Transcript Identity Confirmation

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 Parent Selection
Target Marker-Assisted
Seedling Selection
Developing Useful Markers:
Marker Assisted Breeding
Pipeline
Genomics Applied in Apple Breeding
Target
Traits
Known
QTLs
Breeding
markers
Simple
validation
Allele effects
& distributions
Applications
in breeding
MAPS
MASS
Identity
Confirmation
Traits and Loci Used at UMN and WSU
“A” – Acidity
Md-Exp7 – Texture
(Scab Resistance)
Ma – Acidity, Texture
Md-PG1
– Texture
Md-ACO1 –
Texture (storage)
Md-ACS1 –
Texture (storage)
Traits and Loci Used at UMN and WSU
“Bl” – Overcolor,
blush vs stripe
Rf –
Overcolor
Traits and Loci Used at UMN and WSU
Photo: M. Clark
Md-Exp7, CHVf1 – Scab
Resistance (Texture)
“Bp13”
– Bitter Pit
Ma – Bitter Pit
(Acidity, Texture)
Traits and Loci Used at UMN and WSU
“Bl” – Overcolor
“A” – Acidity
Rf –
Overcolor
Md-Exp7, CHVf1 –
Texture, Scab
Resistance
“Bp13” –
Bitter Pit
Md-PG1
– Texture
Md-ACO1 –
Texture (storage)
Ma – Acidity, Texture,
Bitter Pit
Md-ACS1 –
Texture (storage)
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
• 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
Parent2
Skin color Skin color Rf Skin color
Rf-SSR
SNPs
Bl SNPs
Storage Storage Storage
abilty
ability
ability
Bitterpit 13
ethylene ethylene cell wall
ACS1
ACO1
PG1
MARKER
SSR
SNP
SNP
SCAR
SCAR
SSR
Linkage Group
CrimsonCrisp
PCFW2-134
LG09
LG02
Blbl
LG15
FF
LG10
FN
LG10
PRI669-205
LG09
RfRf
Honeycrisp
Keepsake
F_Honeycrisp
138140
Rfrf
Blbl
FN
NN
292 hom
PixieCrunch
PCFW2-134
PRI669-205
RfRf
Blbl
Snowsweet
Sharon
Fireside
Rfrf
BlBl
FN
NN
MN1628
Frostbite
RfRf
Blbl
FN
NN
MN1837
MN1627
RfRf
BlBl
FF
NN
MN1914
Honeycrisp
RfRf
BlBl
FF
NN
MN1915
Sweet16
RfRf
no call
FF
NN
MN1937
Empress
RfRf
Blbl
FF
FN
MN1942
Honeycrisp
RfRf
blbl or Blbl
FN
FN
MN1955
Honeycrisp
RfRf
Blbl
NN
NN
MN1965
MN1702
rfrf
blbl
FN
NN
MN1971
M_MN1971
rf2rf3
Blbl
FN
NN
MN1980
Honeycrisp
RfRf
Blbl
NN
NN
MN1984
Frostbite
rf2rf2
Blbl
FN
NN
MN1988
Honeycrisp
RfRf
Blbl
NN
NN
MN2000
Keepsake
138158
NorthSpy 138 hom
MN1691
138 hom
Zestar
138 hom
Zestar
138 hom
Zestar
138 hom
NY543
138 hom
Monark
138 hom
Honeycrisp 140168
Honeycrisp 140 hom
Liberty
138 hom
Honeycrisp 140 hom
Freedom
138 hom
Empress
138152
Rfrf
Blbl
NN
NN
FN
Bitterpit
16.1
Bitterpit
16.2
TA16
Major Acid
locus
TA 08
Crispness
Modifies
at harvest
effects of
16
TA16 locus
s s 475880861 s s 475882553 s s 475881697 s s 475876558 s s 475879081
LG13
LG16
na
ss475881704
LG16
LG08
na
LG16
FN
HH
HL
LG16
FN
NN
FN
FN
LL
HL
NN
FN
FN
FN
HL
LL
FN
FN
FN
HL
LL
FN
na
FN
LL
HL
NN
292298
FF
292298
FN
298 hom
FN
289292
FN
289292
FF
289 hom
FN
292 hom
FN
292 hom (291 FN
hom?)
292 hom (291 FN
hom?)
292 hom
292 hom (291 FN
hom?)
292 hom
FN
292298
NN
298 hom faintFN
FN
FN
HL
HH
FN
NN
FN
HL
LL
FN
NN
NN
HL
HL
FN
NN
NN
HH
LL
FF
FF
FF
LL
HL
NN
FF
FF
LL
HL
NN
FN
FN
HL
HL
LL
FN
NN
NN
FN
LL
HH
NN
0
FN
LL
HH
NN
NN
NN
HL
LL
FN
NN
NN
HL
HL
FN
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
Seedling order
replicated in 96
well DNA
extraction plate
Photo by
Kate Evans
Seedling Selection: Culling Decisions
Culling level
set for each
allele in each
genetic test
Genetic tests
selected for each
cross based on
goals and parents
Seedling Selection: Genotype Data Report
•
Culling decision for each seedling combines all genetic tests
• Genotype report color coded
• Maintains order in seedling
tray to facilitate culling
Photo by
Kate Evans
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
• Reliable Markers
Summary
– 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
MSU
Amy Iezzoni (PD)
Jim Hancock
Dechun Wang
Cholani Weebadde
Univ. of Arkansas
John Clark
WSU
Cameron Peace
Dorrie Main
Kate Evans
Univ. of Minnesota
Karina Gallardo
Jim Luby
Vicki McCracken
Chengyan Yue
Nnadozie Oraguzie
Former WSU
Oregon State Univ.
Raymond Jussaume
Alexandra Stone
Mykel Taylor
Cornell
Susan Brown
Kenong Xu
Clemson
Ksenija Gasic
Gregory Reighard
Texas A&M
Dave Byrne
USDA-ARS
Nahla Bassil
Gennaro Fazio Univ. of CA-Davis
Chad Finn
Tom Gradziel
Carlos Crisosto
Plant Research Intl,
Netherlands
Univ. of New Hamp.
Eric van de Weg
Tom Davis
Marco Bink
Questions?