Artemisia L. ITS and ITS2 were evaluated in the PCR and sequencing rate, sequencing peak quality(Q value), and misread rate. One hundred and twelve A. annua samples were collected from 11 populations across over China, which were amplified with universal primers on the ITS and ITS2 regions. Results ITS and ITS2 shared a higher identification efficiency and exhibited 71.43% and 64.11% detectability at the species level, respectively. The Q values of ITS and ITS2 showed that the direct PCR sequencing data were reliable for the ITS2 region and ITS exhibited poor sequencing trace quality. In certain sites, the ITS sequences exhibited reading ambiguities and errors, indicating that the misread and deletion sites in the ITS region would incorrectly inflate the identification ratio. Conclusion ITS2 is a suitable barcode for identification of species in Artemisia L., which enlarges the optimal range of divergence levels for taxonomic inferences using ITS2 sequences.展开更多
With the development of industrialization and aquaculture in Jiangsu and Shandong Provinces along the South Yellow Sea coast,China,eutrophication has greatly intensified in the region,resulting in frequent occurrence ...With the development of industrialization and aquaculture in Jiangsu and Shandong Provinces along the South Yellow Sea coast,China,eutrophication has greatly intensified in the region,resulting in frequent occurrence of diverse harmful algal blooms.An algal bloom formed by a chain-forming dinofl agellate species was recorded in the Haizhou Bay,South Yellow Sea,in September 2020.The causative species was isolated and studied in morphology,molecular phylogeny,pigment profile,presence of paralytic shellfish toxins,and acute toxicity.The loop-shaped apical groove running anticlockwise around the apex,the presence of peridinin as characteristic pigment,as well as a single phylogenic clade of 28S ribosomal DNA(100%posterior probability),defined this species as Gymnodinium impudicum,a non-toxic species that exhibited no obvious biotoxicity to the rotifer Brachionus plicatilis,the copepod Artemia salina,and the shrimp Neomysis awatschensis.Gymnodinium impudicum is typically distributed in coastal waters with high nitrate concentrations,where it reaches a maximum density of 2.6×10~5 cells/L.This is the first report of a G.impudicum bloom in the Yellow Sea;however,G.impudicum blooms may have been misidentified or underreported in Haizhou Bay due to the species morphological similarity with G.catenatum.A combination of multiple methods is recommended to accurately identify new algal bloom species.展开更多
In this study,we performed an inter-laboratory collaborative ring trial to develop and validate specific TaqMan real-time PCR assays for goat-,horse-,and donkey-derived material in meat products.The performances of th...In this study,we performed an inter-laboratory collaborative ring trial to develop and validate specific TaqMan real-time PCR assays for goat-,horse-,and donkey-derived material in meat products.The performances of these assays in different environments and situations were comprehensively evaluated.This ring trial involved the participation of 12 laboratories in Europe and Asia.The results from the participating laboratories were analyzed to determine the specificity,accuracy,false positive rate,limit of detection(LOD),and probability of detection(POD)of the developed assays.Statistical analysis showed that the false positive and negative rates were zero,the LOD was five copies/reaction,and the laboratory standard deviation(σ_(L))was 0.30 for all three assays.Thus,the results demonstrate that the developed methods are robust and suitable for the detection and identification of goat-,horse-,and donkey-derived materials in meat products.展开更多
The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR pr...The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.展开更多
One hundred and thirty-eight echolocation calls of 63 free-flying individuals of five bat species(Rhinolophus ferrumequinum,Myotis formosus,Myotis ikonnikovi,Myotis daubentoni and Murina leucogaster)were recorded(by u...One hundred and thirty-eight echolocation calls of 63 free-flying individuals of five bat species(Rhinolophus ferrumequinum,Myotis formosus,Myotis ikonnikovi,Myotis daubentoni and Murina leucogaster)were recorded(by ultrasonic bat detector(D980))in Zhi’an village of Jilin Province,China.According to the frequency-time spectra,these calls were categorized into two types:FM/CF(constant frequency)/FM(R.ferrumequinum)and FM(frequency modulated)(M.formosus,M.ikonnikovi,M.daubentoni and M.leucogaster).Sonograms of the calls of R.ferrumequinum could easily be distinguished from those of the other four species.For the calls of the remaining four species,six echolocation call parameters,including starting frequency,ending frequency,peak frequency duration,longest inter-pulse interval and shortest inter-pulse interval,were examined by stepwise discriminant analysis.The results show that 84.1%of calls were correctly classified,which indicates that these parameters of echolocation calls play an important role in identifying bat species.These parameters can be used to test the accuracy of general predictions based on bats’morphology in the same forest and can provide essential information for assessing patterns of bat habitat use.展开更多
