Sequence analysis of the internal transcribed spacer 2 (ITS2) region of rDNA for identifying Trichogramma species and evaluating genetic diversity / Análise de sequência do rDNA da região espaçadora do transcrito interno 2 (ITS2) para identificar espécies de Trichogramma e avaliar a diversidade genética

Abstract Species of Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammtidae) are frequently used as biological control agents against Lepidoptera, but practical application of these egg endoparasitoids are complicated because of their complex taxonomy. This study aimed to compare sequences of internal transcribed spacer regions of ribosomal DNA (ITS2-rDNA) of Trichogramma accessions with those deposited in GenBank in order to access the reliability of the ITS2 as a barcode for discriminating species and evaluating the genetic diversity. ITS2-rDNA sequences obtained from seventeen specimens of Trichogramma confirmed previous identifications based on morphological characteristics. Multiple sequence alignment revealed the existence of highly conserved regions in ITS2 sequences while the neighbour-joining dendrogram indicated that the specimens formed three clusters comprising T. manicobai and T. marandobai (group I), T. galloi (group II) and T. pretiosum (group III). The ITS2 marker was shown to be a powerful DNA barcode for discriminating Trichogramma species and could be used to complement the morphological approach.

Resumo Espécies de Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae) são freqüentemente usadas como agentes de controle biológico contra Lepidoptera, esses endoparasitóides de ovos apresentam taxonomia complexa, o que dificulta sua aplicação prática. Este estudo teve como objetivo comparar seqüências de regiões espaçadoras internas transcritas de DNA ribossômico (ITS2-rDNA) de acessos de Trichogramma com aquelas depositadas no GenBank, a fim de avaliar a confiabilidade do ITS2 barcode para discriminar espécies e avaliar a diversidade genética. As seqüências de ITS2-rDNA obtidas de dezessete espécimes de Trichogramma confirmaram identidades anteriores com base em características morfológicas. O alinhamento de múltiplas sequências revelou a existência de regiões altamente conservadas nas sequências ITS2, enquanto o dendrograma indicou que os espécimes formavam três grupos compreendendo T. manicobai e T. marandobai (grupo I), T. galloi (grupo II) e T. pretiosum (grupo III). O marcador ITS2 mostrou ser um poderoso DNA barcode para discriminar espécies de Trichogramma podendo ser usado como complemento da abordagem morfológica.

Evaluation of Genetic Analysis of ESCHERICHIA COLI Isolated from Two Different Environmental Sources: Sewage Water Verses Soiled Bedding Materials of Laboratory Rodents

ABSTRACT The present investigation details an assessment of genetic relationship of E. coli isolates collected from two different environmental sources viz. sewage water and soiled bedding materials of laboratory rodents. Five sewage water samples were collected from the industrial area of Lucknow city and 5 samples of soiled bedding materials of laboratory animals were collected from Animal facility at CSIR-IITR, Lucknow. For this study Random amplified polymorphic DNA markers (RAPD) was chosen as the molecular fingerprinting method. In this study, 10 RAPD primers were used to evaluate the genetic similarity of E. coli. isolates. The RAPD-PCR fingerprints were analyzed and data were scored as 1, 0 matrix. The generated data were fed on Popgene software for calculating genetic diversity and creating dendrogram. The genetic similarity of 85% was recorded from soiled bedding materials and only 71% in sewage water samples in E.coli samples. The dendrogram based generation of clustering of E. coli isolates show two major clusters. Within major cluster sub-cluster were also observed which indicating diversity within isolates of E. coli. The RAPD-PCR based fingerprinting provided a rapid means of discriminating E. coli isolates and considered a relevant tool for molecular typing.

