János Taller

PICcalc: An Online Program to Calculate Polymorphic Information Content for Molecular Genetic Studies

IntroductionMolecular markers have proved to be valuable tools in the characterization andevaluation of genetic diversity within and between species and populations. Markersystems differ in their information content, which depends on polymorphism. Theconcept of polymorphism is used to define genetic variation in a population, whichhas been extensively studied in recent years by several established scientificdisciplines, for example, genetics, ecology, zoology, and microbiology (Mukherjeeet al. 2010; Muneer et al. 2011; Rajkumar et al. 2011). Examples are numerous andobvious. For the practical design of molecular genetic studies, a few questions mustbe considered. How difficult will it be to find usefully polymorphic loci? How manymarkers are needed? How polymorphic must each marker be? These questions canbe answered by measuring the information content of the markers. There are twomeasures of the quality or informativeness of a polymorphism as a genetic marker:heterozygosity (H) and polymorphic information content (PIC). Since its firstapplication by Botstein et al. (1980) PIC has become the most widely applied

Efficiency of Arbitrarily Amplified Dominant Markers (SCOT, ISSR and RAPD) for Diagnostic Fingerprinting in Tetraploid Potato

Three molecular markering techniques: start codon targeted (SCOT), inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers were compared for fingerprinting of 24 varieties and a segregating population of tetraploid potato. The number of scoreable and polymorphic bands produced using the SCOT, ISSR and RAPD primers for varieties was more than that of genotypes. SCOTs markers were more informative, followed by ISSRs marker, than other markers for the assessment of varieties based on polymorphism information content (PIC). There were no significant differences among these markers in terms of the evaluation of genotypes. All marker techniques individually illustrated that Diversity Index and Marker Index for varieties were higher than that of genotypes, and SCOT had superiority to other markers. The resolving power (Rp) of the SCOT, ISSR and RAPD techniques was 71.25, 46.62 and 30.63 for varieties and 21.38, 18.83 and 18.87 for genotypes, respectively. Standard Jaccard’s similarity coefficient of each marker technique revealed that similarity among varieties was less than that of the genotypes. Overall the Shannon index showed that relative genetic diversity of the varieties was high when SCOT markers were used but it was fairly similar when ISSR and RAPD markers were applied. The results of Analysis of Molecular Variance (AMOVA) revealed that variation within groups of varieties of a country was significantly higher than among groups. These results suggest that efficiency of SCOT, ISSR and RAPD markers was relatively the same in fingerprinting of genotypes but SCOT analysis is more effective in fingerprinting of potato varieties. Overall, our results indicate that SCOT, ISSR and RAPD fingerprinting could be used to detect polymorphism for genotypes and for varieties of potato.ResumenSe compararon tres técnicas de marcadores moleculares para caracterizar molecularmente 24 variedades y a una población segregante de papa tetraploide: enfocado a codón de inicio (SCOT), repetición de secuencia inter-simple (ISSR) y amplificación al azar de ADN polimórfico (RAPD). El número de bandas polimórficas registrables producidas usando iniciadores para SCOT, ISSR y RAPD, fue mayor para las variedades que el de los genotipos. Los marcadores SCOT fueron más informativos, seguidos por los de ISSR, más que otros marcadores para el análisis de variedades con base al contenido de información del polimorfismo (PIC). No hubo diferencias significativas entre estos marcadores en términos de la evaluación de genotipos. Todas las técnicas de marcación ilustraron individualmente que el Índice de Diversidad y el índice de marcador para las variedades fue mayor que el de los genotipos, y SCOT tuvo superioridad sobre otros marcadores. El poder de resolución (Rp) de las técnicas SCOT, ISSR y RAPD fue de 71.25, 46.62 y 30.63 para las variedades y de 21.38, 18.83 y 18.87 para los genotipos, respectivamente. El coeficiente estándar de similitud de Jaccard de cada técnica de marcador reveló que la similitud entre variedades era menor que la de los genotipos. En general, el índice Shannon mostró que la diversidad genética relativa de las variedades era alta cuando se usaban los marcadores SCOT, pero era muy similar cuando se aplicaban los marcadores ISSR y RAPD. Los resultados del Análisis de Varianza Molecular (AMOVA) revelaron que la variación dentro de grupos de variedades de un país era significativamente más alta que entre grupos. Estos resultados sugieren que la eficiencia de los marcadores SCOT, ISSR y RAPD fue relativamente la misma en la caracterización de los genotipos, pero el análisis SCOT es más efectivo en la caracterización molecular de las variedades. En general, nuestros resultados indican que la caracterización SCOT, ISSR y RAPD puede usarse para detectar el polimorfismo para genotipos y para variedades de papa.

