DBI Vijay K. Tiwari 1, Oscar Riera-Lizarazu 5, Ajay Kumar 2, Muhammad Javed Iqbal 2, Hilary Gunn 1, KaSandra Lopez 1, Anne Denton 2, Yong Q. Gu 4, Ming-Cheng Luo 3, Gerard Lazo 4, Shahryar F. Kianian 2 and Jeff M. Leonard 1 1 Department of Crop and Soil Science, Oregon State University, Corvallis, OR Department of Plant Sciences, North Dakota State University, Fargo, ND Department of Plant Sciences, University of California, Davis, CA USDA-ARS, Western Regional Research Center, Albany, CA International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru , Andhra Pradesh, India A Quick and High Throughput Approach for Construction of D Genome Radiation Hybrid Maps in Wheat Using Pollen Irradiation Radiation hybrid mapping is an efficient method to generate high-resolution maps that are independent of meiotic recombination. Here, we report a quick, high throughput, and cost-effective approach, based on pollen irradiation, to construct physical maps of all seven D genome chromosomes simultaneously. Panels based on pollen irradiation at 1.5 krad to 2.0 krad have been generated. Subsets of these RH panels were characterized using SSR markers and an average marker retention frequency of 92.6%, 84.1% and 66.5 %, for 1.0, 1.5 and 2.0 krad treatments were estimated, respectively. A portion of one panel representing 89 samples was tested using the DArT marker platform. This resulted in the construction of radiation hybrid maps for all seven D genome chromosomes based upon 650 DArT markers. Total map distances for chromosomes 3D and 7D were cR 2000 and cR 2000, respectively. The total map distance ratio between genetic maps and RH maps (cM/cR 2000 ) for these chromosomes (3D and 7D) were estimated at 1:15 and 1:13, respectively. Marker orders were comparable to previously reported genetic maps. We compared average marker distances from genetic maps and RH maps, based on co-segregating markers and the cM/cR 2000 ratio was found to be 1:18. Markers derived from various BAC contigs are now being tested to estimate the physical map resolution of the developed RH panels. High resolution RH maps will be valuable for analysis of a great number of biologically and agronomically important genes, as well as in the comparative genomics studies of other grass genomes. ABSTRACT Seeds of Hexaploid wheat “Chinese Spring” Greenhouse planting Anthesis stage days Pollen Triticum aestivum n=3x=21 ABD g-irradiation X F1 hybrid: Seed harvested after 25 DAP Dissection and embryo rescue Marker assays and mapping Endosperm tissue collection Marker assays and mapping Pollen irradiation panel 1.Very quick method 2.Small population size 3. Low retention frequencies ( 30-89%) Durum Wheat Triticum turgidum 2n=4x=28 AABB Planting and DNA extraction DNA extraction ChromosomeNumber of markers usedNumber of linkage groupsMap length ( cR 2000 ) 1D D D D D D D Characterization Of Pollen Irradiated Radiation Hybrid Panels Initial characterization of the RH panels developed with different dosages was done using a set of 14 SSR markers Fig.1. Schematic presentation of production of wheat D-genome radiation hybrids (DGRHs). Pollens of the hexaploid wheat (Triticum aestivum, n=3x=21, ABD) landrace ‘Chinese Spring’ treated with gamma-rays were crossed to the tetraploid wheat (T. turgidum, 2n=4x=28, AABB) variety ‘Altar 84’. After observing viable seed set, embryos and endosperms were harvested independently from each seed. Embryos were transferred to embryo rescue media for their regeneration into respective plants. Endosperms were used directly for DNA extraction. Each such embryo- derived plant and endosperm event independently represent a Radiation hybrid mapping panel. Important features of this scheme for the production of a mapping panel is that the irradiated D-genome chromosomes will be in the hemizygous condition and lesions on A and B chromosomes from T. aestivum will be masked by the presence of normal counterparts from T. turgidum. Consequently, this panel will preferentially reveal lesions on D-genome chromosomes. From a mapping perspective, DGRHs permit the simultaneous mapping of all D-genome chromosomes, however polymorphic markers between CS and Altar could be used for mapping of A and B genome chromosomes as well. Status Of Pollen Panels Generated At OSU And NDSU Selection Of Informative Individuals Based On Initial Characterization For High Throughput Genotyping And High Resolution Mapping We opted to work with 2 krad panels based on their lowest marker retention frequencies. 