Molecular characterization of parasite resistant/susceptible Uruguayan Merino lambs

C.G. Ciappesoni, P. Nicolini, L. Kelly, N. Grasso, P. Peraza, A. Cabrera, V. Goldberg (2012) Archivos Latinoamericanos de Producción Animal 20, 34-41

Abstract Gastrointestinal parasites (GIP) are one of the main sanitary and economic limitations for Uruguayan sheep production. Many authors suggest that there would be a possible relationship between microsatellites and sheep parasite resistance. Based on extreme fecal egg worm count expected progeny difference, 50 resistant (R) and 50 susceptible (S) lambs were chosen from a Merino flock. In order to investigate the genetic variability and structure in this flock, four polymorphic microsatellites (McM214, McM130, McM357 and CSRD2138) which are potentially associated with QTL for parasite resistance were analyzed. The number of alleles varied from 8 to 13 showing different frequencies, polymorphic information content values were >0.5 and the observed heterozygosity ranged from 0.735 to 0.773. A considerable genic and genotypic differentiation between R and S subpopulations was observed. McM214 showed a significant deviation from Hardy-Weinberg equilibrium from whole and S population. Fixation Indices (FIS) values indicated also a heterozygote excess in the entire population. McM214 showed linkage disequilibrium with McM357 in the R sample, and with McM130 in the S sample. Population structure analysis proved the origin of two clusters (subpopulations) from two different lines (K=2, similarity coefficient =0.979). Polymorphism of these markers could be used in association analysis with GIP resistance / susceptibility.

Variantes en dos genes candidatos para características de calidad de carne bovina en Argentina.

A. Branda Sica, L.A  Soria, P.M. Corva, E.L. Villarreal, L.M. Melucci, C.A. Mezzadra, A. Schor, y M.C. Miquel (2011) Archivos de Zootecnia (España), 60, 521 - 532

Resumen La calidad de la carne bovina está definida por muchos atributos. Está determinada por factores genéticos y ambientales (edad al sacrificio, alimentación, manejo anterior y posterior a la faena). La tendencia actual es estudiar genes candidatos con el propósito de desarrollar marcadores moleculares que puedan asistir a la selección. El objetivo de este trabajo fue evaluar el efecto de polimorfismos (SNP, single nucleotide polymorphisms) en genes candidatos para terneza y contenido de grasa en novillos engordados en condiciones de pastoreo de Argentina. Se diseñaron métodos moleculares para analizar el SNP 4751 (C/T) en el gen bovino capn1 (subunidad mayor de la μ-calpaína), asociado con terneza y dos polimorfismos (exón 8:G/A, e intrón 9:C/T) en el gen bovino ppargc1a (coactivador 1 alfa del receptor gamma activado por proliferadores peroxisómicos) con efecto sobre contenido de grasa en leche bovina y tipo de fibra en cerdos. Para los análisis de asociación se utilizaron 60 novillos Brangus y 21 Angus con registros de terneza y grasa intramuscular. La terneza (Resistencia al Corte- por Cizalla de Warner-Bratzler) fue determinada en tres tratamientos de maduración (1, 7 y 14 días). Una gran proporción de animales heterocigotos (CT) se observó en el SNP 4751. No se encontró ninguna diferencia entre los genotipos de ese SNP para RC (Resistencia al Corte determinada por Cizalla de Warner-Bratzler). En el SNP del intrón 9 del gen ppargc1a se halló una baja frecuencia de homocigotos TT. No se encontraron diferencias en grasa intramuscular ni en terneza entre los genotipos para dicho SNP. Se identificaron dos nuevos polimorfismos (G/A y C/T) en el exón 8 del gen ppargc1a, a partir de la comparación de secuencias obtenidas de 24 toros de razas distintas (Angus, Brangus, Brahman y Braford). Uno de ellos (G/A) podría provocar la sustitución de serina por asparragina en la posición 364 de la proteína. El alelo A no se encontró en Angus. El SNP C/T es una sustitución conservativa. Es importante que Argentina genere información sobre este tema para optimizar la producción y exportación de carnes de calidad.

Effect of three single-nucleotide polymorphisms in CAPN1 gene on beef tenderness.

