New quantitative trait loci in wheat for flag leaf resistance to Stagonospora nodorum blotch.

Stagonospora nodorum blotch (SNB) is a significant disease in some wheat-growing regions of the world. Resistance in wheat to Stagonospora nodorum is complex, whereby genes for seedling, flag leaf, and glume resistance are independent. The aims of this study were to identify alternative genes for flag leaf resistance, to compare and contrast with known quantitative trait loci (QTL) for SNB resistance, and to determine the potential role of host-specific toxins for SNB QTL. Novel QTL for flag leaf resistance were identified on chromosome 2AS inherited from winter wheat parent 'P92201D5' and chromosome 1BS from spring wheat parent 'EGA Blanco'. The chromosomal map position of markers associated with QTL on 1BS and 2AS indicated that they were unlikely to be associated with known host-toxin insensitivity loci. A QTL on chromosome 5BL inherited from EGA Blanco had highly significant association with markers fcp001 and fcp620 based on disease evaluation in 2007 and, therefore, is likely to be associated with Tsn1-ToxA insensitivity for flag leaf resistance. However, fcp001 and fcp620 were not associated with a QTL detected based on disease evaluation in 2008, indicating two linked QTL for flag leaf resistance with multiple genes residing on 5BL. This study identified novel QTL and their effects in controlling flag leaf SNB resistance.

Functional relationships of phytoene synthase 1 alleles on chromosome 7A controlling flour colour variation in selected Australian wheat genotypes.

Flour colour measured as a Commission Internationale de l'Eclairage (CIE) b* value is an important wheat quality attribute for a range of end-products, with genes and enzymes of the xanthophyll biosynthesis pathway providing potential sources of trait variation. In particular, the phytoene synthase 1 (Psy1) gene has been associated with quantitative trait loci (QTL) for flour b* colour variation. Several Psy1 alleles on chromosome 7A (Psy-A1) have been described, along with proposed mechanisms for influencing flour b* colour. This study sought to identify evolutionary relationships among known Psy-A1 alleles, to establish which Psy-A1 alleles are present in selected Australian wheat genotypes and establish their role in controlling variation for flour b* colour via QTL analysis. Phylogenetic analyses showed seven of eight known Psy-A1 alleles clustered with sequences from T. urartu, indicating the majority of alleles in Australian germplasm share a common evolutionary lineage. In this regard, Psy-A1a, Psy-A1c, Psy-A1e and Psy-A1p were common in Australian genotypes with flour b* colour ranging from white to yellow. In contrast Psy-A1s was found to be related to A. speltoides, indicating a possible A-B genome translocation during wheat polyploidisation. A new allele Psy-A1t (similar to Psy-A1s) was discovered in genotypes with yellow flour, with QTL analyses indicating Psy-A1t strongly influences flour b* colour in Australian germplasm. QTL LOD value maxima did not coincide with Psy-A1 gene locus in two of three populations and, therefore, Psy-A1a and Psy-A1p may not be involved in flour colour. Instead two other QTL were identified, one proximal and one distal to Psy-A1 in Australian wheat lines. Comparison of Psy-A1t and Psy-A1p predicted protein sequences suggests differences in putative sites for post-translational modification may influence enzyme activity and subsequent xanthophyll accumulation in the wheat endosperm. Psy-A1a and Psy-A1p were not involved in flour b* colour variation, indicating other genes control variation on chromosome 7A in some wheat genotypes.

Quantitative trait loci for seedling and adult plant resistance to Stagonospora nodorum in wheat.