To ensure the safety of medications,it is vital to accurately authenticate species of the Apocynaceae family,which is rich in poisonous medicinal plants.We identified Apocynaceae species by using nuclear internal tran...To ensure the safety of medications,it is vital to accurately authenticate species of the Apocynaceae family,which is rich in poisonous medicinal plants.We identified Apocynaceae species by using nuclear internal transcribed spacer 2(ITS2)and psb Atrn H based on experimental data.The identification ability of ITS2 and psb A-trn H was assessed using specific genetic divergence,BLAST1,and neighbor-joining trees.For DNA barcoding,ITS2 and psb A-trn H regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified.The PCR amplification for ITS2 and psb A-trn H sequences was 100%.The sequencing success rates for ITS2 and psb A-trn H sequences were 81%and 61%,respectively.Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psb A-trn H region were downloaded from Gen Bank.Moreover,the analysis showed that the interspecific divergence of Apocynaceae species was greater than its intra-specific variations.The results indicated that,using the BLAST1 method,ITS2 showed a high identification efficiency of 97%and 100%of the samples at the species and genus levels,respectively,via BLAST1,and psb A-trn H successfully identified 95%and 100%of the samples at the species and genus levels,respectively.The barcode combination of ITS2/psb A-trn H successfully identified 98%and 100%of samples at the species and genus levels,respectively.Subsequently,the neighbor joining tree method also showed that barcode ITS2 and psb A-trn H could distinguish among the species within the Apocynaceae family.ITS2 is a core barcode and psb A-trn H is a supplementary barcode for identifying species in the Apocynaceae family.These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.展开更多
The Lusitanian and the Mediterranean pine voles(Microtus lusitanicus Gerbe,1879 and Microtus duodecimcostatus de Selys-Longchamps,1839)are fossorial sister species and have an allopatric pattern of distribution in Por...The Lusitanian and the Mediterranean pine voles(Microtus lusitanicus Gerbe,1879 and Microtus duodecimcostatus de Selys-Longchamps,1839)are fossorial sister species and have an allopatric pattern of distribution in Portugal,which includes a potential sympatry area in the centre of the country.The present study aimed to determine the validity of using presence signs in the field for discrimination of the two species in an area of sympatry(Northern Alentejo)and the characteristics that achieve the best classification accuracy.A total of 175 trapping plots were sampled across the study area.Prior to the set up of traps,ten presence signs were randomly selected for measure-ments of four variables:proportion of soil mounds,mean diameter of mounds,proportion of burrow openings and mean diameter of burrow openings.On the basis of a classification tree analysis,results showed that presence signs can be used to discriminate plots inhabited by one or the other species in the studied sympatry area.The character-istic that most accurately enables species identification is the proportion of burrow openings:for every ten pres-ence signs found in a plot,if more than eight have an opening,then it is inhabited by M.lusitanicus(i.e.mostly burrow openings with few or no mounds present);if eight or fewer have an opening,M.duodecimcostatus is present(i.e.mostly mounds with few or no burrow openings).展开更多
Plant-symbiotic arbuscular mycorrhizal fungi(AMF)are of high global ecological and economic importance,but describing environmental communities of AMF at the species level remains a challenge,despite the need to und...Plant-symbiotic arbuscular mycorrhizal fungi(AMF)are of high global ecological and economic importance,but describing environmental communities of AMF at the species level remains a challenge,despite the need to understand AMF-plant preferences and to apply AMF in sustainable agriculture.Here,the potato-associated AMF species community composition was assessed for three Andean countries along an altitudinal gradient and at different plant stages,by using 454 GS-FLX+sequencing of a 760 bp LSU rRNA gene PCR amplicon.Two methods were compared:defining OTUs based on a simple sequence similarity threshold,or affiliating reference sequences to species based on a high throughput phylogenetic annotation approach using an evolutionary placement algorithm(EPA).The EPA-based approach was not only more precise,but also fundamental to robustly unveil the AMF species community composition.The principal advantage of this approach was also demonstrated by using artificially constructed datasets based on validated public database sequences.The affiliation of sequence reads to species using phylogenetic annotation revealed a surprisingly conserved AMF core-species community structure in Andean potatoes,regardless of different plant stages and environmental factors.In total,41 species were detected and in some cases more than 25 species were found colonizing an individual root system.Acaulospora species were identified as dominant colonizers,co-occurring with Cetraspora nodosa and certain Claroideoglomus and Rhizophagus species in most potato root samples.展开更多