Analysis of phylogenetic relationships in the genus Solanum (Solanaceae) as revealed by RAPD markers

Phylogenetic relationships and genetic variation were examined in the genus Solanum based on the random amplified polymorphic DNA (RAPD) technique. Genetic distances were estimated for 42 accessions from five subgenera [Archaesolanum, Minon (Syn. Brevantherum), Leptostemonum, Potatoe, and Solanum]. This investigation provided new information and reinforced some suggestions from previous phylogenetic studies. Analysis with random markers from the total genome clearly separated Solanum sect. Dulcamara, from the other members of Solanum subg. Potatoe, and indicated that among the analysed Solanum subgenera subg. Solanum is most closely related to it. The results suggest that Solanum sect. Dulcamara should be excluded from Solanum subg. Potatoe. The subclusters formed by S. rostratum and S. citrullifolium appear to be distinct from the subcluster formed by the two accessions of S. sisymbriifolium. This topology indicates that Solanum sect. Androceras and Solanum sect. Cryptocarpum are fairly closely related, although the data suggest that the two sections should not be maintained.

Development of intron targeting (IT) markers for potato and cross-species amplification in Solanum nigrum (Solanaceae).

UNLABELLED PREMISE OF THE STUDY Intron Targeting (IT) primers were developed for potato using expressed sequence tags (EST) and NCBI database records to study genetic diversity. • METHODS AND RESULTS Twenty-nine polymorphic intron targeting (IT) markers were generated and characterized from 30 samples of potato and 22 samples of Solanum nigrum to detect polymorphism. The number of alleles (A) per locus ranged from 2 to 7 in the analyzed populations, and the observed heterozygosity (H(O)) and expected heterozygosity (H(E)) from 0 to 0.833 and 0.750, respectively. All of the primers also amplified in the related species S. nigrum. • CONCLUSIONS The developed markers will provide valuable tools for genetic diversity analysis, genetic mapping, and marker-assisted breeding of potato and related Solanum species.

Leaf-spot disease on European mistletoe (Viscum album) caused by Phaeobotryosphaeria visci: a potential candidate for biological control.

Viscum album (European mistletoe), a perennial, evergreen, hemiparasitic shrub, infects a wide range of woody species. It adversely affects the height and diameter of growth and it is associated with increased mortality of its hosts. There is no effective control methods against it. We have found a specific hyperparasitic fungus, which can completely destroy European mistletoe by infecting its branches, leaves and berries. Both morphological and molecular identification, based on ribosomal internal transcribed spacer sequences (rDNA-ITS), established its identity as Phaeobotryosphaeria visci. Our analysis also revealed unexpected ITS variability, as compared to the previous studies, that needs to be considered in identifying of this pathogen. Because of its efficient pathogenicity this fungus might be a good candidate for biological control of mistletoe.

Study of the origin of the rarely cultivated edible Solanum species: morphological and molecular data

The present study applies RAPD technique and morphometric analysis to study the diversity of some accessions belonging to section Solanum. A total of 252 products were amplified with 23 12-mer arbitrary primer pairs, among which 210 were found to be polymorphic. Sixteen morphological characters were measured and used to compile a dendrogram. Both the morphological and RAPD marker analysis clearly separated the different accessions into similar groups. The results indicate that the analyzed cultivars with unknown origin could be derived from S. retroflexum. We found morphological differences among the S. scabrum subsp. scabrum accession which were not reflected in the molecular data. Presumably these accessions represent cultivated forms selected for their habit, fruit quantity and/or quality and leaf size, respectively.

