Edilson Paiva

Mapping QTLs for aluminum tolerance in maize

Aluminum toxicity is one of the major constraints for plant development in acid soils, limiting food production in many countries. Cultivars genetically adapted to acid soils may offer an environmental compatible solution, providing a sustainable agriculture system. The aim of this work was to identify genomic regions associated with Al tolerance in maize, and to quantify the genetic effects on the phenotypic variation. A population of 168F3:4 families derived from a cross between two contrasting maize inbred lines for Al tolerance was evaluated using the NSRL and RSRL parameters in nutrient solution containing toxic level of aluminum. Variance analyses indicated that the NSRL was the most reliable phenotypicindex to measure Al tolerance in the population, being used for further QTL mapping analysis. RFLP and SSR markers were selected for bulked segregant analysis, and additional SSR markers, flanking the polymorphisms of interest, were chosen in order to saturate the putative target regions. Seven linkage groups were constructed using 17 RFLP and 34 SSR markers. Five QTLs were mapped on chromosomes 2, 6 and 8, explaining 60% of the phenotypic variation. QTL4 and marker umc043 were located on chromosomes 8and 5, close to genes encoding for enzymes involved in the organic acids synthesis pathways, a widely proposed mechanism for Al tolerance in plants. QTL2 was mapped in the same region as Alm2,also associated with Al tolerance in maize. In addition, dominant and additive effects were important in the control of this trait in maize.

Hematoxylin staining as a phenotypic index for aluminum tolerance selection in tropical maize (Zea mays L.)

Abstract Hematoxylin staining is an early indicator of Aluminum (Al) toxicity effects on the apices of young, developing roots grown in nutrient solution. In this work, the potential of this technique as a reliable and reproducible phenotypic index for Al tolerance in tropical maize genotypes was assessed, with its performance systematically compared to two other parameters widely used in breeding programs – relative seminal-root length (RSRL) and net seminal-root length (NSRL). Seeding roots from contrasting genotypes for Al sensitivity stained remarkably different after 24- and 48-h and 7-day exposures to 222 μM Al in nutrient solution, with the Al-dye complex being detected in both the outer (epidermis) and inner (cortex) portions of the roots from the sensitive cultivar. Hematoxylin staining was compared to the RSRL and NSRL parameters using 20 families from the third generation of selfing (S3) following the cross between two contrasting inbred lines that had been previously classified by the RSRL index in an independent procedure. The coloration technique showed the highest capacity to discriminate among tolerant and sensitive genotypes and displayed significant correlation coefficients to the other two indexes. Evaluation of the results from diallel crosses involving nine inbred lines proved that hematoxylin staining was also particularly adequate for identifying expressive hybrid vigor, as demonstrated by the general (GCA) and specific (SCA) combining ability estimates obtained by using the three indexes simultaneously. Hence, hematoxylin staining of Al-stressed root apices appears to be a powerful tool to assist in Al-tolerance selection in tropical maize breeding programs.

Heterotic groups based on yield-specific combining ability data and phylogenetic relationship determined by RAPD markers for 28 tropical maize open pollinated varieties

Twenty eight maize open pollinated varieties (OPVs) were crossed in a diallel scheme and the 378 F1s were evaluated in 10 environments in Brazil. Based on yield-specific combining ability data (SCA), these varieties were classified in four heterotic groups. The consistence of the proposed heterotic groups was confirmed comparing intra- and inter-group F1 values and midparent heterosis. Superior OPVs combinations for use as a source of inbreds in hybrid breeding programs were determined. RAPD markers were used to genotype these varieties. A UPGMA dendogram, based on marker data from 50 primers and 178 polymorphic bands, was obtained. Phylogeny obtained with RAPD markers agreed with known pedigree data. Dent germplasm tended to group separately from flint germplasm. Multidimensional Scaling Analysis on marker data and morphological data showed a higher degree of genetic divergence among the dent germplasm than among the flint germplasm used in this study. Correlation between RAPD marker estimated genetic distance and SCA for yield was low and positive (r = 0.16**).

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene.

Aim:  To avoid the limitations of 16S rRNA‐based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta‐subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies.

