Inheritance of field resistance to Stagonospora nodorum leaf and glume blotch and correlations with other morphological traits in hexaploid wheat (Triticum aestivum L.).

Breeding for wheat varieties resistant to Stagonospora nodorum blotch (SNB) is the most sustainable strategy for controlling the disease. In order to map quantitative trait loci (QTLs) for SNB resistance we analysed 204 recombinant inbred lines of the cross between the winter wheat (Triticum aestivum L.) variety Forno and the winter spelt (Triticum spelta L.) variety Oberkulmer. We determined the level of resistance of adult plants to leaf blotch (SNL) and glume blotch (SNG) as well as morphological traits for 2 years after artificial inoculation with S. nodorum. Using composite interval mapping and LOD > 3.7, we detected ten QTLs for SNG blotch resistance (six inherited from the susceptible parent Forno) and 11 QTLs for SNL resistance (four inherited from Forno) across 2 years. Both resistance traits were moderately correlated (r = 0.52) and had only one common QTL. For SNL resistance, seven QTLs were not associated with QTLs for morphological traits. Among them, QSnl.eth-2D, QSnl.eth-4B and QSnl.eth-7B3 had major effects (R(2) > 13%) and were potential candidates for marker-assisted selection. For SNG, the major QTL on chromosome 5A, explaining 36% of the phenotypic variance for resistance, was associated with the q locus conferring the spelt morphology (long lax ear, long culm and hard glumes). Only QSng.eth-1BS, which explained 7% of the variance for resistance to SNG blotch, was not associated with QTLs for morphological traits. The consequences for breeding programmes are discussed.

QTL analysis of resistance to Fusarium head blight in Swiss winter wheat (Triticum aestivum L.).

Fusarium head blight (FHB) of wheat is a widespread and destructive disease which occurs in humid and semi-humid areas. FHB epidemics can cause serious yield and quality losses under favorable climatic conditions, but the major concern is the contamination of grains with mycotoxins. Resistance to FHB is quantitatively inherited and greatly influenced by the environment. Its evaluation is costly and time-consuming. The genetic basis of FHB resistance has mainly been studied in spring wheat. The objective of this study was to map quantitative trait loci (QTLs) for resistance to FHB in a population of 240 recombinant inbred lines (RILs) derived from a cross between the two Swiss winter wheat cultivars Arina (resistant) and Forno (susceptible). The RILs were genotyped with microsatellite and RFLP markers. The resulting genetic map comprises 380 loci and spans 3,086 cM. The 240 RILs were evaluated for resistance to FHB in six field trials over 3 years. Composite interval mapping (CIM) analyses carried out on FHB AUDPC (i.e. mean values across six environments) revealed eight QTLs which altogether explained 47% of the phenotypic variance. The three main QTLs were mapped on the long arms of chromosomes 6D ( R(2)=22%), 5B ( R(2)=14%) and 4A ( R(2)=10%). The QTL detected on 5B originated from the susceptible parent Forno. Other QTLs with smaller effects on FHB resistance were detected on chromosomes 2AL, 3AL, 3BL, 3DS and 5AL.

Dissection of quantitative and durable leaf rust resistance in Swiss winter wheat reveals a major resistance QTL in the Lr34 chromosomal region.

The Swiss winter bread wheat cv. 'Forno' has a highly effective, durable and quantitative leaf rust ( Puccinia triticina Eriks.) resistance which is associated with leaf tip necrosis (LTN). We studied 240 single seed descent lines of an 'ArinaxForno' F(5:7 )population to identify and map quantitative trait loci (QTLs) for leaf rust resistance and LTN. Percentage of infected leaf area (%) and the response to infection (RI) were evaluated in seven field trials and were transformed to the area under the disease progress curves (AUDPC). Using composite interval mapping and LOD >4.4, we identified eight chromosomal regions specifically associated with resistance. The largest and most consistent leaf rust resistance locus was identified on the short arm of chromosome 7D (32.6% of variance explained for AUDPC_% and 42.6% for AUDPC_RI) together with the major QTL for LTN ( R(2)=55.6%) in the same chromosomal region as Lr34 ( Xgwm295). A second major leaf rust resistance QTL ( R(2)=28% and 31.5%, respectively) was located on chromosome arm 1BS close to Xgwm604 and was not associated with LTN. Additional minor QTLs for LTN (2DL, 3DL, 4BS and 5AL) and leaf rust resistance were identified. These latter QTLs might correspond to the leaf rust resistance genes Lr2 or Lr22 (2DS) and Lr14a (7BL).

Detection of QTLs for Stagonospora glume blotch resistance in Swiss winter wheat.

Stagonospora nodorum is the causal agent of the Stagonospora glume blotch disease in hexaploid wheat. The Swiss winter bread wheat cv. 'Arina' has a highly effective, durable and quantitative glume blotch resistance. We studied 240 single seed descent (SSD)-derived lines of an 'Arina x Forno' F(5:7) population to identify and map quantitative trait loci (QTLs) for glume blotch resistance under natural infestation. Using composite interval mapping (CIM) and LOD>4.5, we detected two chromosomal regions on chromosome arms 3BS and 4BL which were specifically associated with glume blotch resistance. These identified QTLs were designated QSng.sfr-3BS and QSng.sfr-4BL, respectively. QSng.sfr-3BS peaked at the locus Xgwm389 in the telomeric region of the short arm of chromosome 3B and explained 31.2% of the observed phenotypic variance for the resistance within the population. The responsible QSng.sfr-3BS allele originated from the resistant parent 'Arina'. The QTL QSng.sfr-4BL (19.1%) mapped to chromosome arm 4BL ('Forno' allele) very close to two known genes, TaMlo and a catalase ( Cat). Both QTL alleles combined could enhance the resistance level by about 50%. Additionally, they showed significant epistatic effects (4.4%). We found PCR-based microsatellite markers closely linked to QSng.sfr-3BS (gwm389) and QSng.sfr-4BL (gwm251) which make marker-assisted selection (MAS) for Stagonospora glume blotch resistance feasible. We also found one resistance QTL, QSng.sfr-5BL, on the long arm of chromosome 5B which overlapped with QTLs for plant height as well as heading time.

