Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene / Desempenho da técnica tetra-primer PCR em relação a PCR-RFLP na pesquisa de polimorfismos de nucleotídeos únicos (SNPs) rs12979860 (C/T) e rs8099917 (T/G) no gene IFNL4

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)

Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)

Genetic Diversity Analysis of Pogostemon cablin Based on AFLP Markers / 中国实验方剂学杂志

Objective:This paper aims to study the genetic diversity of <italic>Pogostemon cablin</italic> by amplified fragment length polymorphism （AFLP） markers. Method:The 12 pairs of primers were used for AFLP analysis of 212 samples from 14 varieties，and biological analysis software such as POPGENE 32，Arlequinver 3.5，MEGA 7 and NTSYSpc 2.10e were used for polymorphism parameter calculation，principal coordinate analysis and cluster analysis. Result:A total of 2 238 loci were amplified by 12 pairs of primers. 2 226 of them were polymorphic loci， accounting for 99.38%. At the inter-population level，the values of effective alleles（<italic>Ne</italic>），Nei's gene diversity index（<italic>H</italic>），Shannon polymorphic information index（<italic>I</italic>） were 1.365 6±0.066 3， 0.220 7±0.036 4， and 0.343 7±0.050 2，respectively；and 1.118 5±0.038 7，0.071 3±0.023 0，0.109 4±0.035 0，respectively at the intra-population level. Analysis of molecular variance（AMOVA）showed that 71.57% of the total variation of <italic>P. cablin</italic> was of inter-population nature， and 28.43% was of intra-population nature. The 14 populations could be divided into four groups by cluster analysis. Conclusion:The results of AFLP molecular markers showed that abundant genetic diversity was present at inter-population level of <italic>P. cablin</italic>，however，relatively low at intra-population level； the genetic differentiation at the inter-population level was significant，which could provide a reference for the subsequent study of good germplasm selection of <italic>P. cablin</italic>.

DNA fingerprinting and genetic diversity in a threatened alpine medicinal plant, Gentiana crassicaulis (Gentianaceae) using AFLP / 药学学报

Jieji Nabao is a common Tibetan herb. According to our ethnobotanical studies, one of its original plants is identified as Gentiana crassicaulis Duthie ex Burk. (Gentianaceae). Endemic to the Qinghai-Tibet Plateau, this medicinal alpine plant is a threatened species. In this study, 163 individuals from 20 populations of G. crassicaulis were collected throughout its geographical range and amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in this species. A cluster analysis was performed on the AFLP data with Halenia elliptica and Gentiana straminea as the outgroups. From 64 pairs of AFLP primer combinations, 12 pairs were selected for amplification and a total of 315 bands were amplified, of which 254 bands were polymorphic, accounting for 80.63%. High genetic differentiation was detected between populations (87%), and low within populations (13%). The UPGMA (unweighted pair-group method with arithmetic means) tree was topologically consistent with the traditional taxonomic treatments at the species level, and the populations of G. crassicaulis were divided into two branches: one from Yunnan and Guizhou, the other from Tibet, Qinghai, Sichuan and Gansu. PCA analysis and the Mantel test showed that there was a positive correlation between genetic distance and geographical distance. In addition, combined with SSR and SNP markers within cpDNA, the genetic differentiation within the Sichuan population S1 was validated.

Genetic variability of Corynespora cassiicola isolates from Amazonas, Brazil / Variabilidade genética de Corynespora cassiicola isolados do Amazonas, Brasil

Corynespora cassiicola is a cosmopolitan ascomycete widely known as phytopathogen in several crops, and more recently as an emerging pathogen in humans. In this study the genetic variability of 60 isolates of Corynespora cassiicola from different hosts and cities of Amazonas was evaluated, using AFLP molecular markers. Seven genetic groups were identified according to a dendrogram obtained by the Unweighted Pair Group Method using Arithmetical Averages, indicating significant variability among the isolates. Three isolates of different hosts (28, obtained from papaya; 55, obtained from cucumber; and 58, from tomato) remained as single individuals in distinct groups, suggesting marked genetic variation in comparison to the other isolates and possible specificity by the host.(AU)

