Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.).

BACKGROUND: OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. RESULTS: Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. CONCLUSIONS: Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies.

Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (Brassica napus L.).

Single-nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs) are currently the important classes of genetic markers for major crop species. In this study, methods for developing SNP markers in rapeseed (Brassica napus L.) and their in silico mapping and use for genotyping are demonstrated. For the development of SNP and INDEL markers, 181 fragments from 121 different gene sequences spanning 86 kb were examined. A combination of different selection methods (genome-specific amplification, hetero-duplex analysis and sequence analysis) allowed the detection of 18 singular fragments that showed a total of 87 SNPs and 6 INDELs between 6 different rapeseed varieties. The average frequency of sequence polymorphism was estimated to be one SNP every 247 bp and one INDEL every 3,583 bp. Most SNPs and INDELs were found in non-coding regions. Polymorphism information content values for SNP markers ranged between 0.02 and 0.50 in a set of 86 varieties. Using comparative genetics data for B. napus and Arabidopsis thaliana, an allocation of SNP markers to linkage groups in rapeseed was achieved: a unique location was determined for seven gene sequences; two and three possible locations were found for six and four sequences, respectively. The results demonstrate the usefulness of existing genomic resources for SNP discovery in rapeseed.

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling.

BACKGROUND: The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions. RESULTS: By employing time course microarray experiments we identified 68 significantly differentially expressed sequences within the different time points. The majority of differentially expressed genes differed between the near-isogenic line carrying Scmv1 resistance locus at chromosome 6 and the other isogenic lines. Most differentially expressed genes in the SCMV experiment (75%) were identified one hour after virus inoculation, and about one quarter at multiple time points. Furthermore, most of the identified mapped genes were localised outside the Scmv QTL regions. Annotation revealed differential expression of promising pathogenesis-related candidate genes, validated by qRT-PCR, coding for metallothionein-like protein, S-adenosylmethionine synthetase, germin-like protein or 26S ribosomal RNA. CONCLUSION: Our study identified putative candidate genes and gene expression patterns related to resistance to SCMV. Moreover, our findings support the effectiveness and reliability of the combination of different expression profiling approaches for the identification and validation of candidate genes. Genes identified in this study represent possible future targets for manipulation of SCMV resistance in maize.

Variation in DNA methylation patterns of grapevine somaclones (Vitis vinifera L.).

BACKGROUND: In traditional vine areas, the production should present a typicity that partly depends on the grapevine variety. Therefore, vine improvement is considered difficult because of the limited choice in the natural variability of the cultivars within the limits of their characteristics. A possibility to circumvent this problem is the use of somatic variability. In vitro somatic embryogenesis and organogenesis can lead to genotypic and phenotypic variations, described as somaclonal variation, that could be useful for the selection of improved grapevine genotypes. RESULTS: In order to study tissue culture-induced variation of grapevine, we have analysed 78 somaclones obtained from somatic embryos of two distinct cultivars using molecular marker techniques. SSRs were only useful to verify the conservation of the microsatellite genotype between the somaclones and the respective mother clones. AFLP polymorphism between mother clones and somaclones was 1.3-2.8 times higher to that found between clones. However, a majority of the somaclones (45/78) exhibited only few changes. Seven and five somaclones of 'Chardonnay 96' and 'Syrah 174', respectively, which covered at least all polymorphic loci found in AFLP analysis were used for MSAP study. All of the 120 polymorphic fragments were found only in the somaclones. The percentage of full methylation at CCGG recognition sites was slightly higher in somaclones due to more polymorphic bands generated after cleavage by EcoRI/HpaII. Different digestion patterns revealed different methylation status, especially different levels of de-methylation, that are the consequence of the in vitro culture. CONCLUSION: MSAP highlights DNA methylation variation in somaclones compared to mother clones and, therefore, is a powerful tool for genotypic characterisation of somatic embryo-derived grapevines. The detection of the same polymorphic bands in numerous somaclones of different cultivars suggests the possibility of hot spots of DNA methylation variation. SSR profiles of the 'Chardonnay' and 'Syrah' somaclones were the same as of the respective mother clones. The somaclones exhibited a higher AFLP variation than clones obtained via traditional clonal selection in the field. Therefore, somatic embryogenesis through in vitro culture technique could be useful for the selection of improved cultivars with subtle changes but conserving their main characteristics.

Zeaxanthin is bioavailable from genetically modified zeaxanthin-rich potatoes.