Crocodiles,gharials and alligators(order Crocodilia),are aquatic reptiles that live in the tropics of Asia,America,Africa,and Australia.Asian countries such as India,Indonesia,Malaysia,and tropics of Australia are the...Crocodiles,gharials and alligators(order Crocodilia),are aquatic reptiles that live in the tropics of Asia,America,Africa,and Australia.Asian countries such as India,Indonesia,Malaysia,and tropics of Australia are the stronghold of the family Crocodylidae.Among all 23 crocodile species,nine species occur in Asia and its surroundings,including the only member of Gavialidae and Alligatoridae family.They are“mugger”or“Crocodylus palustris,”“saltwater crocodile”or“Crocodylus porosus,”“Philippine crocodile”or“Crocodylus mindorensis,”“New Guinea crocodile”or“Crocodylus novaeguineae,”“Siamese crocodile”or“Crocodylus siamensis,”“gharials”or“Gavialis gangeticus,”“false gharial”or“Tomistoma schlegelii,”and“Chinese alligator”or“Alligator sinensis.”All of these species have been encompassed in“Appendix I”and“Appendix II”of the“Convention on International Trade in Endangered Species of Wild Fauna and Flora,”which prevents any kind of trade involving crocodilian species.However,it has been observed that these crocodiles are illegally poached and trafficked for their lucrative skin,meats,eggs,snouts,and bones in medicinal and cosmetic industries.Although many molecular biologists have come forward for the conservation of these species,lack of knowledge about the available,fast,and dependable techniques makes it difficult for forensic identification of seized or confiscated.It has been a major problem for the implementation of the“Wildlife Protection Law”on illegal trade.This article focuses on molecular techniques developed till date for the rapid and reliable species identification and conservation study of them.展开更多
Background:Anisodus acutangulus(Solanaceae),an important folk medicinal herb in China,produces up to 1.2%alkaloids more than that in other Solanaceae plants such as Hyoscyamus niger,while its evolutionary position in ...Background:Anisodus acutangulus(Solanaceae),an important folk medicinal herb in China,produces up to 1.2%alkaloids more than that in other Solanaceae plants such as Hyoscyamus niger,while its evolutionary position in Hyoscyameae is not very clear.Objective:To explain the evolutionary position of A.acutangulus in the Solanaceae via complete chloroplast genome(cp)sequence.Methods:Complete chloroplast genome of A.acutangulus was obtained and characterized using the Illumina PE150 pair-end sequencing data.Structure of the genome,codon usage,nucleotide variability(Pi)value,distribution of repeats and SSRs between A.acutangulus and other seven Solanaceae species were analyzed.Previously published 22 Solanaceae cp genomes were used to construct phylogenetic tree.Results:The complete cp genome of A.acutangulus is 156082 bp in length,showed the typical quadripartite structure.The complete cp genome of A.acutangulus was highly conserved.A total of 112 unique genes were found in cp genome of A.acutangulus,among which 17 were duplicated.Further,we found eight hotspot regions for genome divergence could be explored as new DNA barcodes for the identification of the Solanaceae species.Phylogenetic analysis showed that A.acutangulus formed a clade with H.niger.Conclusion:A.acutangulus belongs to Hyoscyameae subfamily and the complete cp genome provides valuable information for phylogenetic reconstruction or comparative genomics of A.acutangulus.展开更多
文摘This paper probes into the molecular genetic mechanism of the formation of species, subspecies and variety in evolving progression, and brings forward 5 criteria of an ideal strategy in species identification: stating the specific characteristics at species, subspecies and variety level without any interference of too high polymorphism at individual or population level; keys should be distributed as 0 or 1, e. g. yes or no; satisfying repeatability and simple operation; high veracity and reliability; adaptability to widely various specimen. Respectively, this paper reviews two strategies focusing on detecting the fragment length polymorphism and base replacement and lays out some detail methods under above strategies. It demonstrates that it is not possible to solve all species problems by pursuing identification with only a single gene or DNA fragment. Only based on thorough consideration of all strategies, a method or combined several methods could bring satisfying reliability. For advanced focuses, it requires not only development and optimization of methods under above strategies, but also new originality of creative strategies.
