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.

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

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.

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.

DNase concentration assay to obtain DNA-free RNA from sugarcane leaves.

The success of gene expression studies, protein synthesis, and construction of cDNA libraries directly depends on the purity and integrity of the RNA used in these tests, as even minor amounts of contaminant DNA (<1%) can produce a false positive amplification signal in quantitative real-time PCR. For RNA contaminated with genomic DNA, an essential step in the studies on gene expression is the treatment of the RNA samples with DNase. This study was conducted to test three different concentrations of DNase I (0.02, 0.04, and 0.06 µL/​​ng of RNA), which were chosen based on the results of the RNA sample quantifications and as indicated by the manufacturer, to digest genomic DNA present in the RNA samples extracted from sugarcane leaves with the Concert™ Plant RNA Reagent. The results showed that all three concentrations of DNase significantly reduced DNA concentrations. However, RNA was also degraded on DNase I treatment. In addition, the amount of DNA present in the RNA samples after purification with DNase I was sufficient for its amplification in the tests conducted with conventional PCR. Furthermore, the condition of RNA samples obtained after the treatments allowed for real-time PCR. Therefore, we concluded that 0.02 µL DNase I was the ideal concentration for sugarcane RNA purification, as higher concentrations do not increase the efficiency of the genomic DNA digestion in RNA samples and only make the purification process more expensive. This study provides important information on the effect of high concentrations of DNase I and complements previous studies that have so far tested only the DNase concentration recommended by the manufacturer.

Investigating genetic diversity in sapucaia using inter simple sequence repeat markers.

Sapucaia is a tree species originating from the Brazilian Amazon and is widely distributed in Brazil, especially in the mid-north region (Piauí and Maranhão states). Its seeds are rich in calories and proteins, and possess great potential for commercialization. Little is known about the genetic variability in the germplasm of most Lecythis species. Here, 11 inter-simple sequence repeat primers were used to estimate the genetic variability among 17 accessions, and to determine the levels of genetic variation and the standards of population structure in sapucaia. The accessions were obtained from the active germplasm bank (AGB) of Embrapa Meio-Norte, Teresina, PI, Brazil, and corresponded to four occurrence areas. Ninety-six loci were analyzed among the studied individuals. High variation was found at the species level, where the percentage of polymorphic bands was 94.79%, Nei's genetic diversity (h) was 0.3110, and Shannon's index (I) was 0.4732. In the analyzed populations, the percentage polymorphism ranged from 20.83 to 94.79%, Nei's genetic diversity ranged from 0.0863 to 0.2969, and Shannon's index ranged from 0.1260 to 0.4457. Significant genetic differentiation was detected among the populations (ΦST = 10.66%); however, the greatest genetic differentiation was found within the populations (89.34%), between which there was an intermediate level of gene flow (Nm = 1.10). Accessions BGS 2 and BGS 4 were the most divergent, whereas accessions BGS 14 and BGS 15 were the most similar. Therefore, sapucaia analyzed from the AGB present an elevated level of genetic diversity and may have potential use in genetic breeding programs.

Molecular characterization of accessions of Cratylia argentea (Camaratuba) using ISSR markers.

Cratylia argentea (Desv.) Kuntze (Fabaceae) is a drought-tolerant, perennial legume found primarily in Brazil, Bolivia, and Peru. The shrub is well adapted to acid soils and exhibits high productivity and nutritional value, characteristics that would favor its use as a dry season animal forage supplement in semiarid regions. In plant improvement programs, the production of elite hybrids with superior traits is generally achieved by crossing parents that exhibit the highest level of genetic divergence. Therefore, the aim of the present study was to assess genetic diversity among 13 accessions of C. argentea from the same population maintained in the active germplasm bank of Embrapa Meio-Norte using inter-simple sequence repeat (ISSR) markers. Genetic similarities between C. argentea accessions were estimated from Jaccard coefficients, and a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). The set of 15 primers selected for ISSR analysis generated a total of 313 loci of which 79.23% were polymorphic. The mean number of bands per primer was 20.87, and the amplicons ranged from 280 to 3000 bp in size. Primers UBC834 and UBC827 generated the largest number of polymorphic loci and exhibited 90.91 and 100% polymorphism, respectively. The coefficients of genetic similarity among accessions varied between 0.49 and 0.73. UPGMA cluster analysis allowed the identification of four genotypic groups and demonstrated the existence of considerable variability within the collection. Potential progenitors were selected that would offer good possibilities of obtaining unusual and favorable combinations of genes in a plant breeding program.