The carotenoid zeaxanthin accumulates in the human macula lutea and protects retinal cells from blue light damage. However, zeaxanthin intake from food sources is low. Increasing zeaxanthin in common foods such as potatoes by traditional plant breeding or by genetic engineering could contribute to an increased intake of this carotenoid and, consequently, to a decreased risk of age-related macular degeneration. Our aim was to investigate whether zeaxanthin from genetically modified zeaxanthin-rich potatoes is bioavailable in humans. Three men participated in this randomized, controlled double-blinded, crossover pilot study. All subjects consumed 1,100 g of mashed potatoes, either genetically modified (Solanum tuberosum L. var. Baltica GM47/18; 3 mg zeaxanthin) or wild-type control potatoes (Solanum tuberosum L. var. Baltica; 0.14 mg zeaxanthin). A second treatment was followed after a 7-day wash-out period. The concentration of zeaxanthin was significantly increased in chylomicrons after consumption of genetically modified potatoes and 0.27 mg of the 3 mg zeaxanthin dose could be detected in chylomicrons. Consumption of control potatoes had no effect on concentrations of zeaxanthin in chylomicrons. After normalization of chylomicron zeaxanthin for plasma triacylglycerol, the time course of zeaxanthin concentrations peaked at 7 h after consumption of genetically modified potatoes. There were no significant differences in the concentrations of other major potato carotenoids such as lutein and beta-carotene in chylomicrons after consumption of genetically modified and wild type control potatoes. Thus, consumption of zeaxanthin-rich potatoes significantly increases chylomicron zeaxanthin concentrations suggesting that potentially such potatoes could be used as an important dietary source of zeaxanthin.

Phytophthora infestans-triggered response of growth- and defense-related genes in potato cultivars with different levels of resistance under the influence of nitrogen availability.

The effects of high and low N concentrations on the Solanum tuberosum-Phytophthora infestans interaction were studied in the potato cultivars Bettina, New York 121, Indira and Arkula, which exhibited different levels of resistance. Aboveground biomass and Chl and N content were significantly higher in all cultivars grown in higher N environments, while C:N ratios were lower, confirming successful application of N. High availability of N significantly increased susceptibility of three of the four potato cultivars, and amounts of pathogen within the infected leaflets determined in a quantitative real-time reverse transcriptase-polymerase chain reaction reflected this. Differential gene expression of P. infestans-induced and -repressed genes derived from three subtracted cDNA libraries at 0, 24, 48 and 72 h post-inoculation was studied in parallel. P. infestans attack led to an induction of defense-related and at the same time repression of growth-related potato genes mainly encoding photosynthetic genes. High N supply led to higher transcript abundance of photosynthetic genes such as Chl a/b-binding protein and ribulose bisphosphate carboxylase. N-dependent suppression of defense-related compounds in absence of the pathogen was not observed. Better N nutrition appeared to allow the plants to invest more resources in defense reactions.

Characterization of phenylpropanoid pathway genes within European maize (Zea mays L.) inbreds.

BACKGROUND: Forage quality of maize is influenced by both the content and structure of lignins in the cell wall. Biosynthesis of monolignols, constituting the complex structure of lignins, is catalyzed by enzymes in the phenylpropanoid pathway. RESULTS: In the present study we have amplified partial genomic fragments of six putative phenylpropanoid pathway genes in a panel of elite European inbred lines of maize (Zea mays L.) contrasting in forage quality traits. Six loci, encoding C4H, 4CL1, 4CL2, C3H, F5H, and CAD, displayed different levels of nucleotide diversity and linkage disequilibrium (LD) possibly reflecting different levels of selection. Associations with forage quality traits were identified for several individual polymorphisms within the 4CL1, C3H, and F5H genomic fragments when controlling for both overall population structure and relative kinship. A 1-bp indel in 4CL1 was associated with in vitro digestibility of organic matter (IVDOM), a non-synonymous SNP in C3H was associated with IVDOM, and an intron SNP in F5H was associated with neutral detergent fiber. However, the C3H and F5H associations did not remain significant when controlling for multiple testing. CONCLUSION: While the number of lines included in this study limit the power of the association analysis, our results imply that genetic variation for forage quality traits can be mined in phenylpropanoid pathway genes of elite breeding lines of maize.

Identification of candidate genes associated with cell wall digestibility and eQTL (expression quantitative trait loci) analysis in a Flint x Flint maize recombinant inbred line population.