基金supported by the grants from the 12th Five-year Plan National Key Technology R&D Program of the Ministry of Science and Technology of P.R.China (2012BAK16B01)the National Natural Science Foundation of P.R.China (81330073, 81222041)
文摘Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The current review shows common target genes and screening criteria suitable for species identification, and described various DNA-based molecular biology methods about species identification. Additionally, it discusses the future development of species identification combined with real-time PCR and sequencing technologies.
基金supported by the Program for New Century Excellent Talents in the Ministry of Education in China(NCET-09-0446)lzujbky-2012-k22 to Yu Xia Wu
文摘DNA barcoding is an increasingly prevalent molecular biological technology which uses a short and conserved DNA fragment to facilitate rapid and accurate species identification. Kalidium species are distributed in saline soil habitat throughout Southeast Europe and Northwest Asia, and used mainly as forage grass in China. The discrimination of Kalidium species was based only on morphology-based identification systems and limited to recognized species. Here, we tested four DNA candidate loci, one nuclear locus(ITS, internal transcribed spacer) and three plastid loci(rbc L, mat K and ycf1b), to select potential DNA barcodes for identifying different Kalidium species. Results showed that the best DNA barcode was ITS locus, which displayed the highest species discrimination rate(100%), followed by mat K(33.3%), ycf1b(16.7%), and rbc L(16.7%). Meanwhile, four loci clearly identified the variant species, Kalidium cuspidatum(Ung.-Sternb.) Grub.var. sinicum A. J. Li, as a single species in Kalidium.
文摘Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.
文摘Human and animal hairs have been used in forensic investigations for over a century.Hair is stable under adverse natural conditions;hence,it is often recovered at the crime scene,and it is necessary to determine whether the hair is of human or animal origin.Morphological and genetic characteristics are useful to differentiate human hair from animal hair.In the present study,we analyzed the distinguishing characteristics of hair of various species.In addition,we explore species identification by cytochrome c oxidase I mitochondrial gene analysis.We confirm that both the microscopic and molecular analyses of hairs are useful in forensic investigations.
基金supported by National Natural Science Foundation of China(32060091&31660081)Reserve Talents Project for Young and Middle-Aged Academic and Technical Leaders of Yunnan Province(202105AC160063)。
文摘Roscoea is an alpine or subalpine genus from the pan-tropical family Zingiberaceae,which consists of two disjunct groups in geography,namely the"Chinese"clade and the"Himalayan"clade.Despite extensive research on the genus,Roscoea species remain poorly defined and relationships between these species are not well resolved.In this study,we used plastid genomes of nine species and one variety to resolve phylogenetic relationships within the"Chinese"clade of Roscoea and as DNA super barcodes for species discrimination.We found that Roscoea plastid genomes ranged in length from 163,063 to 163,796 bp,and encoded 113 genes,including 79 protein-coding genes,30 tRNA genes,four rRNA genes.In addition,expansion and contraction of the IR regions showed obvious infraspecifc conservatism and interspecific differentiation.Plastid phylogenomics revealed that species belonging to the"Chinese"clade of Roscoea can be divided into four distinct subclades.Furthermore,our analysis supported the independence of R.cautleoides var.pubescens,the recovery of Roscoea pubescens Z.Y.Zhu,and a close relationship between R.humeana and R.cautloides.When we used the plastid genome as a super barcode,we found that it possessed strong discriminatory power(90%)with high support values.Intergenic regions provided similar resolution,which was much better than that of protein-coding regions,hypervariable regions,and DNA universal barcodes.However,plastid genomes could not completely resolve Roscoea phylogeny or definitively discriminate species.These limitations are likely related to the complex history of Roscoea speciation,poorly defined species within the genus,and the maternal inheritance of plastid genomes.