L.A. Soria, P.M. Corva, A. Branda Sica, A. Schor, L.M. Melucci, E.L. Villarreal, C.A. Mezzadra, R. J.C. Cantet y M.C. Miquel (2009) Archiv Tierzucht 52, 546 - 549

Abstract Meat tenderness is an important trait in beef cattle production, as consumers consider tenderness the most important attribute of beef palatability. There is ample evidence that post mortem proteolysis of myofibrillar proteins is responsible for the decline in shear force during storage. The bovine micromolar calcium-activated neutral protease (CAPN1) gene encodes the large subunit of μ-calpain, which is thought to be one of the most important enzymes involved in post mortem tenderization (KOOHMARAIE 1996). Three single-nucleotide polymorphisms (SNPs) on the CAPN1 gene (316, 530 and 4 751 markers) have been associated with tenderness in different cattle breeds (PAGE et al. 2002, PAGE et al. 2004, WHITE et al. 2005). A more recent study confirmed that markers 316 and 4 751 had an effect on beef tenderness (VAN EENENNAAM et al. 2007). The objective of this research was to determine the existence of polymorphisms and to assess the effect of the reported SNP in the bovine CAPN1 gene on tenderness from a sample of Angus and Brangus steers fattened on pasture.

Association of a novel polymorphism in the bovine PPARGC1A gene with growth, slaughter and meat quality traits in Brangus steers.

L.A. Soria, P.M. Corva, A. Branda Sica, E.L. Villarreal, L.M. Melucci, C.A. Mezzadra, J. Papaleo Mazzucco, G. Fernández Macedo, C. Silvestro, A. Schor, M.C. Miquel (2009) Molecular and Cellular Probes 23, 304–308

The PPARGC1A gene (peroxysome proliferator-activated receptor-g coactivator 1a gene) controls muscle fiber type and brown adipocyte differentiation; therefore, it is a candidate gene for beef quality traits (tenderness and fat content). Two SNPs (Single Nucleotide Polymorphisms) were identified within exon 8 by multiple alignment of DNA sequences obtained from 24 bulls: a transition G/A (SNP 1181) and a transversion A/T (SNP 1299). The SNP 1181 is a novel SNP, corresponding to a non-conservative substitution (AGT/AAT) that could be the cause of amino acid substitution (364Serine/364Asparagine). A Mismatch PCR method was designed to determine genotypes of 73 bulls and 268 steers for SNP 1181. Growth, slaughter and meat quality information were available for the group of steers. Allele A of SNP 1181 was not found in Angus. In 243 steers, no significant differences (P > 0.05) were found for either final live body weight, gain in backfat thickness in Spring, kidney fat weight, kidney fat percentage, Warner–Bratzler shear force at 7 days postmortem, intramuscular fat percentage or meat colour between genotype GG and AG. This SNP could be included in breed composition and population admixture analyses because there are marked differences in allelic frequencies between Bos taurus and Bos indicus breeds.

Biotecnología Vegetal

Antimicrobial activity of pleurocidin is retained in plc-2, a cterminal 12-aminoacid fragment.

A.L. Souza, P. Díaz-Dellavalle, A. Cabrera, P. Larrañaga, M. Dalla-Rizza y Salvatore  Giovanni De-Simone. (2013) Peptides 45, 78–84

Abstract An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.

Insights on gene expression response of a characterized resistant genotype of Solanum commersonii Dun. against Ralstonia solanacearum

R. Narancio, P. Zorrilla, C. Robello, M. Gonzalez, F. Vilaró, C. Pritsch y M. Dalla Rizza (2013) Eur J Plant Pathol 136, 823-835.

Abstract Solanum commersonii is a wild species related to the cultivated potato. Some S. commersonii genotypes have been proven to be resistant to the pathogenic bacteria Ralstonia solanacearum, which causes damage in potato and other economically important crops. Here an expression analysis of the response of a resistant S. commersonii genotype against R. solanacearum was performed using microarrays. The aims of this work were to elucidate the molecular processes involved in the interaction, establish the timing of the response, and contribute to identify genes related to the resistance. The response to the treatment was already initiated at 6 h post-inoculation (hpi) and was established at 24 hpi; during this period, a high number of genes was differentially expressed and several candidate genes for the resistance of S. commersonii to R. solanacearum were identified. At an early stage, the photosynthetic process was highly repressed and several genes encoding proteins related to reactive oxygen species (ROS) production were differentially expressed. The induction of ERF and ACC-oxidase genes related to the ethylene pathway and PR1 related to the salicylic acid pathway suggested the induction of both pathways, and back up the previously reported hemibiotrophic nature of the pathogen. Five genes related to plant defence and observed to be differentially expressed at the first two time points were validated by real time PCR. This work gives a glimpse to the molecular processes involved in S. commersonii resistance and identifies the species as a valuable genetic source for potato breeding against bacterial wilt.

