Robert M. D. Koebner

Temporal trends in the diversity of UK wheat

Abstract The common assertion that scientific plant breeding leads to a narrowing in crop diversity has been examined. We have characterised the dominant UK winter wheat varieties from the period 1934–1994 using two types of PCR-based DNA profiling (AFLPs, amplified fragment length polymorphisms, and SSRs, simple-sequence repeats, microsatellites), seed storage protein analysis and morphological descriptors. The varieties were grouped into a series of decadal groups on the basis of their first appearance on the ’Recommended List’, and by analysis of molecular variance it was shown that an overwhelming proportion of the overall observed variance occurred within, rather than between, decades. A further range of statistical indices provided little evidence for any significant narrowing of overall diversity over the time studied. Principal co-ordinate analysis showed that the diversity in the time periods overlapped and that the most modern group of varieties encompassed the majority of the diversity found in earlier decades. The consistent indication is that plant breeding has resulted, over time, in a qualitative, rather than a quantitative, shift in the diversity of winter wheat grown in the UK.

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

BackgroundWhen 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.ResultsA 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.ConclusionWe 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.

DNA profiling and plant variety registration. III : The statistical assessment of distinctness in wheat using amplified fragment length polymorphisms

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.

The development of a STS marker linked to a yellow rust resistance derived from the wheat cultivar Moro

Abstract.A sequence-tagged-site (STS) marker has been developed for a gene conferring yellow rust resistance originating from the wheat cultivar Moro. The single, dominant, seedling yellow rust resistance gene, designated YrMoro, was mapped to the group 1 chromosomes. The STS marker was developed from an AFLP band which cosegregated with the YrMoro gene. Sequence-specific primers were made which incorporated the selective bases of the AFLP primers, plus 16 and 17 additional bases extending into the AFLP band. This simple, PCR-based marker will allow wheat breeders to pyramid this resistance gene, along with other resistance genes, into a single wheat genotype.

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. 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.

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.

Homoeologous gene silencing in tissue cultured wheat callus

BackgroundIn 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.ResultsEvidence 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.ConclusionOur 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.