Corynespora cassiicola é um ascomiceto cosmopolita amplamente conhecido como fitopatógeno em diversas culturas e, mais recentemente, como patógeno emergente em humanos. Na região Norte do Brasil é responsável por perdas significativas em cultivos tanto em casa de vegetação como em campo aberto. Neste estudo foi avaliada a variabilidade genética de 60 isolados de Corynespora cassiicola procedentes de diferentes hospedeiras e municípios do Amazonas, usando marcadores moleculares AFLP. Foram identificados sete grupos genéticos de acordo com dendrograma obtido pelo método de agrupamento UPGMA, indicando significativa variabilidade entre os isolados. Três isolados de diferentes hospedeiras (isolado 28, obtido de mamoeiro; isolado 55, obtido de pepineiro; e isolado 58, proveniente de tomateiro) permaneceram como indivíduos únicos em grupos distintos, sugerindo variação genética marcante em comparação com os demais e possível especificidade pela hospedeira de origem.(AU)

Application of Specific Fragment Length Polymorphism of Algae rDNA in Identification of Drowning Cases / 法医学杂志

OBJECTIVES@#To identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death by detecting part of 5.8S sequence and second internal transcribed spacer （ITS2） （5.8S+ITS2） of diatom rDNA in water and organs.@*METHODS@#Two cases identified by diatom examination, which received by Nanjing Municipal Public Security Bureau Forensic Center, were taken as the research objects. The difference of the population structure of algae in water and human tissue was analysed by length polymorphism of 5.8S+ITS2 marker.@*RESULTS@#In case 1, similar species of diatom were detected from victim's lung and liver tissues and the water sample. Two kinds of DNA fragments with length of 330 bp and 376 bp were detected from victim's lung tissue and the water sample using 5.8S+ITS2 marker, which could confirm the victim was drowning before death. In case 2, there was no diatom found in victim's lung and liver tissues. Only one kind of DNA fragment with length of 331 bp and low relative fluorescence unit （RFU） was obtained from victim's lung tissue using 5.8S+ITS2 marker, thus the victim was thrown into the water after death.@*CONCLUSIONS@#The experimental results of the two cases in present study are consistent with the actual facts and the result of the diatom microscopic examination. The difference of population structure of specific microorganism in water and human tissue can be detected by 5.8S+ITS2 marker, which can help to identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death.

Analysis of virulence genes and the chromosomal 16S rRNA gene polymorphism of Yersinia enterocolitic in Jiangsu Province,2015 / 中华传染病杂志

Objective To reveal the virulence genes and the polymorphisms of chromosomal 16S rRNA gene of Yersinia enterocolitic strains isolated from different districts in Jiangsu Province,2015. Methods Five virulence genes(ail,virF,yadA,ystA and ystB)of Yersinia enterocolitic strains isolated from different districts in Jiangsu Province were detected by using polymerase chain reaction(PCR),and phylogenetic analysis of chromosomal 16S rRNA gene was performed by amplification and sequencing. Results In this study,73 Yersinia enterocolitic strains were collected in Jiangsu Province in 2015.Among them,56(76.7%)strains carried virulence genes,and ail-virF-yadA -ystA -ystB+were the dominate types in diarrhea patients and other hosts.All strains can be clustering into 4 groups according to the phylogenetic analysis of chromosomal 16S rRNA gene.Conclusions The non-pathogenic Yersinia enterocolitic(ystB+)is the dominant strain in Jiangsu province,and the pathogenic strains are also found in this region.The result of phylogenetic analysis of chromosomal 16S rRNA gene and the profiles of virulence genes are highly consistent.

Pseudomonas spp: contamination sources in bulk tanks of dairy farms / Pseudomonas spp: fontes de contaminação em tanques de expansão em fazendas leiteiras

This study focused on isolating Pseudomonas spp. during milking process in ten dairy farms with manual and mechanical milking systems during dry and rainy seasons, and evaluating DNA homology and patterns of distribution between isolates, in order to identify main sources of milk contamination by Pseudomonas spp. A total of 167 isolates of Pseudomonas spp. were obtained from water, milkers' hands, cows' teats, teat cups, cooling tanks and raw milk. Bacteria of Pseudomonas spp. genus were isolated from 85 and 82 sampling points in dairy farms with manual and mechanical milking system, respectively. A significant difference (p=0.02) on Pseudomonas spp. isolation was observed among samples of surface of cows' teats before and after pre-dipping, but no significant difference (p>0.05) was observed among milking systems or seasons. The possibility of the same Pseudomonas spp. patterns are distributed in different farms and seasons using Amplified Fragment Length Polymorphism (AFLP) technique was demonstrated. Milkers' hands, surface of cows' teats, teat cups and cooling tanks were associated with raw milk contamination with Pseudomonas spp. on farms with manual and mechanical milking system, showing that regardless of the type of milking system and season, proper hygiene procedures of equipment, utensils and workers' hands are essential to avoid contamination of the milk and, therefore, improve milk quality.(AU)

