JOHN INNES CENTRENorwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.
John Snape, Leodie Alibert, Robert Koebner, and Simon Orford.
Under the auspices of the U.K. Wheat Genetic Improvement Network (WGIN) (see Ann Wheat Newslett 50:192), a doubled haploid population developed from the cross 'Avalon/Cadenza' has been chosen to be the reference U.K. wheat mapping population. The aim is to offer to U.K. wheat researchers and breeders a reference map to study QTL and major genes of interest.
The 'Avalon/Cadenza' map is being developed at John Innes Centre and, to date, 203 lines have been genotyped largely with microsatellites and DArTTM markers. The current map includes 90 SSR loci, 3 HMW-glutenin genes, 202 DArTTM markers, and a small number of other markers. The ultimate aim is to develop a saturated map using all publicly available SSRs, and other marker types. In addition to SSRs, Sequence tagged microsatellite (STM) markers, which we now have primer sequences for (Hayden et al. 2006), are being added. Current linkage groups vary in length from 70120 cM, with a marker every 10 to 20 cM. The exceptions are in chromosomes 1D, 2A, 2D, 3D, and 6D, where the linkage groups are smaller.
The map will be soon available on the WGIN website (www.wgin.org.uk), as well as some trait data such as ear emergence, height, and yield taken over the last 2 years in the field.
Lesley Boyd, Clare Lewis, Muge Sayar, James Melichar, Luke Jagger, Hale Tufan, and Nicola Powell.
A number of programs are continuing to characterize the genes/QTL responsible for yellow rust resistance in U.K. wheat cultivars, including the U.K. cultivars Claire, Guardian, and Brigadier.
The genetic diversity studies have also expanded into an assessment of Turkish wheats (durum and bread) as part of collaboration with Prof. M. Sayar, Bogazici University, Istanbul (EU - Marie Curie Fellow) and Dr. J. Braun, CIMMYT, Ankara, Turkey. NBS-profiling is being used to characterize the genetic diversity within the wheat genome associated with NBS (R-gene) sequences. The Turkish cultivars are being compared to a selection of 30 wheat cultivars from across Europe.
James Melichar and Lesley Boyd.
A number of mutants, generated by gamma-radiation in the U.K. cultivar Guardian, were originally selected in the field for enhanced resistance to yellow rust. This enhanced resistance was shown not to express in seedlings, but to be developmentally regulated, expressing at adult plant growth stages.
In addition to the enhancement of resistance to yellow rust a number of the mutants also exhibit enhanced resistance to leaf rust and/or powdery mildew. Doubled-haploid populations have been developed for Guardian and two of the mutants. These are being used to locate both the partial yellow rust APR in Guardian, the mutations responsible for the enhancement of yellow rust resistance, and resistance to leaf rust and powdery mildew. These populations now form part of a European Union-funded program - BioExploit.
Ruth MacCormack and Lesley Boyd.
A Defra-funded program examined the early stages of yellow rust infection to determine what factors optimize infection efficiency of this fungal pathogen. The preinoculation light quantity received by wheat seedlings influenced the ability of the fungal pathogen to find and enter stomata. We are now screening for genetic variation between wheat genotypes for the ability of preinoculation light quantity to effect yellow rust infection efficiency.
Hale Tufan and Lesley Boyd.
A new program within the group started in 2005 funded by the CIGAR - Generation Challenge Program. This program - CEREAL IMMUNITY, forms a collaboration with seven research groups around the world and is lead by Dr. Pietro Piffanelli, AGROPOLIS, Montpellier, France. The program aims to use the Affymetrix wheat micro array to study gene expression in wheat in host and nonhost pathogen interactions and links in with similar studies in rice being carried out by Prof. P. Ronald, UC Davis, USA and Prof. S. Kikuchi, NIAS, Japan.
Simon Orford, Pauline Stephenson, and Robert Koebner.
Simon Orford, Pauline Stephenson, and Robert Koebner.
