Final Report Summary - WHEAT SENESCENCE (Dissection of the senescence and mineral remobilization pathways in wheat) Final scientific report for grant 277036 – Assaf DistelfeldThe research program “Dissection of the senescence and mineral remobilization pathways in wheat” funded by the Marie Curie FP7 program resulted in four publications in international leading plant journals. The program enabled me to establish my lab and to make two major breakthrough; the first is directly related to the research proposal and it includes a comprehensive and in depth characterization the individual GPC genes and their role in the regulation of terminal senescence and mineral remobilization in wheat. We have characterized wheat knockout mutants for the GPC-A1 and GPC-D1 genes in hexaploid wheat and GPC-A1 and GPC-B2 in tetraploid wheat. Our results show a significant delay in senescence in both the gpc-a1 and gpc-d1 single mutants and an even stronger effect in the gpc-1 double mutant in all the tested environments in this study. The accumulation of total N in the developing grains showed a similar increase in the control and gpc-1 plants until 25 days after anthesis (DAA) but at 41 and 60 DAA the control plants had higher N levels than the gpc-1 mutants. At maturity, GPC measurements in all mutants were significantly lower than in control plants while grain weight was unaffected. These results demonstrate that the GPC-A1 and GPC-D1 genes have a redundant function and play a major role in the regulation of monocarpic senescence and nutrient remobilization in wheat (Avni et al. 2014). As a step toward understanding the gene network involved in wheat terminal senescence we performed two RNAseq experiments using gpc-1 double mutants in tetraploid and hexaploid wheat (Cantu et al. 2011; Pearce et al. 2014). The results showed significant differences between gpc-1 double mutant and control plants in the expression level of few hundreds of genes. These genes include transporters from the ZIP and YSL gene families, which facilitate Zn and Fe export from the cytoplasm to the phloem, and genes involved in the biosynthesis of chelators that facilitate the phloem-based transport of these nutrients to the grains. This study provides an overview of the transport mechanisms activated in the wheat flag leaf during monocarpic senescence. It also identifies promising targets to improve nutrient remobilization to the wheat grain, which can help mitigate Zn and Fe deficiencies that afflict many regions of the developing world. The program also allowed me to produce a second breakthrough of establishing one of the most detailed and accurate wheat genetic maps. This genetic map was constructed using cutting-edge technologies and since it relays on a genetic population that is based on a cross between wild × domesticated wheat. Wild emmer wheat (T. turgidum ssp dicoccoides, genomes BBAA) gene-pool is an important source for wheat research and improvement. To utilize this resource, we hybridized wild emmer wheat (subpopulation judaicum, accession Zavitan) with durum wheat (T. turgidum ssp durum, cv. Svevo), and developed an F6 recombinant inbred line (RIL) population. The wheat 90K iSelect SNP genotyping assay was used for genotyping of the RILs, detecting segregation for 16,387 polymorphic markers. The genetic map was constructed based on the genotypic data of 140 RILs using the MultiPoint-ultradense software package and included a total of 14,088 markers grouped into 2,296 genetic loci (unresolvable by recombination) in 14 linkage groups, corresponding to the 14 chromosomes of tetraploid wheat. The map was 2,110 cM long with an average distance of 0.92 cM between adjacent markers. The B genome was slightly more polymorphic (57%) for co-dominant SNP markers than the A genome. The map included 1,012 null-allele markers, in which only one SNP allele was detected, the frequency of these markers in the B genome of wild emmer greatly exceeded that of the A genome (69% and 31%, respectively), which may reflect a greater rate of genomic changes in the B genome. Comparison of our mapped SNP sequences with the barley genome revealed that most of the markers (92.4%) were syntenic. This ultra-dense SNP-based genetic map with a high level of synteny to barley provides a useful framework for genetic analyses of important traits, positional cloning, and marker-assisted selection, as well as for comparative genomics and genome organization studies in wheat and other cereals. To summarize, our map showed the highest resolutions in regions that are fixed in most of the wheat genetic maps and therefore have a great advantage (Avni et al. 2014b; Maccaferri et al. 2014). To improve this resource even further we used 'genotyping by sequencing approach (GBS)' and generated ~900,000 markers (unpublished data). To utilize this resource even further I have initiated an international consortium aimed to sequence wild emmer wheat genome (http://peleglab.wix.com/wewseq ). The consortium is a collaborative effort of 10 labs and its goals are: (1) To assemble a high quality reference genome for wild emmer wheat (AABB genome) (2) To fully annotate the gene space within that reference (3) To establish an open access, web-based database that integrates genomic data (wheat, barley, rice, brachypodium), haplotype data (variation within species), and phenotypic data (yield, quality, disease resistance, climate adaptation, etc.). The expected outcome of this project will enable efficient use of our natural diversity: harnessing the power of molecular biology to assist breeders to increase wheat tolerance to environmental stresses with enhanced yields. This publically available resource will facilitate wheat genetic research worldwide and promote wheat breeding efforts. This project will develop of the knowhow to enable cost-effective genome sequencing and data assimilation for other crop plants.Research carrier development and re-integration – I am fully integrated in the department of Molecular Biology and Ecology of Plants and all the department supported to promote me for tenure. My tenure package is currently under international experts review and I am optimistic about their evaluations. References:1. R. Avni, R. Zhao, S. Pearce, Y. Jun, C. Uauy, F. Tabbita, T. Fahima, A. Slade, J. Dubcovsky, A. Distelfeld (2014a) Functional characterization of GPC-1 genes in hexaploid wheat. Planta 239, pp. 313-324. 2. R. Avni, M. Nave, T. Eilam, H. Sela, C. Alekperov, Z. Peleg, J. Dvorak, A. Korol, A. Distelfeld (2014b) Ultra-dense genetic map of durum wheat × wild emmer wheat developed using the 90K iSelect SNP genotyping assay. Molecular Breeding, pp. 1-14.3. A. Distelfeld, R. Avni, A.M. Fischer (2014) Senescence, nutrient remobilization, and yield in wheat and barley. Journal of Experimental Botany 65, pp. 3783-3798.4. S. Pearce, F. Tabbita, D. Cantu, V. Buffalo, R. Avni, H. Vazquez-Gross, R.R. Zhao, C.J. Conley, A. Distelfeld, J. Dubcovksy (2014) Regulation of Zn and Fe transporters by the GPC1 gene during early wheat monocarpic senescence. Bmc Plant Biology 14.5. M. Maccaferri, A. Ricci, S. Salvi, S.G. Milner, E. Noli, P.L. Martelli, R. Casadio, E. Akhunov, S. Scalabrin, V. Vendramin, K. Ammar, A. Blanco, F. Desiderio, A. Distelfeld, J. Dubcovsky, T. Fahima, J. Faris, A. Korol, A. Massi, A.M. Mastrangelo, M. Morgante, C. Pozniak, A. N'Diaye, S. Xu, R. Tuberosa (2014) A high-density, SNP-based consensus map of tetraploid wheat as a bridge to integrate durum and bread wheat genomics and breeding Plant Biotechnology Journal, pp. 1-16.