JOHN INNES CENTRENorwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.
Karen Butterworth, Alex Reid, Liz Sayers, and Tony Worland.
Photoperiod response is extremely important to wheat varietal adaptability. In many areas of the world, like southern Europe where summers are regularly hot and dry, only the earlier flowering photoperiod insensitive genotypes are able to set and fill grain before plants suffer from summer desiccation.
Photoperiod response is determined primarily by a homoeologous series of genes Ppd-A1, Ppd-B1, and Ppd-D1 (formerly Ppd3, Ppd2, and Ppd1 respectively) located on the short arms of chromosomes 2A, 2B, and 2D. Only Ppd-D1 is believed to exist in commercial European wheat varieties. To compare the effects of Ppd-D1 with alternative, but less utilized, genes Ppd-A1 and Ppd-B1, very precise genetic stocks have been developed to introduce all three photoperiod insensitive genes into the U.K. winter wheat variety Mercia.
When grown in a specially constructed glasshouse where day length was restricted to 10 hours all photoperiod insensitive genotypes flowered significantly earlier than the photoperiod responsive controls. Lines carrying Ppd-D1 were the earliest to flower and lines with Ppd-B1 the latest. Ppd-A1 lines were intermediate in flowering between Ppd-D1 and Ppd-B1. These results were in contrast to earlier work on whole chromosome substitution lines carrying Ppd-A1, Ppd-B1, or Ppd-D1. With this less precise material Ppd-D1 was again shown to be most potent in accelerating ear emergence time, but Ppd-A1 was considered weaker than Ppd-B1. Under field conditions in the U.K. in a Mercia varietal background over 2 years of trials, Ppd-D1 accelerated ear emergence time by around 8 days, Ppd-B1 by 3 days, and Ppd-A1 by 5 days. We anticipate that the Ppd-A1 or Ppd-B1 genes may be of importance in the U.K. where the acceleration of ear emergence time induced by Ppd-D1 is normally too strong. In experiments carried out in cooperation with Dr. M. Rousset, INRA, France, the Ppd genes had a much greater effect of accelerating ear emergence time with Ppd-D1 bringing flowering forward by 17 days, Ppd-A1 by 9 days, and Ppd-B1 by 3 days.
The field experiments conducted in the U.K. showed that whilst accelerating ear emergence time the genes for photoperiod insensitivity had no pleiotropic effects on plant height, 1,000-kernel weight, or ear yield. All three Ppd genes in shortening the life cycle reduce the number of fertile tillers. Both Ppd-A1 and Ppd-D1 significantly reduce the numbers of spikelets developing in the spike by about one and two, respectively. All three photoperiod insensitive genes increased spikelet fertility. In the UK plot yields were reduced as a consequence of the reduction in numbers of fertile tillers. In a farm situation, the genetic reduction in tillering capacity could be compensated for by increasing seed rates.
In the season 2000-01 the lines are being grown in England, France, Germany, Poland, Scotland, and Yugoslavia to determine their adaptive significance in diverse European environments.
Robert Bradburne, Elizabeth O'Connor, Lesley Fish, and John Snape.
Hectoliter weight is an important economic factor determining the selling price of grain in some sectors of the European market. Based on the weight of one liter of unpacked grain, hectoliter weight is likely to be affected not only by seed weight but also by the packing characteristics of the individual grains. The International Triticeae Mapping Initiative (ITMI) RIL mapping population has been extensively mapped using a variety of biochemical and genetic markers and therefore provides a valuable resource for looking at novel phenotypic traits such as hectoliter weight.
The lines were grown in a randomized, replicated field trial and seed from hand threshed tillers were photographed using a digital camera and analyzed using digital image analysis software. In this way, 40 seed per line could be analyzed simultaneously, allowing a representative sample of seed sizes and shapes to be taken from each line. In addition, the 50-kernel weight and hectoliter weight of each of the lines was determined.