Este estudo se propôs a isolar Pseudomonas spp. durante o processo de ordenha em dez fazendas com sistemas manuais e mecanizados, durante as estações seca e chuvosa, além de avaliar a homologia do DNA e seus padrões de distribuição entre os isolados, a fim de se determinar as principais fontes de contaminação do leite. Cento e sessenta e sete isolados de Pseudomonas spp. foram obtidos a partir de amostras de água, mãos de ordenhadores, tetos, teteiras, tanques de resfriamento e leite cru armazenado, sendo 85 e 82 pontos de amostragem em fazendas com sistemas de ordenha manual e mecânico, respectivamente. Diferença estatisticamente significativa foi encontrada entre os isolados observados entre a superfície dos tetos antes e após o pré-dipping (p=0,02), mas nenhuma diferença foi encontrada entre sistemas de ordenha ou estações (p>0,05). A possibilidade do mesmo padrão de Pseudomonas spp. estar distribuído em diferentes fazendas ou estações foi avaliada pela técnica de Polimorfismo do Tamanho de Fragmento Amplificado (AFLP). As mãos de ordenhadores, superfície dos tetos das vacas, teteiras e tanques de resfriamento foram associados com a contaminação do leite cru, demonstrando que independente do tipo de ordenha e estação, a higiene adequada de equipamentos, utensílios e mãos dos ordenhadores é essencial para evitar contaminação do leite, e consequentemente aumentar sua qualidade.(AU)

Análise molecular do EXON 10 do gene receptor de TSH em pacientes com tumores da tireóide / Molecular analysis of EXON 10 of TSH receptor gene in patients with thyroid tumors

Introdução: O câncer da tireoide é a quinta neoplasia mais frequente entre as mulheres. A incidência dessa doença vem aumentando substancialmente nos últimos anos. Objetivos: O objetivo do estudo foi determinar a frequência do polimorfismo D727E no gene tshr e associar aos diferentes fenótipos clínicos de tumores de tireoide. Método: 48 pacientes portadores de tumores da tireoide foram operados em hospital de referência de Belém (Pará, Brasil). O grupo-controle foi composto de 131 indivíduos livres de doenças da tireóide. Resultados: Foram encontrados 26 pacientes com tumores malignos e 22 benignos. A frequência alélica do polimorfismo D727E foi de 17,7% em pacientes com tumor de tireoide e 8% no grupo-controle (p<0,03). O polimorfismo D727E mostrou-se em heterozigose em seis pacientes dos 23 com carcinoma papilar e em um com carcinoma folicular. Um paciente com carcinoma folicular e um com carcinoma indiferenciado não apresentaram o polimorfismo. Sete pacientes dos 15 com bócio colóide apresentaram D727E (seis em heterozigose e um em homozigose). Dois dos seis pacientes com adenoma folicular apresentaram o polimorfismo. Discussão: A análise da distribuição da idade de pacientes com câncer mostrou que houve diferença entre os pacientes com diagnóstico de câncer (48,7±14,7 anos) em relação a pacientes com doença benigna (37,8±11,7 anos). A idade de início dos sintomas, que melhor separa os grupos, foi 46 anos. As variáveis gênero feminino, antecedente familiar e idade >41 anos foram associadas à presença do polimorfismo D727E (p = 0,026), o que não foi observado nos pacientes que não apresentavam o polimorfismo. A ocorrência simultânea dos fatores: gênero feminino, antecedente familiar e idade > 46 anos foi relacionada com maior frequência em pacientes com patologia maligna da tireóide (p=0,0047). Conclusões: Estudos adicionais com um maior tamanho amostral são necessários para investigar com mais força estatística a relevância do polimorfismo D727E na gênese do câncer de tireoide e do bócio nodular coloide.

