STS markers for the wheat yellow rust resistance gene Yr5 suggest a NBS-LRR-type resistance gene cluster.

Two sequence-tagged site (STS) markers for the wheat yellow rust resistance (R) gene Yr5 have been derived through the identification and characterization of linked amplified fragment length polymorphisms (AFLPs). The sequences of the 2 AFLP markers partially overlap with one another, but belong to discrete loci: S19M93-140 completely cosegregates with Yr5, whereas S23M41-310 maps at a distance of 0.7 cM. The DNA sequence of S23M41-310 shows significant homology with the 3' end of nucleotide-binding site (NBS) - leucine-rich repeat (LRR) - type R-genes, in particular with orthologues of the rice bacterial blight R-gene Xa-I. The distinct genetic location of the 2 AFLP loci suggests that Yr5 falls within an R-gene cluster. Because neither sequence forms part of a detectable transcription product, we propose that the Yr5 R-gene cluster includes R-gene analogues and pseudogenes. A Yr5 flanking simple sequence repeat (SSR) marker has also been identified, which allows Yr5 to be effectively incorporated, along with other R-genes for yellow rust, into elite wheat genetic backgrounds, through marker-assisted selection.

Homoeologous gene silencing in hexaploid wheat.

The vast majority of angiosperms are (or were once) polyploid, and as hexaploid bread wheat has undergone two ploidy events separated by approximately 0.5 million years, it represents an elegant model to study gene silencing over time in polyploids. Using an SSCP platform, we have analysed patterns of transcriptional silencing (frequency, genome identity and organ specificity) within 236 single-copy genes, each mapping to one locus on one of the three homoeologous chromosomes within groups 1, 2, 3 and 7 of wheat. In about 27% of unigenes expressed in leaf, and about 26% of those in root, one (rarely two) members of a gene set (homoeoalleles) were not present in the cDNA template. Organ-specific regulation is commonplace, with many homoeoalleles transcribed in leaf but not root (and vice versa). There was little indication of extensive bias towards selective silencing of a particular genome copy. Expression of some of the silenced homoeoalleles was restored in certain aneuploid lines and varieties, and these displayed a significant degree of genetic variation for the silencing of a given homoeoallele. We propose that a substantial proportion of this phenomenon is effected by an epigenetic mechanism, and suggest that this form of genetic variation may be a significant player in the determination of phenotypic diversity in breeding populations.

Development and genetic mapping of sequence-tagged microsatellites (STMs) in bread wheat (Triticum aestivum L.).

The density of SSRs on the published genetic map of bread wheat (Triticum aestivum L.) has steadily increased over the last few years. This has improved the efficiency of marker-assisted breeding and certain types of genetic research by providing more choice in the quality of SSRs and a greater chance of finding polymorphic markers in any cross for a chromosomal region of interest. Increased SSR density on the published wheat genetic map will further enhance breeding and research efforts. Here, sequence-tagged microsatellite profiling (STMP) is demonstrated as a rapid technique for the economical development of anonymous genomic SSRs to increase marker density on the wheat genetic map. A total of 684 polymorphic sequence-tagged microsatellites (STMs) were developed, and 380 were genetically mapped in three mapping populations, with 296 being mapped in the International Triticeae Mapping Initiative W7984 x Opata85 recombinant inbred cross. Across the three populations, a total of 479 STM loci were mapped. Several technological advantages of STMs over conventional SSRs were also observed. These include reduced marker deployment costs for fluorescent-based SSR analysis, and increased genotyping throughput by more efficient electrophoretic separation of STMs and a high amenability to multiplex PCR.

A new approach to extending the wheat marker pool by anchored PCR amplification of compound SSRs.

A study was undertaken to determine the utility in bread wheat of anchored PCR for the development of single locus SSR markers targeted at compound repeat motifs. In anchored PCR, microsatellite amplification is achieved using a single primer complementary to the flanking sequence, and one which anchors to the repeat junction of the compound SSR. The recovery rate of useable markers was found to be similar (43%) to that reported for conventionally generated SSRs. Thus, anchored PCR can be used to reduce the costs of marker development, since it requires that only half the number of primers be synthesised. Where fluorescence-based platforms are used, marker deployment costs are lower, since only the anchoring primers need to be labelled. In addition, anchored PCR improves the recovery of useful markers, as it allows assays to be generated from microsatellite clones with repeat sequences located close to their ends, a situation where conventional PCR amplification fails as two flanking primers cannot be designed. Strategies to permit the large-scale development of compound SSR markers amplified by anchored PCR are discussed.

Temporal flux in the morphological and molecular diversity of UK barley.