The two parents of this population showed significant differences in seed shape and size and even more variation was found within the RI population itself, suggesting transgressive segregation. These data were analyzed with respect to the genetic marker data available for the population. In all, 30 QTLs were found for the eight variables examined, explaining between 12 and 40 % of the phenotypic variation in any one individual trait. There were clusters of QTLs for seed size variables on chromosomes 1A and 7D and for shape variables on the group-2 chromosomes. The distinction between genetic control of seed size and seed shape suggests that it should be possible to breed for altered size or shape independently.
The two most important factors determining hectoliter weight were found to be seed length and seed volume. Both of these showed a significant negative correlation with hectoliter weight suggesting that for the highest hectoliter weight, it is necessary to breed for small, round grains.
We hope that the results from this model population can now be applied to other crosses between high quality British winter wheat varieties which are also under study at the John Innes Centre.
Paul Nicholson, J.P. Carter, F.M. Doohan, A. Mentewab, and H.N. Rezanoor.
The genetic basis of resistance to FHB of a number of sources is being studied using doubled haploid and recombinant inbred lines. Resistance is assessed using several traits to identify individual components of the resistance, including the AUDPC, relative spikelet weight (REW), DON content, and fungal biomass (measured using quantitative PCR). Preliminary results indicate that, in the lines under investigation, resistance is conferred by very few genes of major effect along with other genes of minor effect. Potential mechanisms involved in resistance are also under study. Two antifungal proteins were isolated from grain of Arina and the potential role, if any, of these in the resistance of this variety to FHB is under investigation.
Molecular genetic studies have revealed the presence of, apparently, discrete groups within F. graminearum. Initial studies indicate that there is a potential association of different groups with wheat and maize in Nepal. Studies are being extended to include isolates from Europe and the U.S. and a large number of isolates from China. Additional studies are underway to identify factors involved in the regulation biosynthesis of trichothecene mycotoxin. A number of molecular tools have been developed to facilitate these studies, including RT-PCR assays and isolates tagged with reporter genes controlled by various promoter sequences.
Steve A. Quarrie, Catherine Chinoy, Andy Steed, Les Saker, Paul Farmer, Judy Purves, and David T. Clarkson.
We have studied the genetic control of nitrogen uptake and its effect on productivity in a mapping population of bread wheat in relation to variation in root. Ninety-five doubled haploid lines have been made from the cross 'Chinese Spring/SQ1' and a genetic map prepared with over 300 RFLP, AFLP, microsatellite, isozyme, and morphological markers. These markers provide coverage of about 70 % of the genome, with equal proportions on the A and B genomes but only 11 % on the D genome.
The lines were trialed for 2 years on a very light soil with three rates of nitrogen applied (45, 90, and 170 U/ha) in three replications of two rows per line. As well as measuring agronomic characters at the end of the season, plants were collected during the growing period (in May and June in 1997 and June in 1998) for determining nitrogen content and biomass production. The presence of QTLs was determined using QTL Cartographer model 6 (composite interval mapping) with typically 14 background parameters selected for most traits.
Although the trials were in adjacent parts of the same field in 1997 and 1998, grain yields in 1998 were substantially higher than those in 1997: experiment means 3.3 and 5.7 t/ha in 1997 and 1998, respectively. Thus, the high N treatment increased yield in comparison with the low N treatment by 53 % in 1997 but by only 22 % in 1998. Grain yield was controlled mainly by QTLs on chromosomes 7AL and 5AL in 1997 in all three treatments, but in 1998 yield was controlled by different QTLs in each N treatment and none of them gave QTLs on chromosomes 5AL or 7AL. The only consistent grain yield QTL across the 2 years was located close to the Glu-D1 locus on chromosome 1D for the low-N treatment. Because of the lack of coincidence for yield QTLs across N treatments in 1998, analysis of yield in relation to other traits has focused on data for 1997.
