Genetic diversity of sweet sorghum germplasm in Mexico using AFLP and SSR markers

The objective of this work was to evaluate the diversity and genetic relationships between lines and varieties of the sweet sorghum (Sorghum bicolor) germplasm bank of the National Institute for Forestry, Agriculture and Livestock Research, Mexico, using AFLP and SSR markers. The molecular markers revealed robust amplification profiles and were able to differentiate the 41 genotypes of sweet sorghum evaluated. Analysis of the frequency and distribution of polymorphic fragments allowed for the detection of unique (AFLP) and rare (SSR) alleles in several genotypes (RBSS‑8, RBSS‑9, RBSS‑25, RBSS‑32, and RBSS‑37), indicating that these markers may be associated with a feature that has not yet been determined or may be useful for the identification of these genotypes. The genetic relationships indicated the presence of at least two types of sweet sorghum: a group of modern genotypes used for sugar and biofuel production, and another group consisting of historic and modern genotypes used for the production of syrups. Sweet sorghum genotypes may be used to develop new varieties with higher sugar and juice contents.

Identification and characterization of a resistance gene analog (RGA) from the Caricaceae Dumort family

The majority of cloned resistance (R) genes characterized so far contain a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain, where highly conserved motifs are found. Resistance genes analogs (RGAs) are genetic markers obtained by a PCR-based strategy using degenerated oligonucleotide primers drawn from these highly conserved "motifs". This strategy has the advantage of the high degree of structural and amino acid sequence conservation that is observed in R genes. The objective of the present study was to search for RGAs in Carica papaya L. and Vasconcellea cauliflora Jacq. A. DC. Out of three combinations of primers tested, only one resulted in amplification. The amplified product was cloned in pCR2.1TOPO and than sequenced using M13 forward and reverse primers. Forty-eight clones were sequenced from each species. The 96 sequences generated for each species were cleaned of vector sequences and clustered using CAP3 assembler. From the GENEBANK, one RGA was identified in C. papaya showing a BlastX e-value of 2x10-61 to the gb|AAP45165.1| putative disease resistant protein RGA3 (Solanum bulbocastanum). To the extent of our knowledge this is the first report of a RGA in the Caricaceae Dumort family. Preliminary structural studies were performed to further characterize this putative NBS-LRR type protein. Efforts to search for other RGAs in papaya should continue, mostly to provide basis for the development of transgenic papaya with resistance to diseases.

Cochlear implant: what the radiologist should know

Cochlear implant is the method of choice in the treatment of deep sensorineural hypoacusis, particularly in patients where conventional amplification devices do not imply noticeable clinical improvement. Imaging findings are crucial in the indication or contraindication for such surgical procedure. In the assessment of the temporal bone, radiologists should be familiar with relative or absolute contraindication factors, as well as with factors that might significantly complicate the implantation. Some criteria such as cochlear nerve aplasia, labyrinthine and/or cochlear aplasia are still considered as absolute contraindications, in spite of studies bringing such criteria into question. Cochlear dysplasias constitute relative contraindications, among them labyrinthitis ossificans is highlighted. Other alterations may be mentioned as complicating agents in the temporal bone assessment, namely, hypoplasia of the mastoid process, aberrant facial nerve, otomastoiditis, otosclerosis, dehiscent jugular bulb, enlarged endolymphatic duct and sac. The experienced radiologist assumes an important role in the evaluation of this condition.

Stabilization of fluoroindate glasses by magnesium fluoride and other heavy metal fluorides

Their extended transparency in the IR makes them attractive for use as optical fibers for CO laser power delivery and optical amplification. This paper firstly describes the spectacular stabilizing effect of MgF2 on the binary system InF3-BaF2. The investigation of the InF3-BaF2-MgF2 system led to samples up to 5mm in thickness. Further optimization of this system was achieved by incorporation of limited amounts of other fluorides and resulted in increased resistence to devitrification. The second approach of this work was concerned to the investigation of the pseudo-ternary system InF3-GdF3-GaF3 at constant concentrations of ZnF2-SrF2-BaF2-NaF. Several compositions were studied in this system. The samples presented a better thermal stability when compared to other families of fluoride glasses. Therefore, these glasses seem to be very promising for the fabrication of special optical fibers. Thermal data are reported.