Genetic variation detected by RAPD markers in natural populations of babassu palm (Attalea speciosa Mart.).

The purpose of this study was to analyze the effects of management on the genetic structure of natural populations of Attalea speciosa in the State of Piauí, Brazil, using random-amplified polymorphic DNA (RAPD) markers. Three babassu populations under different management systems were selected. Polymerase chain reactions were performed for 20 RAPD primers. A total of 146 bands were generated, 141 of which were polymorphic (96.58%), with a variation of 4 and 12 loci and an average of 7 bands per primer. A dendrogram revealed a clear separation between the three populations (0.57). Data reliability and node consistency were verified by bootstrap values and the cophenetic correlation coefficient (88.15%). Coefficients of similarity between pairs of genotypes ranged from 0.26 to 0.86, with a mean of 0.57. Nei's genetic diversity index (HE) value of the population sampled in Teresina was 0.212, of Esperantina it was 0.195, and of José de Freitas it was 0.207. After the HE was decomposed, the complete diversity was found to be 0.3213. Genetic differentiation between populations was 0.362, and the estimation of gene flow between populations was low (0.879). Analysis of molecular variance revealed that 59.52% of the variation was contained within populations, and 40.48% was between populations. RAPD markers were effective for genetic diversity analysis within and between natural babassu populations, and exhibited a high level of polymorphism. Genetic diversity was the highest within populations; variability was lower in the managed populations than in the undisturbed populations.

Comparison of eight methods of genomic DNA extraction from babassu.

Babassu (Orbignya phalerata Martius) is one of the most important palms in Brazil because of the largest morphological variation, wide geographic distribution, and high socio-economic importance. The diversity present in babassu germplasm should be protected against loss to ensure their use with high productivity. Study of the available variability in populations of babassu is necessary to develop conservation strategies. The study of genetic variability can be conducted using molecular markers and many of these studies require significant quantity of high-quality DNA. The present study aimed to effect comparison among eight DNA extraction methods in case of O. phalerata. The quality and concentration of nucleic acids were analyzed by spectrophotometry and integrity of DNA was ascertained by agarose gel electrophoresis. The spectrophotometry revealed that some methods resulted in high levels of concentration of nucleic acids, in which values of the ratio A260/280 and A260/230 were outside the range of purity. The agarose gel electrophoresis established the concentration and integrity of DNA. The methods of Murray and Thompson (1980) and Ferreira and Grattapaglia (1998) did not result in satisfactory quantities of DNA. Conversely, the method proposed by Khanuja et al. (1999) resulted in DNA of adequate quality and quantity that could be satisfactorily used for amplification reactions performed with two ISSR primers.

Evaluation of genetic similarity between accessions of Pityrocarpa moniliformis (angico-de-bezerro) using RAPD markers.

Pityrocarpa moniliformis (Benth.) Luckow and Jobson, commonly known as angico-de-bezerro, is a forage legume that occurs naturally in the Caatinga of northeastern Brazil. This fast growing, vigorous, melliferous tree is well adapted to arid terrains and its branches and leaves possess high nutritional value. However, the scarcity of information regarding genetic variability within the species limits its possible exploitation as an animal forage. The aim of the study was to evaluate the genetic similarities of ten accessions of P. moniliformis available in the active germplasm collection of Embrapa Meio-Norte, using the RAPD markers to select those most suitable for cultivation and/or plant breeding. Polymerase chain reaction using ten selected RAPD primers generated 110 amplified loci, 106 (96.4%) of which were polymorphic. Primers A10 and M06 produced the largest number of polymorphic loci (18 and 13 bands, respectively), while primers B18 and K15 generated the smallest number (7 bands each). The dendrogram, constructed using the Jaccard coefficients and considering a cut-off point of 0.41 allowed the separation of the ten accessions into four genotypic groups. The highest genetic similarity coefficient (0.56) was observed between group I accessions BGFAB6 and BGFAB9 and BGFAB 7 and BGFAB 8, while the lowest coefficient (0.11) was observed between accessions BGFAB3 (group IV) and BGFAB10 (group III). The results revealed that genetic variability is present in the accessions of P. moniliformis.