Genetic-diversity assessments, using both phenotypic and molecular-marker data, were made on a collection of 134 barley varieties (both winter and spring types), chosen on the basis of their representation on the NIAB "Recommended List" over the period 1925-1995. Genotypic (AFLP and SSR) and phenotypic (UPOV characters) data were analysed to determine short- and long-term temporal trends in diversity over the period. A consistent pattern emerged demonstrating that only a minor proportion of the overall variance appears to be the result of any temporal drift, although there were strong indications of qualitative shifts in diversity, probably related to the changing relative acreage of winter and spring barleys over the study period. Our overall conclusions are that systematic plant breeding does not inevitably lead to a reduction in the genetic diversity of agricultural crops, and that diverse breeding programmes and the variety delivery systems in place in the UK have generally been successful in maintaining sufficient genetic diversity to allow the steady rise in genetic potential that has been a feature of 20th century crop breeding. The concentration of breeding effort into a smaller number of independent programmes is likely to be prejudicial to the maintenance of the genetic diversity of a crop.

Quantitative trait loci for component physiological traits determining salt tolerance in rice.

Rice (Oryza sativa) is sensitive to salinity, which affects one-fifth of irrigated land worldwide. Reducing sodium and chloride uptake into rice while maintaining potassium uptake are characteristics that would aid growth under saline conditions. We describe genetic determinants of the net quantity of ions transported to the shoot, clearly distinguishing between quantitative trait loci (QTL) for the quantity of ions in a shoot and for those that affect the concentration of an ion in the shoot. The latter coincide with QTL for vegetative growth (vigor) and their interpretation is therefore ambiguous. We distinguished those QTL that are independent of vigor and thus directly indicate quantitative variation in the underlying mechanisms of ion uptake. These QTL independently govern sodium uptake, potassium uptake, and sodium:potassium selectivity. The QTL for sodium and potassium uptake are on different linkage groups (chromosomes). This is consistent with the independent inheritance of sodium and potassium uptake in the mapping population and with the mechanistically different uptake pathways for sodium and potassium in rice under saline conditions (apoplastic leakage and membrane transport, respectively). We report the chromosomal location of ion transport and selectivity traits that are compatible with agronomic needs and we indicate markers to assist selection in a breeding program. Based upon knowledge of the underlying mechanisms of ion uptake in rice, we argue that QTL for sodium transport are likely to act through the control of root development, whereas QTL for potassium uptake are likely to act through the structure or regulation of membrane-sited transport components.

Molecular variation in Leymus species and populations.

Icelandic populations of European lymegrass [Leymus arenarius (L.) Hochst.] were examined using amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of the major ribosomal genes (18S-5.8S-26S rDNA), in comparison with Alaskan populations of its closely related species L. mollis (Trin.) Pilger. The AFLP profiles emerged as two distinct entities, clearly separating the two species, and based on species-specific bands it was simple to distinguish these two morphologically similar species. The rDNA-RFLPs also differentiated the species. Within species, the Icelandic L. arenarius was more homogeneous than the Alaskan L. mollis, and its variation was dispersed over geographically different populations, suggesting a common gene pool. The variation among the Alaskan L. mollis was more extensive and its interrupted pattern may be the result of gene introgression at subspecies level. Within a 40-year-old population of L. mollis established in Iceland from Alaskan material, the molecular profiles separated old and new genotypes. Both AFLP and rDNA revealed the new genotypes to be extremely similar. This rapid change in allele frequency is thought to be the result of adaptation to a new environment.

AFLP fingerprinting reveals pattern differences between template DNA extracted from different plant organs.

AFLP (amplified fragment length polymorphism) fingerprinting of cultivars of bread wheat (Triticum aestivum) and some of its wild relatives has allowed the efficient detection of large numbers of polymorphic amplified fragments. While the reproducibility of fingerprints in repeated experiments is high, pattern differences were observed between fingerprints obtained from seed and leaf DNA template from the same wheat accession. These distinct organ specific amplified DNA fragments were shown to be due neither to genotypic mixtures nor to pathogen contamination. They are likely a result of differences in DNA methylation between organs. Even greater numbers of organ specific amplified fragments were observed when fingerprints obtained from the root and shoot of individual seedlings of the wheat relatives Aegilops mutica and Aegilops speltoides were compared. This phenomenon underlines the importance of ensuring that DNA is extracted from physiologically uniform tissue in phylogenetic studies based on AFLP fingerprints. For this purpose, mature seed is a convenient source.

Predigestion of DNA template improves the level of polymorphism of random amplified polymorphic DNAs in wheat.