Stagonospora nodorum blotch (SNB) caused by Stagonospora nodorum is a severe disease of wheat (Triticum aestivum) in many areas of the world. S. nodorum affects both seedling and adult plants causing necrosis of leaf and glume tissue, inhibiting photosynthetic capabilities, and reducing grain yield. The aims of this study were to evaluate disease response of 280 doubled haploid (DH) individuals derived from a cross between resistant (6HRWSN125) and susceptible (WAWHT2074) genotypes, compare quantitative trait loci (QTL) for seedling and adult plant resistance in two consecutive years, and assess the contribution of QTL on grain weight. Flag leaves and glumes of individuals from the DH population were inoculated with mixed isolates of S. nodorum at similar maturity time to provide accurate disease evaluation independent of morphological traits and identify true resistance for QTL analysis. Fungicide protected and inoculated plots were used to measure relative grain weight (RGW) as a yield-related trait under pathogen infection. The lack of similar QTL and little or no correlation in disease scores indicate different genes control seedling and adult plant disease and independent genes control flag leaf and glume resistance. This study consistently identified a QTL on chromosome 2DL for flag leaf resistance (QSnl.daw-2D) and 4BL for glume resistance (QSng.daw-4B) from the resistant parent, 6HRWSN125, explaining 4 to 19% of the phenotypic variation at each locus. A total of 5 QTL for RGW were consistently detected, where two were in the same marker interval for QSnl.daw-2D and QSng.daw-4B indicating the contribution of these QTL to yield related traits. Therefore, RGW measurement in QTL analysis could be used as a reliable indicator of grain yield affected by S. nodorum infection.

QTL analysis and comparative genomics of herbage quality traits in perennial ryegrass (Lolium perenne L.).

Genetic control of herbage quality variation was assessed through the use of the molecular marker-based reference genetic map of perennial ryegrass (Lolium perenne L.). The restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and genomic DNA-derived simple sequence repeat-based (SSR) framework marker set was enhanced, with RFLP loci corresponding to genes for key enzymes involved in lignin biosynthesis and fructan metabolism. Quality traits such as crude protein (CP) content, estimated in vivo dry matter digestibility (IVVDMD), neutral detergent fibre content (NDF), estimated metabolisable energy (EstME) and water soluble carbohydrate (WSC) content were measured by near infrared reflectance spectroscopy (NIRS) analysis of herbage harvests. Quantitative trait locus (QTL) analysis was performed using single-marker regression, simple interval mapping and composite interval mapping approaches, detecting a total of 42 QTLs from six different sampling experiments varying by developmental stage (anthesis or vegetative growth), location or year. Coincident QTLs were detected on linkage groups (LGs) 3, 5 and 7. The region on LG3 was associated with variation for all measured traits across various experimental datasets. The region on LG7 was associated with variation for all traits except CP, and is located in the vicinity of the lignin biosynthesis gene loci xlpomt1 (caffeic acid-O-methyltransferase), xlpccr1 (cinnamoyl CoA-reductase) and xlpssrcad 2.1 (cinnamyl alcohol dehydrogenase). Comparative genomics analysis of these gene classes with wheat (Triticum aestivum L.) provides evidence for conservation of gene order over evolutionary time and the basis for cross-specific genetic information transfer. The identification of co-location between QTLs and functionally associated genetic markers is critical for the implementation of marker-assisted selection programs and for linkage disequilibrium studies, which will enable future improvement strategies for perennial ryegrass.

Physical location of a HSP70 gene homologue on the centromere of chromosome 1B of wheat ( Triticum aestivum L.).

Cereal centromeres consist of a complex organization of repetitive DNA sequences. Several repetitive DNA sequences are common amongst members of the Triticeae family, and others are unique to particular species. The organization of these repetitive elements and the abundance of other types of DNA sequences in cereal centromeres are largely unknown. In this study, we have used wheat-rye translocation lines to physically map 1BL.1RS centromeric breakpoints and molecular probes to obtain further information on the nature of other types of centromeric DNA sequences. Our results, using the rye-specific centromeric sequence, pAWRC.1, indicate that 1BL.1RS contains a small portion of the centromere from 1R of rye. Further studies used molecular markers to identify centromeric segments on wheat group-1 chromosomes. Selected RFLP markers, clustered around the centromere of wheat homoeologous group-1S chromosomes, were chosen as probes during Southern hybridization. One marker, PSR161, identified a small 1BS segment in all 1BL.1RS lines. This segment maps proximal to pAWRC.1 in 1BL.1RS and on the centromere of 1B. Sequence analysis of PSR161 showed high homology to HSP70 genes and Northern hybridization showed that this gene is constitutively expressed in leaf tissue and induced by heat shock and light stimuli. The significance of this work with respect to centromere organization and the possible significance of this HSP70 gene homologue are discussed.

Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.).

A diminutive rye chromosome (midget) in wheat was used as a model system to isolate a highly reiterated centromeric sequence from a rye chromosome. Fluorescence in situ hybridization (FISH) shows this sequence localized within all rye centromeres and no signal was detected on wheat chromosomes. DNA sequencing of the repetitive element has revealed the presence of some catalytic domains and signature motifs typical of retrotransposon genes and has been called the Bilby family, representing a diverged family of retrotransposon-like elements. Extensive DNA database searching revealed some sequence similarity to centromeric retrotransposons from wheat, barley, and centromeric repetitive sequences from rice. Very low levels of signal were observed when Bilby was used as a probe against barley, and no signal was detected with rice DNA during Southern hybridization. The abundance of Bilby in rye indicates that this family may have diverged from other distantly related centromeric retrotransposons or incorporated in the centromere but rapidly evolved in rye during speciation. The isolation of a rye retrotransposon also allowed the analysis of centromeric breakpoints in wheat-rye translocation lines. A quantitative analysis shows that the breakpoint in IDS.1RL and 1DL.1RS and recombinant lines containing proximal rye chromatin have a portion of the rye centromere that may contribute to the normal function of the centromeric region.

Identification and characterization of wheat-wheatgrass translocation lines and localization of barley yellow dwarf virus resistance.

Stable introgression of agronomically important traits into crop plants through wide crossing often requires the generation and identification of translocation lines. However, the low efficiency of identifying lines containing translocations is a significant limitation in utilizing valuable alien chromatin-derived traits. Selection of putative wheatgrass-wheat translocation lines based on segregation ratios of progeny from gamma-irradiated seed using a standard phenotypic analysis resulted in a low 4% success rate of identifying barley yellow dwarf virus (BYDV) resistant and susceptible translocation lines. However, 58% of the susceptible progeny of this irradiated seed contained a Thinopyrum intermedium chromosome-specific repetitive sequence, which indicated that gamma-irradiation-induced translocations occurred at high rate. Restriction fragment length polymorphism (RFLP) analysis of susceptible lines containing alien chromatin, their resistant sister lines and other resistant lines showed that more than one third of the progeny of gamma-irradiated double monosomic seeds contained wheatgrass-wheat translocations. Genomic in situ hybridization (GISH) analysis of selected lines confirmed that these were wheatgrass-wheat translocation lines. This approach of initially identifying BYDV susceptible deletion lines using an alien chromosome-specific repetitive sequence followed by RFLP analysis of their resistant sister lines efficiently identified resistant translocation lines and localized the BYDV resistance to the distal end of the introgressed Th. intermedium chromosome.

Characterization of wheatgrass-derived barley yellow dwarf virus resistance in a wheat alien chromosome substitution line.