RAPD analysis of the genetic diversity among accessions of Fabaceous forages (Poincianella spp) from the Caatinga.

Among members of the Fabaceae family, native to the Brazilian Caatinga, the species Poincianella pyramidalis and P. bracteosa exhibit particular potential as forage for cattle, sheep and goats. With the aim of establishing genetic relationships within Poincianella, random amplified polymorphic DNA analysis was performed on eight accessions of P. pyramidalis and two accessions of P. bracteosa, originating from the semiarid zone of the state of Piauí, northeastern Brazil, and present in the germplasm bank of Embrapa Meio Norte (Teresina, Piauí, Brazil). Amplification reactions using 11 selected arbitrary sequence primers generated 167 fragments with an overall polymorphism of 70.38%. Five monomorphic loci were generated exclusively in P. pyramidalis accessions, while three unique monomorphic loci were associated with P. bracteosa, and these represented potential species-specific markers. The similarity coefficients between Poincianella accessions were low (mean value 0.59) but with a wide variation (range 0.443 to 0.748). The similarity matrix and the dendrogram constructed using the unweighted pair group method allowed the separation of Poincianella accessions into two major clusters represented by the two distinct species, while the accessions of P. pyramidalis could be separated further into three subgroups. The high level of genetic diversity detected in the genus Poincianella could be used in future breeding programs to produce enhanced cultivars, although the variability could be better exploited if more specimens were collected from other locations within the semiarid region of northeastern Brazil.

Comparison of methods to isolate DNA from Caesalpinia ferrea.

Molecular markers are important for characterizing the genetic diversity of plants and can provide the basis for strategies to protect and conserve endangered populations. However, numerous molecular techniques are used, requiring an evaluation of fast and efficient methods to extract DNA. Since molecular studies of Caesalpinia ferrea are rare, it is important to develop and/or adapt a DNA extraction protocol that produces quality DNA samples to enable the design of strategies for the conservation of this threatened species. This study aimed to compare five methods for DNA extraction and to determine the most efficient protocol for C. ferrea. Sufficient genomic DNA was obtained from the leaves of C. ferrea using all the tested protocols to perform techniques involving molecular markers. Two protocols based on the detergent cetyl trimethyl ammonium bromide, as well as a commercial kit, yielded high concentrations of pure DNA. However, when polymerase chain reaction amplifications were performed, DNA was only successfully amplified from extractions performed with the commercial kit, which produced sufficient genomic DNA of good quality from the leaves of C. ferrea to perform techniques involving molecular markers.

Determination of the genetic diversity among accessions of Senna spectabilis (canafístula) by using RAPD markers.

Senna spectabilis (DC.) H.S. Irwin & Barneby (Fabaceae; Caesalpinioideae), commonly known as "canafístula" or "cassia", is widely used in the semi-arid region of northeastern Brazil as a source of forage and timber. The plant presents a high nutritional content in comparison with other forage species that are native to the Brazilian Caatinga; thus, it represents a valuable resource during periods of drought. The aim of this study was to evaluate the genetic variability among eight accessions of S. spectabilis available in the forage germplasm collection of Embrapa Meio-Norte using the random-amplified polymorphic DNA technique. The 15 primers selected for use in the analysis produced 107 bands, including 59 (55.14%) that were polymorphic. A similarity matrix was generated on the basis of Jaccard coefficients, and a dendrogram was constructed using the unweighted pair group method with arithmetic mean clustering technique. The mean value of the similarity coefficients was 0.73, and the cophenetic correlation coefficient was 83.76%. Accessions CAN. 4 and CAN. 5 presented the greatest genetic similarity, while CAN. 6 and CAN. 8 were the most divergent. The S. spectabilis accessions were classified into two main groups with group I including accessions CAN. 1, CAN. 2, CAN. 4, CAN. 5, CAN. 7, CAN. 8, and CAN. 9, and group II comprising the single accession CAN. 6. The results presented herein revealed that, although the germplasm collection is presently limited, there is sufficient genetic variability among the accessions to permit future breeding programs.

