Marker-Assisted Breeding for Apple at the

University of Minnesota and Washington State University

Jim Luby, Kate Evans, and Cameron Peace

Outline of Presentation Timeline and Flow of Apple Breeding Program

Developing Useful Markers with the Marker Assisted Breeding Pipeline

Identity Confirmation

Marker-Assisted Parent Selection (MAPS)

Marker-Assisted Seedling Selection (MASS)

Timeline and Flow of

Apple Breeding Program

Target Marker-Assisted Seedling Selection

Target Marker-Assisted Parent Selection

Developing Useful Markers:

Marker Assisted Breeding Pipeline

Genomics Applied in Apple Breeding

Target Traits

Known QTLs

Applications in breeding

MAPS

MASS

Identity Confirmation

Breeding markersDNA te

sting

serv

icesQTL

prior

ities

Cross

ing

sche

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Trial

MASS

Simple validation

Allele effects& distributions

Seedli

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selec

tion

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Traits and Loci Used at UMN and WSU

Md-ACO1 – Texture (storage)

Md-Exp7 – Texture (Scab Resistance)

Md-ACS1 – Texture (storage)

Ma – Acidity, Texture

Md-PG1 – Texture

“A” – Acidity

Traits and Loci Used at UMN and WSU

Rf – Overcolor

“Bl” – Overcolor, blush vs stripe

Overcolor

Traits and Loci Used at UMN and WSU

Md-Exp7, CHVf1 – Scab Resistance (Texture)

Ma – Bitter Pit(Acidity, Texture)“Bp13”

– Bitter Pit

Apple scab resistance

Photo: M. Clark

Traits and Loci Used at UMN and WSU

Md-ACO1 – Texture (storage)

Md-Exp7, CHVf1 – Texture, Scab Resistance

Md-ACS1 – Texture (storage)

Ma – Acidity, Texture, Bitter Pit

Rf – Overcolor

Md-PG1 – Texture

“Bp13” – Bitter Pit

“Bl” – Overcolor

“A” – Acidity

Identity Confirmation

Identity Confirmation• LOTS of opportunities for errors in a breeding program

– mislabeled parent trees– pollen collection– crossing– seed harvest and handling– seedling handling and propagation– labeling

malus• Ancestry errors in pedigrees of selections and cultivars – Identity and ancestry for breeding and

intellectual property purposes

• Parentage of Seedlings – 1% to 5% error rate not unusual in a cross

Marker-Assisted

Parent Selection

Parent Selection at UMN• Potential parents list - based on

• Crossing objectives • Available trees or pollen• Phenotype data• Pedigree• Genotype data and relative breeding value at each

locus of interest

Name Parent1 Parent2Skin color Rf-SSR

Skin color Rf SNPs

Skin color Bl SNPs

Storage abilty

ethylene ACS1

Storage ability

ethylene ACO1

Storage ability

cell wall PG1

Bitterpit 13Bitterpit

16.1Bitterpit

16.2

TA16 Major Acid

locus

TA 08 Modifies effects of

TA16 locus

Crispness at harvest

16

MARKER SSR SNP SNP SCAR SCAR SSR ss475880861 ss475882553 ss475881697 ss475876558 ss475879081 ss475881704Linkage Group LG09 LG09 LG02 LG15 LG10 LG10 LG13 LG16 LG16 LG16 LG08 LG16

CrimsonCrisp PCFW2-134 PRI669-205 RfRf Blbl FF FN na na FN HH HL FN

Honeycrisp Keepsake F_Honeycrisp138140 Rfrf Blbl FN NN 292 hom NN FN FN LL HL NN

PixieCrunch PCFW2-134 PRI669-205 RfRf Blbl FN FN FN FN HL LL FN

Snowsweet Sharon Fireside 138158 Rfrf BlBl FN NN 292298 FF FN FN HL LL FN

MN1628 Frostbite NorthSpy 138 hom RfRf Blbl FN NN 292298 FN na FN LL HL NN

MN1837 MN1627 MN1691 138 hom RfRf BlBl FF NN 298 hom FN FN FN HL HH FN

MN1914 Honeycrisp Zestar 138 hom RfRf BlBl FF NN 289292 FN NN FN HL LL FN

MN1915 Sweet16 Zestar 138 hom RfRf no call FF NN 289292 FF NN NN HL HL FN

MN1937 Empress Zestar 138 hom RfRf Blbl FF FN 289 hom FN NN NN HH LL FF

MN1942 Honeycrisp NY543 138 hom RfRf blbl or Blbl FN FN 292 hom FN FF FF LL HL NN