The yield QTL on chromosome 7AL in each N treatment appeared to be associated with plant vigor, as both the rate of dry matter increase and accumulation of N per plant during the vegetative stage (between sampling occasions 1 and 2) gave significant QTLs coincident with those for yield on chromosome 7AL. Another major QTL for N increase per plant was on chromosome 5B. The presence of the vernalization response gene vrn-A1 on chromosome 5AL led to a 3-week range of flowering dates in 1997 and both yield and rate of dry matter increase were significantly negatively associated with flowering date. Furthermore, major QTLs for those traits were coincident with the vrn-A1 locus. Therefore, yield data were reanalyzed after adjusting for flowering date effects using the residuals from linear regressions. This removed the QTLs on chromosome 5AL, but the major QTLs on chromosome 7AL remained.
Root biomass during grain filling was recorded in an experiment using tubes containing a 70-cm column of seived compost, watered regularly. Analysis of root biomass showed a major QTL on chromosome 5AS and another coincident with vrn-A1 on 5AL. In general, root biomass increased with days to flowering. Therefore, root biomass data were also adjusted by regression analysis for flowering date. Adjusting the data removed the QTL coincident with vrn-A1 and produced new QTLs, one coincident with the QTL for yield under low N on chromosome 1D and another on chromosome 5B, coincident with the major QTL for N increase per plant. These results suggest that an extensive root system is important in maintaining N uptake and yield under low fertility conditions, and effects on plant vigor in 1997, not explained by root growth, are present on chromosome 7AL.
Robert Koebner and James Hadfield.
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. The method 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. Plants hemizygous for a known chromosome were selected by microsatellite analysis among F1 individuals obtained by crossing the variety carrying the target gene with the relevant monosomic line, and these were allowed to self-fertilize to generate F2 progeny segregating with respect to the critical chromosome into disomics, monosomics, and nullisomics. The proportion among the progeny in each segregant class depends on the rate of gametic transmission of the monosome. Although this rate is close to 100 % through the pollen as a result of certation, the rate through the female gamete is somewhat chromosome dependent. For mutagenesis of these F2 seed, we used three different agents - fast neutrons from the IAEA reactor in Seibersdof, Austria (now decommissioned), gamma irradiation, and EMS. As the viability of nullisomic individuals tends to be low without any buffering from extra doses of homoeologues, few of the nullisomic selections would be expected to complete their life cycle and produce progeny. Disomic segregants were unlikely to have been selected, as these would have needed to carry simultaneous mutations in both copies of a critical gene. We used microsatellite analysis again, in this case to discriminate the monosomics from any nullisomics.
Targeting three Yr genes (Yr1, Yr5, and Yr10), 219 (out of 543 F1 plants) monosomic 2A, 164 (out of 341) monosomic 2B, and 304 (out of 545) monosomic 1B plants were selected to generate the necessary numbers of hemizygous individuals. Estimated from the test weights, these gave rise to 14,500, 4,500, and 8,500, respectively, F2 seeds for mutagenesis, reflecting differences in the fertility of the different monosomic lines. Following mutagenesis, germination rate was above 80 %, and the numbers of susceptible segregants selected from the three populations were 603, 71, and 241, respectively. In the case of Pm3b, 169 monosomic 1A F1 plants were grown and 200 susceptible F2 individuals were recovered from g-irradiated material and 125 from EMS treatment. We also have generated large numbers of putative mutants from Pm3d with the same technique.
This approach 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 pathway. We believe that this could be used in a systematic way to generate chromosome-specific mutant populations to cover the entire wheat genome, a resource which is not readily available in any other crop species.
From April 2001, the John Innes Centre will restructure its
departmental organization, creating six new theme based Departments
from the original nine. The present Cereals Research Department
will be subsumed into a new Department of Crop Genetics, headed
by Dr. John Snape, which will also include work on Brassica species,
particularly Canola and legumes. Most of the work on wheat at
JIC will still be the responsibility of the department, although
the fungal pathogen programs presently in the Cereals Research
Department with Drs. James Brown, Lesley Boyd, and Paul Nicholson,
will become part of a new department of Disease and Stress Biology.
Work on cereal starch biochemistry and molecular biology, under
Drs. Kay Denyer and Alison Smith will be carried out in the Department
of Metabolic Biology.