An integrative genetic linkage map of winter wheat (Triticum aestivum L.).

We constructed a genetic linkage map based on a cross between two Swiss winter wheat ( Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F(5) single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant ( P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps.

Antifungal activity of a virally encoded gene in transgenic wheat.

The cDNA encoding the antifungal protein KP4 from Ustilago maydis-infecting virus was inserted behind the ubiquitin promoter of maize and genetically transferred to wheat varieties particularly susceptible to stinking smut (Tilletia tritici) disease. The transgene was integrated and inherited over several generations. Of seven transgenic lines, three showed antifungal activity against U. maydis. The antifungal activity correlated with the presence of the KP4 transgene. KP4-transgenic, soil-grown wheat plants exhibit increased endogenous resistance against stinking smut.

Development of a molecular marker for the adult plant leaf rust resistance gene Lr35 in wheat.

The objective of this work was to develop a marker for the adult plant leaf rust resistance gene Lr35. The Lr35 gene was originally introgressed into chromosome 2B from Triticum speltoides, a diploid relative of wheat. A segregating population of 96 F( 2 )plants derived from a cross between the resistant line ThatcherLr35 and the susceptible variety Frisal was analysed. Out of 80 RFLP probes previously mapped on wheat chromosome 2B, 51 detected a polymorphism between the parents of the cross. Three of them were completely linked with the resistance gene Lr35. The co-segregating probe BCD260 was converted into a PCR-based sequence-tagged-site (STS) marker. A set of 48 different breeding lines derived from several European breeding programs was tested with the STS marker. None of these lines has a donor for Lr35 in its pedigree and all of them reacted negatively with the STS marker. As no leaf rust races virulent on Lr35 have been found in different areas of the world, the STS marker for the Lr35 resistance gene is of great value to support the introgression of this gene in combination with other leaf rust (Lr) genes into breeding material by marker-assisted selection.

Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat.

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/ threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.

Genetic and physical characterization of the LR1 leaf rust resistance locus in wheat (Triticum aestivum L.).

The objective of this study was to characterize the leaf rust resistance locus Lr1 in wheat. Restriction fragment length polymorphism (RFLP) analysis was performed on the resistant line Lr1/6* Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F2 populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism between Lr1/6* Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs) Lr1/6* Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F2 populations. One probe (pTAG621) showed very tight linkage to Lr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing the Lr1 resistance gene in different genetic backgrounds showed the same band as Lr1/6* Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant line Lr1/6* Thatcher. This STS, specific for the Lr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked to Lr1 should ultimately provide the basis for positional cloning of the gene.

Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat.

The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), 'Arina' and Lr24/7 (*) "Arina", were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F2 population (150 plants) derived from a cross between the leaf rust resistant Lr24/7 (*) "Arina" and the susceptible spelt (Triticum spelta) variety 'Oberkulmer'. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from 'Agent', a commonly used gene donor for the Lr24 resistance gene.

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat.

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety 'Oberkulmer' was made, and F2 plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.

Genetic diversity in European wheat and spelt breeding material based on RFLP data.

Fifty-two winter wheat (Triticum aestivum L.), nine spring wheat, and 20 spelt (Triticum spelta L.) lines representing part of the European breeding germplasm, were assayed for RFLPs (restriction fragment length polymorphisms) with 56 wheat DNA clones and two barley cDNA clones. Objectives of this study were to (1) determine the level of variation for RFLPs in the wheat and spelt breeding lines, (2) characterize the genetic diversity within the European winter wheat germplasm, and (3) evaluate the usefulness of RFLP markers for pedigree analysis and the grouping of wheat and spelt lines of various origins. Seventy-three of the 166 RFLP loci detected with 58 probes and one restriction enzyme were polymorphic for the 81 lines. The percentage of polymorphic loci was greatest for the B genome (58%) and smallest for the D genome (21%). Among the 81 lines, 271 different RFLP bands were detected. RFLP band frequencies of the winter wheat lines differed considerably (≥0.5) from those of the spring wheat lines at five loci, and from those of the spelt lines at 17 loci. Eight cultivars that had a major impact as progenitors on the development of improved winter wheat cultivars accounted for 93% of the observed RFLP bands in winter wheat. Genetic distance (GD) estimates between two lines ranged between 0.01 and 0.21. Mean GD estimates within winter wheat (0.083), within spring wheat (0.108) and within spelt (0.096) were smaller than between spring and winter wheat (0.114), and greatest between winter wheat and spelt (0.132) and spring wheat and spelt (0.148). Principal coordinate analysis performed on GD estimates revealed a clear separation of wheat and spelt germplasm. Novel spelt lines with various proportions of wheat germplasm were positioned between wheat and traditional spelt lines. The spring wheat lines formed a distinct group at the periphery of the distribution of the winter wheat lines. Subgroupings of the winter wheat lines according to the cluster analysis were in good agreement with their origin, and lines with common ancestors were grouped together.