BACKGROUND: Cell-wall digestibility is the major target for improving the feeding value of forage maize. An understanding of the molecular basis for cell-wall digestibility is crucial towards breeding of highly digestible maize. RESULTS: 865 candidate ESTs for cell-wall digestibility were selected according to the analysis of expression profiles in 1) three sets of brown-midrib isogenic lines in the genetic background of inbreds 1332 (1332 and 1332 bm3), 5361 (5361 and 5361 bm3), and F2 (F2, F2 bm1, F2 bm2, and F2 bm3), 2) the contrasting extreme lines of FD (Flint x Dent, AS08 x AS 06), DD1 (Dent x Dent, AS11 x AS09), and DD2 (Dent x Dent, AS29 x AS30) mapping populations, and 3) two contrasting isogenic inbreds, AS20 and AS21. Out of those, 439 ESTs were assembled on our "Forage Quality Array", a small microarray specific for cell wall digestibility related experiments. Transcript profiles of 40 lines of a Flint x Flint population were monitored using the Forage Quality Array, which were contrasting for cell wall digestibility. Using t-tests (p < 0.01), the expression patterns of 102 ESTs were significantly different between high and low quality groups. Using interval mapping, eQTL (LOD > or = 2.4) were detected for 20% (89 of 439) of the spotted ESTs. On average, these eQTL explained 39% of the transcription variation of the corresponding ESTs. Only 26% (23 of 89) ESTs detected a single eQTL. eQTL hotspots, containing greater than 5% of the total number of eQTL, were located in chromosomal bins 1.07, 1.12, 3.05, 8.03, and 9.04, respectively. Bin 3.05 was co-localized with a cell-wall digestibility related QTL cluster. CONCLUSION: 102 candidate genes for cell-wall digestibility were validated by genetical genomics approach. Although the cDNA array highlights gene types (the tested gene and any close family members), trans-acting factors or metabolic bottlenecks seem to play the major role in controlling heritable variation of gene expression related to cell-wall digestibility, since no in silico mapped ESTs were in the same location as their own eQTL. Transcriptional variation was generally found to be oligogenic rather than monogenic inherited due to only 26% ESTs detected a single eQTL in the present study. One eQTL hotspot was co-localized with cell wall digestibility related QTL cluster on bins 3.05, implying that in this case the gene(s) underlying QTL and eQTL are identical. As the field of genetical genomics develops, it is expected to significantly improve our knowledge about complex traits, such as cell wall degradability. Comprehensive knowledge of the lignin pathway and cell wall biogenesis will allow plant breeders to choose the best genomic targets controlling these characters, for improving forage digestibility through genetic engineering or marker-assisted selection.

Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres.

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.

Comparative expression profiling in meristems of inbred-hybrid triplets of maize based on morphological investigations of heterosis for plant height.

Heterosis, the superior performance of hybrids as compared to their parental mean is an agronomically important phenomenon well-described morphologically. However, little is known about its molecular basis. We investigated four genetically unrelated maize (Zea mays L.) inbred lines and their F(1) crosses both at the phenotype and transcriptome level, focusing on plant height (PHT) component traits. Substantial mid-parent heterosis (MPH) was found for all parent-hybrid triplets for PHT in the range of 37.9-56.4% in the field and 11.1-39.5% under controlled greenhouse conditions. Analyses of heterosis for number and length of internodes showed two to three times higher MPH in the field as compared to the greenhouse. All three traits exhibited high heritabilities, highest for PHT 95-98%. Two methods for gene expression quantification were applied. High-density cDNA uni-gene microarrays containing 11,827 ESTs were utilized for the selection of differentially expressed genes related to heterosis for PHT. For the four triplets with eight possible parent-hybrid comparisons we identified 434 consistently differentially expressed genes with a p < or = 0.05. Microarray results were used to verify the dominance/overdominance hypothesis. In our study, more than 50% genes showed overdominance, 26% partial dominance, 12.6% complete dominance and 10.2% additive gene action. Moreover, more consistently differentially expressed genes were detected in related triplets, sharing one parent, than in unrelated triplets. Quantitative RT-PCR was applied in order to validate microarray results. The role of the differentially expressed genes in relation to heterosis for PHT is discussed.

High levels of linkage disequilibrium and associations with forage quality at a phenylalanine ammonia-lyase locus in European maize (Zea mays L.) inbreds.

Forage quality of maize is influenced by both the content and structure of lignin in the cell wall. Phenylalanine Ammonia-Lyase (PAL) catalyzes the first step in lignin biosynthesis in plants; the deamination of L-phenylalanine to cinnamic acid. Successive enzymatic steps lead to the formation of three monolignols, constituting the complex structure of lignin. We have cloned and sequenced a PAL genomic sequence from 32 maize inbred lines currently employed in forage maize breeding programs in Europe. Low nucleotide diversity and excessive linkage disequilibrium (LD) was identified at this PAL locus, possibly reflecting selective constrains resulting from PAL being the first enzyme in the monolignol, and other, pathways. While the association analysis was affected by extended LD and population structure, several individual polymorphisms were associated with neutral detergent fiber (not considering population structure) and a single polymorphism was associated with in vitro digestibility of organic matter (considering population structure).