Sequence analysis of the internal transcribed spacer 2 (ITS2) region of rDNA for identifying Trichogramma species and evaluating genetic diversity

Abstract Species of Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammtidae) are frequently used as biological control agents against Lepidoptera, but practical application of these egg endoparasitoids are complicated because of their complex taxonomy. This study aimed to compare sequences of internal transcribed spacer regions of ribosomal DNA (ITS2-rDNA) of Trichogramma accessions with those deposited in GenBank in order to access the reliability of the ITS2 as a barcode for discriminating species and evaluating the genetic diversity. ITS2-rDNA sequences obtained from seventeen specimens of Trichogramma confirmed previous identifications based on morphological characteristics. Multiple sequence alignment revealed the existence of highly conserved regions in ITS2 sequences while the neighbour-joining dendrogram indicated that the specimens formed three clusters comprising T. manicobai and T. marandobai (group I), T. galloi (group II) and T. pretiosum (group III). The ITS2 marker was shown to be a powerful DNA barcode for discriminating Trichogramma species and could be used to complement the morphological approach.

Resumo Espécies de Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae) são freqüentemente usadas como agentes de controle biológico contra Lepidoptera, esses endoparasitóides de ovos apresentam taxonomia complexa, o que dificulta sua aplicação prática. Este estudo teve como objetivo comparar seqüências de regiões espaçadoras internas transcritas de DNA ribossômico (ITS2-rDNA) de acessos de Trichogramma com aquelas depositadas no GenBank, a fim de avaliar a confiabilidade do ITS2 barcode para discriminar espécies e avaliar a diversidade genética. As seqüências de ITS2-rDNA obtidas de dezessete espécimes de Trichogramma confirmaram identidades anteriores com base em características morfológicas. O alinhamento de múltiplas sequências revelou a existência de regiões altamente conservadas nas sequências ITS2, enquanto o dendrograma indicou que os espécimes formavam três grupos compreendendo T. manicobai e T. marandobai (grupo I), T. galloi (grupo II) e T. pretiosum (grupo III). O marcador ITS2 mostrou ser um poderoso DNA barcode para discriminar espécies de Trichogramma podendo ser usado como complemento da abordagem morfológica.

Morphological and SSR Assessment of Putative Drought Tolerant M3 and F3 Families of Wheat (Triticum aestivum L.).

In an attempt to develop drought tolerant genotypes of bread wheat, two procedures, i.e., mutation and hybridization were used to induce new genetic variation. Selection for high grain yield/plant (GYPP) and other desirable traits was practiced in the M2 populations of 7 gamma irradiated genotypes and F2 populations of 15 diallel crosses among 6 genotypes of wheat under well watering (WW) and water stress (WS) conditions. Progenies of these selections (53 M3 and 109 F3 families) and their seven parents were evaluated in the field under WW and WS. Significant yield superiority of twelve families (7 M3 ’s and 5 F2 ’s) over their original and better parents, respectively under WS reached 74.71% (SF9). These putative drought tolerant families were assessed on the DNA level using SSR analysis. Fifteen SSR primers were used for PCR amplification of the genomic DNA of these 12 selections and their parents. The SSR analysis proved that the 12 families are genetically different from their 7 parents, with an average polymorphism of 86.67%. The genetic similarities (Gs) ranged from 30% to 88%. Both mutants SF3 and SF4 exhibited very low Gs (42 and 40%, respectively) with their common parent (Giza-168), indicating that gamma rays were very effective in changing the genetic background of Giza-168 towards high GYPP under WS conditions. SSR assay permitted the identification of seven unique bands (5 positive and 2 negative) for three drought tolerant wheat genotypes (SF3, SF4 and Aseel-5). These bands might be considered useful as markers associated with drought tolerance in bread wheat breeding programs.

