Adaptation of a silica membrane-based kit for purification of sequencing-ready BAC DNA

We describe a modification of a protocol for the isolation of BAC DNA using a silica membrane-based kit designed for the isolation of plasmid DNA. The major advantages of this protocol are the expediency of the procedure, the high yield and purity, and the high quality of the BAC DNA that is suitable for direct sequencing

Avaliação da resistência genética à ferrugem (Hemileia vastatrix Berk. et Br.) em cafeeiros F1 de RC oriundos do cruzamento Híbrido de Timor x Catuaí

Neste trabalho, objetivou-se estudar a resistência genética ferrugem em híbridos F1 e Rei provenientes de combinações entre o Híbrido de Timor (originado do cruzamento interespecifico de Coffea arabica com Coffea carzephora) e o cultivar Catual (G. arabica ). C} trabalho foi realizado nos meses de fevereiro e março de 1X98. Foram avaliados, quanto reaçao à ferrugem, os 1 t progenitores (6 Híbridas de Timor: UFV ã7'ó,2, UFV 427-15, tJFV 439-2, UFV X40-22, UFV 445-4b e CIFC 832-i; e 5 Catuai: UFV 2143-193, UFV 2I43-236, UFV 2 44-32, UFV X144-35 e UFV 2144-36), due serviram de testemunhas, os 18 híbridos F1 e os I 07 híbridos RCS provenientes de suas combinaçcães, totalizando I36 tratamentos. Para avaliação da resistência genetica das plantas, foi realizado o teste em discos de falhas (16 discos de cada amostra). Cada disco foi inoculada com aproximadamente 1 mg de uredosporos da raça II de I1. vastatrix Berk. et Br.. As reaçães foram avaliadas 49 dias após a inoculatAo. Foi adotada a seguinte escala: 1- resistente, sem qualquer sinal de infecçâo; 2- resistente, com reação de hipersensibilidade ou com clorose e ausência de esporos; e 3- susceptivel, corn presença pie pústulas uredospóricas. O método de avaliaçdes em discam de folhas em laboratório foi eficiente, com reduzida perda de discos (em média, 0,81 disci por gerbox). Das 10? plantas RCS avaliadas, 81 foram resistentes e apenas 26 apresentaram-se susceptiveis á doença Além das testemunhas Catuai, observou-se queo Híbrida de Timor UFV 427-15 também mostrou-se susceptível â ria estudada, sendo susceptiveis todas as seis plantas RCS provenientes cie seus cruzamentos. Os híbridos F1, provenientes de combinações entre Catuai e Híbrido de Timor, apresentardtn resistência raça Ti, com exceção da híbrido UFV 415-2. Dentre as plantas RC1 resistentes, tomando-se também dor base outras características observadas no campo, siarão selecionadas aquelas que seguïrdo no programa de melhoramento, nesta linha de pesquisaA backcross breeding was conducted to transfer leaf mst resistance genes from resistant hybrids to eomrnercíai cultivars of coffee trees. The genetic resistance of F; hybrid and BC; plants Born a crossing of Timor Hybrid (Cofee arabica x Cofea canephora) and Catuai (C. arabica) were studied. The experiment was conducted from February to March 1998, using leaf discs. After ieaf washing, 16 leaf dises &om web of 136 plants were placed (abaxial side up) in a gerbox. Race Ii of Hemileia vastarrâx was used becaese of its prevalence in the ãeld. Each disc was inoculated with approximately 1 mg of uredospores. Timor Hybríds (UFV 376-2, UFV 445-46, UFV 439-2, UFV 440- 22, CIFC 832-1, and UFV 427-15) and Cantai (UFV 2143-193, UFV 2143-236, UFV 2144-32, UFV 2144-35, and 2144-36) were used' as control. After 49 days, the disease symptoms were scored as the following 3 classes: l- resistant, without symptoms; 2- resistant, with incompatibility reaction or with formation of chlorotíe Spots without sperulation; and 3- susceptible, with large pustules with sporulation. All Catuaí controls were susceptible (class 3). With exception of UFV 427-15, all Timor Hybrid were resistant (class 1 or 2),: All F; hybrid cresses were resistant, with exception of UFV 415-2. Eighty-one out of 107 BC; plants were resistant (23 of class 1 and 58 of class 2). Twenty-six out of ' 10? were susceptíble (class 3), including all 6 EC; plants originated from UFV 427-15. Resistant BC; plants with superior field perfomance were selected for breeding purposes

Sociedade Brasileira de Melhoramento de Plantas Crop Breeding and Applied Biotechnology

