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Double Haploid Application to Allelic Diversity Germplasm

Double Haploid Application to Allelic Diversity Germplasm. Mike Blanco & Andrew Smelser USDA-ARS Plant Introduction Research Unit. Original Allelic Diversity Goals. Develop an unmatched collection of rare maize alleles. Develop 1500-1800 inbreds from 300+ different races of maize.

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Double Haploid Application to Allelic Diversity Germplasm

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  1. Double Haploid Application to Allelic Diversity Germplasm Mike Blanco & Andrew Smelser USDA-ARS Plant Introduction Research Unit

  2. Original Allelic Diversity Goals • Develop an unmatched collection of rare maize alleles. • Develop 1500-1800 inbreds from 300+ different races of maize. • Make these inbreds available to the maize community for basic research.

  3. Allelic Diversity Protocol • Cross an exotic race to PHB47 and PHZ51: PHB47 x (Arequipeno - ARQ 1) PHZ51 x (Arequipeno - ARQ 1) • Backcross the 2 F1’s to make BC1’s: PHB47 x (PHB47/Arequipeno - ARQ 1) PHZ51 x (PHZ51/Arequipeno - ARQ 1) • Self (without selection) to homozygosity

  4. DH Allelic Diversity Goals • Determine if Double Haploid (DH) technology can be utilized for more rapid release of Allelic Diversity (AD) germplasm • Evaluate induction & doubling frequencies among races, and effect of recurrent parent used

  5. Methods & Materials • Used AD Races • BC1’s with PHZ51 • BC1’s with PHB47 • 50 Races (74 Populations) from 11 countries were induced in 2008 and doubled, selected, and selfed in 2009

  6. Methods & Materials • Used RWSxRWK-76 To Induce • 9-10% Induction Rate (Geiger 2009) Lines provided to us by Dr. Lubberstedt On average 14 days earlier than B73

  7. Methods & Materials • Summer 2008 • Crosses made to AD races with Hohenheim inducer (Dr. Lubberstedt & Staff) • Winter 2008/2009 • Identified potential haploid kernels in lab • Summer 2009 • Doubled & Selfed haploids (partial & full tassels)

  8. Screening for Haploids Possible Haploid Contaminant

  9. Screening Haploids Hybrid Haploid Purple Scutellum

  10. Screening Problems Morocho Backcrossed to PHB47 Montaña Backcrossed to PHB47 R1-nj Marker Inhibition ColoredSeed

  11. Identifying “Haploids” by Seedling Vigor

  12. Induction Percentages are based on visual kernel sorting by use of kernel marker

  13. Results of Haploid Induction

  14. Results of Haploid Induction

  15. Results of Haploid Induction

  16. Results of Haploid Induction

  17. Summary • Haploid Induction: • Within the same 24 accessions • Backcrossed to PHZ51 - 6.4% Induction Rate • Backcrossed to PHB47 - 5.4% Induction Rate • 5 Races (out of 50) had 9% to 14.4% Induction Rate • There are differences among races in response to haploid induction (p-value .0009) • There is no difference in the frequency of haploid induction when accessions are crossed to either PHZ51 or PHB47 (p-value .9210)

  18. Doubling Methods • Stealth Herbicide (Microtubule Inhibitor) • Active Ingredient: Pendimethalin • One field application applied at 90% the recommended rate at the 3 leaf stage • Colchicine (small study-14 pops) • Injection of 0.125% colchicine solution with 0.5% dimethylsulfoxide at 3-4 leaf stage

  19. Pollinating Double Haploids Sterile Partially Fertile

  20. Pollinating Double Haploids

  21. Results of Doubling are based on ears with seed production DH Ears from Altiplano backcrossed to PHB47

  22. Results of Doubling(Recurrent Parent) Same 24 Accessions

  23. Results of Doubling(Kernels/Ear) Same 24 Accessions

  24. Results of Doubling(Treatment) Same 14 populations

  25. Results of Doubling(Kernels/Ear) Same 14 populations

  26. Results of Induction & Doubling (Overall) 50 Races (74 Populations) Does not include colchicine treatment

  27. Summary (Observations) • DH technology was effectively used in AD germplasm to develop DH lines • Haploid induction averaged 6% (range 2.6-14.4%) among the 74 populations and haploid kernels were successfully identified with 85% accuracy • Doubling averaged 7% (range 0-24%) among the 74 populations

  28. Summary (Observations) • Differences among the populations were found for haploid induction and doubling, but there does not appear to be major differences when crossed to PHZ51 or PHB47. • Colchicine application was more effective for doubling (19%) than Stealth herbicide field application (6%)

  29. Summary (Limitations) • Induction: Kernel markers are not effective with colored kernels, or if inhibitor genes are present . • Doubling: The rate of genomic doubling needs to be improved from 7% (to ~20-25%) to reduce the number of rows required for AD germplasm.

  30. Summary (Challenges) • Develop methods to identify haploid kernels among the colored kernels/inhibitor genes • Improve doubling percentages • Identify and develop germplasm for spontaneous doubling

  31. Summary (Challenges) • Improve environmental conditions for haploid growth and doubling • Explore effectiveness of DH vs traditional selfing to capture exotic alleles, and utilize diversity • To our knowledge, this is the first large scale evaluation of different races (50) using DH technology

  32. Acknowledgements • AD & DH Support • Thomas Lubberstedt (ISU Dept. Agronomy) • Everton Brenner (ISU Dept. Agronomy) • USDA-ARS GEM Raleigh, NC State Staff • AgiWise, L.L.C. • AgReliant Genetics, L.L.C. • Monsanto Company • Pioneer Hi-Bred Int., Inc. • Syngenta Seeds, Inc.

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