ISSR analysis of six medicinal plants in Potentilla L / 中草药

Objective: To analyze the genetic relationship among the medicinal plants of Potentilla L. Methods: Six species of germplasm resources in Potentilla L. were analyzed by ISSR molecular markers. To make up the systematic diagram of genetic relationship by Popgene 1.32 software, cluster by UPGMA method, and establish the dendrogram. Results: A total of 105 ISSR bands were scored for 11 primers, among which 89 were polymorphic bands. The average percentage of polymorphic bands was 84.76%. Genetic similarity coefficient changed from 0.4476 to 0.7905. Conclusion: By cluster analysis, it shows that some of the medicinal plants in Potentilla L. from the same region are in the same group and demonstrates the rule of geographical distribution in the tested materials. This study will provide the foundation for identification and development of medicinal plants in Potentilla L., and guide collection and evaluation of germplasm resources.

ISSR analysis on genetic diversity in Phyllanthus urinaria / 中草药

Objective To detect the genetic diversity of 30 populations of Phyllanthus urinaria from some provinces of China. Methods ISSR was used to analyze the genetic diversity of 30 populations of P. urinaria from Chongqing, Shaanxi, Henan, Yunnan, Guangxi, Guangdong, Zhejiang, Guizhou, Fujian, and Hainan Provinces of China. Results Twenty four primers selected from 60 ISSR primers were used for amplification and a total of 264 DNA bands were obtained, including 245 polymorphic bands and 19 common bands. At species level, the average percentage of polymorphic bands (PPB) was 92.80%. The range of the genetic similarity (GS) value was 0.579 5-0.916 7, with a higher genetic diversity. Cluster analysis showed that the P. urinaria population was roughly divided into three branches: inland branch, coastal branch, and transition branch which located in Yunnan Province; Individual populations presented obvious specificity. Conclusion The genetic diversity of germplasm resources in P. urinaria is higher.

Genetic diversity analysis of rice cultivars (Oryza sativa L. ) differing in salinity tolerance based on RAPD and SSR markers

Thirty rice cultivars were evaluated for salinity tolerance during the seedling stage and were divided into five tolerance groups including tolerant (T), moderately tolerant (MT), moderately susceptible (MS), susceptible (S) and highly susceptible (HS) which comprised 5, 10, 9, 4 and 2 cultivars respectively. Genetic diversity of all rice cultivars was evaluated using random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers. The cultivars were evaluated for polymorphisms after amplification with 20 random decamer primers and 20 SSR primer pairs. A total of 161 RAPD markers and 190 SSR alleles were produced which revealed 68.94 percent and 89.47 percent polymorphism respectively. Mean genetic similarity coefficient was 0.82 for RAPD and 0.70 for SSR. Cluster analysis based on RAPD markers was effective in grouping cultivars based on their salt tolerance ability. Group IA1, IB and IV contained three T, three S and two HS rice cultivars respectively. The MT and MS cultivars which showed similar physiological responses to salinity were resolved into two groups: Group IA2 and Group II comprising ten and eight MT/MS cultivars respectively. Cluster analysis based on SSR markers separated rice cultivars into groups based on genetic relatedness which did not correspond to salinity tolerance level. The results from this study provided some useful implications for salt tolerance breeding programs. The evaluation of genetic similarity and cluster analysis together with salt tolerance ability provides some useful guides for assisting plant breeders in selecting suitable genetically diverse parents for the crossing program.

Diversidade genética entre acessos de espinheira-santa (Maytenus ilicifolia Mart. ex Reis.) coletados no estado do Rio Grande do Sul, Brasil / Genetic diversity of "espinheira-santa" (Maytenus ilicifolia Mart. ex Reis.) accessions collected in Rio Grande do Sul State, Brazil