Genetic Variability of Thermal Nymphaea (Nymphaeaceae) Populations Based on ISSR Markers: Implications on Relationships, Hybridization, and Conservation

The globally widespread genus Nymphaea exhibits a wide range of morphological and taxonomical diversity. The intrusion of a cultivated variety by progressive propagation and its affect on aquatic habitat is demonstrated in this case study. We have studied the genetic diversity, population, and stand structure of the neophyte Nymphaea × ‘Panama Pacific’ as well as other species found in Lake Hévíz and dikes nearby using inter-simple sequence repeat (ISSR) markers. The ISSR assay revealed a low genetic variability for the small populations of Nymphaea caerulea, Nymphaea lotus var. thermalis, and a medium level for Nymphaea alba, Nymphaea rubra var. longiflora, and Nymphaea × ‘Panama Pacific’. The evolutionary genetic status of individuals found in the overlapping cultivation area of Nymphaea × ‘Panama Pacific’ and N. caerulea was affirmed to be of hybrid origin by reticulate network analysis and with morphological parameters. The Bayesian analysis of hybrid classes and the segregation of the ISSR markers also confirmed the hybrid origin of the individuals in question and revealed that they are falling into F2 or latter genotype frequency classes, indicating the viability and fertility of the hybrids. The set of analyzed species by phylogenetic network analysis of ISSR data has been divided into three major groups according to their evolutionary patterns (subg. Barachyceras, Lotos, and Nymphaea). Our results are in accordance with these three major subgenera within Nymphaea.

Molecular characterization of atrazine resistance in common ragweed (Ambrosia artemisiifolia L.)

Common ragweed (Ambrosia artemisiifolia L.) is the most frequent weed in the Carpathian Basin and is spreading fast in other parts of Europe. In recent years, besides the wild type, a mutant genotype resistant to atrazine herbicides has evolved and is now widespread in many areas. The present study demonstrates that the atrazine resistance of ragweed is maternally inherited, and is caused by a point mutation inthe psb A chloroplast gene. The promoter 5′-untranslated region and the open reading frame regions of the gene were analysed, and a homology search was performed. Both the atrazine-resistant and susceptible types of cpDNA were present in atrazine-resistant plants, while the mixed presence of both genotypes in the same plant, known as heteroplasmy, was not unequivocally detectable in susceptible plants.

Next generation sequencing based development of intron-targeting markers in tetraploid potato and their transferability to other Solanum species.

Intron-targeting (IT) markers were developed from next generation sequencing (NGS) derived transcript sequencing data from the potato cultivar White Lady. The applicability of the IT markers was analyzed in other potato genotypes, and their transferability was studied in other Solanum species: section Archaesolanum (5 species), sect. Solanum (6 species) and a Solanum nigrum population (11 genotypes). Out of 250 randomly chosen transcript sequences, 144 intron harboring loci could be identified for which primer pairs were designed on exons flanking the putative introns. The usefulness of the IT primers was experimentally analyzed on a subset of 40 randomly chosen loci. Statistical analysis of diversity parameters was performed using the ATETRA and POPGENE software packages. By localizing the detected 17 polymorphic loci 11 of the 12 potato chromosomes could be identified. Specificity of the designed IT primers was tested by sequence analysis of amplified IT fragments in a randomly chosen locus. The results revealed the efficiency of NGS derived IT marker development and indicated their utility in diverse molecular analyses including their applicability for cross-species studies.

Development of Molecular Tools for Distinguishing Between the Highly Similar Rx1 and Rx2 PVX Extreme Resistance Genes in Tetraploid Potato

The Rx1 and Rx2 are extreme resistance genes, which have been introgressed from different species into potato cultivars and breeding lines. These two genes have a 98% and 96% sequence similarity at the nucleotide as well as at the amino acid level, respectively. Except one extra amino acid in the Rx2 gene, the high variations of the amino acid chain are due to single and double nucleotide variations, which are scattered throughout the coding regions. The high level of sequence similarity makes it complicated to identify these genes and to distinguish them from other highly similar genes, like the Gpa2 or from paralogous sequences by a single polymerase chain reaction (PCR). Here, we report the development of markers for the simple and rapid identification of the Rx1 as well as the Rx2 gene. Further, a multiplex PCR reaction is recommended for the simultaneous detection of both genes in a single reaction. Since these genes reside on different chromosomes, following their inheritance by the multiplex PCR method could help the easy incorporation of both genes into breeding lines. The detection method shown here could be routinely used in marker-assisted selection for Potato virus X extreme resistance and could enhance the effectiveness of potato breeding programs. Besides potato breeding, this method could also be effectively applied to mapping experiments as well as in research studies of resistance.