Random amplified polymorphic DNA (RAPD) analysis in wheat has proven to be poor in its levels of both reproducibility and polymorphism. By digesting the template, prior to performing PCR, with frequently cutting restriction enzymes, the level of polymorphism was improved. RAPD profiles from certain primers were not affected by this pretreatment of the template, but other primers produced distinct profiles from each of several restriction enzymes assayed. Some polymorphisms were specific to one or more restriction digests, but none involved the simple loss of bands from the unrestricted template profile. Genotypic comparisons enabled the selection of primer-restriction enzyme combinations that enabled polymorphic and mappable patterns to be produced both between wheat varieties and between wheats with and without chromosomal segments deriving from related species.

Cytogenetic and molecular mapping of the wheat-Aegilops longissima chromatin breakpoints in powdery mildew-resistant introgression lines.

The amount of alien chromatin introgressed in eight wheat/Ae. longissima Pm13 recombinant lines, involving breakpoints on the short arms of wheat chromosomes 3B and 3D, was evaluated by cytogenetic and molecular approaches. For each line the residual homologous synaptic ability of the recombinant chromosome in its proximal wheat and distal alien portion was estimated through meiotic analyses. Subsequently, telocentric and RFLP mapping were used to assess the genetic distance from the wheat centromere to the wheat/Ae. longissima breakpoints. One 3B recombinant line was distinguished from the other four by the chromosome pairing and telocentric mapping analyses. RFLP analysis succeeded in differentiating the remaining four lines into two groups. Chromosome pairing and telocentric mapping of the three 3D recombinant lines suggested that all had distinct breakpoints. However, the RFLP data could not discriminate between the two more proximal translocations. Physical locations for some RFLP loci were determined by a comparison of genotypes and C-banding karyotypes. This showed a considerable expansion of the genetic map compared to its physical length.

RFLP analysis of an Aegilops ventricosa chromosome that carries a gene conferring resistance to leaf rust (Puccinia recondita) when transferred to hexaploid wheat.

RFLP analysis has been used to characterise XM(v), a chromosome of Aegilops ventricosa present in a disomic addition line of wheat. This chromosome is known to carry a major gene conferring resistance to leaf rust (Lr). The analysis demonstrated that XM(v) is translocated with respect to the standard wheat genome, and consists of a segment of the short arm of homoeologous group 2 attached to a group 6 chromosome lacking a distal part of the short arm. Lr was located to the region of XM(v) with homoeology to 2S by analysis of a leaf rust-susceptible deletion line that was found to lack the entire 2S segment. Confirmation and refinement of the location of Lr was obtained by analysis of a spontaneous resistant translocation in which a small part of XM(v) had been transferred to wheat chromosome 2A.

Conversion of a RAPD-generated PCR product, containing a novel dispersed repetitive element, into a fast and robust assay for the presence of rye chromatin in wheat.

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.

Generation of PCR-based markers for the detection of rye chromatin in a wheat background.

Oligonucleotide primers were developed to detect the presence of four rye sequences using a PCR assay. These assays give a rye-specific signal from wheat DNA template which contains various rye chromosomes or chromosome segments. The sequences identified were associated with the nucleolar organiser region, the 5S-Rrna-R1 locus, the telomere, and a widely dispersed, rye-specific repetitive element Ris-1. The primers amplified from the well-established loci Nor-R1 and 5S-Rrna-R1 on rye chromosome arm 1RS, and also located a 5s-Rrna locus on chromosome 3R. The telomere-associated sequence was present on every rye chromosome, and was also present, at a low copy number, in both wheat and barley. These assays will be particularly useful for introgression programmes aimed at reducing the rye content of the 1BL.1RS wheat-rye translocation. When multiplexed, the primers will enable a rapid, simultaneous assay for a number of distinct rye loci, which can be derived from a small portion of mature endosperm tissue.

Genetic variation for waterlogging tolerance in the Triticeae and the chromosomal location of genes conferring waterlogging tolerance in Thinopyrum elongatum.

A number of Triticeae species were tested for tiller production, shoot dry matter production, and root penetration in waterlogged soil, and Thinopyrum elongatum and Elytrigia repens were shown to have better tolerance than wheat using these criteria. Tests of a number of wheat-alien amphiploids showed that there was at least partial expression of this exotic genetic variation in a wheat genetic background. The presence of chromosomes 2E and 4E of Th. elongatum was associated with a positive effect on root growth in waterlogged conditions. The positive effect of the 4E chromosome addition was mimicked by tetrasomic lines carrying extra doses of wheat homoeologues 4B and 4D, and it was concluded that the beneficial effect contributed by the presence of 4E was probably due to an increased dosage of group 4 chromosomes. However, the positive effect of adding chromosome 2E to wheat could not be reproduced by added doses of chromosomes 2A, 2B, or 2D, suggesting that this alien chromosome carries gene(s) for tolerance not present on its wheat homoeologues. This gene(s) was further located to the long arm of chromosome 2E by testing ditelosomic addition lines.

