Paulo Sarmanho da Costa Lima

Application of a double-enrichment procedure for microsatellite isolation and the use of tailed primers for high throughput genotyping

The number of microsatellite loci and their allelic diversity contribute to increase accuracy and informativity of genetic estimates, however, the isolation of microsatellite loci is not only laborious but also quite expensive. We used (GATA)n and (GACA)n tetranucleotide probes and single- and double-enrichment hybridization to construct and screen a genomic library with an increased proportion of DNA fragments containing repeat motifs. Repeats were found using both types of hybridization but the double-enrichment procedure recovered sequences of which 100% contained (GATA)n and (GACA)n motifs. Microsatellite loci primers were then designed with an M13R-tail or CAG-tag to produce scorable PCR products with minimal stutter. The approach used in this study suggests that double-enrichment is a worthwhile strategy when isolating repeat motifs from eukaryotic genomes. Moreover, the use of tailed microsatellite primers provides increased resolution for compound microsatellite loci, with a significant decrease in costs.

VARIABILIDADE DE CARACTERíSTICAS FÍSICAS E QUÍMICAS DE FRUTOS DE GERMOPLASMA DE BACURI DA REGIÃO MEIO-NORTE DO BRASIL

ABSTRACT – The objective of this study was to evaluate physical and chemical characteristics of bacury fruits collected fromselected plants of occurrence in the Mid-North region. The following fruit characteristics were analysed: length e width; averageweight; average polp weight; length to width ratio; peel thickness; percentage of peel; percentage of polp; percentage of seed; numberof seeds/fruit; number of partenocarpic section/fruit; total soluble solids content; titratable acidity, and total soluble soli ds content totitratable acidity ratio. Fruits were collected from 26 selected plants mapped in nine locations in Piaui and Maranhao States. Physicaland chemical fruit characterizatics were evaluated at the Plant Physiology Laboratory of Embrapa Meio-Norte, in Teresina, PI, Brazil.Fruit samples used varied in size according to the availability of fruit production from each selected plant. The variance analysisindicated significant effects for collect local and selected plants for all characteristics analysed, except for the number of partenocarpicsection/fruit for which did not have local effect. Average fruit weight and average polp weight; average fruit weight and fruit width;average polp weight and fruit width; fruit length and peel thickness; fruit length and percentage of peel; peel thickness and p ercentageof peel; and average fruit weight and fruit length showed high values for phenotypic correlations (r

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.

Consumption Rates and Performance of Erinnyis ello L. on Four Cassava Varieties

O consumo foliar de mandioca (Manihot esculenta Crantz) e a caracterizacao da injuria potencial por Erinnyis ello L. foram determinados em quatro variedades de mandioca. Lagartas de primeiro instar foram criadas individualmente em folhas de quatro variedades de mandioca. Foi usado o delineamento experimental de blocos ao acaso com nove repeticoes. Folhas frescas com areas previamente determinadas foram fornecidas as larvas diariamente. O consumo foliar em cada instar e o peso das lagartas e pupas foram registrados. Nao houve diferenca significativa no consumo foliar nas quatro variedades (P=0,82). O total de area foliar consumida (cm2) para as variedades testadas foi: 1030,5 (Fio de Ouro), 971,5 (Aipim Bravo), 968,6 (Urubu), 956,1 (Jaburu), sendo de 981,7 o consumo medio das quatro variedades. Aproximadamente 94% do consumo ocorreu durante os dois ultimos instares. Variedades nao afetaram o desenvolvimento larval (P=0,96). Os pesos medios de lagartas (g) em cada variedade foram: 4,9 (Jaburu), 4,8 (Fio de Ouro), 4,7 (Urubu), 4,6 (Aipim Bravo) e 4,8 (media das quatro variedades). Dois modelos, um relacionando a area foliar consumida e desenvolvimento larval (y=abx )e outro do crescimento larval (y=5/1+ea+bx), foram estabelecidos. Os dados obtidos indicam que as variedades testadas neste estudo foram nutricionalmente semelhantes para o desenvolvimento de E. ello. Como o consumo foliar de E. ello foi semelhante para todas as variedades, a mesma taxa de consumo pode ser usada para o calculo dos niveis de dano economico para manejar E. ello nessas variedades.

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.

Does vegetational diversification reduce coffee leaf miner Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae) attack?

The effects of increasing plant diversity on the population of the coffee leaf-miner Leucoptera coffeella (Guérin-Mèneville) were investigated in two organic coffee production systems. One system consisted of coffee intercropped with banana trees (shaded system) and the other one of coffee intercropped with pigeon pea (unshaded system). The increase in plant diversity on both systems was achieved via introduction of green manures such a perennial pea nut, sunn hemp and Brazilian lucerne. The population of L. coffeella, predation and parasitism of L. coffeella mines were biweekly evaluated during eight months. The increase in plant diversity on both systems did not affect the attack of L. coffeella on coffee leaves and the mine parasitism rate. However, there was a positive and significant relationship between increasing plant diversity and coffee leaf mine predation by wasps on unshaded coffee system and a negative relationship on shaded coffee system.

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.

Transference of a crystal protein gene from bacillus thuringiensis and its expression in bradyrhizobium sp. cells

The plasmid pHT409 that harbours the cryIA(a) gene for the production of a δ-endotoxin (crystal protein) from Bacillus thuringiensis was transferred into Bradyrhizobium sp. A conjugal transfer system aiming to introduce the plasmid into the Bradyrhizobium sp. host from colonies of an Escherichia coli donor strain (DH5α::pHT409) has been developed. As a result exconjugants were obtained in which the transferred plasmid has been detected by both microbiological and electrophoresis techniques. The cryIA(a) gene when inside the new host had a low expression level which was detected by immunoblotting.