Comparison of maize brown-midrib isogenic lines by cellular UV-microspectrophotometry and comparative transcript profiling.

The molecular mechanisms underlying cell wall digestibility in maize (Zea mays L.) have been studied in three sets of maize brown-midrib isogenic lines in the genetic background of inbreds 1332 (1332 and 1332 bm3), 5361 (5361 and 5361 bm3), and F2 (F2, F2 bm1, F2 bm2, and F2 bm3). Two complementary approaches, SSH (suppression subtractive hybridization) and microarray-based expression profiling, were used to isolate and identify candidate genes in isogenic lines for bm mutants. Metabolic pathway analysis revealed that transcriptional events caused by altering the expression of a single bm gene involve all metabolic and signaling pathways. 53 ESTs were differentially expressed in all three isogenic bm3 comparisons, whereas 32 ESTs were consistently differentially expressed in different bm isogenic lines in F2 background. About 70% ESTs isolated by SSH were not present on the unigene microarray, demonstrating the usefulness of the SSH procedure to identify genes related to cell wall digestibility. Together with lignin analysis by cellular UV-microspectrophotometry, expression profiling in isogenic bm lines proved to be useful to understand alterations at the sub-cellular and molecular level with respect to lignin composition. The down-regulation of COMT affected the expression of CCoAOMT genes and caused a reduced content both of G and S units in bm3 mutants.

Molecular plant breeding: achievements in green biotechnology and future perspectives.

Since one decade ago, transgenic crop plants are globally grown; in 2004, it was estimated to cover a total of 81 Mio ha in 17 countries. At present, four plant species (soybean, maize, cotton and rapeseed) dominate with two traits (herbicide tolerance and insect resistance). The traits on which research concentrates and the constructs which might come next onto the market are outlined. The procedure on how to clone such genes of interest, e.g. via map-based cloning, and some other helpful approaches of green biotechnology, like high throughput techniques and functional markers, are summarised, and a rough calculation about the market value of transgenic crops in US dollars is quoted.

Mapping of extreme resistance to PVY (Ry (sto)) on chromosome XII using anther-culture-derived primary dihaploid potato lines.

The inheritance of extreme resistance to PVY (Ry (sto)) by a single dominant locus was confirmed by obtaining a 1:1 segregation ratio in a virus inoculation test with 28 resistant (Ryry) to 29 susceptible (ryry) anther culture-derived dihaploid lines (2n=2x=24) from cv. "Assia" (2n=4x=48) having extreme resistance derived from Solanum stoloniferum in simplex constitution (Ryryryry). Twelve Ry (sto) markers selected in AFLP assays using bulked segregant analysis were applied to 106 tested potato cultivars from Germany, The Netherlands and Poland and 19 potato cultivars were identified by these markers as extremely resistant to PVY in alignment with phenotypic data. The locus for extreme resistance (Ry (sto)) to PVY was mapped on chromosome XII co-segregating with the SSR marker STM 0003. The utility of anther-culture derived dihaploid potatoes for genetic marker development was demonstrated. Marker transferability from diploids to tetraploids provides an optimistic potential for marker-assisted selection in potato breeding programs.

Identification by suppression subtractive hybridization of genes that are differentially expressed between near-isogenic maize lines in association with sugarcane mosaic virus resistance.

The molecular mechanisms underlying the development and progression of sugarcane mosaic virus (SCMV) infection in maize are poorly understood. A study of differential expression was conducted to identify genes involved in resistance to SCMV. In this study, we combined suppression subtractive hybridization and macroarray hybridization to identify genes that are differently expressed in the near isogenic lines F7+ (SCMV resistant) and F7 (susceptible). Altogether, 302 differentially expressed genes were identified in four comparisons based on constitutively expressed and inducible genes, and on compatible and incompatible plant-virus interactions. Apart from genes related to metabolism, most of the functionally classified genes identified belonged to three pathogenesis-related categories: cell rescue, defense, cell death and ageing, signal transduction, and transcription. The latter three groups accounted for 56-66% of the genes classified. Some 19% (60 of 302) of the identified genes had previously been assigned to 29 bins distributed over all ten maize chromosomes. Among the mapped genes, 31% (18 of 58) were located within the Scmv2 and Scmv1 regions on chromosomes 3 and 6, respectively, which have been associated with resistance to SCMV. Promising candidate genes for Scmv1 have been identified, such as AA661457 (receptor-like kinase Xa21-binding protein 3). The implications of the genomic distribution of differentially expressed genes identified by this isogenic comparison are discussed in the context of breeding for resistance.