Characterization of Thinopyrum bessarabicum chromosome segments in wheat using random amplified polymorphic DNAs (RAPDs) and genomic in situ hybridization.

Ten random amplified polymorphic DNA (RAPD) markers specific to chromosome 5E(b) of Thinopyrum bessarabicum were detected. Genomic in situ hybridization and standard cytological observations revealed that six of the markers are located on the 5E(b) short arm and four are located on the 5E(b) long arm. These RAPD markers have been used to confirm the identity of putative 5E(b) (5A) and 5E(b) (5D) substitution individuals. The potential of RAPDs for the detection of wheat/alien recombinants is discussed.

Chromosomal rearrangements in the rye genome relative to that of wheat.

An RFLP-based genetic map of Secale Cereale has provided evidence for multiple evolutionary translocations in the rye genome relative to that of hexaploid wheat. DNA clones which have previously been mapped in wheat indicated that chromosome arms 2RS, 3RL, 4RL, 5RL, 6RS, 6RL, 7RS and 7RL have all been involved in at least one translocation. A possible evolutionary pathway, which accounts for the present day R genome relative to the A, B and D genomes of wheat, is presented. The relevance of these results for strategies designed to transfer useful genes from rye, and probably other related species, to wheat is discussed.

RFLP mapping of genes affecting plant height and growth habit in rye.

RFLP mapping of chromosome 5R in the F3 generation of a rye (Secale cereale L.) cross segregating for gibberellic acid (GA3)-insensitive dwarfness (Ct2/ct2) and spring growth habit (Sp1/sp1) identified RFLP loci close to each of these agronomically important genes. The level of RFLP in the segregating population was high, and thus allowed more than half of the RFLP loci to be mapped, despite partial homozygosity in the parental F2 plant. Eight further loci were mapped in an unrelated F2 rye population, and a further two were placed by inference from equivalent genetic maps of related wheat chromosomes, allowing a consensus map of rye chromosome 5R, consisting of 29 points and spanning 129 cM, to be constructed. The location of the ct2 dwarfing gene was shown to be separated from the segment of the primitive 4RL translocated to 5RL, and thus the gene is probably genetically unrelated to the major GA-insensitive Rht genes of wheat located on chromosome arms 4BS and 4DS. The map position of Sp1 is consistent both with those of wheat Vrn1 and Vrn3, present on chromosome arms 5AL and 5DL, respectively, and with barley Sh2 which is distally located on chromosome arm 7L (= 5HL).

Isolation and characterization of wheat-rye recombinants involving chromosome arm 1DS of wheat.

The introgression of genetic material from alien species is assuming increased importance in wheat breeding programs. One example is the translocation of the short arm of rye chromosome 1 (1RS) onto homoeologous wheat chromosomes, which confers disease resistance and increased yield on wheat. However, this translocation is also associated with dough quality defects. To break the linkage between the desirable agronomic traits and poor dough quality, recombination has been induced between 1RS and the homoeologous wheat arm IDS. Seven new recombinants were isolated, with five being similar to those reported earlier and two havina new type of structure. All available recombinantsw ere characterized with DNA probes for the loci Nor-R1, 5SDna-R1, and Tel-R1. Also, the amount of rye chromatin present was quantified with a dispersed rye-specific repetitive DNA sequence in quantitative dot blots. Furthermore, the wheat-rye recombinants were used as a mapping tool to assign two RFLP markers to specific regions on chromosome arms 1DS and 1RS of wheat and rye, respectively.

Determination of the transmission frequency of chromosome 4S (l) of Aegilops sharonensis in a range of wheat genetic backgrounds.

The transmission of chromosome 4S (l) from Aegilops sharonensis was observed in a range of wheat genetic backgrounds. Chromosome 4S (l) was transmitted at a very high frequency (at least 97.8%) in all crosses. The genetic background appears to only have a small effect on transmission. The frequency of transmission of chromosome 4S (l) was the same in each genetic background through both the male and female gametes.

Genetic characterisation of a further homoeoallelic series of grain esterase loci,Est-6, in wheat.

Isoelectric focussing in alkaline pH gels has permitted the identification of a new homoeoallelic series of genes,Est-6, encoding grain esterases in bread wheat,Triticum aestivum. Nullisomic analysis located these genes to the short arms of the homoeologous group 2 chromosomes. A search for polymorphism withinEst-6 revealed null alleles at each ofEst-A6,Est-B6 andEst-D6. A further homoeolocus,Est-M6, is present on chromosome arm2MS ofAegilops comosa.