基金Supported by the Natural Science Foundation of Fujian Province(Nos.2014J01133,2017J01638)the National Natural Science Foundation of China(No.31272668)the Program for New Century Excellent Talents in Fujian Province University and the Foundation for Innovative Research Team of Jimei University,China(No.2010A004)
文摘Thirty-five new microsatellite loci from the sea cucumbers H olothurian scabra(Jaeger, 1833) and Apostichopus japonicas(Selenka, 1867) were screened and characterized using the method of magnetic bead enrichment. Of the twenty-four polymorphic loci tested, eighteen were consistent with Hardy-Weinberg equilibrium after a modified false discovery rate(B-Y FDR) correction, whereas six showed statistically significant deviations(CHS2 and CHS11: P <0.014 790; FCS1, FCS6, FCS8 and FCS14: P <0.015 377). Furthermore, four species of plesiomorphous and related sea cucumbers(Holothurian scabra, Holothuria leucospilota, Stichopus horrens and Apostichopus japonicas) were tested for mutual cross-amplification using a total of ninety microsatellite loci. Although transferability and universality of all loci were generally low, the results of the cross-species study showed that the markers can be applied to identify individuals to species according to the presence or absence of specific microsatellite alleles. The microsatellite markers reported here will contribute to the study of genetic diversity, assisted breeding, and population conservation in sea cucumbers, as well as allow for the identification of individuals to closely related species.
基金This work was supported by research grants from the Fundamental Research Funds for the Central Universities(No.201762014)the National Natural Science Foundation of China(No.31772414)the National Natural Science Foundation of Qingdao City(No.20-3-4-16-nsh).
文摘The dried shellfish products with rich nutrients and low-calorie content are favorite food in China,especially in coastal areas.However,the species of dried shellfish products in the market are usually unknown,as the taxonomic features were removed during the production process.This study described the application of DNA barcoding technique to the identification of 100 dried shellfish(scallop,squid,octopus and cuttlefish)products in markets.Samples were authenticated by comparing mitochondrial cytochrome oxidase subunit I(COI)gene and 16S ribosomal RNA(16S rRNA)gene sequences with public reference taxonomic databases.The results showed that all the 100 products can be identified at species level.Sixty four scallop adductor products were processed using the bay scallop,Argopecten irradians,and one was from Portuguese oyster,Crassostrea angulata.All the 27 squid,2 cuttlefish and 6 octopus products were produced by the Jumbo flying squid,Dosidicus gigas.The neighbour-joining tree is in agreement with the results of DNA barcoding analysis.The 64 scallop samples formed one A.irradians cluster,leaving Sca65 clustered with the reference oyster sequence C.angulata(MH997922).All the 35 cephalopod(squid,octopus and cuttlefish)samples formed a D.gigas cluster.This investigation revealed a low variety of dried shellfish products sold on the market,and highlighted the high rate of mislabeling and species substitution.Our present work provides a practical method for tracing and authenticating shellfish products.
基金Supported by the Project of New 20 Items of Colleges and Universities in Jinan City (2021GXRC057). Taishan Industrial Leading Talent Project (Efficient Ecological Agriculture Innovation) (LJNY202001).
文摘Due to its unique advantages, molecular marker technology is widely applied in the research of forest tree species. This paper reviewed the application of molecular marker technology in tree species resource diversity, germplasm identification, genetic map construction, gene mapping and marker-assisted selection (MAS) breeding. In addition, it elaborated the great significance of molecular marker technology to promote the sustainable development of forestry production in China.