Generation and characterization of interspecific hybrids of Lotus uliginosus × Lotus corniculatus

A. Castillo, M. Rebuffo, M. Dalla Rizza, G. Folle, F. Santinaque, O. Borsani, y  J. Monza (2012) Crop Science 52, 1–11

Abstract A method is described for obtaining interspecific hybrids between commercial lines of Lotus uliginosus Schkuhr and Lotus corniculatus L. Hybridization was possible between these species using embryo rescue. Two strategies were used to confirm 40 F1 hybrids, that is, flow cytometry and microsatellite simple sequence repeats (SSRs). The L. uliginosus and L. corniculatus parents have significantly different genome size (2.5 ± 0.02 and 2.22 ± 0.02 pg, respectively) and the offspring from the cross had intermediate values. Two SSR primers were polymorphic between the genotypes tested, TM1150 and EH380069. Maternal effect was detected in L. uliginosus genotypes, asdetermined by greater frequency of F1 hybrids. Recombinant traits were observed in the F1 hybrid progeny. Ninety F1 hybrids resulting from L. uliginosus × L. corniculatus crosses showed different levels of fertility in the polycross; <3% F1 plants produced more than 1000 seeds without embryo rescue while 10% of plants did not flower the first year. The F1 hybrid plants grown under greenhouse conditions exhibited shoot phenotypes similar to L. uliginosus (maternal) whereas F2 phenotypes were similar to L. corniculatus (paternal) in field trials. Rhizome presence was observed in 60% of F2 hybrid plants. The diameters of the root crowns of these plants, however, were similar to that of L. corniculatus.

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis.

M.T. Federici, J. Marcondes, S.C. Picchi, E. Stuchi, A.L. Fadel, L.M. Laia, M.V.F. Lemos, y E.G.M.  Lemos. (2012) Electronic Journal of Biotechnology. DOI: 10.2225/vol15-issue3-fulltext-4

 Abstract Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels.

Activity of Naturally Derived Antimicrobial Peptides against Filamentous Fungi Relevant for Agriculture

P. Larrañaga, P. Díaz-Dellavalle, A. Cabrera, D. Alem, C. Leoni, L.A. Souza, S.G. De-Simone, Dalla-Rizza, M. (2012) Sustainable Agriculture Research 1, No.2.

Abstract The search for environmentally biocompatible and cost-effective methods to control filamentous fungi in agriculture is becoming increasingly urgent. In vitro antimicrobial activity of three synthetic peptides was investigated against some filamentous fungi with agricultural relevance. The peptides were an analog of Temporin called Temporizina, a fragment from Pleurocidin termed Plc-2, and a peptide identified from sesame seeds named Pses3. Antimicrobial activity of these peptides towards filamentous fungi has not been previously reported. Seven plant pathogenic or mycotoxigenic fungal species, isolated from plant tissues were assayed: Alternaria solani, Colletotrichum gloesporioides, Fulvia fulvum, Fusarium oxisporum, Aspergillus niger, A. ochraceus and Penicillium digitatum. Values of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were determined and compared with the commercially available fungicide Captan as a positive control. The peptides showing greatest inhibition were Pses3 and Plc-2 and C. gloesporioides was the most sensitive of the evaluated fungi. The MIC values for Plc-2 and Pses3 peptides ranged from 0.64 μM to 10.25 μM. These values were much lower than those observed for Captan, suggesting the potential of these peptides as fungicides. In particular, Pses3 is a novel peptide derived from sesame seeds not reported in databases.

Molecular and cytogenetic characterization of a collection of bahiagrass (Paspalum notatum Flügge) native to Uruguay.

