Silvana Regina Rockenbach Marin

Genotypic diversity among brazilian isolates of Macrophomina phaseolina revealed by RAPD

Macrophomina phaseolina has been considered one of the most prevalent soybean (Glycine max) pathogens in Brazil. No genetic resistance has been determined in soybean and very little is known about the genetic diversity of this pathogen in tropical and sub-tropical regions. Fifty-five isolates from soybean roots were collected in different regions and analyzed through RAPD for genetic diversity. The UPGMA cluster analysis for 74 loci scored permitted identification of three divergent groups with an average similarity of 99%, 92% and 88%, respectively. The three groups corresponded to 5.45%, 59.95% and 34.6%, respectively of all isolates used. A single plant had three different haplotypes, while 10.9% of the analyzed plants had two different haplotypes. In another study the genetic similarity was evaluated among isolates from different hosts [soybean, sorghum (Sorghum bicolor), sunflower (Helianthus annuus), cowpea (Vigna unguiculata), corn (Zea mays) and wheat (Triticum aestivum)] as well as two soil samples from native areas. Results showed that more divergent isolates originated from areas with a single crop. Isolates from areas with crop rotation were less divergent, showing high similarity values and consequently formed the largest group. Amplification of the ITS region using primers ITS1 and ITS4 produced only one DNA fragment of 620 bp. None of the isolates were differentiated through PCR-RFLP. Our results demonstrated genetic variability among Brazilian isolates of M. phaseolina and showed that one single root can harbor more than one haplotype. Moreover, cultivation with crop rotation tends to induce less specialization of the pathogen isolates. Knowledge of this variation may be useful in screening soybean genotypes for resistance to charcoal rot.

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Abstract Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC3F2:3 and F2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11795, OPAE-08837, OPR-07548 and OPY-072030) were identified in the BC3F2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean.

Detection and partial characterization of a carlavirus causing stem necrosis of soybean in Brazil

No ano agricola de 2000/2001, ocorreu um surto de nanismo e necrose da haste em soja (Glycine max) plantada em duas areas do Brasil Central e na safra seguinte, esta anomalia foi constatada em outras regioes produtoras, mesmo distantes mais de 2.000 km de onde fora inicialmente constatada. Estudos envolvendo ensaios de transmissao (enxertia, mecânica e insetos vetores), microscopia eletronica, purificacao, sorologia e ensaios moleculares indicaram que a enfermidade foi causada por um carlavirus transmitido por mosca branca, possivelmente relacionado ao Cowpea mild mottle virus (CpMMV).

Implications of ethylene biosynthesis and signaling in soybean drought stress tolerance

BackgroundEthylene is a phytohormone known for inducing a triple response in seedlings, leaf abscission and other responses to various stresses. Several studies in model plants have evaluated the importance of this hormone in crosstalk signaling with different metabolic pathways, in addition to responses to biotic stresses. However, the mechanism of action in plants of agricultural interest, such as soybean, and its participation in abiotic stresses remain unclear.ResultsThe studies presented in this work allowed for the identification of 176 soybean genes described elsewhere for ethylene biosynthesis (108 genes) and signal transduction (68 genes). A model to predict these routes in soybean was proposed, and it had great representability compared to those described for Arabidopsis thaliana and Oryza sativa. Furthermore, analysis of putative gene promoters from soybean gene orthologs permitted the identification of 29 families of cis-acting elements. These elements are essential for ethylene-mediated regulation and its possible crosstalk with other signaling pathways mediated by other plant hormones.From genes that are differentially expressed in the transcriptome database, we analyzed the relative expression of some selected genes in resistant and tolerant soybean plants subjected to water deficit. The differential expression of a set of five soybean ethylene-related genes (MAT, ACS, ACO, ETR and CTR) was validated with RT-qPCR experiments, which confirmed variations in the expression of these soybean target genes, as identified in the transcriptome database. In particular, two families of ethylene biosynthesis genes (ACS and ACO) were upregulated under these experimental conditions, whereas CTR (involved in ethylene signal transduction) was downregulated. In the same samples, high levels of ethylene production were detected and were directly correlated with the free fraction levels of ethylene’s precursor. Thus, the combination of these data indicated the involvement of ethylene biosynthesis and signaling in soybean responses to water stress.ConclusionsThe in silico analysis, combined with the quantification of ethylene production (and its precursor) and RT-qPCR experiments, allowed for a better understanding of the importance of ethylene at a molecular level in this crop as well as its role in the response to abiotic stresses. In summary, all of the data presented here suggested that soybean responses to water stress could be regulated by a crosstalk network among different signaling pathways, which might involve various phytohormones, such as auxins, ABA and jasmonic acid. The integration of in silico and physiological data could also contribute to the application of biotechnological strategies to the development of improved cultivars with regard to different stresses, such as the isolation of stress-specific plant promoters.

Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii

Cercospora kikuchii esta envolvido na desfolha da soja (Glycine max), normalmente em associacao com Septoria glycines, no final do ciclo da cultura. Setenta e dois isolados, obtidos principalmente de sementes com mancha purpura e oriundas de diferentes regioes do Brasil, mostraram variabilidade fenotipica. O teor de cercosporina e a velocidade de crescimento de colonias foram bastante variaveis entre os isolados. Uma forte correlacao foi identificada entre o teor de cercosporina e virulencia. Diferencas geneticas, entre e dentro da populacao analisada, foram observadas por RAPD com a analise de 86 loci. As analises de RAPD permitiram agrupar os isolados em sete grupos. Nenhuma relacao foi identificada entre os grupos de RAPD e a origem geografica ou teor de cercosporina. A sequencia da regiao espacadora do DNA ribossomal (ITS1-5,8S-ITS2) foi determinada em 13 isolados escolhidos nos diferentes agrupamentos. A similaridade das sequencias obtidas comparadas as sequencias de C. kikuchii depositadas no GenBank (AY266160, AY266262, AY152577 e AF291708) variou de 97 a 100%. Este trabalho demonstrou que os isolados brasileiros de C. kikuchii de diferentes origens sao variaveis quanto a virulencia, aos padroes de RAPD e ao teor de cercosporina. O teor de cercosporina pode ser um bom parâmetro na escolha de um isolado adequado para selecao de cultivares tolerantes ou resistentes a esse patogeno. Considerando que ele e facilmente transmitido por sementes nao e surpresa encontrar os mesmos haplotipos em diferentes regioes. A migracao poderia ser favorecida por sementes infetadas como demonstrado na analise de RAPD.

Quantitative differential expression of alpha and beta ryanodine receptor genes in PSE (Pale, Soft, Exudative) meat from two chicken lines: broiler and layer

ABSTRACT Total RNA isolated from Pectoralis major muscle from PSE (L* 24h >53.0, pH 0.05) in α-ryr gene expression regardless of line studied. The β-ryr RQ results suggested that in PSE samples an alteration might occur in the regular ratio (1:1) of α-RyR/β-RyR normally found in avian muscles. These results provided the first evidence of PSE meat occurrence as a result of the differential expression of ryanodine receptor genes which might lead to an increased in Ca 2+ availability at the cell milieu. Key words: malignant hyperthermia, poultry, gene expression, real time PCR * Author for correspondence: mshimo@uel.br

Introduction of the rd29A: AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration

The loss of soybean yield to Brazilian producers because of a water deficit in the 2011–2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2–17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress.

Characterization of powdery mildews strains from soybean, bean, sunflower, and weeds in Brazil using rDNA-ITS sequences.

