mike blanco & andrew smelser usda-ars plant introduction research unit

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DOUBLE HAPLOID APPLICATION TO ALLELIC DIVERSITY GERMPLASM Mike Blanco & Andrew Smelser USDA-ARS Plant Introduction Research Unit

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DOUBLE HAPLOID APPLICATION TO ALLELIC DIVERSITY GERMPLASM

Mike Blanco & Andrew SmelserUSDA-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.

Make these inbreds available to the maize community for basic research.

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

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

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

Methods & Materials

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

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)

Contaminant

Possible Haploid

Screening for Haploids

Hybrid

Haploid

Purple Scutellum

Screening Haploids

Screening Problems

R1-nj Marker Inhibition

Colored

Seed

Morocho Backcrossed to PHB47

Montaña Backcrossed to PHB47

Identifying “Haploids” by Seedling Vigor

Induction Percentages are based on visual

kernel sorting by use of kernel marker

Results of Haploid Induction

Backcrossed toRace Accession # Country PHB47 PHZ51Cristalino Amarallo PI 516163 Argentina 5.6%Altiplano PI 485364 Bolivia 4.9% 14.4%Kcello Ecuatoriano Ames 28740 Bolivia 7.0% 7.3%Patillo Grande Ames 28748 Bolivia 5.9% 6.5%Pisankalla NSL 286568 Bolivia 7.7% 8.6%Karapampa NSL 286824 Bolivia 6.5% 9.5%Yungueño NSL 286578 Bolivia 5.5% 4.7%Pojoso Chico NSL 286760 Bolivia 6.0% 4.0%Morado PI 485373 Bolivia 6.5%Chake-Sara Ames 28744 Bolivia 5.6%Niñuelo PI 485371 Bolivia 6.8%Coroico NSL 286500 Bolivia 3.8%Patillo Ames 28736 Bolivia 5.5%

Results of Haploid Induction

Backcrossed toRace Accession # Country PHB47 PHZ51BR105 (Suwan) Ames 26251 Brazil 6.2% 6.1%BR106 (Tuxpeño) Ames 26252 Brazil 7.5% 8.5%Curagua Grande PI 485412 Chile 4.2% 2.6%Camelia NSL 42755 Chile 7.6%Montaña PI 445252 Colombia 5.2% 5.1%Yucatán PI 445514 Colombia 4.7% 5.5%Andaqui PI 444284 Colombia 4.8%Cabuya PI 445323 Colombia 4.5%Pira PI 445528 Colombia 6.8%CUBA164 (Mixed Creole) PI 489361 Cuba 2.6% 4.5%Chandelle Ames 28574 Cuba 6.7%

Results of Haploid Induction Backcrossed to

Race Accession # Country PHB47 PHZ51Mishca PI 488016 Ecuador 4.9% 10.0%Candela NSL 287040 Ecuador 3.2% 3.9%Yunquilano PI 485436 Ecuador 4.7%Nal-tel amarillo de tierra baja Ames 28487 Guatemala 5.9%Olotón Ames 28539 Guatemala 4.8%San Marceno Ames 28511 Guatemala 4.1%Early Caribbean Ames 28579 Martinique 7.2% 8.2%Elotes Occident PI 484828 Mexico 5.2% 5.6%Dulcillo de Noreste PI 490973 Mexico 3.7% 4.8%Bofo Ames 28481 Mexico 6.2% 4.6%Onaveño PI 484880 Mexico 5.2% 4.8%Cónico Norteño PI 515577 Mexico 7.3%Raton PI 484454 Mexico 9.7%

Results of Haploid Induction Backcrossed to

Race Accession # Country PHB47 PHZ51Elotes Occidentales PI 628414 Mexico 6.7%Gordo PI 484406 Mexico 3.8%Cónico PI 484601 Mexico 3.2%Confite Puntiagudo Ames 28653 Peru 7.4% 5.2%Morocho PI 571413 Peru 3.7% 7.6%Perla PI 571479 Peru 5.6% 7.4%Morado Canteño PI 515026 Peru 3.4% 5.1%Morocho PI 503511 Peru 4.4%Ancashino PI 514763 Peru 4.1%Cuzco PI 485274 Peru 9.1%San Juan Huancavelica PI 503711 Peru 5.5%Aragüito NSL 283561 Venezuela 7.7%Costeño Ames 28614 Venezuela 4.7%

Summary

Haploid Induction:Within the same 24 accessions

Backcrossed to PHZ51 - 6.4% Induction RateBackcrossed 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)

Doubling Methods

Stealth Herbicide (Microtubule Inhibitor)Active Ingredient: PendimethalinOne 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

Pollinating Double Haploids

Sterile

Partially Fertile

Pollinating Double Haploids

Results of Doubling are based on ears with

seed production

DH Ears from Altiplano backcrossed to PHB47

Results of Doubling(Recurrent Parent)

Recurrent Avg.  

ParentDoublin

g Range

PHB47 8%0%-20%

PHZ51 6%0%-16%

Same 24 Accessions

Results of Doubling(Kernels/Ear)

Recurrent Total Avg Frequency DistributionParent # of Ears k/ear 1-5 k 6-10 k 11-15 k 16-20 k 21+ kPHB47 115 10.2 60.0% 22.6% 4.4% 5.2% 7.8%PHZ51 91 9.0 52.7% 17.6% 6.6% 5.5% 17.6%

Same 24 Accessions

Results of Doubling(Treatment)

  Avg.  Treatme

ntDoublin

g RangeHerbicid

e 6%0%-12%

Colchicine 19%

0%-36%

Same 14 populations

Results of Doubling(Kernels/Ear)

Total Avg Frequency Distribution

Treatment # of Ears k/ear 1-5 k 6-10 k 11-15 k 16-20 k 21+ k

Herbicide 66 11.7 57.6% 24.2% 6.1% 4.5% 7.6%Colchicine 82 17.1 39.0% 22.0% 11.0% 2.4% 25.6%

Same 14 populations

Results of Induction & Doubling (Overall)

Induction DoublingAvg

. RangeAvg

. Range

6%3%-14% 7%

0%-24%

50 Races (74 Populations)

Does not include colchicine treatment

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

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%)

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.

Summary (Challenges)

Develop methods to identify haploid kernels among the colored kernels/inhibitor genes

Improve doubling percentages

Identify and develop germplasm for spontaneous doubling

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

Acknowledgements

AD & DH SupportThomas Lubberstedt (ISU Dept. Agronomy)Everton Brenner (ISU Dept. Agronomy)USDA-ARS GEM Raleigh, NC State StaffAgiWise, L.L.C.AgReliant Genetics, L.L.C.Monsanto CompanyPioneer Hi-Bred Int., Inc.Syngenta Seeds, Inc.