R. Reyno, R. Narancio, P. Speranza, J. Do Canto, B. Lopez-Carro, P. Hernández, J. Burgueño, D. Real, M. Dalla Rizza (2012) Genetic Resources and Crop Evolution 59, 1823-1832

Abstract Paspalum notatum is a subtropical grass present throughout America, and one of the main constituents of the natural grasslands in Uruguay. An apomictic autotetraploid (2n = 4x = 40) is the most frequent cytotype. The occurrence of sexual diploids (2n = 2x = 20) has also been reported as well as the occasional presence of apomictic triploids and pentaploids in Argentina. In this study, ISSR (inter simple sequence repeats) molecular markers were used to analyze the genetic variability of 210 P. notatum individuals from a collection from Uruguay. Cytometric analyses and chromosome counts were used to assess the ploidy level of the individuals. All plants from Uruguay analyzed were tetraploid. Intra- and inter-population variability was found both in genomic DNA content and at the genotypic level. Several multilocus genotypes were shared among individuals within populations and among populations over moderate geographical ranges, at the same time, very dissimilar genotypes were found within the same population. Part of the genetic variance among populations can be explained by a broader scale geographic structure which is partly coincident with the traditionally recognized grassland management regions. In spite of the apparently high degree of genetic admixture within populations, groups of related genotypes seem to follow a broader geographical structure in the area under study. These results suggest that an efficient collection strategy for this apomictically reproducing species should include carefully planned intra- and inter-population sampling. A broader scale regional sampling strategy should also be considered although further studies will be required to define genetic structure at this level.

Response to photoxidative stress induced by cold in japonica rice is genotype dependent.

V. Bonnecarrère, O. Borsani, P. Diaz, F. Capdevielle, P. Blanco, J. Monza (2011) Plant Science 180, 726–732

Abstract Two japonica rice genotypes, INIA Tacuarí and L2825CA, were analyzed for tolerance to low temperature during early vegetative growth. Effect on photosynthesis, energy dissipation, pigment content, xanthophyll-cycle pool conversion, hydrogen peroxide accumulation, oxidative damage and antioxidant enzyme activities were determined to better understand potential mechanisms for cold tolerance. Photoinhibition was measured using chlorophyll fluorescence and oxidative damage by lipid peroxidation and electrolyte leakage. Both genotypes were demonstrated to be cold tolerant which was consistent with their reduced levels of photoinhibition and oxidative damage compared with a cold-sensitive genotype during chilling stress. The strategy for cold tolerance differed between the two genotypes, and involved different mechanisms for disposal of excess energy. The presence of high lutein concentrations and the existence of active non-harmful energy dissipation processes through the xanthophyll cycle appeared to be responsible for chilling tolerance in INIA Tacuarí. On the other hand, increased cold tolerance of L2825CA relative to INIA Tacuarí was related to the higher constitutive superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6).

Genetic diversity in a natural population of the halophytic legume Prosopis  strombulifera revealed by AFLP fingerprinting.

A. Llanes, V. Bonnecarrère, F. Capdevielle, S. Vidal (2011) Boletín de la Sociedad Argentina de Botánica 46, 305-312

 Abstract Prosopis strombulifera (Lam.) Benth. is a spiny shrub with the maximum tolerance limits reported for halophytic plants. This species is frequently found in the salinized areas in south-western of Córdoba and San Luis provinces, Argentina. Little is known about the genetic diversity within this species in a native population. Genetic diversity in 60 plants of P. strombulifera in south-western San Luis was investigated using AFLP analysis. Polymorphism was found among the samples tested. Four combinations of primers led to the identification of an average of 250 polymorphic bands and the data were used for cluster analysis. P. strombulifera genotypes are clearly separated in subclusters and reflect the diversity within the collection area. This study is a contribution to describe the intra-specific diversity in a natural population of P. strombulifera, and the polymorphism obtained is comparable with other populations of Prosopis species. Results demonstrate the importance of identifying different intra-population genotypes as components of a gene bank of P. strombulifera.

Molecular characterization of Lotus corniculatus L. cultivars using transferable microsatellite markers.

D. Alem, R. Narancio, P. Díaz Dellavalle, M. Rebuffo, R. Zarza, y M. Dalla Rizza. (2011)  Ciencia e investigación agraria 8, 453-461.

