Characterisation of the low temperature tolerant locus (Ltp1) on wheat chromosome 5D

Wheat production will need to increase by 60% in the next 50 years. However, this increase will need to happen against a background of climatic change. Temperatures are predicted to fluctuate globally by several degrees by the end of this century. The wheat growing area of nearly one billion people will be affected by major temperature fluctuations and an associated reduction in yield. Higher temperatures during wheat's flowering can have a major effect on subsequent grain yield. The climate is also going to become increasingly unpredictable with more extremes of weather in other regions, including the UK. Cold weather during floral development is also associated with sudden reductions in wheat yields. Cold wet weather occurring during floral development during mid-1980s resulted in significant sterility, leading to a 70% reduction in yields in the UK. Wheat yields need to increase against this background of climate change.

Clearly it is possible to screen wild relatives of wheat for those exhibiting greater temperature tolerance than wheat. Such characters can then be transferred from the wild relative to wheat through prebreeding. However, there are problems with this approach. Firstly, studies have shown that the relatives are more tolerant of temperature simply because their genome structure is simpler than wheat, not because they necessarily carry genes which provide them with a higher level of temperature tolerance. Secondly, there are particular stages in wheat's floral development that are more sensitive to temperature effects than others. Wheat varieties and wild relatives may also seem apparently more temperature tolerant simply because they have altered the timing of their pre-meiotic and meiotic stages, to avoid the extremes of temperature. Of course, such a strategy does not alleviate the problem of dealing with marked changes in temperatures during day and night. Thus, developing wheat whose floral development stages are truly more tolerant of temperatures is an important priority. Selection of plants truly tolerant of temperatures would require the identification of large numbers of plants at the temperature sensitive stage of floral development, then subjecting them to a range of temperatures, before scoring the subsequent effect on the level of seed set. This approach would be hugely time consuming and laborious. Realistically, it needs to be performed on a limited number of plants.

Our project addresses this problem. It will provide tools for selecting wheat that is more tolerant of temperatures during floral development. Our strategy is based around the fact that our recent research has narrowed down a temperature sensitivity stage of floral development covering pre-meiosis when replication occurs. Previous research showed that the long arm of 5DL also carries a locus which provides cells at this pre-meiotic stage with a tolerance to low temperatures. Our recent studies confirmed and extended these observations, namely that the long arm of wheat chromosome 5D (5DL) carries a locus, Ltp1 which provides cells at this stage with a tolerance to low (and high) temperatures, subsequently affecting whether chromosomes pair homologously or homoeologously. We have undertaken preliminary mapping and have defined the locus to the proximal region of 5DL, which includes the location of the Ph1 homoeoallele. We will refine the mapping to identify Ltp1 controlling low temperature tolerance during pre-meiosis, and generate resources to characterise the 5DL Ph1 homoeoallele. The resources generated will be used to assess whether these loci are also controlling tolerance to high temperature. The characterisation of Ltp1 will provide us with tools for screening a large number of wheat varieties to identify those with different variants of the locus. Such plants can be assessed for level of their temperature tolerance, to identify the variant providing the plant with the highest level of temperature tolerance.

Our studies show that wheat is particularly sensitive to both low and high temperatures during pre-meiosis. They have also confirmed that the long arm of chromosome 5D (5DL) carries a locus, Ltp1, providing pre-meiotic tolerance to low temperature. In the absence of this locus, homologues totally fail to pair during the meiotic telomere bouquet stage, instead pairing homoeologously after this stage. Since the Ph1 is functional, the paired homoeologues fail to crossover, leading to unbalanced gametes, and sterility. Our overall aim is to characterise Ltp1. The strategy is initially similar to that used to successfully define the Ph1 locus. M2 plants derived from 250 and 300Gy irradiated seed will be screened for 5DL deletions using DNA markers derived from the 5DL. The M2 plants with 5DL deletions will be phenotyped for whether they are tolerant of low temperatures. The DNA markers will define the approximate size and location of the deletion break points on 5DL. The phenotyped deletions will define a minimum overlap region still possessing the low temperature tolerance locus. The gene content of this minimum overlap region will be established using the newly available IWGSC-NRGene wheat genome sequence. Those genes specifically expressed during pre-meiosis will be identified. The TILLING population recently developed at JIC in collaboration with UC Davis will then be exploited. Mutations in the genes have been identified and displayed on a browser. All lines carrying mutations in the pre-meiotic genes will be screened for sensitivity to low temperatures during pre-meiosis. This will identify candidate genes for tolerance to low temperatures. We will also exploit a Ltp1 mutant to confirm that the Ltp1 candidates are indeed mutated. Cell biology approaches will also be used to accurately stage when low temperature affects the meiocytes during pre-meiosis. The deletion lines identified for 5DL will also be screened for pre-meiotic tolerance to high temperature.