Introduction: Cancer of the thyroid gland is the fifth most common malignancy among women. The disease’s incidence has been increasing over the years. Objectives: The aim of this study, was to determine the frequency of polymorphism D727E tshr gene and its association with different clinical phenotypes of thyroid cancer. Results: 48 patients with thyroid surgical diseases were operated in the city of Belém (Pará, Brazil). The control group was composed by 131 individuals free thyroid diseases. We observed 26 patients with thyroid malignant disease and 22 benign. The allele frequency of polymorphism D727E was 17.7% in patients with thyroid tumor and 8% in the control group (p <0.03). Among malignant thyroid tumors, D727E mutation was found in heterozygosis in 6 of the 23 patients with papillary thyroid carcinoma and in one of two patients with follicular carcinoma. Among the considered benign thyroid tumors: 7 out of 15 patients with nodular goiter colloid showed the mutation (6 heterozygous and 1 homozygous). Two of 6 patients with follicular adenoma showed polymorphism D727E. Discussion: The analysis of the age distribution of patients with thyroid cancer showed that there were differences between patients with cancer diagnosis (48.7 ± 14.7 years), compared to patients with benign disease (37.8 ± 11.7 years). The age of onset of the symptoms that best separates the groups was 46 years. The variables gender (female), family history and age > 41 years were associated with the presence of D727E polymorphism (p = 0.026), which was not observed in patients without this polymorphism. The simultaneous occurrence of gender (female), family history and age > 46 years was associated with increased frequency in patients with malignant thyroid pathology (p = 0.0047). Conclusions: Additional studies with a larger sample size are needed to investigate harder statistical relevance of polymorphism D727E in the genesis of thyroid cancer as well as nodular goiter.

Genetic diversity of Brassica oleracea var. capitata gene bank accessions assessed by AFLP

The genetic diversity of 20 cabbage (Brassica oleracea var. capitata, including sub.var. alba and rubra) cultivars and landraces from the Gene bank of Crop Research Institute was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two cultivars of Brassica pekinensis (syn. Brassica rapa var. pekinensis) were used as outliers in the study. Thirty AFLP primer combinations produced a total of 1084 fragments. A total of 806 fragments, 364 (45 percent) of them polymorphic, were found across 20 Brassica oleracea var. capitata accessions. The accessions were clustered into two main groups. Special subgroups, reflecting place of origin, were observed within these groups. Ten selective primer pairs were found to be most informative because each of these uniquely identified all of the accessions used. Furthermore, two accessions of Brassica pekinensis were clearly differentiated from the Brassica oleracea var. capitata accessions. AFLP is an efficient tool for determination of genetic diversity of cabbage gene bank accessions.

Genotypic characterization of Staphylococcus aureus isolated from bovines, humans, and food in Indonesia

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.

Identification of the Kiwifruit Germplasms in Jiangxi Province by AFLP and Its Classification Significance / 中国生物化学与分子生物学报

The kiwifruit germplasms in Jiangxi province were identified by amplified fragment length polymorphism(AFLP)markers．Four primer pairs that had been selected from 64 ones had detected a total of 190 bands among 31 germplasms of kiwifruit,one hundred and seventy nine bands that representing 94．2％of total bands were polymorphic．The identification rates of 31 germplasms were up to 100％．The result suggests that applying AFLP markers to analyze the kiwifruit germplasms is feasible．Then Clustering analysis the AFLP resuit by UPGMA．The dendrogram indicated that the Dice similarity coefficients of 31 germplasms of kiwifruit ranged from 0．50 to 0．85,it suggested that their genetic relationships weren't near．Thirty one germplasms could divide into four groups according to the Dice similarity coefficient 0．56．Sect．Leiocarpae and Sect．Maculatae clustered into one group；A．Melliana of Sect．Strigosae was one group；A．Chinensis var. Rufopulpa,A．Eriantha,A．Fulvicoma var．Lenata,A．Styracifolia and A．Jiangxiensis in Sect．Stellatae clustered into the same group；but the two germplasms of Sect．Stellatae,A．Deliciosa and A．Chinensis clustered into the other group．Interestingly,the‘Ganmi-5’was in the same rank with A．Chinensis from the dendrogram,but it was the variety of A．Chinensis according to the traditional classification．It is necessary that the‘Ganmi-5’should be further researched in classification．The genetic relationships among the kiwifruit germplasms in Jiangxi province were identified and characterized by themolecular method,which was consistent with the traditional classification in certain degree and provided new evidences for the classification of the kiwifruit germplasms．

Comparison of RAPD, AFLP, and EF-1alpha Sequences for the Phylogenetic Analysis of Fusarium oxysporum and Its formae speciales in Korea

Although Fursarium oxysporum causes diseases in economically important plant hosts, identification of F. oxysporum formae speciales has been difficult due to confusing phenotypic classification systems. To resolve these complexity, we evaluated genetic relationship of nine formae speciales of F. oxysporum with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and translation elongation factor-1 alpha (EF-1alpha) gene. In addition, the correlation between mycotoxin content of fusaric acid and isolates based on molecular marker data was evaluated using the modified Mantel's test. According to these result, these fusaric acid-producing strains could not identify clearly, and independent of geographic locations and host specificities. However, in the identification of F. oxysporum formae speciales, especially, AFLP analysis showed a higher discriminatory power than that of a the RAPD and EF-1alpha analyses, all three techniques were able to detect genetic variability among F. oxysporum formae speciales in this study.