Molecular characterization of Platonia insignis Mart. ("bacurizeiro") using inter simple sequence repeat (ISSR) markers.

Platonia insignis Mart. (Clusiaceae) is widespread throughout the Amazon and adjacent areas. The fruits (known locally as "bacuri") have significant commercial potential, but the species is under threat from agro-industrial expansion. The genetic variability within 72 genotypes of P. insignis belonging to ten populations collected in the Brazilian states of Maranhão and Piauí, and maintained in the germplasm collection of Embrapa Meio-Norte, has been determined, and the organization of genetic diversity within populations, between populations and among geographic groups verified. Eighteen selected inter simple sequence repeat primers allowed amplification of 236 loci of which 221 (93.64%) were polymorphic, indicating a high level of genetic diversity. At the population level, the Shannon and Nei diversity indices ranged from 0.082 to 0.323 and from 0.120 to 0.480, respectively. The global coefficient of genetic differentiation (G(ST)) was 0.4730 indicating that differentiation between populations was significant, a finding that was confirmed by analysis of molecular variance (Φ(ST) = 0.28). UPGMA cluster analysis revealed that the genotypes could be stratified into groups that were well defined and consistent with those identified in the dendrogram constructed using pair wise Φ(ST) values. The high genetic diversity established in this study may facilitate the management and conservation of the germplasm of P. insignis.

RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil.

We evaluated genetic variability of mango (Mangifera indica) accessions maintained in the Active Germplasm Bank of Embrapa Meio-Norte in Teresina, Piauí, Brazil, using RAPDs. Among these accessions, 35 originated from plantings in Brazil, six from the USA and one from India. Genomic DNA, extracted from leaf material using a commercial purification kit, was subjected to PCR with the primers A01, A09, G03, G10, N05, and M16. Fifty-five polymorphic loci were identified, with mean of 9.16 ± 3.31 bands per primer and 100% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis demonstrated five genotypic groups among the accessions examined. The genotypes Rosa 41, Rosa 48 and Rosa 49 were highly similar (94% similarity), whereas genotypes Sensation and Rosa 18 were the most divergent (only 7% similarity). The mango accessions were found to have considerable genetic variability, demonstrating the importance of analyzing each genotype in a collection in order to efficiently maintain the germplasm collection.

Molecular characterization of cajá, Spondias mombin (Anacardiaceae), by RAPD markers.

The arboreal species Spondias mombin L. (Anacardiaceae) is widely distributed in Brazil, where the fruits, known by the common name of cajá, are an important commercial commodity. We evaluated genetic variability among 32 cajá accessions of the Germplasm Collection of Embrapa Meio-Norte using RAPD technique. Reaction conditions for efficient RAPD amplifications were optimized in preliminary tests, and primers were selected from a set designed by the University of British Columbia on the basis of high levels of polymorphism and adequate band resolution. The 21 primers employed in the final analysis produced 145 fragments, 79% of which were polymorphic. Based on the RAPD data, a dendrogram was constructed using the unweighted pair group method with arithmetic mean clustering technique. The 32 cajá accessions were classified into three main groups with a mean genetic similarity of 68.8%. Group I comprised 26 accessions (74.1% similarity), and group II included five accessions (74.0% similarity), while group III consisted of one accession (BGC 06), which exhibited the lowest similarity coefficients. Accessions BGC 06 and BGC 31 were the most unrelated and, hence, most suitable for initial crossings in order to obtain high levels of segregation. We concluded, based on the repeatability and reproducibility tests, that the RAPD technique is reliable and efficient for revealing the genetic diversity of cajá accessions, which will be useful for genetic improvement programs.