Estratégias de pré-concentração em eletroforese capilar (CE): parte 1. Manipulação da velocidade eletroforética do analito

Capillary electrophoresis has become a well-established and routine-based separation technique. It is based on the differences between charged analyte mobility in aqueous or organic electrolytes. Its major limitation is the sensitivity due to small sample injection volumes and the narrow diameter of the capillaries, especially when UV detection is used. There are a number of ways to increase the concentration sensitivity. This report shows some on-line preconcentration strategies to perform it in free solution capillary electrophoresis that are based on manipulation of the analyte electrophoretic velocity during the sample introduction (stacking, field amplification and transient isotachophoresis).

Bioconcentração e biomagnificação de metilmercúrio na baía de Guanabara, Rio de Janeiro

Methylmercury was determined in water and aquatic biota from Guanabara Bay. Trophic transfer of methylmercury was observed between trophic levels from prey (microplankton, mesoplankton and fish with different feeding habits) to top predator (pelagic demersal fish). Top predator fish presented the highest methylmercury concentrations (320.3 ± 150.7 mg kg-1 dry wt.), whereas microplankton presented the lowest (8.9 ± 3.3 mg kg-1 dry wt.). The successive amplification of methylmercury concentrations and its bioconcentration factor with increasing trophic levels from base to top indicate that biomagnification may be occurring along the food web. Results suggest the importance of feeding habits and trophic level in the bioaccumulation of methylmercury by aquatic biota.

RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.

Molecular and pathogenic variability of Drechslera isolates from oats

Severe epidemics of leaf blotch and black leaf spot of oat (Avena sativa) caused by Drechslera avenae and Drechslera sp., respectively, are frequently observed in the State of Paraná, Brazil. Although some morphological differences between the isolates causing two different symptoms were noticed, the genetic relationship between them was not clear. Twenty-four isolates of D. avenae and Drechslera sp, collected between 1996-98, were assessed for the genetic variability by molecular and pathogenic analyses. The amplification products using primer pair ITS4/ITS5 showed a fragment length of approximately 600 bp for all the isolates except for one black spot isolate, where the fragment length was approximately 550 bp. Restriction enzymes Hinf I and Taq I, that cut in the ITS region, produced similar restriction patterns for all the isolates, whereas four others produced variable restriction patterns. RAPD analysis also showed distinctive patterns for some isolates. No clear difference between the black spot and the leaf blotch isolates was observed either by the molecular or by the pathogenicity analysis. Nonetheless, the rDNA analysis suggests that Drechslera probably comprises at least three distinct taxa. The results indicate that the difference observed between the isolates originating from two types of symptoms is due to intra-specific variants of D. avenae.

Identification of rapd marker linked to blast resistance gene in a somaclone of rice cultivar Araguaia

The gene Pi-ar confers resistance to Pyricularia grisea race IB-45 in a somaclone derived from immature panicles of the susceptible rice (Oryza sativa) cultivar Araguaia. RAPD technique was used to identify molecular markers linked to this gene utilizing bulked segregant analysis. Initially, the two parental DNAs from the resistant donor SC09 and 'Araguaia' were analyzed using random primers. Of the 240 primers tested, 203 produced amplification products. The two parental DNAs along with the resistant and susceptible bulks of F2 population were screened using 48 primers that differentiated resistant and susceptible parents. Even though eight primers differentiated the resistant bulk from the susceptible bulk, as well as somaclone SC09 and 'Araguaia', only one primer, OPC02 ('GTGAGGCGTC'), was found to be tightly linked (1.7cM) to the resistance gene of somaclone SC09.

Telomere and microsatellite primers reveal diversity among Sclerotinia sclerotiorum isolates from Brazil

Sclerotinia sclerotiorum, the causal agent of white mold, is a problem of winter bean (Phaseolus vulgaris) production in Brazil under center-pivot irrigation. Isolates of S. sclerotiorum were obtained from a center-pivot-irrigated field near Guaíra-SP, Brazil. Mycelial compatibility group (MCG) studies revealed the presence of only two MCG. PCR/RFLP analysis of the ITS1-5.8S-ITS2 ribosomal subunit regions of these field isolates of S. sclerotiorum failed to show any genetic differences between these two MCGs. DNA amplification with a chromosomal telomere sequence-based primer and one microsatellite primer revealed genetic polymorphisms among isolates within the same MCG. Isolates taken from beans and two other crops from another region of Brazil showed the same two MCG and had identical banding patterns for the telomere and microsatellite primers. These findings support the use of telomere sequence-based primers for revealing genotypic differences among S. sclerotiorum isolates.

