Pinpointing Ab10 Chromosome Using DNA Markers
Zach
1
Nelson ,
Joan
2
Peterson ,
Carolyn
3
Lawrence
1George
Washington Carver Intern, Iowa State University, Ames Iowa
2Dept. of Agronomy, Iowa State University, Ames Iowa
3USDA-ARS, Dept. of Genetics, Development and Cell Biology, Iowa State University, Ames Iowa
Abstract
Results and Conclusion
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
For this project, I ran three different DNA markers on 26
genotypes. Preliminary results were not satisfactory so we reevaluated 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.
Figure 1. Maize ears from Southwest accessions.
Figure 3 Zach preparing DNA, setting up PCR, and
taking gel images.
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.
M
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17a 17b17c18a 18b18c18d 19a19b19c M
Gel Patterns
SSR marker p-umc1359
Figure 4 Image of PCR products visualized in agarose gel
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 SSRspecific 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.
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)
Table 2. List of DNA Samples
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 1. List of SSR markers tested.