cDNA-AFLP Reveals Variation of Sequence Region trnF-ndhJ in Cytoplasmic Male Sterile Chinese Cabbage-pak-choi / 生物化学与生物物理进展

By means of cDNA amplified fragment length polymorphism(cDNA-AFLP) technique, a fragment P1708 was amplified from Polima cytoplasmic male sterile Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinenesis Makino, syn, B. rapa L. ssp. chinenesis) 'Bpol97-05A'. RT-PCR showed that this fragment was specifically expressed in male sterile material. Sequencing and BLAST search in GenBank database indicated that P1708 had 100% homolog with chloroplast ndhJ-trnF gene region except a 54 bp insertion. Gene specific primer pairs were synthesized according to ndhJ-trnF gene region and two fragments about 1 900 bp were amplified respectively using genomic DNA templates of Polima cabbage and male fertile oilseed rape. The sequencing results showed that the gene region ndhJ-trnF of Polima cabbage contained two 54 bp repeats and some variation sites. The repeat part shared the same sequence as trnF gene except three bases at 5′ ends. For the insertion of 108 bp sequence, a new open reading frame was created.

Comparison of Pulsed-field Gel Electrophoresis, Amplified Fragment Length Polymorphism, and Infrequent Restriction Site-Polymerase Chain Reaction for Molecular Typing of Escherichia coli and Staphylococcus aureus Strains / 감염

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) has been regarded a standard method for genotyping in epidemiologic studies. However, it is tedious and time-consuming to perform. Two alternative genotyping methods have recently been developed using the polymerase chain reaction (PCR):amplified fragment length polymorphism (AFLP) and infrequent restriction site-polymerase chain reaction (IRS-PCR). These methods have not yet been applied yet to common pathogens such as Staphylococcus aureus. The purpose of this study was to determine the applicability of AFLP and IRS-PCR for the genotyping of E. coli and S. aureus isolates. METHODS: We performed PFGE, AFLP, and IRS-PCR on clinical isolates of E. coli (n=27) and S. aureus (n=30). We assessed each method in terms of discriminatory power, quality, and efficiency. RESULTS: In E. coli, the discriminatory powers of IRS-PCR and AFLP were comparable to that of PFGE. PFGE discerned 24 (88.8%) out of 27 strains, IRS-PCR discerned 22 (81.5%) out of 27, and AFLP discerned 25 (92.6%) out of 27. In the case of S. aureus, PFGE discerned 27 (90%) out of 30 strains, while both IRA-PCR and AFLP discerned 12 (40%) out of 30. The test-ing took four days to complete with PFGE, two days with AFLP, and was completed within one day with IRS-PCR. IRS-PCR showed better resolution than both PFGE and AFLP. CONCLUSION: In cases of E. coli, AFLP and IRS-PCR could be good alternatives for epidemiologic typing, as they offer better efficiency and comparable discriminatory power to PFGE. On the other hand, IRS-PCR and AFLP do not seem to be suitable for the strain-to-strain differentiation of S. aureus.

AFLP Analysis on genetic diversity of germplasm resource in Dryopteris fragrans / 中草药

Objective To determine the genetic diversity of germplasm resource in Dryopteris fragrans by amplified fragment length polymorphism(AFLP) technique.Methods Forty-six samples from six populations of D.fragrans were analyzed by AFLP DNA markers,and the genetic diversity was evaluated by NTSYS-PC 2.10 and PopGen32.Results The percentage of polymorphic bands(PPB) reached to 76.67%,observed number of alleles(Na) was 1.766 7, effective number of alleles(Ne) was 1.504 6,Nei′s gene diversity index(H) was 0.292 1,Shannon information index(I) was 0.431 6,and genetic differentiation index(Gst) was 0.412 5.Conclusion Genetic diversity is high,gene diversity in populations(Hs) is 0.170 5,and gene diversity among populations(Dst) is 0.119 8.Because of environmental specificity,its resource on the spot should be protected.

