Marcelino Pérez de la Vega

Preliminary study on genetic variability of Dicrocoelium dendriticum determined by random amplified polymorphic DNA

Genetic variability of adult specimens of Dicrocoelium dendriticum has been studied using random amplified polymorphic DNA (RAPD). The worms were collected from the infected livers of different sheep from several localities in León province (NW Spain). DNA fragments were amplified by means of decamer primer oligonucleotides of arbitrary sequence. Some primers produce complex and highly variable patterns of amplified DNA in D. dendriticum. Intra- and inter-population genetic variability of adult parasites were analyzed, scoring polymorphic and monomorphic reproducible bands by means of the Jaccard similarity, and dendrograms showing genetic relationships between individuals were obtained using the FITCH method. Genetic variability seems to be high in this parasite and genetic similarity within a population (worms infecting a single animal) is similar to the average similarity between worms from different sheep. These results suggest that each sheep is infected by numerous genetically different parasites from one or more populations of infected ants.

Identification of pathovars and races of Pseudomonas syringae, the main causal agent of bacterial disease in pea in North-Central Spain, and the search for disease resistance

A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.

Characterization of Pseudomonas syringae pv. syringae isolates associated with bacterial blight in Lathyrus spp. and sources of resistance

The diploid annual legume species, Lathyrus sativus L., also known as grass pea, and L. cicera L., also known as chickling pea, are neglected species although they are widely grown as pulse crops for human food and also as grain feed or as forage in many of the harshest agro-environments of the world. The surface devoted to these two crops is increasing in Spain, where they were traditionally grown. The presence of bacterial blight among these crops was detected in fields located in the North part of the Central Spanish Meseta during the 2008–9 and 2009–10 seasons. Metabolic-biochemical tests, inoculation experiments in different hosts and molecular markers identified the causal agent as Pseudomonas syringae pv. syringae. This work represents the first description of the occurrence of P. syringae pv. syringae in Lathyrus spp. Bacteria isolated from these two plant species were also pathogenic and very aggressive in other cool season legume species. Rep-PCR patterns showed a close similarity among the P. syringae pv. syringae samples collected within each Lathyrus species, and that the bacterial isolates from Lathyrus spp. were very similar to strains of this pathogen isolated from common and hairy vetch, and from pea. Multilocus sequencing typing confirmed the similarity of one of these isolates with database P. syringae pv. syringae sequences. Most of the L. cicera landraces evaluated for resistance to P. syringae pv. syringae were susceptible or highly susceptible, but some promising resistance sources to this pathogen were found under controlled conditions.

Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse.

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

Obtaining retrotransposon sequences, analysis of their genomic distribution and use of retrotransposon-derived genetic markers in lentil (Lens culinaris Medik.)

Retrotransposons with long terminal repeats (LTR-RTs) are widespread mobile elements in eukaryotic genomes. We obtained a total of 81 partial LTR-RT sequences from lentil corresponding to internal retrotransposon components and LTRs. Sequences were obtained by PCR from genomic DNA. Approximately 37% of the LTR-RT internal sequences presented premature stop codons, pointing out that these elements must be non-autonomous. LTR sequences were obtained using the iPBS technique which amplifies sequences between LTR-RTs. A total of 193 retrotransposon-derived genetic markers, mainly iPBS, were used to obtain a genetic linkage map from 94 F7 inbred recombinant lines derived from the cross between the cultivar Lupa and the wild ancestor L. culinaris subsp. orientalis. The genetic map included 136 markers located in eight linkage groups. Clusters of tightly linked retrotransposon-derived markers were detected in linkage groups LG1, LG2, and LG6, hence denoting a non-random genomic distribution. Phylogenetic analyses identified the LTR-RT families in which internal and LTR sequences are included. Ty3-gypsy elements were more frequent than Ty1-copia, mainly due to the high Ogre element frequency in lentil, as also occurs in other species of the tribe Vicieae. LTR and internal sequences were used to analyze in silico their distribution among the contigs of the lentil draft genome. Up to 8.8% of the lentil contigs evidenced the presence of at least one LTR-RT similar sequence. A statistical analysis suggested a non-random distribution of these elements within of the lentil genome. In most cases (between 97% and 72%, depending on the LTR-RT type) none of the internal sequences flanked by the LTR sequence pair was detected, suggesting that defective and non-autonomous LTR-RTs are very frequent in lentil. Results support that LTR-RTs are abundant and widespread throughout of the lentil genome and that they are a suitable source of genetic markers useful to carry out further genetic analyses.