ABSTRACT Genetic resistance in common bean (Phaseolus vulgaris L.) is the most effective control of anthracnose caused by Colletotrichum lindemuthianum (Sacc. et Magnus) Lams.-Scrib. In a previous report, two genetic symbols, Q and B, were assigned to describe the gene(s) controlling the resistance of the cultivar AB 136 to races 31 and 69 of the pathogen, respectively. In the present study, progeny tests with two identical sets of F 2:3 population were used to obtain the first experimental evidence whether one single gene controls the resistance to both races 31 and 69. The absence of recombinations between the putative genes (Q and B) allowed the conclusion that a single dominant gene controls the resistance of the cultivar AB 136 to both races 31 and 69. The symbol Co-6 was assigned to the gene. In addition, linkage analysis with RAPD marker indicated that Co-6 also controls the resistance of AB 136 to other races of the pathogen, or that different genes are present in the same linkage block

Genetic and Molecular Characterization of the I Locus of Phaseolus vulgaris

The I locus of the common bean, Phaseolus vulgaris, controls the development of four different phenotypes in response to inoculation with Bean common mosaic virus, Bean common mosaic necrosis virus, several other related potyviruses, and one comovirus. We have generated a high-resolution linkage map around this locus and have aligned it with a physical map constructed with BAC clones. These clones were obtained from a library of the cultivar “Sprite,” which carries the dominant allele at the I locus. We have identified a large cluster of TIR–NBS–LRR sequences associated within this locus, which extends over a distance >425 kb. Bean cultivars from the Andean or Mesoamerican gene pool that contain the dominant allele share the same haplotypes as revealed by gel blot hybridizations with a TIR probe. In contrast, beans with a recessive allele display simpler and variable haplotypes. A survey of wild accessions from Argentina to Mexico showed that this multigene family has expanded significantly during evolution and domestication. RNA gel blot analysis indicated that the TIR family of genes plays a role in the response to inoculations with BCMV or BCMNV

Comparative study of different molecular markers for classifying and establishing genetic relationships in Coffea canephora

The genetic variability characterization of the accessions of the germplasm collection, using molecular markers, is being applied as a complementary strategy to the traditional approaches to redefine the plant genetic resources. In this study, we compared the informativeness and efficiency of the molecular markers RAPD, AFLP and SSR in the analysis of 94 accessions of Coffea canephora germplasm held by the breeding program of the Brazilian Agricultural Research Corporation (Embrapa), Rondônia State, Brazil. For this, we considered the marker’s discriminatory power and level of polymorphism detected and also the genetic relationships and clustering (dendrogram) analysis. The RAPD marker yielded low-quality data and problems in the discrimination of some accessions, being less recommended for genetic studies of C. canephora. The SSRs had a higher level of information content and yielded high-quality data, while AFLP was the most efficient marker system because of the simultaneous detection of abundant polymorphism markers per few reactions. Our results indicate that AFLP and SSR, allies to the intrinsic characteristics of each technique, are the most suitable molecular markers for genetic studies of C. canephora. However, the choice of AFLP or SSR in the species characterization should be made in agreement with some characteristics that are discussed in this work

The effects of encoding data in diversity studies and the applicability of the weighting index approach for data analysis from different molecular markers.

The use of molecular markers to study genetic diversity represents a breakthrough in this area, because of the increase in polymorphism levels and phenotypic neutrality. Codominant markers, such as microsatellites (SSR), are sensitive enough to distinguish the heterozygotes in genetic studies. Despite this advantage, there are some studies that ignore this feature and work with encoded data because of the simplicity of the evaluation, existence of polyploids and need for the combined analysis of different types of molecular markers. Thus, our study aims to investigate the consequences of these encodings on simulated and real data. In addition, we suggest an alternative analysis for genetic evaluations using different molecular markers. For the simulated data, we proposed the following two scenarios: the first uses SNP markers, and the second SSR markers. For real data, we used the SSR genotyping data from Coffea canephora accessions maintained in the Embrapa Germplasm Collection. The genetic diversity was studied using cluster analysis, the dissimilarity index, and the Bayesian approach implemented in the STRUCTURE software. For the simulated data, we observed a loss of genetic information to the encoded data in both scenarios. The same result was observed in the coffee studies. This loss of information was discussed in the context of a plantbreeding program, and the consequences were weighted to germplasm evaluations and the selection of parents for hybridization. In the studies that involved different types of markers, an alternative to the combined analysis is discussed, where the informativeness, coverage and quality of markers are weighted in the genetic diversity studies