Lolium grasses are widely distributed and used, and their economic importance is significant. We report the development of Lolium single nucleotide polymorphism (SNP) markers that were derived from barley EST data and that were suitable for allele quantification (AQ) via pyrosequencing (PSQ). Polymorphism information content (PIC) values for 22 developed markers ranged between 0.23 and 0.93 (average PIC value 0.80). The markers were used to characterize a collection of 2829 Lolium accessions. Eleven SNP markers could be mapped in the Lolium perenne VrnA mapping population. This is the first report on fingerprinting a comprehensive genebank collection by quantification of SNP alleles. The markers clearly distinguish between L. perenne and L. multiflorum and will prove as very useful for the management of germplasm collections.

Our investigations combine chromosome 5A copy number variation associated with relative 5A Q gene expression and morphological and agronomic data to characterize the occurrence of speltoid plants in winter wheat cultivars.

Breeding for field resistance to Septoria tritici blotch (STB), caused by Mycosphaerella graminicola (anamorph: Septoria tritici), is the most suitable strategy for controlling this important disease of wheat. Although many Stb genes for resistance to single pathogen isolates have been identified in wheat, knowledge of their efficiency against natural fungal populations is lacking. In a quantitative-trait-locus (QTL) mapping approach in six environments and four locations, field resistance to STB was studied in a doubled-haploid population derived from a cross between the field-resistant cultivar Solitär and the susceptible cultivar Mazurka. After plant height as a disease escape trait was accounted for, five QTL with effects on STB response on chromosomes 5A, 6D and 7D explained 20 % of the genotypic variance; QTL × environment interactions were minor. Field resistance was conferred exclusively by alleles from Solitär, which was previously shown to carry the isolate-specific genes Stb6 and Stb11 as well as minor QTL detected with seven fungal isolates. Surprisingly, neither the Stb6 nor Stb11 isolate-specific genes nor minor QTL previously detected in Solitär were found to be involved in its field resistance. The study suggests that resistance breeding for STB should not rest solely on the deployment of Stb genes. Field tests are indispensable to show their efficacy and durability and to identify genes conferring partial field resistance to STB.

Breeding for resistance to Septoria tritici blotch (STB), caused by Mycosphaerella graminicola (anamorph: Septoria tritici), is an essential component in controlling this important foliar disease of wheat. Inheritance of seedling resistance to seven worldwide pathogen isolates has been studied in a doubled-haploid (DH) population derived from a cross between the field resistant cultivar Solitär and the susceptible cultivar Mazurka. Multiple quantitative trait locus (QTL) mapping revealed major and minor genetic effects on resistance as well as several epistatic relationships in the seedling stage. Solitär conferred resistance to isolate IPO323, governed by Stb6 on chromosome 3A, as well as to IPO99015, IPO92034, Hu1 and Hu2 controlled by a QTL on chromosome arm 1BS, possibly corresponding to Stb11 and minor QTL on chromosomes 1B, 3D, 6B and 7D. Resistance of Mazurka to IPO90015 and BBA22 was caused by a QTL located in a region on 4AL which harbours Stb7 or Stb12. QTL specific to pycnidial coverage on 3B and specific to necrosis on 1A could be discovered for isolate IPO92034. Pairwise epistatic interactions were reliably detected with five isolates. Although their contributions to the total variance are generally low, the genotypic effect of the QTL by QTL interaction of 4AL (Stb7 or Stb12) and 3AS (Stb6) made up almost 15% of disease expression. Altogether, the results suggest a complex inheritance of resistance to STB in the seedling stage in terms of isolate-specificity and resistance mechanisms, which have implications for marker-assisted breeding in an attempt to pyramid STB resistance genes.