Maytenus ilicifolia Mart. ex Reis., popularmente conhecida como espinheira-santa, é espécie autóctone pertencente à família Celastraceae, usada para tratamento de úlceras gástricas e gastrites. Devido à importância medicinal, houve aumento no extrativismo das populações naturais, tornando-a uma espécie prioritária para a conservação, a fim de evitar a erosão genética. Buscou-se com este trabalho analisar a diversidade genética de 20 acessos de M. ilicifolia coletados em diferentes localidades no Rio Grande do Sul. Utilizando marcadores moleculares do tipo AFLP, foram testadas oito combinações de primers, que geraram 455 bandas eletroforéticas, 100 por cento polimórficas. As combinações de primers E-ACC/M-CAA, E-ACG/M-CTA, E-ACG/M-CTC apresentaram o maior número de bandas eletroforéticas, 71 cada, totalizando 46,80 por cento do polimorfismo total. Os valores de similaridade genética calculada pelo coeficiente simple matching foram utilizados para gerar o dendrograma de similaridade pelo método UPGMA. Foi obtido alto coeficiente de correlação cofenética (r=0,94), demonstrando elevada representatividade dos dados de similaridade genética no dendrograma. Pela AMOVA verificou-se que 89,33 por cento da diversidade total ocorreram entre indivíduos dentro das populações. A caracterização molecular de acessos de Maytenus ilicifolia por meio de AFLP foi eficiente para identificar diversidade genética. Através da análise de similaridade genética o banco de germoplasma poderia ser composto com os acessos que apresentaram menor similaridade e maior número de alelos, permitindo com que estes fornecessem ampla cobertura do genoma que compõem Maytenus ilicifolia. Os acessos que ficaram agrupados em mesmo cluster e com número reduzido de alelos podem ser descartados deste banco. A diversidade genética intrapopulacional identificada por esse marcador foi muito maior do que aquela entre populações.

Maytenus ilicifolia Mart. ex Reis., popularly known as "espinheira-santa", is an autochthonous species belonging to the Celastraceae family. This species is used to treat ulcers and gastritis. Due to its medicinal importance, the exploitation of natural populations has increased, making the conservation of this species essential to prevent genetic erosion. The aim of this work was to analyze the genetic diversity of 20 M. ilicifolia accessions collected in different localities of Rio Grande do Sul State. Using AFLP-type molecular markers, eight primer combinations were tested, producing 455 electrophoretic profiles, with 100 percent polymorphism. The primer combinations E-ACC/M-CAA, E-ACG/M-CTA and E-ACG/M-CTC presented the largest number of electrophoretic profiles, 71 each, totaling 46.80 percent of the total polymorphism. The values of genetic similarity estimated by Simple Matching Coefficient were used to produce the dendrogram of similarity by the UPGMA method. A high cophenetic correlation coefficient (r = 0.94) was obtained, demonstrating high representativeness of the data of genetic similarity in the dendrogram. Using AMOVA, 89.33 percent of the total diversity were observed among individuals from the same population. The molecular characterization of Maytenus ilicifolia accessions by AFLP allowed the identification of genetic diversity. The genetic similarity analysis indicated that the germplasm bank could be composed of the accessions presenting the lowest similarity and the largest numbers of alleles, providing a comprehensive coverage of Maytenus ilicifolia genome. The accessions that were grouped into one same cluster and with a reduced number of alleles could be disposed of this bank. The genetic diversity identified by this marker within populations was much greater than that between populations.

Isozyme, ISSR and RAPD profiling of genotypes in marvel grass (Dichanthium annulatum).

Genetic analysis of 30 accessions of marvel grass (Dichanthium annulatum Forsk.), a tropical range grass collected from grasslands and open fields of drier regions, was carried out with the objectives of identifying unique materials that could be used in developing the core germplasm for such regions as well as to explore gene (s) for drought tolerance. Five inter-simple sequence repeat (ISSR) primers [(CA)4, (AGAC)4, (GACA)4 ]; 27 random amplified polymorphic DNA (RAPD) and four enzyme systems were employed in the present study. In total, ISSR yielded 61 (52 polymorphic), RAPD 269 (253 polymorphic) and enzyme 55 isozymes (44 polymorphic) bands. The average polymorphic information content (PIC) and marker index (MI) across all polymorphic bands of 3 markers systems ranged from 0.419 to 0.480 and 4.34 to 5.25 respectively. Dendrogram analysis revealed three main clusters with all three markers. Four enzymes namely esterase (EST), polyphenol oxidase (PPO), peroxidase (PRX) and superoxide dismutase (SOD) revealed 55 alleles from a total of 16 enzyme-coding loci. Of these, 14 loci and 44 alleles were polymorphic. The mean number of alleles per locus was 3.43. Mean heterozygosity observed among the polymorphic loci ranged from 0.406 (SOD) to 0.836 (EST) and accession wise from 0.679 (IG3108) to 0.743 (IGKMD-10). Though there was intermixing of few accessions of one agro-climatic region to another, largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 iterations also showed large numbers of nodes (11 to 17) having strong clustering (>50 bootstrap values) in all three marker systems. The accessions of the arid and drier regions forming one cluster are assigned as distinct core collection of Dichanthium and can be targeted for isolation of gene (s) for drought tolerance. Variations in isozyme allele numbers and high PIC (0.48) and MI (4.98) as observed with ISSR markers indicated their usefulness for germplasm characterization.