基金National Natural Science Foundation of China(No.81473303)Major Scientific and Technological Special Project for "Significant New Drugs Creation"(No.2014ZX09304307001)
文摘Objective To select a more suitable DNA barcode to identify the species in Artemisia L. Methods ITS, ITS2, and three other major universal barcode candidates(mat K, rbc L, and psb A-trn H) were evaluated in the identification efficiency using a total of 1433 sequences downloaded from Gen Bank representing 343 species in Artemisia L. ITS and ITS2 were evaluated in the PCR and sequencing rate, sequencing peak quality(Q value), and misread rate. One hundred and twelve A. annua samples were collected from 11 populations across over China, which were amplified with universal primers on the ITS and ITS2 regions. Results ITS and ITS2 shared a higher identification efficiency and exhibited 71.43% and 64.11% detectability at the species level, respectively. The Q values of ITS and ITS2 showed that the direct PCR sequencing data were reliable for the ITS2 region and ITS exhibited poor sequencing trace quality. In certain sites, the ITS sequences exhibited reading ambiguities and errors, indicating that the misread and deletion sites in the ITS region would incorrectly inflate the identification ratio. Conclusion ITS2 is a suitable barcode for identification of species in Artemisia L., which enlarges the optimal range of divergence levels for taxonomic inferences using ITS2 sequences.
基金Supported by the Science and Technology Basic Resources Investigation Program of China(No.2018FY100200)the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDA23050302)+1 种基金the National Natural Science Foundation of China(Nos.41776127,42006135)the Sino-Australian Centre for Healthy Coasts(No.2016YFE0101500)。
文摘With the development of industrialization and aquaculture in Jiangsu and Shandong Provinces along the South Yellow Sea coast,China,eutrophication has greatly intensified in the region,resulting in frequent occurrence of diverse harmful algal blooms.An algal bloom formed by a chain-forming dinofl agellate species was recorded in the Haizhou Bay,South Yellow Sea,in September 2020.The causative species was isolated and studied in morphology,molecular phylogeny,pigment profile,presence of paralytic shellfish toxins,and acute toxicity.The loop-shaped apical groove running anticlockwise around the apex,the presence of peridinin as characteristic pigment,as well as a single phylogenic clade of 28S ribosomal DNA(100%posterior probability),defined this species as Gymnodinium impudicum,a non-toxic species that exhibited no obvious biotoxicity to the rotifer Brachionus plicatilis,the copepod Artemia salina,and the shrimp Neomysis awatschensis.Gymnodinium impudicum is typically distributed in coastal waters with high nitrate concentrations,where it reaches a maximum density of 2.6×10~5 cells/L.This is the first report of a G.impudicum bloom in the Yellow Sea;however,G.impudicum blooms may have been misidentified or underreported in Haizhou Bay due to the species morphological similarity with G.catenatum.A combination of multiple methods is recommended to accurately identify new algal bloom species.
基金National Key Research and Development Program of China(2017YFC1601700)Shanghai Science and Technology Commission Standard Special Fund(19DZ2205000)Shanghai Science and Technology Commission Technology Platform Research Fund(20DZ2291900).
文摘In this study,we performed an inter-laboratory collaborative ring trial to develop and validate specific TaqMan real-time PCR assays for goat-,horse-,and donkey-derived material in meat products.The performances of these assays in different environments and situations were comprehensively evaluated.This ring trial involved the participation of 12 laboratories in Europe and Asia.The results from the participating laboratories were analyzed to determine the specificity,accuracy,false positive rate,limit of detection(LOD),and probability of detection(POD)of the developed assays.Statistical analysis showed that the false positive and negative rates were zero,the LOD was five copies/reaction,and the laboratory standard deviation(σ_(L))was 0.30 for all three assays.Thus,the results demonstrate that the developed methods are robust and suitable for the detection and identification of goat-,horse-,and donkey-derived materials in meat products.
基金the National Natural Science Foundation of China (Grant No. 30471099)Development Plan of the State Key Fundamental Research of China (Grant No. 2006CB101600)the National High Technology and Development Program of China (Grant No. 2006AA10A113)
文摘The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.
基金supported by the National Natural Science Foundation of China (Grant No.30370261,30570311)Project of New Century Outstanding Youth Foundation in Ministry of Education of China (No.NCET-04-0309)+1 种基金Key Program of Ministry of Education,China (No.104257)Outstanding Youth Foundation of Jilin Province (No.20030114).
