References Cox, D.R., Burmeister, M., Price, E.R., Kim, S., and R.M. Myers Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science 250: Erayman, M.D., Sandhu D., Sidhu M., Dilbirligi, P.S., Baenziger P.S., and K.S. Gill Demarcating the gene-rich regions of the wheat genome. Nucleic Acids Res. 32: Johnson, E.B., V.J. Nalam, R.S. Zemetra, and O. Riera-Lizarazu Mapping the compactum locus in wheat (Triticum aestivum L.) and its relationship to other spike morphology genes of the Triticeae. Euphytica 163: Krattinger, S., Wicker,T., and B. Keller Map-based cloning of genes in Triticeae in Genetics and Genomics of the Triticeae. Springer Science, NY, NJ. p Peng, Z-S., Martinek, P., Kosuge, K., Kuboyama, T., Watanabe, N., Genetic mapping of a mutant gene producing three pistils per floret in common wheat. J. Appl. Genet 49: Vijay K. Tiwari 1, Kasandra Lopez 1, Oscar Riera-Lizarazu 2, Shahryar F. Kianian 3, Jeffrey M. Leonard 1 ABSTRACT At 16,000 Mb, the wheat genome is 40-fold larger than the rice genome and carries up to 80% repetitive DNA. Genetic maps based upon meiotic recombination poorly resolve regions of low recombination which occupy up to 30% of total chromosome length. Map-based cloning of wheat genes in recombination-poor regions is therefore difficult. Here we report a quick approach based on pollen irradiation to construct physical maps of chromosome regions for both Compactum and Three Pistil loci. The club head phenotype is determined by the dominant Compactum (C) locus located on the pericentromeric region of chromosome 2D. It has proven difficult to map as it lies in an area of low recombination. Three Pistils (Pis1) is a wheat floral mutant producing three pistils per floret instead of the usual single pistil. Pis1 has been mapped to chromosome 2DL. We irradiated pollen of hexaploid wheat (AABBDD) carrying the genes of interest with 1.5 krad gamma-rays prior to fertilization of tetraploid (AABB) wheat. The quasi-pentaploid F 1 plants (AABBD) carried radiation-induced fragments of D genome chromosomes. Measures of marker coretention were used to estimate linkage and build physical maps. Phenotypes, resulting from loss or retention of either C or Pis1, were observed directly in F 1 s. Separate F 1 mapping panels for C and Pis1 were generated. Since D-genome chromosomes were single copy in pentaploid F 1 s, most chromosome-specific markers were informative. We outline the approach and present preliminary physical maps of both genes Physical Mapping of Compactum and Three Pistil Loci on Wheat Chromosome 2D Using Radiation Hybrids DBI Department of Crop and Soil Science, Oregon State University, Corvallis, OR 2 International Crops Research Institute for the Semi-Arid Tropics, Patancheru, Andhra Pradesh, India 3 Department of Plant Sciences, North Dakota State University, Fargo ND Fig. 4. Marker retention along Chromosome 2D. 187 Pis1/Altar and 93 Corringin/Langdon F 1 s were genotyped, with 12 SSR markers each. Marker retention along chromosome 2D ranged from 80.6% to 96.8% in the two populations. Although largely uniform, both populations showed higher retention of marker wmc144 at 67 cM. Within the lines, marker retention averaged 85.3% and 86.7% in the two populations with ranges of 11.1% to 100% and 7.7% to 100%. 72/187 (38.5%) and 30/93 (32.2%) of the lines were mapping informative for 2D in the Pis1/Altar and Corringin/Langdon populations respectively. Table 1. Germination Rates of Pis1/Altar and Corringin/Langdon F 1 s. F 1 seeds planted directly in soil had reduced germination rates. To increase plant recovery, embryos from the Pis1/Altar population were excised and rescued on media. Embryo rescue increased recovery of viable plants to 25-28%. Summary These preliminary results mapping two genes on wheat chromosome 2D support the utility of the radiation hybrid physical mapping procedure. The location of Pis1 on 2DL is in agreement with genetic mapping efforts and will be refined with the addition of more phenotypes. In the next two months, sufficient phenotypes from the Compactum mapping population should allow mapping of C as well. Three markers completely linked to C in a genetic map based on 260 lines were separated by 