Abstract Lotus corniculatus L. is the most important agricultural species in the genus Lotus and is the most widely distributed Lotus species worldwide. L. corniculatus genotypes form complex groups that are difficult to recognize both morphologically and biochemically. Given the extensive and expensive process of isolating Simple Sequence Repeats (SSR)(also called microsatellites), the possibility of using microsatellites already identified in related species is highly attractive. The aim of this work was the identification and validation of transferable microsatellite markers in L. corniculatus, and using those markers to study the genetic variability among four cultivars. Each cultivar of L. corniculatus was represented by 15 genotypes. Ten microsatellite markers were evaluated, and from those, four were selected based on their discriminative values observed among cultivars. We detected 29 alleles for the four markers, and there was an average of 7.25 alleles per locus. The marker TM0197 had the fewest number of alleles (5) and TM0083 had the highest number of alleles (10). The polymorphic information content (PIC) for the selected markers varied from 0.19 to 0.35, and the markers were therefore classified as highly informative. Based on the markers, we found high variability between individuals of the same cultivar. The use of transferable microsatellite markers could be useful to differentiate individuals at a relatively low cost, showing a great potential for use in breeding programs.

Antifungal activity of medicinal plant extracts against phytopathogenic fungus Alternaria spp.

P. Díaz Dellavalle, A. Cabrera, D. Alem, P. Larrañaga, F. Ferreira, y M. Dalla Rizza. (2011) Chilean Journal of Agricultural Research 7, 231-239

Abstract The aim of the study was to evaluate the antifungal activity of extracts of 10 plant species used in traditional Uruguayan medicine against the phytopathogenic fungus Alternaria spp. The plants were selected on the basis of their reported ethnobotanical uses. Aqueous, saline buffer and acid extracts of different plant species were screened in vitro for their antifungal activity against Alternaria spp. For the antifungal evaluation we used a microspectrophotometric assay. Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts were determined. Three solvents were assayed on different tissues of the plants and among the 29 evaluated extracts, 31% of the extracts inhibited growth, similar to the effects of a chemical fungicide. Acid extracts of the plants were more effective than the aqueous or buffer extracts against Alternaria spp. The MIC values of the extracts were determined ranging between 1.25 and 25 μg mL-1. The MFC values of the extracts ranged between 1.25 μg mL-1 (Rosmarinus officinalis L.) and 10 μg mL-1 (Cynara scolymus L.). MICs and MFCs values obtained from leaves (Salvia officinalis L. and R. officinalis) and seeds extracts (Salvia sclarea L.) were quite comparable to values obtained with the conventional fungicide captan (2.5 μg mL-1). The extracts of Salvia sclarea, S. officinalis and R. officinalis could be considered as potential sources of antifungal compounds for treating diseases in plants. These extracts showed maximum activity, even at very low concentrations, and the same fungicide effects as chemical fungicide. We conclude from this that these extracts exhibit amazing fungicidal properties that support their traditional use as antiseptics.

Establishment of micropropagation and cell suspension culture conditions on Achyrocline flaccida (Weinm.) DC. (Asteraceae).

V. Bonnecarrère,  L. Berna, A. Castillo (2009) Agrociencia 13,1-6

Abstract Achyrocline flaccida (Weinm.) DC. (Asteraceae) is a medicinal plant species, commonly known as yellow marcela. It is a rich source of flavonoids and other secondary metabolites with antioxidant properties. Their inflorescences are used as remedies in folk medicine for the treatment of a variety of human ailments. In fact, the permanence of this species is threatened by the increased interest of medicinal herb collectors. Thus, techniques which could provide vegetative propagated material for commercial use are necessary, and in vitro-propagation is a valuable method for producing large numbers of genetically uniform, pathogen-free plants in a short time. Moreover, theproduction of Achyrocline secondary metabolites is crucial for research and commercial large scale production since they are controllable systems and easy to scale up. Besides productive aims, cell culture suspensions are valuable tools to investigate metabolic pathways involved in secondary metabolites synthesis and to discover new bioactive molecules. The aims of this study were the optimization of a method to propagate in vitro plant of A. flaccida and the establishment of cell suspension cultures to determine the optimal culture conditions in order to improve cell growth as a first step toward secondary metabolites production. It was concluded that DKW without growth regulators is the optimal medium for micropropagation of this species. Friable callus formation was optimized in MS supplied with 0.5 mg L-1 2,4-D while cell suspensions were better obtained and maintained in DKW supplied with 1mg L-1 2,4-D.

Biomolecules as host defense weapons against microbial pathogens.