282 lines from 2 krad panel were screened. Based on that we picked highly informative lines for further high throughput genotyping to construct radiation hybrid maps of all D genome chromosomes, simultaneously.  Out of 94 lines we further selected 81 lines along with parents (CS and Altar), F1 endosperm DNA, F1 reconstructed DNA and nulli and tetra lines for all seven D-genome chromosomes. These set of lines were then sent for DArT genotyping.  We applied 10 SSR markers per chromosomes for all seven D –genome chromosomes. Average Resolution Of RH Mapping Panels And Maps We used two approaches to estimate resolution of the mapping panel used in this study.  Our system present a whole genome mapping panel, so we used contigs based marker information from wheat chromosome 3B. Based on the frequency of breaks between two markers of known distances we estimate the resolution of our mapping panel to be ≈550 kb.  Based on the number of obligate breaks induced /Mb of the chromosome (7D), we estimated the resolution of 7D chromosome to be ≈ 500 kb. However this is an underestimate as more breaks will be discovered with more markers added.  Assuming that radiation-induced chromosome breakage is random and uniform, resolution of a physical map can be easily increased by adding more lines. Radiation dose (krad) Number of plants generated Number of lines tested Marker retention % per plant (range) % of individuals containing all of tested markers Number of lines generated Number of lines tested Marker retention % per plant (range) % of individuals containing all of tested markers Plants/ tissue derived from embryosEndosperms 1 krad (78-100) krad (13-100) (50-100) krad (33-100)8.7 Total500272≈ Table 1. Showing status and properties of pollen irradiated plant and endospermic panels Fig.2. % DNA marker retention frequency based on 14 SSR markers for 3 different pollen panels derived Fig.3. Marker retention frequencies across the all three genomes (3a.) and within the D genome chromosomes (3b.) based on selected RH lines using DArT and SSR markers. Wheat genome 3a.3b. Development Of Pollen Irradiated Radiation Hybrid Mapping Panel Table 2.Represents map based information of all 7 D radiation hybrid maps Genetic map Bin map RH map Bin map Genetic map RH map 4a. 4b. 4c. Fig. 4. RH maps of 1D, 3D and 7D chromosomes and their comparisons with respective genetic and deletion bin maps. (4a) and (4b): RH map of 1D and 3D are flanked by genetic map on their left and by bin maps on the right side. (4c): RH map of 7DS-4 at the extreme right, correlated with 7D bin map in the middle, on the left side of the bin map there is RH map of the centromeric region and is compared with 7D genetic map, showing that 8 cM distance of genetic map is corresponding to cR to RH map of the region. RH Mapping A total of 740 (640 DArT + 70 SSR) markers were used to create RH maps of D genome chromosomes using just 81 informative lines. RH mapping software CARTHAGENE was used to order the markers at the threshold distance of 0.3 and LOD score of 10. RH maps of 1D, 3D and 7D chromosomes Status Of The Project  We are in the process of assembling panels of varying levels of resolution.  Retrotransposon element junctions (REJ) are generated as a new single-copy and locus-specific marker system for hexaploid wheat. NimbelGen array using RJM markers is being developed for high throughput genotyping. References  Somers DJ, Isaac P, Edwards K: A high-density microsatellite consensus map for bread wheat (Triticum aestivum l.). Theor Appl Genet 109: (2004).  Sourdille P, Singh S, Cadalen T, Brown-Guedira GL, Gay G, Qi L, Gill BS, Durfour P, Murigneux A, Bernard M: Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (Triticum aestivum l.). Funct Integr Genomics 4:12-25 (2004).   cM/cR ratio was found to be quite variable across the length of the chromosome.  At the end of the chromosome average cM/cR 2000 ratio calculated was 1:15  However the ratio(cM/cR 2000 ) increased abnormally towards the centromeric region and was found to be 1:84 (7D). Acknowledgements Funding from the National Science Foundation, Plant Genome Research Program grant No. DBI is gratefuly acknowledged.