1. Mike Blanco & Andrew Smelser USDA-ARS Plant Introduction Research Unit

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

7. 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. ContaminantPossible Haploid

9. Hybrid Haploid Purple Scutellum

10. R1-nj Marker Inhibition Colored Seed Morocho Backcrossed to PHB47 Montaña Backcrossed to PHB47

13. Backcrossed to RaceAccession #CountryPHB47PHZ51 Cristalino AmaralloPI 516163Argentina5.6% AltiplanoPI 485364Bolivia4.9%14.4% Kcello EcuatorianoAmes 28740Bolivia7.0%7.3% Patillo GrandeAmes 28748Bolivia5.9%6.5% PisankallaNSL 286568Bolivia7.7%8.6% KarapampaNSL 286824Bolivia6.5%9.5% YungueñoNSL 286578Bolivia5.5%4.7% Pojoso ChicoNSL 286760Bolivia6.0%4.0% MoradoPI 485373Bolivia6.5% Chake-SaraAmes 28744Bolivia5.6% NiñueloPI 485371Bolivia6.8% CoroicoNSL 286500Bolivia3.8% PatilloAmes 28736Bolivia5.5%

14. Backcrossed to RaceAccession #CountryPHB47PHZ51 BR105 (Suwan)Ames 26251Brazil6.2%6.1% BR106 (Tuxpeño)Ames 26252Brazil7.5%8.5% Curagua GrandePI 485412Chile4.2%2.6% CameliaNSL 42755Chile7.6% MontañaPI 445252Colombia5.2%5.1% YucatánPI 445514Colombia4.7%5.5% AndaquiPI 444284Colombia4.8% CabuyaPI 445323Colombia4.5% PiraPI 445528Colombia6.8% CUBA164 (Mixed Creole)PI 489361Cuba2.6%4.5% ChandelleAmes 28574Cuba6.7%

15. Backcrossed to RaceAccession #CountryPHB47PHZ51 MishcaPI 488016Ecuador4.9%10.0% CandelaNSL 287040Ecuador3.2%3.9% YunquilanoPI 485436Ecuador4.7% Nal-tel amarillo de tierra bajaAmes 28487Guatemala5.9% OlotónAmes 28539Guatemala4.8% San MarcenoAmes 28511Guatemala4.1% Early CaribbeanAmes 28579Martinique7.2%8.2% Elotes OccidentPI 484828Mexico5.2%5.6% Dulcillo de NorestePI 490973Mexico3.7%4.8% BofoAmes 28481Mexico6.2%4.6% OnaveñoPI 484880Mexico5.2%4.8% Cónico NorteñoPI 515577Mexico7.3% RatonPI 484454Mexico9.7%

16. Backcrossed to RaceAccession #CountryPHB47PHZ51 Elotes OccidentalesPI 628414Mexico6.7% GordoPI 484406Mexico3.8% CónicoPI 484601Mexico3.2% Confite PuntiagudoAmes 28653Peru7.4%5.2% MorochoPI 571413Peru3.7%7.6% PerlaPI 571479Peru5.6%7.4% Morado CanteñoPI 515026Peru3.4%5.1% MorochoPI 503511Peru4.4% AncashinoPI 514763Peru4.1% CuzcoPI 485274Peru9.1% San Juan HuancavelicaPI 503711Peru5.5% AragüitoNSL 283561Venezuela7.7% CosteñoAmes 28614Venezuela4.7%

17. 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. 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% dimethyl sulfoxide at 3-4 leaf stage

19. Sterile Partially Fertile

21. DH Ears from Altiplano backcrossed to PHB47

22. RecurrentAvg. ParentDoublingRange PHB478%0%-20% PHZ516%0%-16% Same 24 Accessions

23. RecurrentTotalAvgFrequency Distribution Parent# of Earsk/ear1-5 k6-10 k11-15 k16-20 k21+ k PHB4711510.260.0%22.6%4.4%5.2%7.8% PHZ51919.052.7%17.6%6.6%5.5%17.6% Same 24 Accessions

24. Avg. TreatmentDoublingRange Herbicide6%0%-12% Colchicine19%0%-36% Same 14 populations

25. TotalAvgFrequency Distribution Treatment# of Earsk/ear1-5 k6-10 k11-15 k16-20 k21+ k Herbicide6611.757.6%24.2%6.1%4.5%7.6% Colchicine8217.139.0%22.0%11.0%2.4%25.6% Same 14 populations

26. InductionDoubling Avg.RangeAvg.Range 6%3%-14%7%0%-24% 50 Races (74 Populations) Does not include colchicine treatment

27. 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. 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. 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. Develop methods to identify haploid kernels among the colored kernels/inhibitor genes Improve doubling percentages Identify and develop germplasm for spontaneous doubling

31. 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. 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.