M. Dalla Rizza, P. Diaz Dellavalle, R. Narancio, A. Cabrera y F. Ferreira (2008) Recent Patents on DNA & Gene Sequences 2, 82-96

Abstract Antimicrobial peptides have been considered a new source of biomolecules in several fields of research/innovative applications: they would adjust to an ideal behavior seeking to overcome clinicians, microbiological, human-animal-plant-environmental concerns. Antimicrobial peptides can be considered as ancient weapons found in living organisms suggesting they have played a fundamental role in his successful co-evolution with pathogens. Acting on microorganism membrane or having intracellular targets, they can also act as effectors of the innate immune response resulting on non-specific mechanisms of action. Two elements have speeded the research on pathogen control alternatives: a verified increase of antibiotic resistance and the relevance of finding amenable environmental compounds in plant health. As a result of its importance, great efforts have been accomplished to find, characterize, combine and synthesize effective antimicrobial peptides. This review intends to emphasize the generation of biomolecules, whether native or synthetic analogues, that have been matter of recent patents. Developments of biomolecules suitable for therapeutic scopes and agricultural use have several challenges such as intrinsic toxicity, in vivo stability and suitable formulation contemplating the cost of production. Thus, biotechnological procedures using microbial systems or transgenic crop as plant factories might help to solve these challenges.

Breeding system of the aerial flowers in an amphicarpic clover species: Trifolium polymorphum

D. Real, M. Dalla Rizza, R. Reyno, y K.H. Quesenberry (2007) Crop Science 47, 1401–1406

Abstract Two perennial Trifolium, T. polymorphum Poir. and T. argentinense Speg., are American clovers unique within the genus for being amphicarpic. There is no consensus in the literature regarding the breeding system of the aerial fl owers of T. polymorphum, therefore, the breeding system was studied. In 1997 T. polymorphum was collected in Uruguay and evaluated at INIA Tacuarembó. In 2001, 10 field patches were marked and in 2004, 20 plants per patch were characterized with simple sequence repeat markers. Patch J10 showed a particular molecular profile, therefore, 198 open-pollinated progenies freely visited by honeybees were studied. In 2005, at the University of Florida, Gainesville, different handpollination treatments were conducted within an accession from Paraguay. Trifolium polymorphum was able to cross-pollinate with all the known pollen donors molecularly marked that surrounded plants from patch J10 (30%), also with some nonmarked native ones from the vicinity (10%) as well as with itself (60%), when allowed to be visited by honeybees. However, when there are no pollinators, the selfi ng rate is minimal. The proposed classifi cation for the breeding system is an allogamous, self-compatible species that benefi ts from pollinators to set seed.

Genetic diversity and DNA content of three South American and three Eurasiatic Trifolium species

M. Dalla Rizza, D. Real, R. Reyno, V. Porro, J. Burgueño, E. Errico y K.H. Quesenberry (2007) Genetics and Molecular Biology 30, 1118-1124

Abstract Six species of Trifolium (T. polymorphum Poir., T. riograndense Burkart, T. argentinense Speg., T. medium L., T. pratense L. and T. repens L.) were analyzed using inter-simple sequence repeats (ISSR) markers. Six selected primers generated 186 polymerase chain reaction (PCR) products exploring 112 loci in 34 genotypes analyzed with molecular sizes ranging from 200 to 1300 bp. These primers were able to discriminate among and within species, with the PCR products being on average 41.6% species-specific and 59.9% polymorphic at the within species level. Nuclear DNA content was determined by flow cytometry and revealed variation among species. The 1Cx genome size values were calculated and were found to range from 0.46 pg (T. pratense) to 0.96 pg (T. polymorphum). Genome size values of South American species were higher than those of Eurasiatic origin. The analyses of the molecular data grouped the six species in agreement with their geographical origin and clearly differentiate T. polymorphum from T. argentinense. The Eurasiatic group showed the highest average of species-specific bands (45.3%) and the South American group exhibited the highest amount of total bands (59.7). The highest level of intra-species polymorphisms was detected in T. argentinense (92.9%), followed by T. medium (89.5%).