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.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Print-capture PCR for detection of tomato begomoviruses from plants and whiteflies

Print-capture (PC) Polymerase chain reaction (PCR) was evaluated as a novel detection method of plant viruses. Tomato (Lycopersicon esculentum) plants infected with begomovirus (fam. Geminiviridae, gen. Begomovirus) and viruliferous whiteflies were used to study the efficiency of the method. Print-capturing steps were carried out using non-charged nylon membrane or filter paper as the solid support for DNA printings. Amplified DNA fragments of expected size were consistently obtained by PCR from infected plants grown in a greenhouse, after direct application of printed materials to the PCR mix. However, virus detection from a single whitefly and from field-grown tomato samples required a high temperature treatment of printed material prior to PCR amplification. Comparison of nylon membrane and filter paper as the solid support revealed the higher efficiency of the nylon membrane. The application of print-capture PCR reduces the chances of false-positive amplification by reducing manipulation steps during preparation of the target DNA. This method maintains all the advantages of PCR diagnosis, such as the high sensitivity and no requirement of radioactive reagents.

Variability in ribosomal DNA genic and spacer regions in Verticillium dahliae isolates from different hosts

Using PCR-based assays with specific primers for amplification of the ribosomal DNA intergenic spacer region (IGS) and a portion of the mitochondrial DNA small subunit ribosomal RNA gene (mtDNA SSU rRNA), the genetic variability among Verticillium dahliae isolates from olive (Olea europaea) and other host species from Argentina and Brazil was estimated. The derived UPGMA-generated phenograms based upon the restriction fingerprinting data of rDNA IGS products revealed genetic differences, correlating with the host of origin. Isolates infecting olive genetically distinct from those from cocoa (Theobroma cacao) and sunflower (Helianthus annuus). Digestion of mitochondrial DNA SSU rRNA PCR products revealed less variability, distinguishing only one isolate from sunflower. Ribosomal DNA ITS restriction patterns were identical for all isolates of V. dahliae, irrespective of host of origin. These preliminary results may have relevance for Verticillium wilt control practices, possibly reflecting a different evolutionary origin, or reproductive isolation of the pathogen in olive, distinct from populations of other hosts.

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

The objective of this research was to develop a primer for a polymerase chain reaction specific for Xylella fastidiosa strains that cause Pierce's Disease (PD) in grapes (Vitis vinifera). The DNA amplification of 23 different strains of X. fastidiosa, using a set of primers REP1-R (5'-IIIICGICGIATCCIGGC-3') and REP 2 (5'-ICGICTTATCIGGCCTAC-3') using the following program: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min and 72 ºC/1 min and 30 s) 72 ºC/5 min, produced a fragment of 630 bp that differentiated the strains that cause disease in grapes from the other strains. However, REP banding patterns could not be considered reliable for detection because the REP1-R and REP 2 primers correspond to repetitive sequences, which are found throughout the bacterial genome. The amplified product of 630 bp was eluted from the agarose gel, purified and sequenced. The nucleotide sequence information was used to identify and synthesize an specific oligonucleotide for X. fastidiosa strains that cause Pierce's Disease denominated Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3') which was used jointly with the REP-2 primer at the following conditions: 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. The DNAs isolated from strains of X. fastidiosa from other hosts [almond (Prumus amygdalus), citrus (Citrus spp.), coffee (Coffea arabica), elm (Ulmus americana), mulberry (Morus rubra), oak (Quercus rubra), periwinkle wilt (Catharantus roseus), plums (Prunus salicina) and ragweed (Ambrosia artemisiifolia)] and also from other Gram negative and positive bacteria were submitted to amplification with a pair of primers Xf-1/REP 2 to verify its specificity. A fragment, about 350 bp, was amplified only when the DNA from strains of X. fastidiosa isolated from grapes was employed.