A Quick Method For Detection Of Gamma Ray-induced Genomic Deletions In Wheat DBI-0822100 Vijay K....
1
A Quick Method For Detection Of Gamma Ray-induced Genomic Deletions In Wheat DBI- 0822100 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. α-gliadins are wheat gluten proteins that cause celiac disease. More than 100 clustered genes produce the storage proteins, although most allergenic epitopes are encoded on the D- genome. Half-seed protein extracts of RM 2 seeds derived from parents of RH 1 lines carrying deletions on 6DS were screened by acid-PAGE for loss of specific bands. One line was found missing two protein bands derived from 6D (A) and another deficient in bands derived from 1B (B). A B Fig 2. Screening RM 2 lines for homozygous deletions. Three genes affecting seed size, CKX- 2, DDM-4, and GW2, were targeted. •CKX2 (Cytokinin Oxidase/Dehydrogenase) is a degrader of cytokinin, a hormone associated with cell division and differentiation. Increased levels of cytokinins have been shown to increase grain number per spike. •DDM-4 (Decrease in DNA Methylation) is responsible for transposon and transgene silencing, through DNA methylation. •GW2 (Grain Width and Weight) has been found to be negatively associated with grain weight and width through the regulation of cell division DNA was extracted from multiple individual progeny of selfed RM 1 plants that produced deletions in the RH 1 s and was screened by PCR with gene-specific primers. Typical gels show deletions (indicated by arrows) for CKX-2(A) and GW2 (B). A B Target Gene RH 0 line Deletions/RM 2 s GECN Cytokinin Oxidase (CKX) CKX-m1 0/20 ≥2 CKX-m2 1/19 2 CKX-m3 0/11 ≥2 CKX-m4 0/19 ≥2 CKX-m5 0/20 ≥2 CKX-m6 0/18 ≥2 Decrease in DNA Methylatio n (DDM) DDM-m1 0/20 ≥2 DDM-m2 0/19 ≥2 DDM-m3 1/15 2 DDM-m4 0/19 ≥2 DDM-m5 0/15 ≥2 Grain Weight and Width (GW2) GW2-m1 0/19 ≥2 GW2-m2 0/8 ≥1 GW2-m3 0/20 ≥2 GW2-m4 6/20 1 GW2-m5 0/5 ≥1 GW2-m6 0/18 ≥2 GW2-m7 0/19 ≥2 GW2-m8 0/20 ≥2 GW2-m9 0/19 ≥2 GW2- m10 0/15 ≥2 GW2- m11 0/8 ≥1 Alpha Gliadin AG-m1 1/50 3 AG-m2 0/8 ≥1 AG-m3 0/43 >3 AG-m4 1/38 3 AG-m5 0/10 ≥1 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). Summary Screening quasi-pentaploid radiation hybrids (RH 1 s) was effectively used to identify Chinese Spring lines homozygous for radiation-induced deletions of specific genes. Three of the genes may affect seed size and the other group of genes affect protein quality. All mutant lines are being grown to measure phenotypes. The low frequency of recovery of homozygous mutants suggests that screening of the pentaploid hybrids is a viable alternative to blind mass screens of multiple RM 2 lines. At least one mutant was found for each of the four targets. The initial target lines were identified based on a screen of 94 35-krad RH 1 lines. We have made >1100 RH 1 lines so we now have resources to isolate multiple independent deletions of each gene. The number of germ-line cells (GECN) was estimated from 1 to >3 in various lines. This calculation may be confounded by seed sampling bias from chimeric RM 1 plants and unknown rates of transmission of chromosomes harboring deletions. Experiments with larger sample sizes are planned to refine this measurement. CKX-2 and DDM-4 were successfully mapped to chromosomes 1B and 4D respectively. Mapping of GW2 is underway. This protocol demonstrates that by screening a single plate of RH 1 lines made as part of the D- genome Radiation Hybrid Mapping Project, RM 2 lines homozygous for targeted deletions can be easily isolated for loss-of-function studies. 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 seeds Tetraploid 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 wPt-5127 wPt-732417 w Pt-732257 0.0 DDM -4 12.8 wPt-0710 wPt-667352 w Pt-666805 wPt-0472 24.0 w Pt-731623 29.0 w Pt-669279 wPt-666306 36.9 w Pt-8793 64.4 wm c-818 374.4 w Pt-4721 w Pt-3753 0.0 w Pt-741980 w Pt-8320 398.9 tPt-1772 421.4 w Pt-742499 433.3 w Pt-1684 445.1 C KX2-2 468.2 w Pt-8279 605.5 w Pt-3451 655.2 RH map 1B Bin 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 297:1871-1873 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. 122:211- 223 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:10.1007/s11033-010-0367-9) Radiation Mutant (RM 1 )
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Transcript of A Quick Method For Detection Of Gamma Ray-induced Genomic Deletions In Wheat DBI-0822100 Vijay K....
A Quick Method For Detection Of Gamma Ray-induced Genomic Deletions In Wheat
DBI-0822100 Vijay K. Tiwari1, Hilary Gunn1, Oscar Riera-Lizarazu2, Shahryar F. Kianian3, Jeffrey M, Leonard1
1Department of Crop and Soil Science, Oregon State University, Corvallis, OR2International 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 RH1 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 RH1 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 RH1 plants were largely sterile, we traced back to the irradiated ‘Chinese Spring’ stock used to produce a given RH1 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.
