Quantification of Pyrenophora teres in infected barley leaves using real-time PCR.

Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R(2)=0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.

SSR allelic diversity changes in 480 European bread wheat varieties released from 1840 to 2000.

A sample of 480 bread wheat varieties originating from 15 European geographical areas and released from 1840 to 2000 were analysed with a set of 39 microsatellite markers. The total number of alleles ranged from 4 to 40, with an average of 16.4 alleles per locus. When seven successive periods of release were considered, the total number of alleles was quite stable until the 1960s, from which time it regularly decreased. Clustering analysis on Nei's distance matrix between these seven temporal groups showed a clear separation between groups of varieties registered before and after 1970. Analysis of qualitative variation over time in allelic composition of the accessions indicated that, on average, the more recent the European varieties, the more similar they were to each other. However, European accessions appear to be more differentiated as a function of their geographical origin than of their registration period. On average, western European countries (France, The Netherlands, Great Britain, Belgium) displayed a lower number of alleles than southeastern European countries (former Yugoslavia, Greece, Bulgaria, Romania, Hungary) and than the Mediterranean area (Italy, Spain and Portugal), which had a higher number. A hierarchical tree on Nei's distance matrix between the 15 geographical groups of accessions exhibited clear opposition between the geographical areas north and south of the arc formed by the Alps and the Carpathian mountains. These results suggest that diversity in European wheat accessions is not randomly distributed but can be explained both by temporal and geographical variation trends linked to breeding practices and agriculture policies in different countries.