Progress 06/15/03 to 06/14/06
Outputs
EMS mutagenized populations of maize were generated at both Purdue and at Iowa State. Establishing a TILLING service for the maize community required creating, maintaining or curating any mutant populations for TILLING beyond what the community already had in hand. However the mutation densities in the existing material were not high enough to allow efficient screening in a cost effective manner. This project worked towards these goals. Pollen from maize tassels was mutagenized and then applied to females of the same inbred. The net results were M1 populations in which every seed was a unique collection of mutations because it arose from fertilization by a single mutagenized pollen grain and every seed was heterozygous for any mutations created. In the case of the W22 populations, we were also able to use strains marked with the endosperm mutations bronze1 (bz1) and shrunken1 (sh1) as females and mutagenize pollen from a homozygous Bz1 Sh1 strain. This scenario allowed
us to measure directly how effective our mutagenesis was in each EMS treatment by scoring bz1* mutations at Bz1 or sh1* mutations at Sh1 in our M1 seed. These seed appeared as bronze, plump or purple, shrunken exceptions on the M1 ears (self pollination contaminants would be both bronze and shrunken). We generated 35 treatment groups containing ~20,000 M1 seed of W22 and ~30,000 M1 seed of B73. We further tested these treatments by randomly selecting ~400 M1 seed for each inbred representing all the treatment groups, planting them, selfing to create M2 ears and then screening 40 M2 kernel families from each of these ears in a sandbench to score for photosynthetic mutations, seedling defects or embryo lethality. Using the empirical criteria developed in the Arabidopsis TILLING Project, treatments that produced >30% of families segregating recessive lethality (fewer than 24 kernels germinating, p<0.05) were advanced to the next stage of production, a total of ~12,000 W22 and ~16,000 B73
seed. Eight bronze kernels appeared among the 12,000 W22 kernels, lower than the canonical 1 per 1000 M2 families (or M1 kernels, in this case) [22], but within statistical error. These extremely limited data (lethality among M2 families and the M1 mutations at Bz1) suggest this W22 mutagenesis will have a mutation density higher than the present lines (8 damaging alleles/17.7% = ~48 mutations in Bz1 over 1416 bp of coding exon or ~2.9 mutations per kb per 1000 plants). The M1 seed from these treatments were planted in Summer 2004 to create TILLING POPULATION B. Tissue was collected and lyophilized for DNA, and the plants were selfed. ~50% were both male and female fertile and produced M2 ears, ~60% of which had >40 kernels on them (our minimum acceptable seed set), a total of 8556 potentially useable lines. Unfortunately, increasing all 8556 of these newer lines to M3 families was beyond the reduced resources of this USDA award. We have initiated M3 increases on a limited scale at
Purdue and implemented a Priority Increase strategy so that we can begin to bring these populations on-line more quickly.
Impacts
These populations have allowed us to provide a cost-effective TILLING service in maize, a valuable functional genomics resource.
Publications
- Weil, C. F., R. Monde, B. Till, L. Comai and S. Henikoff (2005) Mutagenesis and functional genomics in maize. Maydica 50:415-424
- Weil, C. F. (2005) Single base hits score a home run in wheat. Trends in Biotech. 23:220-222
- Weil, C. F. and R. Monde (2006) Getting the point:mutations in maize. Plant Genome (in press).
- Monde, R. and C. Weil, (2007) Reverse genetics in maize, invited review for Israel Journal of Plant Science.
- Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, Greene EA, Comai L, Henikoff S. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol. 4(1):12
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Progress 10/01/04 to 09/30/05
Outputs
A population of maize was mutagenized to create M1 mutant seed heterozygous for all mutations created. These seed were planted, M1 tissue harvested and freeze-dried and the plants selfed to create a population of mutagenized B73 inbred and another of the W22 inbred. Tests are in progress using TILLING on five known gene targets to accurately assess the mutation density in these populations; however, preliminary data from the W22 population suggest these new populations have nearly double the mutation density of the current TILLING populations and will serve as an outstanding resource both for TILLING/reverse genetics and as a community resource for forward screens.
Impacts
These populations will help make TILLING more cost effective in maize and provide a central resource for a wide range of mutant screens by the entire communtiy.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Mutagenized populations of maize inbred lines W22 and B73 were created prior to the start of the award by pollen mutagenesis using EMS. The effectiveness of 15 different treatments of W22 and 35 treatments of B73 have now been tested by randomly sampling 40 M1 kernels for each treatment, planting them in winter nurseries in Hawaii and Puerto Rico and selfing to create M2 ears, then sandbench screening M2 families. Treatments that produced ≥30% families segregating for embryo and seedling lethal phenotypes were advanced to production in Summer 2004. 20,000 W22 M1 seed were planted at Purdue and 30,000 B73 M1 seed at Iowa State. Coleoptiles and emerging early leaves were harvested, frozen and lyophilized for DNA preparations. Those plants that matured and were fertile were then selfed to create M2 ears. We now have in hand 10,000 M2 ears and tissue samples for each of the two inbred lines. I addition, 1500 M2 lines of W22 were advanced to M3 populations by random
intermating within M2 families to create sufficient seed to distribute to TILLING clients when the service opens (probably early 2005). Current plans are to start advancing the M2 lines created this summer to M3 starting next summer, as well as DNA preparation and arraying.
Impacts
The result of this work will be a substantial improvement in the materials available for TILLING and the mutant collections available to the maize community, particularly in these uniform backgrounds where the majority of other genomics resources are being concentrated. In addition, these mutant lines will be available for wider screening by the community.
Publications
- Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, Greene EA, Comai L, Henikoff S. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol. 4(1): 12
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Progress 06/15/03 to 12/12/03
Outputs
Maize populations of the inbred W22 and the inbred B73 were mutagenized with EMS at Purdue University and an additional population of B73 was mutagenized at Iowa State University in the summer of 2003. The net production of mutant M1 seed were 38,000 M1 seed of W22 and 52,000 M1 seed of B73. In addition, 5000 W22 M1 plants have been selfed to M2, and DNA made from tissue samples collected from the M1 plants, processed into DNA and arrayed. Over 3,000 B73 plants have undergone the same. W22 samples from prior work have been tested for mutation density by the Arabidopsis TILLING project and have mutation densities consistent with approximately four lesions per 1500 bp target per 4-fold pool plate. Material mutagenized this summer was treated for a longer period with EMS and we anticipate its mutation density will be higher. Tests of the B73 mutation density are presently being carried out.
Impacts
The result of this work will be a substantial improvement in the materials available for TILLING and the mutant collections available to the maize community, particularly in these uniform backgrounds where the majority of other genomics resources are being concentrated. In addition, these mutant lines will be available for wider screening by the community.
Publications
- No publications reported this period
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