73 cR in our RH physical map based on 93 lines, validating the approach in areas of low recombination. Because F 1 phenotypes are genotyped and phenotyped directly, this mapping protocol proceeds quickly. It is unclear if higher marker retention near wmc144 is due to marker anomalies, centromere retention, or selection for a required gene(s). Further development of markers in this region may answer this question. Additional markers will allow estimation of the potential mapping resolution of these panels. Assuming radiation-induced chromosome breakage is random and uniform, resolution of physical maps can be easily increased by adding more lines. For genes in areas of low recombination and with phenotypes amenable to F 1 scoring, radiation hybrid mapping using irradiated-pollen-derived deletion mutants may be a viable option for map- based cloning. Fig.1. Effect of Radiation Dose on Seed Set. Seed set of F 1 seeds decreased as gamma-ray radiation dosage of pollen increased. Since deletions were expected to increase with radiation dosage, a treatment of 1.5 kRAD was selected as the highest rate that would allow recovery of viable F 1 plants. ABD ABD ABD AB Hexaploid Pollen Gamma-Rays X Tetraploid gamete METHODS Hexaploid wheat spikes at the point of anthesis were irradiated with varying dosages of gamma rays. The pollen was immediately used to fertilize emasculated tetraploid plants. Gamma ray-induced deletions of the hexaploid-derived chromosomes were recovered in the quasi-pentaploid F 1 s. Deletions in the A or B genome were masked by tetraploid-derived homologous chromosomes, but deletions in D-genome chromosomes are detectable by PCR as null alleles. Deletions in the targeted genes are detectable by phenotyping the F 1 s. F 1 mapping populations are being genotyped and phenotyped. Measures of marker cosegregation are used to build physical distance-based radiation hybrid maps. Targeted Gene Quasi-pentaploid F1 PopulationPis1/AltarCorringin/Langdon Florets pollinated Seed set Plants recovered (%)55 (20.7)105 (33.2) B C A D Fig. 5. Radiation Hybrid map of 2D. Two maps were constructed using the 30 Corringin/Langdon lines (A) or the 72 Pis1/Altar lines (B). Distances on RH maps are in cR1500 (centiRays). Although preliminary, orders of markers in both maps largely agree with the consensus genetic map (C) and the bin map (D). Pis1 was linked to cfd168. Insufficient phenotypes in the Corringin/Langdon population at this time only allowed an estimation of its position between cfd116 and gwm358 until more phenotypes are available. cR/cM ratios were 35:1 and 5:1 for maps A and B, respectively. The large cR/cM ratio of the Corringin/Langdon map, B, may reflect the fact that Compactum occurs in an area of low recombination and therefore reduced genetic distances. Estimated position of Compactum Wt single pistil wheat Pis1 mutant with three pistils Fig, 2. Target genes and phenotyping. Two genes located on chromosome 2D, Pis1 and Compactum, were targeted to test the procedure. Common wheat produces a single seed from a single pistil per floret (A, B). The Pis1 mutant retains the normal three stamens but produces three pistils(C,D). All pistils are fertile and often set three seeds per floret. We used pollen from irradiated spikes of the Pis1 mutant to pollinate the tetraploid, single-seeded variety ‘Altar. F 1 s from unirradiated Pis1 plants produce three pistils. Club wheat differs from common wheat through the action of the Compactum (C) locus which results in a compact spike (E). C is completely linked to SSR loci Xcfd116, Xgwm358, Xwmc144, Xcfd17, and Xbarc145. Because these markers are located on bin C-2DL3 and bin C-2DS1 the locus could reside on the long or short arm of 2D. Irradiated pollen from the club head Corringin was used to fertilize tetraploid Langdon. Unirradiated F 1 s are club head. C A D B Corringin F 1 with deletion Langdon F1F1 E Fig 3. Identifying deletions. Since D-genome chromosomes are in single copy, D-genome markers are dominant and null PCR reactions indicate deletions. A universal control primer is multiplexed in all reactions as a positive control for the PCR reaction Control primer D genome marker deletions C B A D