MN1955 Honeycrisp Monark 138 hom RfRf Blbl NN NN 292 hom (291 hom?)FN FF FF LL HL NN

MN1965 MN1702 Honeycrisp 140168 rfrf blbl FN NN 292 hom (291 hom?)FN FN FN HL HL FN

MN1971 M_MN1971 Honeycrisp 140 hom rf2rf3 Blbl FN NN 292 hom LL NN

MN1980 Honeycrisp Liberty 138 hom RfRf Blbl NN NN 292 hom (291 hom?)FN NN FN LL HH NN

MN1984 Frostbite Honeycrisp 140 hom rf2rf2 Blbl FN NN 292 hom FN 0 FN LL HH NN

MN1988 Honeycrisp Freedom 138 hom RfRf Blbl NN NN 292298 NN NN NN HL LL FN

MN2000 Keepsake Empress 138152 Rfrf Blbl NN NN 298 hom faintFN NN NN HL HL FN

MN2006 Sawa Honeycrisp 138140152 Rfrf BlBl FF NN 292298 FN 0 FN LL LL NN

MN2008 Summerred Zestar 138152 Rfrf FF NN 289298 FF NN na HH

MN2016 Zestar Arlet 138 hom RfRf BlBl FF NN 289298 FN NN NN HH HL FF

MN2033 Honeycrisp Zestar 138 hom RfRf no call FF NN 289292 FN FN FN HL HL FN

MN2042 Honeycrisp Burgundy 138 hom RfRf Blbl FN NN 292298 FN NN NN HL HL FN

MN2046 Honeycrisp Zestar 138 hom RfRf Blbl FF FN 292298 NN NN NN HL HL FN

MN2095 Etter Pink Honeycrisp 140168 rfrf FN NN 289292 HL

MN2099 Etter Pink Honeycrisp 138168 Rfrf FN NN 292298 HL

Parent Selection at UMN• Prospective cross list

• based on favorable phenotype and genotype combinations

• Logistics • final crossing “call” made in field based on flowering

time, tree availability, pollen quality, etc.

Marker-Assisted

Seedling Selection

Seedling Selection: Sample Collection

96 count seedling tray for sample collection

Photo by Kate Evans

Seedling order replicated in 96 well DNA extraction plate

Genetic tests selected for each cross based on goals and parents

Seedling Selection: Culling DecisionsCulling level set for each allele in each genetic test

Seedling Selection: Genotype Data Report

Photo by Kate Evans

• Genotype report color coded

• Maintains order in seedling tray to facilitate culling

• Culling decision for each seedling combines all genetic tests

Seedling Selection: Culling

• Verbal repetition of seedling location and culling instruction to reduce errors

• Usually best with three people

• Culling requires 2-3 person/hours per 1000 seedlings

Summary• Reliable Markers – Developed for 9 loci and 8 target traits in our germplasm– Functional SNP haplotypes and PCR-based markers (SSR,

Indel)

• Identity confirmation – RosBREED SNP markers used to confirm ancestry of ~ 900 parents,

selections, seedlings and ancestors at UMN, WSU and Cornell– PCR-based Markers used on over 20,000 seedlings at WSU and UMN

• Marker-Assisted Parent Selection – Used to design 120 crosses from 2011 through 2013.

• Marker-Assisted Seedling Selection – Over 20,000 seedlings screened in 2011 through 2013– Culling from 25 to 90% depending on cross and markers

Many Thanks!

Acknowledgements

This project is supported by the Specialty Crop Research Initiative of USDA’s

National Institute of Food and Agriculture

MSUAmy Iezzoni (PD)

Jim HancockDechun Wang

Cholani Weebadde

WSUCameron Peace

Dorrie MainKate Evans

Karina Gallardo Vicki McCracken

Nnadozie OraguzieFormer WSU

Raymond JussaumeMykel Taylor

CornellSusan BrownKenong Xu

Clemson Ksenija Gasic

Gregory Reighard

Texas A&MDave Byrne

Univ. of CA-DavisTom Gradziel

Carlos Crisosto 

Univ. of New Hamp.Tom Davis

Univ. of MinnesotaJim Luby

Chengyan Yue

Oregon State Univ.Alexandra Stone

Plant Research Intl, Netherlands

Eric van de WegMarco Bink

USDA-ARSNahla Bassil

Gennaro FazioChad Finn

Univ. of ArkansasJohn Clark

Questions?