文摘One hundred and thirty-eight echolocation calls of 63 free-flying individuals of five bat species(Rhinolophus ferrumequinum,Myotis formosus,Myotis ikonnikovi,Myotis daubentoni and Murina leucogaster)were recorded(by ultrasonic bat detector(D980))in Zhi’an village of Jilin Province,China.According to the frequency-time spectra,these calls were categorized into two types:FM/CF(constant frequency)/FM(R.ferrumequinum)and FM(frequency modulated)(M.formosus,M.ikonnikovi,M.daubentoni and M.leucogaster).Sonograms of the calls of R.ferrumequinum could easily be distinguished from those of the other four species.For the calls of the remaining four species,six echolocation call parameters,including starting frequency,ending frequency,peak frequency duration,longest inter-pulse interval and shortest inter-pulse interval,were examined by stepwise discriminant analysis.The results show that 84.1%of calls were correctly classified,which indicates that these parameters of echolocation calls play an important role in identifying bat species.These parameters can be used to test the accuracy of general predictions based on bats’morphology in the same forest and can provide essential information for assessing patterns of bat habitat use.
基金supported by the CAMS Initiative for Innovative Medicine(No.2016-12M-2-003)the Special Subsidies for Public Health Services of TCM("The National Survey of TCM Resources",DSS,MOF,No.66/2017)。
文摘To ensure the safety of medications,it is vital to accurately authenticate species of the Apocynaceae family,which is rich in poisonous medicinal plants.We identified Apocynaceae species by using nuclear internal transcribed spacer 2(ITS2)and psb Atrn H based on experimental data.The identification ability of ITS2 and psb A-trn H was assessed using specific genetic divergence,BLAST1,and neighbor-joining trees.For DNA barcoding,ITS2 and psb A-trn H regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified.The PCR amplification for ITS2 and psb A-trn H sequences was 100%.The sequencing success rates for ITS2 and psb A-trn H sequences were 81%and 61%,respectively.Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psb A-trn H region were downloaded from Gen Bank.Moreover,the analysis showed that the interspecific divergence of Apocynaceae species was greater than its intra-specific variations.The results indicated that,using the BLAST1 method,ITS2 showed a high identification efficiency of 97%and 100%of the samples at the species and genus levels,respectively,via BLAST1,and psb A-trn H successfully identified 95%and 100%of the samples at the species and genus levels,respectively.The barcode combination of ITS2/psb A-trn H successfully identified 98%and 100%of samples at the species and genus levels,respectively.Subsequently,the neighbor joining tree method also showed that barcode ITS2 and psb A-trn H could distinguish among the species within the Apocynaceae family.ITS2 is a core barcode and psb A-trn H is a supplementary barcode for identifying species in the Apocynaceae family.These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.
基金support was provided by Funda玢o para a Ciência e Tecnologia(SAPIENS Project POCI/BIA-BDE/57053/2004 and a PhD grant PRAXIS/SFRH/BD/21403/2005).
文摘The Lusitanian and the Mediterranean pine voles(Microtus lusitanicus Gerbe,1879 and Microtus duodecimcostatus de Selys-Longchamps,1839)are fossorial sister species and have an allopatric pattern of distribution in Portugal,which includes a potential sympatry area in the centre of the country.The present study aimed to determine the validity of using presence signs in the field for discrimination of the two species in an area of sympatry(Northern Alentejo)and the characteristics that achieve the best classification accuracy.A total of 175 trapping plots were sampled across the study area.Prior to the set up of traps,ten presence signs were randomly selected for measure-ments of four variables:proportion of soil mounds,mean diameter of mounds,proportion of burrow openings and mean diameter of burrow openings.On the basis of a classification tree analysis,results showed that presence signs can be used to discriminate plots inhabited by one or the other species in the studied sympatry area.The character-istic that most accurately enables species identification is the proportion of burrow openings:for every ten pres-ence signs found in a plot,if more than eight have an opening,then it is inhabited by M.lusitanicus(i.e.mostly burrow openings with few or no mounds present);if eight or fewer have an opening,M.duodecimcostatus is present(i.e.mostly mounds with few or no burrow openings).