A large collection of German rye leaf rust isolates was analysed to characterize the diversity, spatial variation and temporal dynamics of virulences. Virulence-avirulence phenotypes (=pathotypes) were determined on 23 host differentials. We found 93 pathotypes among 177 single-uredinial isolates in 2000, 201 pathotypes among 437 isolates in 2001, and 125 pathotypes among 213 isolates in 2002. In total, the 827 analyzed isolates represented 317 pathotypes. Frequency of virulences on the individual differentials varied from 2% to 97%. Eight of the differentials showed a high resistance level with virulence frequencies < 10%. Virulence complexity of the isolates ranged from 3 to 21 with a mean of nine. The percentages of highly virulent isolates (> 14 virulences) increased from 4 to 15% during the sampling period. A high level of virulence diversity was observed within and between individual sampling sites with Simpson indices around 0.9. Evenness indices ranged from 0.88 to 0.92. Four of the five most frequent pathotypes were found in each year but their frequency never exceeded 10%. Isolates with unusual virulence combinations could be clearly separated by principal component analysis. Location-specific pathotype frequencies were revealed in each year, but the frequency patterns varied across years. On four fields a considerable increase of highly virulent pathotypes occurred within 6 weeks during the epidemic. The high diversity of pathotypes as well as the fast accumulation of highly virulent pathotypes favour the adaptation of the pathogen to race-specific host resistances. More durable resistance might be achievable by combining new effective race-specific resistances with adult-plant and/or race-non-specific quantitative resistances.

Speltoid spikes are characterized by pyramidal spike morphology featuring an elongated rachis and tenacious glumes. Speltoids are considered undesirable spike aberrants in wheat breeding leading to increased heterogeneity within a cultivar candidate. As a consequence, the presence of speltoids may result in rejection of a cultivar candidate during official field trials or denial of cultivar certification during seed multiplication. A reliable method is, thus, required to assess the occurrence of speltoids, early on in a wheat breeding program. The domestication gene Q located on the long arm of wheat chromosome 5A is known to suppress the speltoid phenotype in wheat. Here, a quantitative pyrosequencing assay was developed to distinguish between normal wheat plants, which possess two copies of the Q allele, and aberrants, which are either aneuploids lacking the correct number of chromosome 5A copies or plants which carry the primitive q allele. An accurate and reproducible determination of the Q gene copy number was achieved for different wheat genotypes based on homoeologous sequence quantification with two primer combinations at the Q locus. Single plants with one to four copies of the Q allele could be detected by quantitative pyrosequencing which corresponded to the occurrence of speltoid (1 Q allele), normal (2 Q alleles), and compact (more than 2 Q alleles) spikes. Q and q specific alleles could be differentiated at SNP position 2299 of the Q gene. This SNP is assumed to be related to the emergence of free-threshing wheat forms. To our knowledge this is the first report for detection of aneuploids and differentiation of Q alleles in bread wheat using pyrosequencing technology. In future, quantitative pyrosequencing assay can be applied in wheat breeding programs to carry out marker-assisted selection against the presence of speltoid spike aberrants.

A panel of 13 sugar beet lines and one genotype each of the Beta vulgaris cultivars red beet and Swiss chard, and B. vulgaris ssp. maritima were used to identify polymorphisms in alignments of genomic DNA sequences derived from 315 EST- and 43 non-coding RFLP-derived loci. In sugar beet lines, loci of expressed genes showed an average SNP frequency of 1/72 bp, 1 in 58 bp in non-coding sequences, increasing to 1/47 bp upon the addition of the remaining genotypes. Within analysed DNA fragments, alleles at different SNP positions displayed linkage disequilibrium indicative of haplotype structures. On average 2.7 haplotypes were found in sugar beet lines, and haplotype conservation in expressed genes appeared to exceed 500 bp in length. Seven different genotyping techniques including SNP detection by MALDI-TOF mass spectrometry, pyrosequencing and fluorescence scanning of labelled nucleotides were employed to perform 712 segregation analyses for 538 markers in three F-2 populations. Functions were predicted for 492 mapped sequences. Genetic maps comprised 305 loci covering 599.8 cM in population K1, 241 loci distributed over 636.6 cM in population D2, and 166 loci over 507.1 cM in population K2, respectively. Based on 156 markers common to more than one population an integrated map was constructed with 524 loci covering 664.3 cM. For 377 loci the genome positions of the most similar sequences from A. thaliana were identified, but little evidence for previously presented ancestral genome structures was found.

We established a chromosome specific DNA library of the Aegilops markgrafii chromosome B. Eight microdissected chromosomes B obtained from a monosomic T. aestivum-Aegilops markgrafii addition line were PCR-amplified and the DNA was cloned in Escherichia coli DH5α. Clones were characterized by dot blot hybridization with total Ae. markgrafii DNA. 62% of clones represented repetitive sequences and 38% low or single copy sequences. The estimated length of excised inserts varied between less than 200 bp and more than 500 bp. The average size of inserts was 310 bp.