ABSTRACT Wheatgrass (Thinopyrum intermedium) possesses a high level of resistance to barley yellow dwarf virus (BYDV) subgroup I and subgroup II strains. A wheat line (P29), in which the 7D chromosome has been substituted with a group 7 chromosome from T. intermedium, was examined for the level of resistance to two subgroup I and two subgroup II BYDV strains. In P29 plants inoculated with the subgroup I PAV strains, the titer of virus in leaf and stem tissue was typically reduced 42 to 52% when compared with the BYDV-susceptible cv. Abe. P29 and 'Abe' had the same content of PAV in roots. These results and the absence of detectable virus in inoculated T. intermedium plants indicate that the complete resistance to subgroup I possessed by the wheatgrass has not been introgressed into P29. In contrast, P29 was completely resistant throughout the plant to the subgroup II strains, NY-RPV and NY-RMV, demonstrating that the complete resistance to subgroup II in T. intermedium was incorporated into P29. Further analysis of this resistance to NY-RPV showed that NY-RPV can replicate in mesophyll protoplasts of P29 and 'Abe', suggesting that this resistance is not operating at the single-cell level. Molecular marker analysis confirmed that the T. intermedium chromosome present in P29 is a different group 7 wheatgrass chromosome than that present in L1, a wheat line with BYDV resistance properties similar to those of P29.

Structural organization of an alien Thinopyrum intermedium group 7 chromosome in U.S. soft red winter wheat (Triticum aestivum L.).

Barley yellow dwarf virus (BYDV) resistance in soft red winter wheat (SRWW) cultivars has been achieved by substituting a group 7 chromosome from Thinopyrum intermedium for chromosome 7D. To localize BYDV resistance, a detailed molecular genetic analysis was done on the alien group 7 Th. intermedium chromosome to determine its structural organization. Triticeae group 7 RFLP markers and rye specific repetitive sequences used in the analysis showed that the alien chromosome in the P29 substitution line has distinguishing features. The 350-480 bp rye telomeric sequence family was present on the long arm as determined by Southern and fluorescence in situ hybridization. However, further analysis using a rye dispersed repetitive sequence indicated that this alien chromosome does not contain introgressed segments from the rye genome. The alien chromosome is homoeologous to wheat chromosomes 7A and 7D as determined by RFLP analysis. Presence of the waxy gene on chromosomes 7A, 7B, and 7D but its absence on the alien chromosome in P29 suggests some internal structural differences on the short arm between Th. intermedium and wheat group 7 chromosomes. The identification of rye telomeric sequences on the alien Thinopyrum chromosome and the homoeology to wheat chromosomes 7A and 7D provide the necessary information and tools to analyze smaller segments of the Thinopyrum chromosome and to localize BYDV resistance in SRWW cultivars.

Analysis of rye B-chromosome structure using fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) has been used to analyse the structure of the rye B chromosome. Genomic in situ hybridization (GISH) demonstrates the high level of overall similarity between A and B chromosomes of rye, as well as the presence of a number of specific sequences. The B-specific repeat families D1100 and E3900 have been analysed in terms of their physical location and possible contiguity. Rye Bs contain members of the rye-specific dispersed repetitive family R173, as well as centromeric regions similar to those of the As. The B chromosomes analysed in our study lack detectable rDNA sequences. Anomalous results have been obtained with a number of subtelomeric repetitive probes from rye. Bs usually lack these sequences, but evidence is presented that in some cases A-B translocation events may relocate such sequences from the As to the Bs. These data are discussed in the context of current models for the origin of the B chromosome.

The molecular identification of the midget chromosome from the rye genome.

The diminutive "midget" chromosome is found in plants containing a wheat nuclear genome with a substituted rye cytoplasm. This cytoplasmic substituted line arose during successive backcrossing of a wheat/rye amphiploid to wheat as the recurrent male parent. Southern and in situ hybridization with a dispersed repeat sequence specific for rye, R173, indicates that the midget chromosome originates from within the rye genome. Various DNA markers previously mapped to group 1 chromosomes of wheat and barley were used to trace the origin of the midget chromosome from within the rye genome. Ten short arm and 36 long arm probes were used and one marker was identified, which hybridizes to the midget chromosome and maps to the proximal region of the long arm of chromosome 1R. An additional marker was generated from a genomic library of the line containing the midget chromosome. This also maps to the long arm of 1R. The results indicate that the midget chromosome contains a small segment of the long arm of chromosome 1R.