1. Sierra Wolfenbarger John Fowler Rex Cole

2. Like most plants it completes an “alternation of generation” Diploid to Haploid to Diploid Haploid stage is called the gametophyte stage (e.g.: pollen and embryo sac)

3. Corn is a model organism and discoveries could relate to other plants Maize is one of the top three food crops in the world With a better understanding of the fertilization process, higher yields from this crop could be pursued, resulting in more corn for everyone

4. Haploid gametophytes require active gene functions Pollen grain Embryo sac Pollen tube

5. Not much is known about gametophyte genes Due to haploidy, mutations in active genes are often lethal Impossible to study if mutation is not heritable Pollen grain Embryo sac Pollen tube Pollen grain Embryo sac Pollen tube

6. Wx1+wx1 Parent Plant (diploid) wx1 Gametophyte (haploid) Wx1+ A Trisome is a special chromosome arm that gives the gametophyte an extra copy of a set of genes trisome

7. wx1 Gametophyte (haploid) Wx1+ trisome wx1 Gametophyte (haploid) Without the trisome, mutation is lost The trisome rescues the mutant gene and the gametophyte stage Survive Results in death

8. The trisomes monitored by kernel marker Example translucent kernel trisomeNo trisome wx1/wx1wx1/wx1/Wx1+

9. An Activator transposon (Ac) is used to disrupt the coding sequence of genes Ac is monitored by a separate marker Example purple spotting wx1 Wx1+ Trisome Ac transposon chromosome

10. The Ac can duplicate, creating a new transposon insertion monitored by spotting phenotype of the kernels New mutation potentially interesting 2 Ac1Ac wx1 Wx1+ wx1 Wx1+

11. Isolate and identify newly found gametophyte genes Adapt TAIL-PCR protocol to Maize Gather more data to support gametophyte mutant phenotypes Verify plant genotypes using PCR Perform crosses with mutants to confirm effect on gametophytes

12. The lab has already recovered a number of mutations (duplicated Ac’s), with trisomes present These plants were crossed with a *tester line, and then 2Ac + trisome seeds were selected by phenotype * Tester= homozygous recessive for all kernel markers used

13. These trisome +2Ac seeds are a set of putative gametophyte mutants The progeny of these seeds are evaluated to see if a transmission phenotype is present Based on ratios of kernel phenotype

14. Trisome no AcTrisome +2AcTrisome+ 1Ac No trisome +1AcNo trisome+ 2AcNo trisome No Ac

15. Trisome no AcTrisome +2AcTrisome+ 1Ac No trisome +1AcNo trisome+ 2AcNo trisome No Ac

16. Trisome no AcTrisome +2AcTrisome+ 1Ac No trisome +1AcNo trisome+ 2AcNo trisome No Ac

17. Putative gametophyte mutants were planted 3 with trisome 9s and 7 with trisome 1L Experiment: Cross PCR-verified plants to confirm mutant effects on gametophytes

18. Plants are checked to make sure they contain trisome Polymerase Chain Reaction (PCR) Wx1+ primers for B centromere wx1

19. Mutant 1 line Wx1+wx1 + control H2O

20. Mutant 2 line Wx1+wx1 + control H2O

21. Only 2 of 10 mutant lines contain the trisome H2O +control H2O 500bp Mutant 1 line Mutant 2 line Wx1+wx1Wx1+wx1

22. Wx1+ wx1 Results in higher frequency of trisome marker + 2Ac

23. Identify the mutant gene using Thermal Asymmetric Interlaced PCR (TAIL-PCR) Adapt procedure to maize Ac TAIL-PCR uses both nested Ac-specific primers and degenerate arbitrary primers alternating high and low annealing temperature cycles Arbitrary degenerate primer Known primer

24. Second step of TAIL-PCR (TAIL- 2 ) Uses diluted DNA from 1 st Tail reaction (TAIL- 1 ) with nested Ac primer TAIL- 1 primer Arbitrary degenerate primer Nested primer

25. 500 bp 100bp 1000bp Mutant 1 line Original Ac line H2O

26. 5’end of Ac transposonflanking sequence

27. Chr 10 Flanking sequence

28. Gene flanking sequence matches Chr 10 Flanking sequence

29. Trisome confirmed in 2 of 10 mutant lines Tester crosses made with these 2 lines Future: harvest and sort seeds to confirm mutant transmission ratios

30. Trisome confirmed in 2 of 10 mutant lines Tester crosses made with these 2 lines Future: harvest and sort seeds to confirm mutant transmission ratios TAIL-PCR protocol adapted to Maize Mapped 2 new transposon sites using TAIL-PCR Future: additional TAIL-PCR to find Ac’s of interest (from 2 gametophyte mutant lines)

31. Trisome confirmed in 2 of 10 mutant lines Tester crosses made with these 2 lines Future: harvest and sort seeds to confirm mutant transmission ratios TAIL-PCR protocol adapted to Maize (Ac-TAIL) Mapped 2 new transposon sites using Ac-TAIL Future: additional TAIL PCR to find Ac’s of interest (from 2 gametophyte mutant lines) OVERALL: Ac-TAIL will be very useful as more gametophyte mutants are found

32. Howard Hughes Medical Institute National Science Foundation John Fowler Kevin Ahern Fowler Lab: Rex Cole Zuzana Vejlupkova Maria Ivanchenko Chintan Joshi Nathan Synder Luisa Snyder