A Quick Method For Detection Of Gamma Ray-induced Genomic Deletions In Wheat DBI Vijay K. Tiwari 1, Hilary Gunn 1, Oscar Riera-Lizarazu 2, Shahryar F. Kianian 3, Jeffrey M, Leonard 1 1 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 Abstract Radiation-induced genomic deletions provide a quick and inexpensive approach to produce a large mutant pool. The density of mutations generated allows for genome-wide saturation with relatively small populations. In this report, we present a quick method for generating, screening, and validating wheat deletion mutants. We grew γ-ray irradiated seeds of hexaploid wheat ‘Chinese Spring’ (AABBDD) to flowering prior to fertilization of tetraploid wheat line ‘Altar’ (AABB). Quasi-pentaploid RH 1 plants carried radiation-induced fragments of ‘Chinese Spring’ chromosomes. Panels of deletion mutants derived from 35-, 40-, and 45-krad dosages were tested using SSR markers across all three genomes. Average marker loss of these panels was 3.0, 3.5 and 4.0%, respectively. Any D- genome marker or polymorphic A- or B-genome marker can be used to identify deletion mutants in the RH 1 lines. As proof of concept, panels were screened for deletions in three yield-related genes using gene-specific primers. The average loss for gene-specific markers was similar to that of the SSRs. Because quasi-pentaploid RH 1 plants were largely sterile, we traced back to the irradiated ‘Chinese Spring’ stock used to produce a given RH 1 line harboring targeted-gene mutations. Approximately 20 selfed seeds from the pertinent irradiated ‘Chinese Spring’ line were planted in the greenhouse for the identification of homozygous knockouts for subsequent phenotypic characterization. These analyses will also yield genetically effective cell number (GECN) estimates for this system. The gene-specific markers used in the screenings are also being mapped on wheat chromosomes using ‘Chinese Spring’ wheat deletion stocks and mapping populations. This technique should allow rapid identification of gene knockouts for reverse genetic applications. Abstract Radiation-induced genomic deletions provide a quick and inexpensive approach to produce a large mutant pool. The density of mutations generated allows for genome-wide saturation with relatively small populations. In this report, we present a quick method for generating, screening, and validating wheat deletion mutants. We grew γ-ray irradiated seeds of hexaploid wheat ‘Chinese Spring’ (AABBDD) to flowering prior to fertilization of tetraploid wheat line ‘Altar’ (AABB). Quasi-pentaploid RH 1 plants carried radiation-induced fragments of ‘Chinese Spring’ chromosomes. Panels of deletion mutants derived from 35-, 40-, and 45-krad dosages were tested using SSR markers across all three genomes. Average marker loss of these panels was 3.0, 3.5 and 4.0%, respectively. Any D- genome marker or polymorphic A- or B-genome marker can be used to identify deletion mutants in the RH 1 lines. As proof of concept, panels were screened for deletions in three yield-related genes using gene-specific primers. The average loss for gene-specific markers was similar to that of the SSRs. Because quasi-pentaploid RH 1 plants were largely sterile, we traced back to the irradiated ‘Chinese Spring’ stock used to produce a given RH 1 line harboring targeted-gene mutations. Approximately 20 selfed seeds from the pertinent irradiated ‘Chinese Spring’ line were planted in the greenhouse for the identification of homozygous knockouts for subsequent phenotypic characterization. These analyses will also yield genetically effective cell number (GECN) estimates for this system. The gene-specific markers used in the screenings are also being mapped on wheat chromosomes using ‘Chinese Spring’ wheat deletion stocks and mapping populations. This technique should allow rapid identification of gene knockouts for reverse genetic applications. Table 1. Recovery of mutants and calculation of genetically effective cell number (GECN). Eight to fifty RM 2 seeds from five to eleven independent RM 1 lines were screened by PCR for deletion of three gene sequences or by acid-PAGE for missing protein bands. Five of 22 lines tested yielded a mutant. Χ 2 tests were used to measure the goodness-of-fit of the data to 3:1, 7:1, or 11:1 segregation patterns predicted by one, two, or three germline-cell models. One line fit a single cell model (white), two lines fit a two-cell model (blue), and two lines fit a three-cell model (green). The majority of lines yielded no mutants, therefore we could only estimate the lower bound of GECN. Because the frequency of mutant recovery from only one line supported a model of GECN=1, we assume that generally GECN=2 or more in wheat. Our harvesting procedure might bias collection of seeds from the chimeric RM 1 plant such that mutations detected in the RH 1 s are under-represented in the RM 2 generation. Fig. 4. Mapping of CKX2 and DDM-4. DNA extracted from endosperm of a cross using gamma-ray irradiated pollen of Chinese Spring and Altar was assayed with DArT markers (see poster 125 for further details). By assaying the same mapping population with gene-specific markers, CKX-2 (A) and DDM-4 (B) were mapped to chromosomes 1B and 4D respectively. Map distances are in cR2000 (centiRays). Fig. 1. Development and screening of mutant panel. Seeds of Chinese Spring were irradiated with 35 krad of gamma rays,(1) grown to flowering (Radiation Mutants, RM 1 ), and crossed with tetraploid Altar(2). DNA extracted from the Radiation Hybrid F 1 s (RH 1 s) was screened with gene-specific primers and deletions were identified as null PCR reactions.(3) Frequency of detected deletions in the RH 1 s was 3.0%, 3.3% and 6.6% for 35, 40, and 45 krad treatments, respectively. The parental Chinese Spring RM 1 line harboring the deletion was identified (4) and the Radiation Mutant progeny (RM 2 s) of selfed plants were screened with the same gene-specific primers.(5) All D-genome chromosomes were monosomic in the RH 1 s for ease of detection, but polymorphic primers can be used to detect deletions in the A and B genome as well. 1. Gamma rays Chinese Spring seedsTetraploid Altar X Radiation Hybrid (RH 1 ) 3. Screen for deletions self Radiation Mutant (RM 2 ) 5. Screen progeny of deletion-containing lines to identify homozygotes 2. Cross with tetraploid A B D A B RH map 1BBin map 1B RH map 4D Bin map 4D A B 4. Identify CS parental line References Gendrel, Anne-Valerie, Zachary Lippman, Cristy Yordan, Vincent Colot, Robert A. Martienssen. (2002) Dependence of Heterochromatic Histone H3 Methylation Patterns on the Arabidopsis Gene DDM1. Science 297: Su, Z., C. Hao, L. Wang, Y. Dong, X. Zhang. (2011) Identification and development of a functional marker of TaGW2 associated with grain weight in bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 122: Van Herpen et al. (2006) Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes. BMC genomics 7:1 Zhang, J., W. Liu, X. Yang, A. Gao, X. Li, X. Wu, L. Li. (2010) Isolation and characterization of two putative cytokinin oxidase genes related to grain number per spike phenotype in wheat. Mol. Biol. Rep. (doi: /s ) Radiation Mutant (RM 1 )