Fig.3 Screen of RM2s for loss of gene product. α-gliadins are wheat gluten proteins that cause celiac disease. More than 100 clustered genes produce the storage proteins, although most allergenic epitopes are encoded on the D-genome. Half-seed protein extracts of RM2 seeds derived from parents of RH1 lines carrying deletions on 6DS were screened by acid-PAGE for loss of specific bands. One line was found missing two protein bands derived from 6D (A) and another deficient in bands derived from 1B (B).
A B
Fig 2. Screening RM2 lines for homozygous deletions. Three genes affecting seed size, CKX-2, DDM-4, and GW2, were targeted. •CKX2 (Cytokinin Oxidase/Dehydrogenase) is a degrader of cytokinin, a hormone associated with cell division and differentiation. Increased levels of cytokinins have been shown to increase grain number per spike. •DDM-4 (Decrease in DNA Methylation) is responsible for transposon and transgene silencing, through DNA methylation. •GW2 (Grain Width and Weight) has been found to be negatively associated with grain weight and width through the regulation of cell division DNA was extracted from multiple individual progeny of selfed RM1 plants that produced deletions in the RH1s and was screened by PCR with gene-specific primers. Typical gels show deletions (indicated by arrows) for CKX-2(A) and GW2 (B).
A B
Target Gene RH0 line Deletions/RM2s GECN
Cytokinin Oxidase (CKX)
CKX-m1 0/20 ≥2CKX-m2 1/19 2CKX-m3 0/11 ≥2CKX-m4 0/19 ≥2CKX-m5 0/20 ≥2CKX-m6 0/18 ≥2
Decrease in DNA
Methylation (DDM)
DDM-m1 0/20 ≥2
DDM-m2 0/19 ≥2
DDM-m3 1/15 2
DDM-m4 0/19 ≥2
DDM-m5 0/15 ≥2
Grain Weight and Width
(GW2)
GW2-m1 0/19 ≥2GW2-m2 0/8 ≥1GW2-m3 0/20 ≥2GW2-m4 6/20 1GW2-m5 0/5 ≥1GW2-m6 0/18 ≥2GW2-m7 0/19 ≥2GW2-m8 0/20 ≥2GW2-m9 0/19 ≥2
GW2-m10 0/15 ≥2 GW2-m11 0/8 ≥1
Alpha Gliadin
AG-m1 1/50 3
AG-m2 0/8 ≥1
AG-m3 0/43 >3
AG-m4 1/38 3
AG-m5 0/10 ≥1
Table 1. Recovery of mutants and calculation of genetically effective cell number (GECN). Eight to fifty RM2 seeds from five to eleven independent RM1 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. Χ2 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 RM1 plant such that mutations detected in the RH1s are under-represented in the RM2 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).
SummaryScreening quasi-pentaploid radiation hybrids (RH1s) was effectively used to identify Chinese Spring lines homozygous for radiation-induced deletions of specific genes. Three of the genes may affect seed size and the other group of genes affect protein quality. All mutant lines are being grown to measure phenotypes. The low frequency of recovery of homozygous mutants suggests that screening of the pentaploid hybrids is a viable alternative to blind mass screens of multiple RM2
lines. At least one mutant was found for each of the four targets. The initial target lines were identified based on a screen of 94 35-krad RH1 lines. We have made >1100 RH1 lines so we now have resources to isolate multiple independent deletions of each gene. The number of germ-line cells (GECN) was estimated from 1 to >3 in various lines. This calculation may be confounded by seed sampling bias from chimeric RM1 plants and unknown rates of transmission of chromosomes harboring deletions. Experiments with larger sample sizes are planned to refine this measurement. CKX-2 and DDM-4 were successfully mapped to chromosomes 1B and 4D respectively. Mapping of GW2 is underway. This protocol demonstrates that by screening a single plate of RH1 lines made as part of the D-genome Radiation Hybrid Mapping Project, RM2 lines homozygous for targeted deletions can be easily isolated for loss-of-function studies.
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, RM1), and crossed with tetraploid Altar(2). DNA extracted from the Radiation Hybrid F1s (RH1s) was screened with gene-specific primers and deletions were identified as null PCR reactions.(3) Frequency of detected deletions in the RH1s was 3.0%, 3.3% and 6.6% for 35, 40, and 45 krad treatments, respectively. The parental Chinese Spring RM1 line harboring the deletion was identified (4) and the Radiation Mutant progeny (RM2s) of selfed plants were screened with the same gene-specific primers.(5) All D-genome chromosomes were monosomic in the RH1s 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 seeds Tetraploid Altar
X
Radiation Hybrid (RH1)
3. Screen for deletions
self
Radiation Mutant (RM2)
5. Screen progeny of deletion-containing lines to identify homozygotes
2. Cross with tetraploid
A
B
D
A
B
wPt-5127 wPt-732417wPt-7322570.0
DDM-412.8wPt-0710 wPt-667352wPt-666805 wPt-047224.0
wPt-73162329.0wPt-669279 wPt-66630636.9wPt-879364.4
wmc-818374.4
wPt-4721 wPt-37530.0
wPt-741980 wPt-8320398.9tPt-1772421.4wPt-742499433.3wPt-1684445.1CKX2-2468.2
wPt-8279605.5
wPt-3451655.2
RH map 1B Bin map 1B
RH map 4D Bin map 4D
A B
4. Identify CS parental line
ReferencesGendrel, 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 297:1871-1873
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. 122:211-223
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:10.1007/s11033-010-0367-9)
Radiation Mutant (RM1)
