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Testing the Role of Conserved Genes in Pollen Development

Testing the Role of Conserved Genes in Pollen Development. Luisa Snyder Mentor: Dr. John Fowler Lab Mentor: Dr. Rex Cole Dept. Botany and Plant Pathology Summer 2008. Pollen. The way that flowering plants undergo sexual reproduction Allows exchange of gametes among plants.

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Testing the Role of Conserved Genes in Pollen Development

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  1. Testing the Role of Conserved Genes in Pollen Development Luisa Snyder Mentor: Dr. John Fowler Lab Mentor: Dr. Rex Cole Dept. Botany and Plant Pathology Summer 2008

  2. Pollen • The way that flowering plants undergo sexual reproduction • Allows exchange of gametes among plants sols.unlv.edu/Schulte/Anatomy/Repro/Repro.html specialcomment.wordpress.com/2007/04/

  3. Pollen Shape Diversity en.wikivisual.com/index.php/Sporopollenin

  4. www.umanitoba.ca/.../Lab8/biolab8_1.html www.yugatech.com/photos/?a=flowers&i=672 90ways.com/sciarchive/sci25.php Despite diversity there are many common pollen characteristics across species www.answers.com/topic/maize www.simple-expressions.org/2007_12_01_archive...

  5. Project Purpose • To identify genes involved in the development and function of pollen. • Potentially may lead to: - Improved crops - Containment for genetically engineered plants staffwww.fullcoll.edu/tmorris/an_inconvenient... www.coextra.eu/country_reports/news1018_en.html

  6. Male Gametophyte Life Cycle Mitosis Quartet Pollen +/m Heterozygous +/+ Wild Type

  7. Arabidopsis thaliana http://www.prep.biotech.vt.edu/expinfo/expinfo_anatomy.html

  8. Hypothesis • A core set of pollen genes provide important functions in all angiosperm pollen. Rice Maize Arabidopsis www.physorg.com/news117381801.html www.gramene.org/species/zea/maize_intro.html www.botanical.com/botanical/mgmh/r/rice--15.html

  9. Predictions The genes in the pollen core set should be: - Conserved and highly expressed in pollen across Arabidopsis, rice, and maize. 2. Mutation in the genes in the pollen core set should cause a pollen defect. Arabidopsis Pollen Rice Pollen Maize Pollen www.biomedcentral.com/1471-2229/5/22/figure/F1 epigenome.eu/en/3,41,0 www.lec.lancs.ac.uk/ccm/ed.htm

  10. Pollen Genes Criteria Ancient Gene • Putative Orthologous Group (POG) • Highly expressed in Arabidopsis, maize, and rice pollen Arabidopsis gene Rice gene Maize gene Mutant gene is available Orthologous genes No duplicate genes in Arabidopsis

  11. Candidate Pollen Genes

  12. Experimental Approach and Methods Microscopy Determine if there is a phenotypic defect due to mutation in the candidate genes. Gel Electrophoresis PCR Determine if there is a transmission defect due to a mutation in the candidate genes. www.sparknotes.com/.../lifecycle/section2.rhtml www.molecularstation.com/molecular-biology-im... www.territorioscuola.com/wikipedia

  13. Testing for Phenotypic Defect At5g17290- Autophagy protein ATG5 At5g20690- Transmembrane protein kinase

  14. Results of Pollen Phenotypic Defect

  15. Testing for Transmission Defect Reciprocal Crosses: X +/m pollen +/+ stigma X +/m stigma +/+ pollen

  16. Transmission Defect Prediction of the Results No Transmission Defect Stigma + + Half wild type plants + Mostly wild type plants Pollen m Half heterozygous plants

  17. Genotyping by PCR At1g64110- ATPase family protein Wild Type Heterozygous At1g64110

  18. Example: PCR Test for Transmission Defect At1g64110- ATPase family protein Stigma +/+ Pollen +/m X Controls Frequency: 12 Heterozygous 12 Wild type 1:1 = No transmission defect

  19. Summary: Transmission Defect Experiments* * At least 18 plants tested for each gene

  20. Conclusions-So Far Hypothesis A core set of pollen genes provide important functions in all angiosperm pollen. Prediction Mutation in the genes in the pollen core set should cause a pollen defect. Observation None of my four candidate genes showed a defect Possible Explanations - Transmission defect might be mild - Not enough plants - Unrecognized duplicate genes - 3 out of 4 have likely duplicates - Not enough candidate genes tested

  21. However

  22. Testing Transmission Defect Reciprocal Crosses: X +/m pollen +/+ stigma X +/m stigma +/+ pollen Female Gametophyte

  23. (An Unexpected) Female-Specific Transmission Defect ! At5g17290- Autophagy protein ATG5 Controls Using heterozygous pollen 9:9 WT Band Mut Band Het : WT Controls Using heterozygous stigma 9:20 WT Band Mut Band Het : WT c2 = 4.17 p<0.05 Statistically Significant!

  24. Future Research Currently working with four more candidate genes Follow up on pollen tube branching phenotype in the genes At1g64110 and At5g17290 Test the next generation for the female specific transmission defect in the gene At5g17290 - If confirmed, determine the phenotypic defect in the female gametophyte

  25. Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern Dr. John Fowler Dr. Rex Cole Fowler Lab: Zuzana Vejlupkova Nathan Snyder Dr. Maria Ivanchenko Dr. Lol Cooper Sierra Wolfenbarger

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