Analysis on genetic diversity of different geographical populations of Houttuynia cordata / 中草药

Objective To study the genetic diversity of different geographical populations of Houttuynia cordata in China.Methods The genetic diversity of 15 geographical populations of H.cordata from 13 provinces in China was estimated using amplified fragment length polymorphism(AFLP) markers.The data of amplified bands were analyzed by the software POPGENE and MEGA.Results The ten AFLP primers employed produced a total of 110 discernable and reproduceable amplified fragments.The percentage of polymorphic bands within different populations was 70.51%.Genetic diversity analysis showed that effective number of alleles(Ne) was 1.210,Nei′s gene diversity(H) was 0.119,and Shannon′s genetic diversity index(I) was 0.186.The coefficient of genetic distance was 0.008 9—0.181 8 among populations.A UPGMA dendrogram based on Nei′s(1972) genetic distance visualized that the 15 populations were grouped into three different clusters,Emei population was one individual cluster group and the other populations were grouped into two different clusters corresponding to the different geographical areas.Conclusion The genetic diversity within different geographical populations of H.cordata in China is plentiful.

AFLP Analysis on genetic diversity for germplasm resources of Fritillaria thunbergii cultivated in Zhejiang Province / 中草药

Objective To study the genetic diversity of Fritillaria thunbergii,a traditional Chinese herb in Zhejiang Province in China.Methods The genetic diversity of six representational populations of F.thunbergii including 32 individuals was investigated by amplified fragment length polymorphism(AFLP) maker technique.Results The genetic diversity was revealed as follow: the Nei′s genetic diversity index(He) 0.169 0?0.175 7,Shannon′s information index(I) 0.269 8?0.245 3,percentage of polymorphic loci(PPB) was 76.85% at the species level;Ht 0.169 0?0.030 9,and Hs 0.150 8?0.024 0,I 0.233 3?0.261 9, PPB was 50.38% at population level.The genetic differentiation index(Gst) was 0.107 6,Nm 4.147 0.The result of dendrogram of six populations indicated that Dongyang and Yongkang populations shared the minimum genetic distance(0.015 0),they were classified into a group,and Xiangshan and Jinyun populations shared the maximum genetic distance(0.032 4).Conclusion The genetic diversity of F.thunbergii cultivated in Zhejiang Province is very rich,which could ensure the long-term survival of F.thunbergii.But the genetic diversity of F.thunbergii is relatively higher in population levels while lower at the species levels and the degree of genetic differentiation occured among the populations is not significant.The germplasm resources are relatively stable among these six populations.These populations could be used to breed the fine strains of F.thunbergii as the bases.

Application of AFLP markers for analyzing genetic difference among cultivated Pinellia ternata / 中草药

Objective The DNA fingerprints of cultivated Pinellia ternata collected from different geographic regions were generated by using AFLP markers to find the feasibility in analyzing their genetic diversity, relationship, and germplasm identification. Methods The DNA polymorphism of 51 cultivated germplasm of P. ternata collected from 17 different habitats and four cultivars of P. pedatisecta (outgroup) were detected by AFLP molecular markers. Results The DNA fingerprints of 51 individuals of P. ternata were obviously distinguished by eight pairs of high polymorphic and efficient primer combinations screened from 64 primer combinations. The phylogenetic clustering results revealed that all the tested cultivars were fully differentiated, and individuals from the same regions were mainly clustered together. Moreover, cultivars from East-China, including Zhejiang and Jiangsu Provinces, displayed clear genetic distinction from other regions. The clustering results were strongly supported by Bootstrap test. Conclusion AFLP Markers can be potentially used in analyzing of genetic diversity, relationship, and germplasm identification of this medicinal plant, and the germplasm from regions of East-China, including Zhejiang and Jiangsu Provinces, displays the relative separate genetic characters from other regions.

AFLP Analysis on diversity of germplasm resource in cultivated and wild Cistanche deserticola / 中草药

Objective To determine the genetic diversity of germplasm resource in cultivated and wild Cistanche deserticola. Methods Fifty-eight samples from three populations of cultivated and wild C. deserticola were analyzed by amplified fragment length polymorphism (AFLP) DNA markers, and the gene- tic diversity was evaluated by PopGen32. Results The average percentage of polymorphic loci (PPL) of cultivated C. deserticola is 79.16%. The PPL of wild population is 89.53%. Average Neis gene diversity index (He) from four populations was 0.193 8, Shannons genetic diversity index (I) was 0.300 4, and genetic differentiation index (Gst) was 0.097 9. Conclusion The diversities of cultivated and wild C. deserticola are both higher and theres no differentiation between them. It shows that genetic diversity of inner-species is higher, which is not the reason for endangerment. Therefore, wild nursery and artificial cultivating are the best measures for the conservation and sustainable utilization in C.deserticola.