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. This account relates to the key achievements and challenges, reflects on the lessons learned and outlines future research priorities.

The utility of combining simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker genotyping was determined for genetically mapping a novel aphid (Aphis craccivora) resistance locus in cowpea breeding line SARC 1-57-2 and for introgressing the resistance into elite cultivars by marker-assisted backcrossing (MABC). The locus was tagged with codominant SSR marker CP 171F/172R with a recombination fraction of 5.91% in an F-2 population from Apagbaala' x SARC 1-57-2. A SNP-genotyped biparental recombinant inbred line population was genotyped for CP 171F/172R, which was mapped to position 11.5 cM on linkage group (LG) 10 (physical position 30.514Mb on chromosome Vu10). Using CP 171F/172R for foreground selection and a KASP-SNP-based marker panel for background selection in MABC, the resistance from SARC 1-57-2 was introduced into elite susceptible cultivar Zaayura'. Five BC4F3 lines of improved Zaayura' that were isogenic except for the resistance locus region had phenotypes similar to SARC 1-57-2. This study identified a novel aphid resistance locus and demonstrated the effectiveness of integrating SSR and SNP markers for trait mapping and marker-assisted breeding.

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. ponticum recombinant lines involving different segments of the alien 7AgL chromosome arm, carrying or lacking Lr19. Differential PCR products were isolated and sequenced. From one such sequence (AG15), tightly linked to Lr19, a 4,121-bp full-length cDNA was obtained. Its deduced 1,258 amino acid sequence has the characteristic NBS-LRR domains of plant R gene products and includes a coiled-coil (CC) region typical of monocots. The genomic DNA sequence showed the presence of two exons and a short intron upstream of the predicted stop codon. Homology searches revealed considerable identity of AG15 with the cloned wheat resistance gene Pm3a and a lower similarity with wheat Lr1, Lr21, and Lr10. Quantitative PCR on leaf-rust-infected and non-infected Lr19 carriers proved AG15 to be constitutively expressed, as is common for R genes.

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.RESULTS:Evidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested.CONCLUSION:Our results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.

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). There was no evidence for any homoeologous silencing at seven of the fifteen genes, but in the remaining eight, at least one of the three homoeologues varied qualitatively for expression in either the root or the seedling leaf. The identity of the non-expressed homoeologue was generally consistent, but when the expression profiles of eight informative genes were compared, only two varieties shared the same pattern of silencing. A small-scale study suggested that silencing patterns were largely inherited across self-pollinated generations, and some evidence is presented for the epigenetic segregation of these patterns in a population bred from parents having contrasting silencing profiles. Epigenetic variation exerts a significant effect on phenotype, so given the ubiquity and variability in homoeologous silencing observed in wheat, we suggest that it is likely to play a considerable role in generating phenotypic variation. Thus epigenetic profiling may need to be incorporated as part of the analytical tool kit for predictive wheat breeding.

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. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.RESULTS:A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors.CONCLUSION:We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually propagated. Of significant biological interest is the conclusion that the mode of regeneration has no significant effect on the balance between sequence and methylation state change induced by the tissue culture process.

This paper examines the fate of alleles and changes of genetic diversity in old ( ca 1930s) versus more modern ( ca 1990s) UK bread wheat varieties using 14 mapped DNA microsatellite (simple sequence repeat, SSR) loci and morphological markers. The allelic constitution of varieties belonging to three time periods (early, intermediate, late) was determined. While at certain loci one or more SSR alleles were gained between early and late periods, at others the allelic representation remained constant, although a shift in allelic frequencies could sometimes be detected. No locus showed a clear, net loss in the total number of alleles over the time period. In a further group of loci, there was neither clear gain nor loss, but rather a dynamic flux of alleles. A comparison of the allelic constitution of the UK variety set with a larger genetic pool (non-UK varieties) showed that some loci were rather similar in allelic constitution, while others possessed additional diversity. Certain SSR alleles appeared to be associated with old or modern varieties, possibly indicating associations with chromosome regions under selection pressure. The same exercise was conducted on the basis of 14 of the morphological characteristics recorded in the course of distinctness, uniformity and stability testing of varieties. Overall, this analysis generated a similar picture of changes in diversity to that obtained from the microsatellite data.

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.

This chapter contains sections titled: Introduction Molecular Markers in Genetic Diversity Studies in Wheat and Barley Molecular Markers for Cultivar Identification Marker Assisted Selection Marker-Based Genotyping in Crop Breeding and Genetics The Future of Molecular Genotyping in Crop Breeding and Genetics Literature Cited

The use of AFLP analysis to produce DNA profiles from a set of 55 wheat varieties, commonly grown in the UK over the past 60 years, is described. Using six different primer pairs, 90 polymorphic bands were readily recognised and recorded. These AFLP bands are not significantly clustered and hence can be used with some confidence, even though they are not mapped. Statistical approaches to the analysis of the data were developed such that the discrimination between the varieties achieved by the use of the six primer pairs, both separately and in combination, could be derived and compared to that achieved by a common set of morphological descriptors. Various criteria for the definition of distinctness in terms of the number of band differences required between pairs of varieties were also compared. In general, higher levels of discrimination were achieved by the inclusion of greater numbers of bands in the analysis. The optimal number of polymorphic bands appears to be between v and 2v, where v is the number of varieties under test. Discrimination levels were adversely affected if the number of bands was below v/2. Distinctness levels achieved by the use of molecular markers can be calibrated so that they reproduce those seen with morphological characters. The results are discussed in relation to the possible use of DNA profiling methods for distinctness, uniformity and stability testing.