The Kirkhouse Trust: Successes and Challenges in Twenty Years of Supporting Independent, Contemporary Grain Legume Breeding Projects in India and African Countries.

This manuscript reviews two decades of projects funded by the Kirkhouse Trust (KT), a charity registered in the UK. KT was established to improve the productivity of legume crops important in African countries and in India. KT's requirements for support are: (1) the research must be conducted by national scientists in their home institution, either a publicly funded agricultural research institute or a university; (2) the projects need to include a molecular biology component, which to date has mostly comprised the use of molecular markers for the selection of one or more target traits in a crop improvement programme; (3) the projects funded are included in consortia, to foster the creation of scientific communities and the sharing of knowledge and breeding resources.

A candidate for Lr19, an exotic gene conditioning leaf rust resistance in wheat.

Lr19, one of the few widely effective genes conferring resistance to leaf rust in wheat, was transferred from the wild relative Thinopyrum ponticum to durum wheat. Since Lr19 confers a hypersensitive response to the pathogen, it was considered likely that the gene would be a member of the major nucleotide-binding site (NBS)-leucine-rich repeat (LRR) plant R gene family. NBS profiling, based on PCR amplification of conserved NBS motifs, was applied to durum wheat-Th.

Homoeologous gene silencing in tissue cultured wheat callus.

Background: In contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses.

Variation for homoeologous gene silencing in hexaploid wheat.

The absence of expression of individual members of a homoeologous set of genes in a polyploid is a well-established phenomenon. However, the extent to which such 'homoeologous silencing' can vary between individual genotypes within a species is unexplored. We have used the single-strand conformation polymorphism assay to identify homoeologue non-expression at 15 single-copy genes across a panel of 16 wheat varieties, representative of the genetic diversity present in modern northern European winter wheat (Triticum aestivum).

Identification of variation in adaptively important traits and genome-wide analysis of trait-marker associations in Triticum monococcum.

Einkorn wheat Triticum monococcum (2n=2x=14, A(m)A(m)) is one of the earliest domesticated crops. However, it was abandoned for cultivation before the Bronze Age and has infrequently been used in wheat breeding. Little is known about the genetic variation in adaptively important biological traits in T.

Powdery mildew resistance in 155 Nordic bread wheat cultivars and landraces.

The occurrence and distribution of seedling resistance genes and the presence of adult plant resistance to powdery mildew, was investigated in a collection of 155 Nordic bread wheat landraces and cultivars by inoculation with 11 powdery mildew isolates. Eighty-nine accessions were susceptible in the seedling stage, while 66 accessions showed some resistance. Comparisons of response patterns allowed postulation of combinations of genes Pm1a, Pm2, Pm4b, Pm5, Pm6, Pm8 and Pm9 in 21 lines.

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.

Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.).

Background: When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes.

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.

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.

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.

The conversion of wheat RFLP probes into STS markers via the single-stranded conformation polymorphism technique.

We describe a flexible and general strategy for converting a wheat RFLP-based assay into a PCR-based sequence-tagged site (STS), and have applied it to derive markers for a powdery mildew resistance gene present in a wheat-rye translocation. The concept is based on deriving PCR primers that amplify all of the homoeoloci defined by a single-copy cDNA sequence, and separating the resulting mixture of homoeoamplicons via single-stranded conformation polymorphism (SSCP) gels, which are able to detect minor differences between related DNA sequences. After their separation, the individual homoeoamplicons were sequenced and these were used to define nucleotide polymorphisms that could be exploited to design locus-specific PCR primers.

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.

21st century wheat breeding: plot selection or plate detection?

The publicly reported limited application of marker-assisted selection (MAS) in wheat breeding programmes to date is reviewed and contrasted with the current situation, in which it has increasingly become technically feasible to tag almost any gene with a microsatellite assay. Although this capability is starting to have an impact on the conduct of large breeding programmes, a much more profound change in breeding strategy will become possible when single nucleotide polymorphism (SNP) technology has matured sufficiently so that the throughput of molecular marker-based genotyping is able to keep pace with the numbers of plants that breeders routinely handle in the field. We explore the extent to which the genomics revolution might generate a change in the conventional breeding paradigm, which has operated with such success for the best part of the 20th century, and identify the need for a substantial reduction in assay price before MAS is likely to make more than a marginal impact on present practice.

The impact of molecular markers on the wheat breeding paradigm.

We briefly review the limited application of marker assisted selection in past wheat breeding programmes, and contrast the current situation, where increasingly it has become feasible to tag almost any gene with a microsatellite assay. Although this capability is having an impact on the conduct of large breeding programmes, a much more profound change in breeding strategy will become possible when SNP technology has matured sufficiently so that the throughput of molecular marker-based genotyping will be able to keep pace with the numbers of plants that breeders can handle in the field. We discuss the considerations that will need to be addressed in the generation of a new breeding paradigm to take advantage of the genomics revolution.

Large-scale mutagenesis directed at specific chromosomes in wheat.

A novel approach has been developed to allow for the efficient selection of loss-of-function wheat mutants in the M1 generation, following either physical or chemical mutagenesis. This has generated an order of magnitude increase in the efficiency of identification of mutants, and also greatly increases the likelihood that selected individuals reflect mutation events at the target locus, rather than at genes acting elsewhere in the disease resistance pathway. The approach relies only on prior knowledge of the chromosomal location of the target gene, and uses the polyploidy of wheat to construct populations for mutagenesis in which large numbers of individuals are hemizygous for the target gene.

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.

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.

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.

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.

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.

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.

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.

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.

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.