As part of our continuing contribution to the Wheat Genetic Improvement Network (see Ann Wheat Newslet 50:192), we have further advanced an immortal population of EMS mutagenized spring wheat cultivar Paragon by single-seed descent. The initial M1 population numbered ~3,500 individuals, from which two M2 seeds were sown per M1 plant. The population is currently being sown in the field as ~7,000 single-ear rows representing the M5 generation. A number of fixed phenotypic mutants have been isolated, for example reversion from spring to winter habit, dwarfness, ear morphology, awnedness, spelt etc. From summer 2006, this field multiplied seed will be made available to collaborators for gene discovery and functional gene analysis. Interested researchers can make contact via the WGIN website (www.wgin.org.uk).
Andrew Bottley and Robert Koebner.
Using an SSCP platform, we have been analyzing patterns of transcriptional silencing (frequency, genome identity, and organ specificity) within unigene homoeologous sets, by assaying gDNA and cDNA amplicons derived from 236 such genes mapping to one of homoeologous 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) homoeologs were not represented in the cDNA template. Organ-specific regulation is commonplace, with many homoeologs transcribed in leaf but not root (and vice versa). We have detected little indication of bias towards selective silencing of a particular genome copy. Surprisingly, the expression of some of these non-transcribed homoeologs was restored in certain aneuploid lines and varieties. A simple repressor mechanism could explain about one-third of these cases, but for the remaining two-thirds, an epigenetic mechanism of silencing is suspected. We suggest that this form of genetic variation may be a significant player in the determination of phenotypic diversity in breeding populations.
Nicola Hart and Robert Koebner.
As research into crop improvement continues to yield the DNA sequences of agronomically important genes, the opportunity to study the outcome of mutagenesis at the DNA level is becoming available. We are investigating the size, nature and frequency of induced genetic lesions following g irradiation and EMS mutagenesis of the bread wheat cultivar Paragon. The major focus is on the Rht-1 semidwarfing genes, which meet certain necessary criteria (known DNA sequence, single copy, known chromosomal location, and recognizable effect on phenotype). We have developed sets of primers to amplify the full length (in ~500-bp segments) of Rht-B1 and Rht-D1 and are using an SSCP platform to search for sequence alterations in the various amplicons across an EMS population of 7,000 individuals. To date, seven independent mutants (from a screen of about 2,000 individuals) in the 5' amplicon of Rht-B1 have been identified, four of which predict an amino-acid change. Global levels of mutation in the population are also being explored using a retrotransposon-based S-SAP assay.
Simon Griffiths, Tracie Foote, and Graham Moore.
Insights into the control of chromosome pairing in polyploid wheat have been recently realized through a molecular and cell biological characterization of the Ph1 locus. In most species, chromosome pairing is first initiated via telomere interactions, manifested by the clustering of telomeres at the start of meiosis. However, we have seen in wheat that the telomeres of homologs pair correctly whether or not Ph1 is present. So what is Ph1 affecting in the rest of the chromosome? It has long been known that premature and asynchronous chromatin condensation affects wheat F1 hybrids; meanwhile in model organisms such as yeast, premature chromatin condensation results when cdc2 is over-expressed. In the absence of Ph1, the condensation of meiotic chromosomes is particularly asynchronous and premature. Differential condensation of homologs implies asynchrony in their chromatin conformation, thereby increasing the possibility of illegitimate pairing. Correct pairing at the telomere, followed by illegitimate association of sites along the arms would generate the multivalent structures observed in Ph1-deficient genotypes. Interestingly, there is a known correlation between condensation and recombination sites, which may explain why multivalents fail to resolve in the absence of Ph1. What happens at centromeres? In the presence of Ph1 the centromeres pair prior to meiosis and go on to form seven distinct clusters, just as the telomeres are clustering at the start of meiosis. In contrast, in the absence of Ph1, although the centromeres still pair, the number of clusters is only rarely reduced to the seven, reflecting the fact that the centromeres themselves are less condensed.
Thus we have a cell biological explanation of how Ph1 functions within wheat itself, so how do wide hybrids behave? The presence of multiple B chromosomes (which are highly heterochromatic) can compensate for the absence of Ph1, resulting in suppression of homoeolog pairing and recombination. Their presence also delays S phase, leaving the chromosomes more condensed at an equivalent meiotic stage. This supports the model that Ph1 functions through the control of chromatin condensation. In wheat-rye hybrids we have shown that in the presence of Ph1, chromosomes are more condensed at the point of pairing initiation, than in its absence. As in wheat itself, in Ph1 hybrids all the centromeres fuse to seven clusters at the start of meiosis, while in ph1 hybrids, they rarely form as few as seven clusters.