Improved resolution of nonsilica-based size-exclusion HPLC column for wheat flour protein analyses

M. Dalla Rizza, P. Díaz Dellavalle, D. Vázquez, y M. Castro (2005) Cereal Chemistry 82, 287–289

The use of size-exclusion high-performance liquid chromatography (SE-HPLC) has become an important method adopted for cereal protein quality characterization within different breeding programs. Several types of SE-HPLC columns have been used for the study of wheat flour proteins. Different attributes have been stated for available SE-columns. They include solubility of the proteins and elution conditions, minimal interaction with the column support, resolution of the peaks, and column life in a routine procedure (Huebner and Bietz 1985; Singh et al 1990; Batey et al 1991; Ciaffi et al 1996). These considerations must be taken into account before starting new analysis approaches. Denaturing agents such SDS cause negative effects on silicabased support. Therefore, attempts to remove the SDS from the sample preparation and elution buffers have been made (Batey et al 1991). Since this work was published, many other researchers have adopted hydrophobic elution conditions for SE-HPLC wheat protein characterization to improve peak resolution (Gupta et al 1993; Larroque et al 1997; Huebner and Bietz 1999). Other research groups use hydrophilic conditions (Dachkevitch and Autran

1989; Ciaffi et al 1996; Carceller and Aussenac 1999; Tronsmo et al 2002). All of them agree on the convenience of using SDS for protein extraction. Moreover, Batey et al (1991) recommended the use of >0.3% SDS in the extracting buffer to avoid problems later in the chromatographic performance. The aim of this note is to present the advantages of using a novel size-exclusion column, Superdex 200 (Amersham Pharmacia Biotech, Uppsala, Sweden). This column allows the use of the same SDS level in the extracting and elution buffer, improving resolution. The main peaks were characterized by SDS-PAGE confirming good resolution of the separation process.

Characterization of Pyricularia grisea population from Uruguay by molecular analysis.

P. Taheri, V. Bonnecarrère, M. Höfte (2004) Comm. Agr. Biol. Sci. 69, 207-210

Abstract Blast disease, caused by hemibiotrophic fungus Pyricularia oryzae Sacc. (teleomorph: Magnaporthe oryzae (Hebert) Barr.), previously known as Magnaporthe grisea, is the most serious disease of rice and causes high yield losses in most rice growing regions every year. Blast is an endemic disease in all Iranian and Uruguayan rice fields, with yield losses up to 90 % that has been reported from some rice growing regions. Various types of blast infection can be observed in a field, including symptoms on leaves, collar, neck, panicle, and grain. Although the fungus is only known to reproduce asexually in nature, it is infamous for its diversity. P. oryzae is not only pathogenic on rice, but also on other cultivated and wild gramineous hosts. However, the species is considered to consist of host-limited forms. Knowledge of different rice blast pathogen populations and factors affecting genetic structure of the isolates in some of main rice growing countries, such as Uruguay, is still scarce. However, understanding disease epidemiology and plant-pathogen interactions, and finally sustainable disease management is highly dependent on  knowledge of the genetic diversity of the pathogen. Diversity can be studied using a wide array of molecular techniques. AFLP method is based on selective amplification of restricted fragments generated from total genomic DNA. Because of rapidity, replicability, high resolution, and adequate discriminatory power above and below species level in a variety of taxa including bacteria, fungi, plants, and animals, AFLP markers have emerged as a major type of genetic markers with broad spectrum of application, especially in analyzing population structure. Also, the more loci which are screened, the lower the probability of making a mistake by chance factors (ref. 20 of my phytopath. Paper). AFLP is used increasingly to study genetic diversity of several plant pathogenic fungi. The genetic variability of 55 P. oryzae isolates from Iran and 32 isolates from Uruguay, was analysed using amplified fragment length polymorphism (AFLP). Cluster analysis using different methods and principal co-ordinate analysis (PCO), based on the AFLP data from 679 monomorphic and polymorphic bands generated with eight primer combinations, was performed. The resulted grouping of the isolates revealed 4 separate AFLP groups among a total of 87 isolates. Within each AFLP group, two or more haplotypes were detected with a genetic similarity of 100 %. Overall genetic similarity was greater than 50 % between Iranian and Uruguayan populations. Little evidence for gene flow between the two populations of the pathogen. Analysis of Molecular Variance (AMOVA) revealed that rice varietal type and geographic region were the dominant factors determining genetic structure of P. oryzae populations; but rice cultivar had not significant effect.