基金the European Community's Seventh Framework Programme FP7/2007 under grant agreement no.227522。
文摘Plant-symbiotic arbuscular mycorrhizal fungi(AMF)are of high global ecological and economic importance,but describing environmental communities of AMF at the species level remains a challenge,despite the need to understand AMF-plant preferences and to apply AMF in sustainable agriculture.Here,the potato-associated AMF species community composition was assessed for three Andean countries along an altitudinal gradient and at different plant stages,by using 454 GS-FLX+sequencing of a 760 bp LSU rRNA gene PCR amplicon.Two methods were compared:defining OTUs based on a simple sequence similarity threshold,or affiliating reference sequences to species based on a high throughput phylogenetic annotation approach using an evolutionary placement algorithm(EPA).The EPA-based approach was not only more precise,but also fundamental to robustly unveil the AMF species community composition.The principal advantage of this approach was also demonstrated by using artificially constructed datasets based on validated public database sequences.The affiliation of sequence reads to species using phylogenetic annotation revealed a surprisingly conserved AMF core-species community structure in Andean potatoes,regardless of different plant stages and environmental factors.In total,41 species were detected and in some cases more than 25 species were found colonizing an individual root system.Acaulospora species were identified as dominant colonizers,co-occurring with Cetraspora nodosa and certain Claroideoglomus and Rhizophagus species in most potato root samples.
文摘Crocodiles,gharials and alligators(order Crocodilia),are aquatic reptiles that live in the tropics of Asia,America,Africa,and Australia.Asian countries such as India,Indonesia,Malaysia,and tropics of Australia are the stronghold of the family Crocodylidae.Among all 23 crocodile species,nine species occur in Asia and its surroundings,including the only member of Gavialidae and Alligatoridae family.They are“mugger”or“Crocodylus palustris,”“saltwater crocodile”or“Crocodylus porosus,”“Philippine crocodile”or“Crocodylus mindorensis,”“New Guinea crocodile”or“Crocodylus novaeguineae,”“Siamese crocodile”or“Crocodylus siamensis,”“gharials”or“Gavialis gangeticus,”“false gharial”or“Tomistoma schlegelii,”and“Chinese alligator”or“Alligator sinensis.”All of these species have been encompassed in“Appendix I”and“Appendix II”of the“Convention on International Trade in Endangered Species of Wild Fauna and Flora,”which prevents any kind of trade involving crocodilian species.However,it has been observed that these crocodiles are illegally poached and trafficked for their lucrative skin,meats,eggs,snouts,and bones in medicinal and cosmetic industries.Although many molecular biologists have come forward for the conservation of these species,lack of knowledge about the available,fast,and dependable techniques makes it difficult for forensic identification of seized or confiscated.It has been a major problem for the implementation of the“Wildlife Protection Law”on illegal trade.This article focuses on molecular techniques developed till date for the rapid and reliable species identification and conservation study of them.
基金This work was financially supported by the National Key R&D Program of China(2018YFC1706200)National Natural Science Fund of China(81522049,31571735,82003888)+1 种基金Zhejiang Provincial Ten Thousands Program for Leading Talents of Science and Technology In-novation(2018R520)Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents.
文摘Background:Anisodus acutangulus(Solanaceae),an important folk medicinal herb in China,produces up to 1.2%alkaloids more than that in other Solanaceae plants such as Hyoscyamus niger,while its evolutionary position in Hyoscyameae is not very clear.Objective:To explain the evolutionary position of A.acutangulus in the Solanaceae via complete chloroplast genome(cp)sequence.Methods:Complete chloroplast genome of A.acutangulus was obtained and characterized using the Illumina PE150 pair-end sequencing data.Structure of the genome,codon usage,nucleotide variability(Pi)value,distribution of repeats and SSRs between A.acutangulus and other seven Solanaceae species were analyzed.Previously published 22 Solanaceae cp genomes were used to construct phylogenetic tree.Results:The complete cp genome of A.acutangulus is 156082 bp in length,showed the typical quadripartite structure.The complete cp genome of A.acutangulus was highly conserved.A total of 112 unique genes were found in cp genome of A.acutangulus,among which 17 were duplicated.Further,we found eight hotspot regions for genome divergence could be explored as new DNA barcodes for the identification of the Solanaceae species.Phylogenetic analysis showed that A.acutangulus formed a clade with H.niger.Conclusion:A.acutangulus belongs to Hyoscyameae subfamily and the complete cp genome provides valuable information for phylogenetic reconstruction or comparative genomics of A.acutangulus.
