1. Abstract Maize, (Zea mays L. ssp., corn) Abnormal Chromosome ten (Ab10) is an uncommon variant of chromosome 10. Ab10 causes a segregation abnormality during female meiosis by conveying loci linked to all chromosome knobs into the next generation at an increased rate. This could cause a reduction in the genetic diversity represented in seed stock. In order to diagnose chromosome 10 constitution, we are testing Simple Sequence Repeat (SSR) markers 26 genetic lines of known chromosomal constitution including Southwest Maize accessions. We will post results from this work as well as from research conducted by other interns in the Lawrence group online at a website to be made available through http://www.lawrencelab.org/ at the end of the 2007 summer internship. Abstract Maize, (Zea mays L. ssp., corn) Abnormal Chromosome ten (Ab10) is an uncommon variant of chromosome 10. Ab10 causes a segregation abnormality during female meiosis by conveying loci linked to all chromosome knobs into the next generation at an increased rate. This could cause a reduction in the genetic diversity represented in seed stock. In order to diagnose chromosome 10 constitution, we are testing Simple Sequence Repeat (SSR) markers 26 genetic lines of known chromosomal constitution including Southwest Maize accessions. We will post results from this work as well as from research conducted by other interns in the Lawrence group online at a website to be made available through http://www.lawrencelab.org/ at the end of the 2007 summer internship. Hypotheses 1) Molecular markers can be developed to diagnose chromosome 10 constitutions for maize. (2) The presence of Ab10 causes segregation distortion which, in turn, causes a change in the genetic profiles of maize accessions in the National Plant Germplasm collection. Hypotheses 1) Molecular markers can be developed to diagnose chromosome 10 constitutions for maize. (2) The presence of Ab10 causes segregation distortion which, in turn, causes a change in the genetic profiles of maize accessions in the National Plant Germplasm collection. Materials & Methods Three SSR markers (Table 1) were tested on twenty-six genetic lines (Table 2) of known chromosomal constitution using DNA extract prepared in 2006. Standard PCR protocol was followed: 1. Reaction mixture was prepared including buffer, dNTPs, MgCl2, SSR marker (primers), and Taq. 2. Reaction mix, DNA, and mineral oil were aliquoted into the microplate. 3. PCR reaction carried out according to protocol and SSR- specific Tm. 4. Agarose gel (4%) was prepared and loaded with PCR products. 5. Gel was run for two hours or longer. 6. Bands were visualized over a UV light images were taken. 7. Band patterns were analyzed for discerning N10 and Ab10. Materials & Methods Three SSR markers (Table 1) were tested on twenty-six genetic lines (Table 2) of known chromosomal constitution using DNA extract prepared in 2006. Standard PCR protocol was followed: 1. Reaction mixture was prepared including buffer, dNTPs, MgCl2, SSR marker (primers), and Taq. 2. Reaction mix, DNA, and mineral oil were aliquoted into the microplate. 3. PCR reaction carried out according to protocol and SSR- specific Tm. 4. Agarose gel (4%) was prepared and loaded with PCR products. 5. Gel was run for two hours or longer. 6. Bands were visualized over a UV light images were taken. 7. Band patterns were analyzed for discerning N10 and Ab10. Pinpointing Ab10 Chromosome Using DNA Markers Zach Nelson 1, Joan Peterson 2, Carolyn Lawrence 3 1 George Washington Carver Intern, Iowa State University, Ames Iowa 2 Dept. of Agronomy, Iowa State University, Ames Iowa 3 USDA-ARS, Dept. of Genetics, Development and Cell Biology, Iowa State University, Ames Iowa Pinpointing Ab10 Chromosome Using DNA Markers Zach Nelson 1, Joan Peterson 2, Carolyn Lawrence 3 1 George Washington Carver Intern, Iowa State University, Ames Iowa 2 Dept. of Agronomy, Iowa State University, Ames Iowa 3 USDA-ARS, Dept. of Genetics, Development and Cell Biology, Iowa State University, Ames Iowa M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17a 17b17c18a 18b18c18d 19a19b19c M SSR marker p-umc1359 Gel Patterns Figure 4 Image of PCR products visualized in agarose gel Results and Conclusion For this project, I ran three different DNA markers on 26 genotypes. Preliminary results were not satisfactory so we re- evaluated our techniques. To improve our results, we decided to not use the multi channel pipette, and instead used a single channel pipette and transferred all aliquots individually. Secondly, we changed to a hot start Taq polymerase and thirdly, we rechecked our master mix volumes to ensure the right proportions. PCR of the first and second markers did not produce many bands, but the third marker, p-umc1359, did produce easily visualized bands in some lines (Fig. 4). We determined that our gel patterns were not diagnostic for chromosome 10 constitution. Results and Conclusion For this project, I ran three different DNA markers on 26 genotypes. Preliminary results were not satisfactory so we re- evaluated our techniques. To improve our results, we decided to not use the multi channel pipette, and instead used a single channel pipette and transferred all aliquots individually. Secondly, we changed to a hot start Taq polymerase and thirdly, we rechecked our master mix volumes to ensure the right proportions. PCR of the first and second markers did not produce many bands, but the third marker, p-umc1359, did produce easily visualized bands in some lines (Fig. 4). We determined that our gel patterns were not diagnostic for chromosome 10 constitution. References Mroczek, R.J., Melo, J.R., Luce, A.C., Hiatt, E.N. and Dawe, R.K. 2006. The Maize Ab10 Meiotic Drive System Maps to Supernumerary Sequences in a Large Complex Haplotype. Genetics 174(1): 145-154. Acknowledgements National Science Foundation for funding Table 2. List of DNA Samples Figure 1. Maize ears from Southwest accessions. Figure 3 Zach preparing DNA, setting up PCR, and taking gel images. Table 1. List of SSR markers tested. Figure 2. Schematic diagram of N10 and Ab10 chromosomes. R, L, O, W, and Sr are loci that have been identified (Mroczek et al., 2006)