Isozyme Variation and Genetic Structure of Populations of Avena barbata from Argentina

Genetic diversity was analysed in 52 Argentinian populations of Avena barbata, a tetraploid grass introduced in America from Spain during the colonization period. Nine isozyme systems were studied and 14 loci identified, five of which were polymorphic. Cluster analysis based on Hedrick’s index revealed a high similarity among populations. The total diversity (Pt) in the 52 populations was 0.144, the mean diversity (Ps) was 0.04, while between population diversity (Dst) was 0.103. The resulting coefficient of differentiation (Gst) was 0.714, indicating that diversity among populations was an important contributor to the total variability. Genetic diversity was structured into multilocus associations; 122 different complexes were found among 3311 individuals, but only two complexes occurred at a high frequency. The distribution pattern of these frequent multilocus genotypes was associated with environmental factors, mainly rainfall and temperature. The comparison of these results with those of previous studies on A. barbata from Spain indicated that Spanish and Argentinian populations are closely similar in allelic composition on a locus-by-locus basis but different in multilocus genotypic composition. We concluded that selection was the main force involved in the reorganization of the Spanish genepool into novel multilocus associations adapted to specific habitats in Argentina.

Isozyme markers and genetic variability in three species of Centrosema (Leguminosae)

Isozyme patterns and their genetic control in three Centrosema species are described. Seven isozymatic systems (aspartate aminotransferase, glucose-6-phosphate isomerase, phosphoglucomutase, anodal peroxidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase) were studied in 18 populations and several breeding lines of C. acutifolium, C. brasilianum and C. pubescens, using starch gel electrophoresis techniques. All systems, except glucose-6-phosphate isomerase, are described for the first time in these species. A total of 17 isozyme loci were scored; this represents the largest set of Mendelian loci known up to now in Centrosema species. Isozyme polymorphism and variability within and between populations and species were relatively high and allowed discrimination among species

A genome-wide identification and comparative analysis of the lentil MLO genes

Powdery mildew is a widespread fungal plant disease that can cause significant losses in many crops. Some MLO genes (Mildew resistance locus O) have proved to confer a durable resistance to powdery mildew in several species. Resistance granted by the MLO gene family members has prompted an increasing interest in characterizing these genes and implementing their use in plant breeding. Lentil (Lens culinaris Medik.) is a widely grown food legume almost exclusively consumed as dry seed with an average world production of 4.5 million tons. Powdery mildew causes severe losses on certain lentil cultivars under particular environmental conditions. Data mining of the lentil CDC Redberry draft genome allowed to identify up to 15 gene sequences with homology to known MLO genes, designated as LcMLOs. Further characterization of these gene sequences and their deduced protein sequences demonstrated conformity with key MLO protein characteristics such as the presence of transmembrane and calmodulin binding domains, as well as that of other conserved motifs. Phylogenetic and other comparative analyses revealed that LcMLO1 and LcMLO3 are the most likely gene orthologs related to powdery mildew response in other species, sharing a high similarity with other known resistance genes of dicot species, such as pea PsMLO1 and Medicago truncatula MtMLO1 and MtMLO3. Sets of primers were designed as tools to PCR amplify the genomic sequences of LcMLO1 and LcMLO3, also to screen lentil germplasm in search of resistance mutants. Primers were used to obtain the complete sequences of these two genes in all of the six wild lentil relatives. Respective to each gene, all Lens sequences shared a high similarity. Likewise, we used these primers to screen a working collection of 58 cultivated and 23 wild lentil accessions in search of length polymorphisms present in these two genes. All these data widen the insights on this gene family and can be useful for breeding programs in lentil and close related species.

Prospects: The Importance of Common Bean as a Model Crop

Common bean is the most important grain legume crop for food consumption worldwide and has a role in sustainable agriculture as a main source of proteins and nutrients. This book provides insights into some of the key achievements made in the study of common bean, as well as a timely overview of topics that are pertinent for future developments in legume genomics. At the conclusion of this volume, it is as important to take a look back so as to put a forward view in proper perspective.

The Common Bean Genome

Common bean (Phaseolus vulgaris L.) is broadly adapted to environments with moderate growing temperatures, about 400 mm of precipitation and a growing season of 60–120 days. The popularity of the crop originates from the fact that it is relatively easy to produce, it is flavorful and versatile, and it is a good source of nutrition. The two major types of common bean are dry edible beans and snap or garden beans. Precise economic valuation of the common bean crop is difficult to obtain on a global scale because other species are often included in the statistical data collected in different countries, but with production of 18.9 million T for all types, it is the most widely produced grain legume and ranked third after soybean and groundnut for oilseed and grain legumes combined. Common bean is produced in both developed and developing countries and is an important source of protein, carbohydrates, some vitamins, and micronutrients. Common bean first became known to the scientific world with the Columbian exchange beginning in 1493, but little is known about the genetic diversity of the early introductions to Europe. Systematic breeding of common bean began in the nineteenth century in Europe and the USA. Common bean was the subject of Mendel’s genetics research, was used by Johannsen to investigate quantitative inheritance, and has the distinction of being the first plant species where a quantitative trait locus was identified. Contemporary research on common bean in the recent past has been conducted in about 21 academic disciplines with plant physiology, medicine, microbiology, and food science, resulting in the most publications. Plant breeding, genetics, plant pathology, and J. R. Myers (&) Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA e-mail: james.myers@oregonstate.edu K. Kmiecik 7202 Portage Rd, De Forest, WI 53532, USA e-mail: kakmiecik@sbcglobal.net