During the next 50 years, as much wheat grain will be required as has been produced since the beginning of agriculture, against a background of changes in climatic conditions. Global temperatures are likely to increase by several degrees by the end of the century, with wheat yields falling by 6% for every degree increase in temperature. In the UK and France, wheat yields have also failed as a result of low temperatures during the flowering stage. One billion wheat consuming poor will be in regions where temperatures are expected to undergo major fluctuations. Thus, the political impact of producing more temperature tolerant wheat is enormous. There are numerous incidents of political instability as a result of higher wheat prices, for example, the 'Arab Spring' initially started as a knock-on effect of localised heat and drought affecting wheat yields in Russia.

The G20 Wheat Initiative established by the G20 ministers has highlighted identifying temperature tolerance as an important trait going forward. Wheat pre-meiosis is particularly sensitive to temperature fluctuation (both high and low). We have recently confirmed the mapping of both high and low pre-meiotic temperature tolerance to chromosome 5DL. The 'Case for Support' indicates that the 5DL Ltp1 locus may confer tolerance to both. Screening for high temperature tolerance is extremely labour intensive, and the phenotype is compounded by other factors. Screening for low temperature tolerance is easier. The successful outcome of present proposal will be the identification of the locus Ltp1 controlling this pre-meiotic tolerance. This information will be exploited after this grant finishes to screen the Watkin's diversity panel, thereby identifying a limited number of accessions carrying the full range of alleles present at this locus. These specific accessions can be then scored for temperature tolerance. Through BBSRC WISP (Wheat improvement ISP), populations are available in which these landraces have been already crossed onto an elite wheat. Thus, the pre-meiotic temperature tolerant allele may have been already transferred into an elite background. A simple KASP marker assay would identify those progeny carrying the optimal allele. BBSRC WISP involved private breeders (Limagrain, KWS-UK, RAGT, Syngenta, Elsoms) who have been provided with the IP free germplasm generated and are growing the germplasm in their international nurseries. The current proposal would inform the breeders which lines carry the pre-meiotic temperature tolerant loci. Prof Moore is on the management board of the CGIAR WHEAT breeding (CIMMYT (International maize and wheat improvement centre) and ICARDA (International Centre for Agricultural Research in Dry Areas)) programme for the resource-poor in the developing World. CIMMYT germplasm is again being exploited within Designing future wheat (DFW), and CIMMYT will be informed as to which germplasm carry the optimal premeiotic temperature tolerant alleles.

The expected outcome of this project will be the creation of tools to enable breeders to better screen accessions for temperature tolerance. The immediate beneficiaries will be those involved directly in wheat pre-breeding. The programme will also enhance the development of a skills base in cytogenetics, a dying art in the UK, which can be utilized by the next generation of scientists involved in wheat prebreeding and the larger wheat community.

This research will make a contribution to food security and sustainable agriculture, key objectives of BBSRC's strategic plan. Wheat has been identified as a key crop by BBSRC and it has financially supported a new DFW programme. Ultimately, the farmers will benefit by having durable wheat cultivars more tolerant of changeable abiotic conditions. This in turn will have major societal benefits through reducing political instability as a result of fluctuations in wheat supply, and hence wheat prices, due to changing climatic conditions.