Genetic diversity of Paenibacillus polymyxa populations isolated from the rhizosphere of four cultivars of maize (Zea mays) planted in Cerrado soil

Abstract A tropical Brazilian soil (Cerrado) was planted with four cultivars of maize (CMS04, CMS11, CMS22 and CMS36) and the genetic diversity of the Paenibacillus polymyxa populations present in their rhizospheres was determined after 90 days of sowing. For that, a total of 67 isolates were identified as P. polymyxa by classical biochemical tests and were analyzed for DNA polymorphism with the randomly amplified polymorphic DNA (RAPD) and amplification of repetitive DNA sequences (rep) methods. The amplification patterns obtained using three arbitrary primers and the primer BOXA1R were used separately to construct dendrograms based on the unweighted pair groups method with arithmetic means (UPGMA). Fifty-four genotypic groups were formed when data from different PCR amplifications were combined, showing a high level of genetic polymorphism among P. polymyxa strains. A dendrogram based also on combined PCR data, followed by cluster analysis with minimum-variance criteria (Ward) and Euclidean distance, showed that P. polymyxa strains could be divided into two main clusters. One cluster was formed predominantly by strains from maize cultivars CMS04 and CMS36, while the other cluster was formed predominantly by strains of maize cultivars CMS11 and CMS22. Multivariate analysis of variance (MANOVA) allowed the correlation between the genetic structure of P. polymyxa populations and the different cultivars of maize to be studied. The results showed that the strains isolated from the rhizospheres of the different maize cultivars were significantly different.

Combined analysis of supernatant-based feeding bioassays and PCR as a first-tier screening strategy for Vip-derived activities in Bacillus thuringiensis strains effective against tropical fall armyworm

Aims: To assess whether feeding bioassays using culture‐supernatant proteins could be combined with PCR into a first‐tier screening strategy for Vip3A‐like genes efficient against tropical Spodoptera frugiperda. 
Methods and Results: Out of 12 Bacillus thuringiensis strains studied, the total protein concentrated from the culture supernatant of only the strain HD125 yielded a significantly increased armyworm mortality and an intense band of the predicted size for VIP3A protein in SDS‐PAGE. However, PCR and sequencing data indicated Vip‐like genes are ubiquitous in tropical B. thuringiensis isolates. Interestingly, the HD125 strain was also the only one displaying a single‐band amplification pattern and the highest sequence identity to the reported Vip3A(a) gene. 
Conclusions: Results suggest the insecticidal effectiveness of putative VIPs in B. thuringiensis isolates can be preliminarily estimated by the use of supernatant‐derived total protein in feeding experiments, though only in a limited manner. 
Significance and Impact of the Study: A simple and cost‐effective first‐tier screening strategy for VIP‐derived activities in B. thuringiensis collections can be developed by combining PCR and feeding bioassays. Moreover, the employed primers showed to be useful as a tool for strains differentiation at DNA level, and for characterization and isolation of Vip‐like genes in tropical B. thuringiensis germplasm.

Androgenetic haploids and SSR markers as tools for the development of tropical maize hybrids

The traditional process of obtaining maize hybrids involves the generation of inbred lines through successive generations of selfing and subsequent testcrosses in order to identify the best combining ability by allelic complementation. A fast alternative to obtain inbred lines is to induce the formation of haploids followed by chromosome doubling. However, even with the aid of haploid-inducing genetic sources, this strategy has not been widely used in maize breeding programs, partly due to difficulties inherent to haploid generation and identification. In order to evaluate the possibility of using dihaploids to generate homozygous maize tropical lines, we used the androgenetic haploid inducer line W23 as a female parent in crosses with the tropical single-cross hybrid BRS1010. Within the progeny of these crosses, 462 seeds were phenotypically selected as putative haploids by the purple-colored endosperm and colorless embryo conditioned by the R1-nj gene. Among these, only four individuals were confirmed as being haploids using SSR markers, chromosome counting and flow cytometry, showing that the phenotypic marker was not efficient in detecting haploids in the tropical maize genotype used. All four haploids as well as some diploid plants presented reduced size, corroborating the difficulties for haploid identification by phenotypic evaluation. Genetic diversity analysis revealed by SSR markers divided the haploids in two groups represented by flint and dent maize inbred lines, which could be helpful in identifying complementary dihaploid lines. The present article demonstrates that a combination of haploid production and SSR fingerprinting is a feasible strategy for maize hybrid development in tropical germplasm.

