István Cernák

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

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.

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.

Nuclear intron-targeting markers in genetic diversity analysis of black nightshade (Solanum sect. Solanum, Solanaceae) accessions

Different molecular markers are routinely used in studies of potato (Solanum tuberosum) and the genus Solanum in general. Genome sequence databases provide potential to design new markers for various applications. Here we present the application of a recently developed core set of nuclear intron-targeting (indel) markers. These markers are based on the fact that in the plant genome introns are more variable than exons; therefore primers flanking exons can reveal polymorphisms related to introns. We detected such variation among accessions of the eight different species of black nightshades (Solanum sect. Solanum). Members of this group are important sources of food, mostly in Africa, while others are poisonous weeds with near global distribution. The tested 29 primers were designed previously for potato based on Solanaceae EST and other genomic databases and targeted 16 different genes. Our results showed that Solanum intron-targeting markers are not very polymorphic but identified considerable structure among accessions indicating fairly high interspecies differentiation. Further analyses showed that inbreeding is unlikely to be the major driving force in determining the genetic structure of the analyzed species. All phylogenetic analyses resolved the species included in our study as distinct clades with high support values, but provided weak information about their internal relationships. In summary, indel markers would be useful for the assignment of new Solanum germplasm to taxonomic groups or to identify certain taxa. They could also be used to address important question about genetic diversity and should yield results comparable to other markers covering the whole genome.

Application of direct PCR in rapid rDNA ITS haplotype determination of the hyperparasitic fungus Sphaeropsis visci (Botryosphaeriaceae)

BackgroundThe plant pathogenic fungus, Sphaeropsis visci a dark-spored species of Botryosphaeriaceae, which causes the leaf spot disease of the European mistletoe (Viscum album). This species seems to have potential as a tool for biological control of the hemiparasite. For the rapid detection of S. visci haplotypes we tested a direct PCR assay without prior DNA purification. This approach was based on a polymerase enzyme from the crenarchaeon Sulfolobus solfataricus engineered by fusion protein technology, which linked the polymerase domain to a sequence non-specific DNA binding protein (Sso7d).FindingsMost isolates of Sphaeropsis visci grouped together in our phylogenetic analyses, indicating that isolates had a previously reported haplotype sequence, which is commonly found in the analyzed Hungarian population. This haplotype was also reported from diseased mistletoe bushes from other European countries. We further identified unique single nucleotide polymorphisms (SNPs) in the ITS region, which were specific to the only well resolved clade in the phylogenetic analysis.ConclusionsThe diPCR approach allowed amplification of ITS rRNA gene directly from small amounts of fungal samples without prior DNA extraction. This simple bioassay in plant disease management enables collection of genomic data from fungal plant pathogen populations.

What types of powdery mildew can infect wheat-barley introgression lines?

This work is a detailed study of the infection of fungal biotrophic pathogens causing powdery mildew diseases on introgression lines originating from the intergeneric hybridisation between wheat and barley (Triticum aestivum L. × Hordeum vulgare L.). Powdery mildew fungi are among the most widespread biotrophic pathogens of plants also and infect dicot and monocot species. Most powdery mildew species are strictly host specific. They colonize only a narrow range of species or one particular host species. The intergeneric hybridisation between wheat and barley could result in expansions of host ranges of the barley powdery mildew. Our experiments covered natural infections in the field and artificial infections under greenhouse conditions. Formae speciales of powdery mildew were identified on the basis of the sequencing results of ribosomal internal transcribed spacer sequences (rDNA-ITS). We identified Blumeria graminis f.sp. tritici isolate 14 (HM484334) on the wheat parent and all wheat-barley introgression lines and B. g. f. sp. hordei isolate MUMH1723 (AB 273556) on the barley parent, respectively. The wheat-barley introgression lines were inoculated with barley powdery mildew under greenhouse conditions. According to our results the added barley chromosomes (or segments) do not cause host range expansion of barley powdery mildew.