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

Develop an unmatched collection of rare maize alleles. Develop inbreds from 300+ different races of maize. Make these inbreds available to the maize community for basic research.

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

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

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

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

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)

ContaminantPossible Haploid

Hybrid Haploid Purple Scutellum

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

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

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

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

Backcrossed to RaceAccession #CountryPHB47PHZ51 Elotes OccidentalesPI Mexico6.7% GordoPI Mexico3.8% CónicoPI Mexico3.2% Confite PuntiagudoAmes 28653Peru7.4%5.2% MorochoPI Peru3.7%7.6% PerlaPI Peru5.6%7.4% Morado CanteñoPI Peru3.4%5.1% MorochoPI Peru4.4% AncashinoPI Peru4.1% CuzcoPI Peru9.1% San Juan HuancavelicaPI Peru5.5% AragüitoNSL Venezuela7.7% CosteñoAmes 28614Venezuela4.7%

Haploid Induction: Within the same 24 accessions Backcrossed to PHZ % Induction Rate Backcrossed to PHB % 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)

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

Sterile Partially Fertile

DH Ears from Altiplano backcrossed to PHB47

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

RecurrentTotalAvgFrequency Distribution Parent# of Earsk/ear1-5 k6-10 k11-15 k16-20 k21+ k PHB %22.6%4.4%5.2%7.8% PHZ %17.6%6.6%5.5%17.6% Same 24 Accessions

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

TotalAvgFrequency Distribution Treatment# of Earsk/ear1-5 k6-10 k11-15 k16-20 k21+ k Herbicide %24.2%6.1%4.5%7.6% Colchicine %22.0%11.0%2.4%25.6% Same 14 populations

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

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

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%)

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.

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

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

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.