Genetic variation among pathogens causing "Helminthosporium" diseases of rice, maize and wheat

Vinte amostras de especies de fungos, agentes da helmintosporiose em cereais, foram obtidas de diferentes regioes geograficas, sendo nove constituidas de Bipolarisoryzae, isoladas de cultura do arroz (Oryza sativa), sete de B.sorokiniana coletadas de trigo (Triticum aestivum), duas de B.maydis e duas de Exserohilumturcicum provenientes de milho (Zea mays). As amostras foram analisadas atraves das tecnicas de PCR-RFLP e RAPD. O polimorfismo de tamanho observado entre as amostras na regiao ITS1-ITS2 e o espaco compreendido da regiao 5,8S do rDNA indicou diferencas geneticas entre as amostras, enquanto o fenograma construido atraves do metodo de UPGMA apos a digestao com as enzimas de restricao, indicaram polimorfismo inter e intraespecifico. Os perfis de RAPD indicaram um expressivo grau de polimorfismo entre as diferentes especies. Entre as amostras da mesma especie ocorreu um baixo indice de polimorfismo. O fenograma, obtido pelo metodo de UPGMA, permitiu diferenciar as quatro especies analisadas e agrupou as mesmas conforme a especie hospedeira. Os perfis de RAPD obtidos revelaram ausencia de correlacao entre os fatores climaticos e a origem geografica dos isolados de B. sorokiniana e B. oryzae. Especies teleomorficas revelaram alto nivel de similaridade com seus correspondentes anamorfos.

Prospecting sugarcane genes involved in aluminum tolerance

Aluminum is one of the major factors that affect plant development in acid soils, causing a substantial reduction in yield in many crops. In South America, about 66% of the land surface is made up of acid soils where high aluminum saturation is one of the main limiting factors for agriculture. The biochemical and molecular basis of aluminum tolerance in plants is far from being completely understood despite a growing number of studies, and in the specific case of sugarcane there are virtually no reports on the effects of gene regulation on aluminum stress. The objective of the work presented in this paper was to prospect the sugarcane expressed sequence tag (SUCEST) data bank for sugarcane genes related to several biochemical pathways known to be involved in the responses to aluminum toxicity in other plant species and yeast. Sugarcane genes similar to most of these genes were found, including those coding for enzymes that alleviate oxidative stress or combat infection by pathogens and those which code for proteins responsible for the release of organic acids and signal transducers. The role of these genes in aluminum tolerance mechanisms is reviewed. Due to the high level of genomic conservation in related grasses such as maize, barley, sorghum and sugarcane, these genes may be valuable tools which will help us to better understand and to manipulate aluminum tolerance in these species.

Effect of Baculovirus spodoptera isolates in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae and their characterization by RAPD

Foram utilizados 22 isolados de virus amostrados em diferentes regioes produtoras de milho do Brasil. Os virus foram purificados e suas suspensoes fornecidas a lagartas sadias do 3o e 4o instar de Spodoptera frugiperda (J.E. Smith). A mortalidade foi avaliada diariamente, e as lagartas infectadas foram congeladas logo apos sua morte, o que em geral ocorreu do 5o ao 7o dia apos ingestao do virus. Os isolados foram usados em seis concentracoes (103 a 108 poliedros/ml) e uma testemunha (agua). Os percentuais de mortalidade, duracao do periodo larval e periodo pupal, peso de pupa e a concentracao letal (CL50) foram determinados para todos os isolados. Foram observadas diferencas significativas entre todos os isolados e concentracoes testadas para todos os parâmetros avaliados, e tambem foi constatada a presenca da interacao isolado x concentracao, exceto para periodo pupal. Os padroes de amplificacao de 54 marcadores RAPD, sendo 41 polimorficos, foram utilizados para avaliar a distância genetica e a sua correlacao com os indices de mortalidade das lagartas. A divergencia genetica calculada pelo coeficiente Jaccard utilizando os dados moleculares permitiu dividir os isolados em dois grupos, com um elevada confiabilidade. O agrupamento nao apresentou associacao com a taxa de mortalidade causada pelos isolados ou com sua distribuicao geografica. No entanto, um fragmento de RAPD OPW04.2280 apresentou-se altamente associado com a mortalidade das lagartas e com a CL50, explicando 23% e 65% da variacao fenotipica para essas caracteristicas entre os isolados virais, respectivamente.