Reproductive and Molecular Evidence for Allogamy in Lotononis bainesii Baker

D. Real, M. Dalla Rizza, K.H. Quesenberry y M. Echenique (2004) Crop Science 44, 394-400

Abstract Reproductive characteristics can influence seed production and the amount and distribution of genetic variation within populations. Lotononis bainesii Baker is a subtropical forage legume from southern Africa that earlier researchers reported as having a cleistogamous form of reproduction. More recent reports suggest that species in the genus Lotononis Section Listia reproduce chasmogamously. Research from this study suggests that this species needs pollinators to produce seed and that genotypes exist that are self-incompatible. The use of sequence characterized amplified regions (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers demonstrated that L. bainesii is highly allogamous.

Development and application of a dap-B-based IVET system to study colonization of rice by the endophytic nitrogen-fixing bacteria Pseudomonas stutzeri

H. Rediers, V. Bonnecarrère,  P. Rainey, K. Hamonts, J. Vanderleyden, R. De Monts (2003) A15. Appl. Micr. Env. 69, 6864-6874

Abstract Pseudomonas stutzeri A15 is a nitrogen-fixing bacterium isolated from paddy rice. Strain A15 is able to colonize and infect rice roots. This strain may provide rice plants with fixed nitrogen and hence promote plant growth. In this article, we describe the use of dapB-based in vivo expression technology to identify P. stutzeri A15 genes that are specifically induced during colonization and infection (cii). We focused on the identification of P. stutzeri A15 genes that are switched on during rice root colonization and are switched off during free-living growth on synthetic medium. Several transcriptional fusions induced in the rice rhizosphere were isolated. Some of the corresponding genes are involved in the stress response, chemotaxis, metabolism, and global regulation, while others encode putative proteins with unknown functions or without significant homology to known proteins.

Analysis of Uruguayan weedy rice genetic diversity using AFLP molecular markers

M.T. Federici, D. Vaughan, N. Tomooka, A. Kaga, Xin Wang Wang, Koji Doi, M. Francis G. Zorrilla, N. Saldain (2001) Electronic Journal of Biotechnology http://www.ejb.org/content/vol4/issue3/full/3

Abstract Weedy rice is a serious problem in Uruguayan rice fields since intensification of rice production started about 10 years ago. The genetic diversity of 26 weedy accessions of weedy rice and 6 Uruguayan cultivars were analyzed using AFLP (amplified fragment length polymorphisms) methodology. Abundant polymorphisms were found among samples tested. Using different methods of analysis three groups of samples were revealed. A relationship was found between three groups and morphological traits. One group had a black hull, purple apex and long awn (wild type traits) while another group had straw hull and apex, and short or no awn (domestication traits). The third group included the cultivars analyzed and some weedy rice samples. The weedy rice in this third group is presumed to most closely mimic cultivated rice and may have recently evolved. The results suggest that weedy rice adapts either to the natural environment or to cultivation. The former type with black hull and long awn may be easy to control because it can easily be seen. The later group may be difficult to control, particularly since the weedy rices within the cluster consisting of cultivars suggest that weedy rices are continually evolving in Uruguayan rice fields. The AFLP technique is very effective for assessing genetic diversity within weedy rice and will be very useful for fingerprinting of local cultivars of rice.

Phytotoxic Protein PcF, Purification, Characterization, and cDNA Sequencing of a Novel Hydroxyproline-containing Factor Secreted by the Strawberry Pathogen Phytophthora cactorum

G. Orsomando, M. Lorenzi, N. Raffaelli, M. Dalla Rizza, B. Mezzetti, y S. Ruggieri (2001) DOI 10.1074/jbc.M101377200

Abstract A novel protein factor, named PcF, has been isolated from the culture filtrate of Phytophthora cactorum strain P381 using a highly sensitive leaf necrosis bioassay with tomato seedlings. Isolated PcF protein alone induced leaf necrosis on its host strawberry plant. The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBankTM/EBI data banks showed that PcF shared no significant homology with other known sequences. The 52-residue PcF protein, which contains a 4-hydroxyproline residue along with three S–S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectric point of 4.4. The molecular mass of isolated PcF is 5,622 6 0.5 Da as determined by mass spectrometry and matches that calculated from the deduced amino acid sequence with cDNA sequencing. The cDNA sequence indicates that PcF is first produced as a larger precursor, comprising an additional N-terminal, 21-residue secretory signal peptide. Maturation of this protein involves the hydroxylation of proline 49, a feature that is unique among other known secreted fungal phytopathogenic proteins.