Marker-assisted backcrossing using microsatellites and validation of SCAR Phs marker for resistance to white mold in common bean

Molecular markers may accelerate selection through the identification of plants with higher proportion of recurrent parent genome, as well as identifying those plants bearing target alleles like quantitative traits loci (QTLs) for white mold resistance. The objectives of this work were: 1) to employ microsatellite markers (SSR) in order to accelerate the recovery of recurrent parent genome 2) to validate sequence characterized amplified region (SCAR) Phs associated with a QTL that confers resistance to white mold, as previously identified in bean populations. Lines G122 and M20 were crossed, which generated 267 F1 plants from backcross (BC) BC1 and 113 plants from backcross BC2.SSR polymorphic markers were adopted. The relationship between BC plants and the recurrent parent was estimated based on the recurrent genome proportion (PR) in each BC plant, and the Sorensen-Dice genetic similarity (sg ir). To determine how much the phenotypic variation is explained by SCAR Phs, 56 F1:2BC1 progenies were evaluated on the field following a random block design with two replications through the straw test method. SSR markers are efficient in identifying individuals with a greater proportion of the recurrent genome. SCAR Phs was not efficient for the indirect selection of common beans for white mold resistance.

Genetic analysis of species in the Genus Catasetum (ORCHIDACEAE) using RAPD Markers

In this work, RAPD molecular markers were used to access the genetic variability and to study the inter and intraespecifc relationship in a group of 37 species, including 56 individuals. A total of 15 RAPD primers were selected for DNA amplification. From a total of 221 bands analyzed, 209 (95 percent) were polymorphics. The level of interespecifc genetic similarity ranged from 37 percent between Catasetum complanatum and Catasetum laminatum to 83 percent between Catasetum triodon and Catasetum uncatum. The intraspecifc genetic similarity varied 88 percent for the individuals of Catasetum triodon to 93 percent between the individuals of Catasetum atratum and Catasetum macrocarpum. These results would contribute to understand the genetic relationship in Catasetum, to define the strategies to establish a germplasm core collection for the genus and to provide support for breeding programs.

Neste trabalho, marcadores moleculares de RAPD foram utilizados para acessar a variabilidade genética e estudar as relações interespecíficas e intraespecífica em um grupo de 37 espécies, compreendendo 56 plantas individuais. Um total de 15 primers foram selecionados para amplificação do DNA. De um total de 221 bandas analisadas, 209 (95 por cento) foram polimórficas. O nível de similaridade genética interespecífica variou de 37 por cento entre Catasetum complanatum e Catasetum laminatums a 83 por cento entre Catasetum triodon e Catasetum uncatum. A similaridade genética intraespecífica variou de 88 por cento entre os indivíduos de Catasetum triodon a 93 por cento entre os indivíduos de Catasetum atratum e Catasetum macrocarpum. Os resultados deste trabalho contribuem para o entendimento das relações interespecíficas no gênero Catasetum, para definir estratégias para o estabelecimento de um banco de germoplasma e para dar suporte a programas de melhoramento.