Soybean powdery mildew (Erysiphe diffusa) was considered a minor disease in Brazil in the decades immediately after its identification. However, since the outbreak in 1996/97 in all cultivated areas the disease has become a constant threat to farmers and losses of up to 25% have been reported. The report of a new species, E. glycines, infecting soybean in Japan, and the occurrence of the disease in other plant species (Phaseolus vulgaris, Helianthus annuus,Sonchus oleraceus,Hypochaeris brasiliensis, and Bidens pilosa) commonly found growing nearby soybean fields, raised questions in relation to the taxonomy of the powdery mildew strains found in or around soybean fields in Brazil. Analysis of the internal transcribed sequence (ITS) of the rDNA was undertaken to ascertain the pathogen species associated to each of the hosts. Powdery mildew strains isolated from Glycine max were identified as E. diffusa. Strains from P. vulgaris were very similar to E. diffusa, with 4 nt differences, and differed from Erysiphe poligony by 11 nt. Strains from H. annuus and S. oleraceus grouped with the species Golovinomyces cichoracearum, while strains from H. brasiliensis and B. pilosa were similar to Podosphaera fusca and Neoerysiphe cumminsiana, respectively. To our knowledge this is the first molecular identification of powdery mildew in Brazil based on rDNA sequence comparison. In addition, this study presented evidence for the occurrence of N. cumminsiana in America.

Effect of crop rotation on specialization and genetic diversity of Macrophomina phaseolina

Charcoal rot, caused by Macrophomina phaseolina, is one of the most important diseases of soybean. Genetic variability among soybean isolates has been observed but the effect of host specialization on genetic variability has not been reported. In this work, isolates from soybean, corn and sunflower were evaluated based on cross inoculations and number of microsclerotia/g of roots. The highest and lowest significant (P<0.005) production of microsclerotia was obtained in soybean (863 microsclerotia/g of roots) and sunflower (578 microsclerotia/g of roots), respectively, regardless of the origin of the isolate. Additionally, the effect of a 20-year crop rotation on genetic variability based on RAPD was studied. Eighty-nine isolates from five populations were obtained from soil samples under four crop rotation systems and an uncropped soil. Seven clusters were obtained considering a similarity level of 85%. Analysis indicated that M. phaseolina is a highly diverse species and also revealed a strong effect of the rotation system on genetic diversity. AMOVA was conducted for the RAPD data. From the total genetic variability, 21% (P<0.0001) could be explained by the differences between populations while 79% could be explained by differences within populations ( Φst = 0.2110; P<0.0001). The mean coefficient of gene differentiation (GST) estimated among the five populations indicated 27% of differentiation between populations similar to the AMOVA results where Φst= 0.2110. Total gene diversity estimated indicated high levels of variability (HT =0.3484). Results suggest that genetic differentiation of M. phaseolina can be altered by crop rotation.

Characterization of Molecular and Physiological Responses Under Water Deficit of Genetically Modified Soybean Plants Overexpressing the AtAREB1 Transcription Factor

Drought is one of the major factors limiting crop productivity worldwide. Currently, the techniques of genetic engineering are powerful tools for the development of drought-tolerant plants, once they allow for the modification of expression patterns of genes responsive to drought. Within this context, transcription factors recognize specific DNA sequences in the regulatory region of target genes, and thereby regulate their expression. AREB is a transcription factor in the basic leucine zipper family, which binds to the ABRE element in the promoter region of genes induced by abscisic acid and drought. In this study, soybean plants transformed with the 35S:AtAREB1 construct were submitted to drought under greenhouse conditions. AtAREB1 expression was observed in the transgenic lines 1Ea2939 and 1Eb2889, but not in the event 1Ea15 and, under control of the CaMV 35S promoter, did not cause dwarfism and resulted in a higher survival rate of transformed plants after drought and rehydration. Moreover, 1Ea2939 and 1Eb2889 plants presented a greater total number of pods and seeds and increased dry matter content of seeds. The best performance of the transgenic lines 1Ea2939 and 1Eb2889 relative to BR 16 plants (wild type) and to event 1Ea15 might be related to mechanisms of drought prevention through reduced stomatal conductance and leaf transpiration under control conditions. Changes in the expression profile of phosphatases and kinases may also be involved. Such results suggest that the constitutive overexpression of the transcription factor AtAREB1 leads to an improved capacity of the soybean crop to cope with drought with no yield losses.