Determination of detection and quantification limits for SNP allele frequency estimation in DNA pools using real time PCR.

The quantification of single nucleotide polymorphism (SNP) allele frequencies in pooled DNA samples using real time PCR is a promising approach for large-scale diagnostics and genotyping. The limits of detection (LOD) and limits of quantification (LOQ) for mutant SNP alleles are of particular importance for determination of the working range, which, in the case of allele-specific real time PCR, can be limited by the variance of calibration data from serially diluted mutant allele samples as well as by the variance of the 100% wild-type allele samples (blank values). In this study, 3sigma and 10sigma criteria were applied for the calculation of LOD and LOQ values. Alternatively, LOQ was derived from a 20% threshold for the relative standard deviation (%RSD) of measurements by fitting a curve for the relationship between %RSD and copy numbers of the mutant alleles. We found that detection and quantification of mutant alleles were exclusively limited by the variance of calibration data since the estimated LOD(calibration) (696 in 30 000 000 copies, 0.0023%), LOQ(20%RSD) (1470, 0.0049%) and LOQ(calibration) (2319, 0.0077) values were significantly higher than the LOD(blank) (130, 0.0004%) and LOQ(blank) (265, 0.0009%) values derived from measurements of wild-type allele samples. No significant matrix effects of the genomic background DNA on the estimation of LOD and LOQ were found. Furthermore, the impact of large genome sizes and the general application of the procedure for the estimation of LOD and LOQ in quantitative real time PCR diagnostics are discussed.

Analysis of differentially expressed genes in a susceptible and moderately resistant potato cultivar upon Phytophthora infestans infection.

SUMMARY To gain deeper understanding of the host-pathogen interaction in the system potato-Phytophthora infestans, subtractive hybridization in combination with cDNA array hybridization was used. Leaflets of a moderately resistant and a susceptible potato cultivar were inoculated with P. infestans. The infection of the potato leaves was quantified by real-time quantitative PCR. Using infected and control tissue, two cDNA libraries highly enriched for P. infestans-induced genes were prepared. Within 531 clones randomly picked and sequenced from the libraries, 285 unigenes were found, from which 182 clones were selected for further analysis by cDNA array hybridization. Sixteen hours post inoculation genes were not induced significantly, whereas 72 h post inoculation induction of gene expression was clearly detectable. In both cultivars, 143 genes were induced moderately (>/= two-fold), and 35 of the selected genes appeared to be strongly induced (>/= seven-fold). Among these clones were mainly genes associated with stress and/or defence mechanisms. The strongest gene induction was found in 4-week-old susceptible plants. In the moderately resistant cultivar, transcripts of a number of genes accumulate with plant age; as a result, induction of gene expression upon infection was less pronounced. Down-regulation of three genes was observed in both cultivars, upon infection. Transcript levels of these three genes increased in uninfected plants within 4 weeks of growth. Other differences in defence responses of the two cultivars could be determined and their effects are discussed.

Combined use of linked markers for genotyping the Pm1 locus in common wheat.

Genotyping of 98 wheat cultivars/lines was carried out with molecular markers that are linked to the Pm1 locus: two bi-allelic (dominant) markers: the sequence-tagged site Xsts638-7A and the amplified fragment length polymorphism XE39M58-77-7A; and the multi-allelic simple sequence repeat marker Xgwm344-7A. Employing segregation data recorded in the population Chinese Spring x Virest (Pm1e), genetic mapping revealed that Xgwm344-7A and XE39M58-77-7A were distally linked to Pm1e in the repulsion phase with respective linkage distances of 0.9 cM and 4.8 cM, while Xsts638-7A was found to co-segregate with Pm1e in the coupling phase. The genotyping results of Xsts638-7A and XE39M58-77-7A confirmed disease scoring, except for the accessions of cultivars Omega, Remus and Weihenstephan Stamm M1N. The SSR marker Xgwm344 amplified 15 different fragments ranging from 102 bp to 147 bp, with 15 entries being null-allelic at the 7A and 7B homoeoloci. It was found that wheat lines having resistance alleles at the Pm1 locus mainly show the null allele at the Xgwm344-7A locus. Due to their fast-evolving nature, the use of multi-allelic SSRs for genotype determination may be complicated. However, the combined use of multiple linked marker alleles seems to be a promising approach for genotyping a broad range of plant materials.

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat.

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.