Caracterização genética de Brycon orbignyanus utilizando o sistema seminatural / Genetic characterization of Brycon orbignyanus using the semi-natural system

Avaliou-se o efeito do sistema seminatural na diversidade genética de um estoque de Brycon orbignyanus, utilizado em programas de repovoamento, com o marcador molecular RAPD. Vinte e quatro reprodutores, 12 machos e 12 fêmeas e 95 larvas da progênie foram analisados. Os nove primers utilizados produziram 90 fragmentos, dos quais 94,4 por cento foram polimórficos. Houve diferença significativa na frequência de 20 dos 90 fragmentos entre os reprodutores e sua progênie sem a presença de fragmentos exclusivos. O índice de diversidade genética de Shannon, a porcentagem de fragmentos polimórficos e a diversidade genética de Nei foram mais altos nos indivíduos da progênie. A similaridade genética foi maior nos indivíduos do estoque de reprodutores. A análise de variância molecular mostrou que a maior parte da variação está dentro de cada grupo (89,1 por cento) e não entre os grupos (10,9 por cento). A identidade e a distância genética entre os estoques foram de 0,944 e 0,057, respectivamente. Assim, a utilização do sistema seminatural evitou a mortalidade de reprodutores B. orbignyanus e conservou a variabilidade genética da progênie.

The effect of the semi-natural system on the genetic diversity of a Brycon orbignyanus stock, used in stock enhancement programs, was evaluated with the RAPD molecular marker. Twenty-four broodstocks - 12 males and 12 females - and 95 larvae of the offspring were analyzed. The nine used primers produced 90 fragments, of which 94.4 percent were polymorphic. There was significant difference in the frequency of 20 out of the 90 fragments between the broodstocks and their offspring without the presence of exclusive fragments. The Shannon genetic diversity index, the percentage of polymorphic fragments and the Nei gene diversity were higher in the offspring individuals. Genetic similarity was higher in broodstock individuals. The analysis of molecular variance results showed that the major part of the genetic variation is within the groups (89.1 percent) and not between them (10.9 percent). The identity and genetic distance between the groups were 0.944 and 0.057, respectively. Like this, the use of the semi-natural system avoided the mortality of B. orbignyanus broodstocks and conserved the genetic variability of the offspring.

Change in genetic size of small-closed populations: lessons from a domestic mammal population

The aim of this study was to monitor changes in genetic size of a small-closed population of Iranian Zandi sheep, by using pedigree information from animals born between 1991 and 2005. The genetic size was assessed by using measures based on the probability of identity-by-descend of genes (coancestry, f, and effective population size, Ne), as well as measures based on probability of gene origin (effective number of founders, f e, effective number of founder genomes, f g, and effective number of non-founder genomes, f ne). Average coancestry, or the degree of genetic similarity of individuals, increased from 0.81 percent to 1.44 percent during the period 1993 to 2005, at the same time that Ne decreased from 263 to 93. The observed trend for f e was irregular throughout the experiment in a way that f e was 68, 87, 77, 92, and 80 in 1993, 1996, 1999, 2002, and 2005, respectively. Simultaneously, f g, the most informative effective number, decreased from 61 to 35. The index of genetic diversity (GD) which was obtained from estimates of f g,decreased about 2 percent throughout the period studied. In addition, a noticeable reduction was observed in the estimates of f ne from 595 in 1993 to 61 in 2005. The higher than 1 ratio of f e to f g indicated the presence of bottlenecks and genetic drift in the development of this population of Zandi sheep. From 1993 to 1999, f ne was much higher than f e, thereby indicating that with respect to loss of genetic diversity, the unequal contribution of founders was more important than the random genetic drift in non-founder generations. Subsequently, random genetic drift in non-founder generations was the major reason for f e> f ne. The minimization of average coancestry in new reproductive individuals was recommended as a means of preserving the population against a further loss in genetic diversity.

Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffea liberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.