We have now completed a molecular characterization of the Ph1 locus. This has revealed that, following polyploidization, a subtelomeric heterochromatin block became inserted into a group of cdc2-like genes on 5B. As it is now clear that Ph1 is involved in the regulation of chromatin condensation, it seems likely that this insertion event generated a functional and/or regulatory change at the 5B cdc2-like gene family. At the moment, the exact nature of how this rearranged locus functions remains to be determined. The wider implication of this discovery is that a newly synthesized allopolyploid needs to tightly control the meiotic checkpoint to ensure synchronized control of chromatin replication and condensation at meiosis. In particular, it is necessary that homologs condense in a coordinate way in order to ensure their correct pairing and the resolution of recombination events during the course of meiosis.
RAGT SEEDS LTD.
Maris Centre, Hauxton Rd., Cambridge CB2 2LQ, United Kingdom.
Peter Jack (RAGT Seeds Ltd), Mike Field (Advanta Seeds Ltd), Peter Werner (CPB Twyford Ltd), Chris Chapman (Nickerson (UK) Ltd), Tina Henriksson (SW Seed Ltd), David Feuerhelm (Elsoms Seed Ltd), Tina Barsby (Biogemma UK Ltd), Graham Jellis (HGCA), Alex Waugh (NABIM), Sue Salmon (CCFRA), James Brosnan (SWRI), Andy Phillips (Rothamsted Research), Michael Holdsworth (University of Nottingham), John Snape (John Innes Centre), and Peter Kettlewell (Harper Adams University College).
This large (£2.15 M BPS, $3.77 M USD) multidisciplinary project (HFN LINK Project LK0975) started in October 2005 and runs until March 2010, seeks to identify genes and environmental stimuli that control variation in Hagberg falling number (HFN), an industry standard measure of starch integrity. Two independent phenomena are involved, preharvest sprouting (PHS) and prematurity amylase (PMA), with differing underlying genetic components and environmental triggers. In both cases, controlled conditions for expression of the character will be developed and used to help identify candidate genes involved and to map these against genetic markers identified in a broad range of elite U.K. germ plasm. The objective is to generate and validate DNA markers, ideally within the controlling gene(s), to enable breeders to select against undesirable PHS and PMA alleles in conventional crossing programs.
The breeding partners will supply and genotype a range of mapping populations, test their HFN performance across multiple locations over multiple years, and help test candidate gene leads. The academic groups will be responsible for various upstream activities. Harper Adams will develop a screening system to reliably induce PMA, largely based on what is known concerning the environmental factors which induce it. The potential of the established association between large grain size and PMA will be assessed to explore manipulation of grain growth as an alternative to an environmental screen. The screening system will be used to phenotype the mapping populations as a prelude to identifying molecular markers linked to genes or QTL for resistance. Rothamsted Research will complement this work by investigating the molecular basis for PMA production in developing grain, using a combination of amylase-GUS reporter lines, laser capture microdissection of individual grain tissues, microarray analysis and quantitative RT-PCR. TILLING will be attempted to identify novel alleles of genes involved in PHS and PMA from cultivar collections and mutagenized populations of wheat. The University of Nottingham will focus on PHS induction, by examining the relationship between dormancy in wheat embryos and PHS susceptibility. This will involve an examination of the developmental windows of dormancy induction, maintenance and loss during maturation, an analysis of genotype variation in depth of dormancy, relating depth of dormancy to PHS in a scalable way, comparing PHS induction under controlled environments with field performance, and the development of a lab-based smart screen for the analysis of PHS. The John Innes Centre will identify and analyze genetic variation for HFN with additional input from breeding companies' field trials, DNA marker analyses, and novel germplasm. An array of varieties and mapping populations representing HFN diversity among modern U.K. winter wheats will be physiologically assayed for resistance to PHS and PMA at two sites over three years, which will facilitate gene discovery via gene mapping. Collaborative development of both screening methods and identification of resistance/candidate genes will integrate the effort to supply the industry with intelligent phenotype and genotype selection tools, to promote the breeding of new wheat varieties with stable HFN and enhanced grain quality.
The project is being coordinated by RAGT Seeds Ltd and is strongly
endorsed by the entire supply chain, especially the breeding community.