Transcript Slide 1

QTL and QTL allele
validation in apple
Sujeet Verma, Cameron Peace, Jim Luby, Kate Evans, Susan
Brown, Matt Clark, Cari Schmitz, Yingzhu Guan, Ben Orcheski,
Umesh Rosyara, Nahla Bassil, Eric van de Weg
25 July 2013
ASHS Palm Desert, CA
Outline of Presentation
 What to Target First?
 QTL Validation: LG16Ma for acidity
(and crispness and other traits)
 QTL Allele Validation for LG16Ma
(Ma-indel DNA test)
 Other Jewels for Apple
What to Target First?
- deciding which of the known QTLs
is worth attempting to validate for breeding
Some Apple QTLs Known Previously
(focusing on Mendelian Trait Loci and QTLs with candidate genes)
Ma locus – “high” vs “medium” acidity,
also major QTL for crispness,
juiciness, consumer liking, others
2MBAc “Apple
flavor” locus
Md-MYB1 gene
for red skin vs.
non-red skin
Md-MYB10 gene
for red flesh
Md-PG1 gene
for firmness
Vf locus – scab
resistance
Md-ACS1 gene
for storability
Second major
acidity locus
Md-ACO1 gene
for storability
Firmness QTL –
Md-Exp7 gene
etc…
RosBREED’s Socio-Economics Surveys
What trait is my
next breeding
target?
We like disease
resistant apple
trees!
We like firm
tart cherries
that pit well!
I like tasty
strawberries!
Processors
Breeder
survey 2010
Household data
Producer survey Market Intermediary
analysis 2010;
Fall 2011survey Fall 2010consumer survey 2012
Winter 2012
Winter 2011
Trait Values
What Do People Really Want in Fruit
of a New Apple Cultivar?
And the survey says...
Some Apple QTLs Known Previously
Ma locus – “high” vs
“medium” acidity, also
major QTL for crispness,
juiciness, consumer
liking, others
Md-PG1 gene
for firmness
2MBAc “Apple
flavor” locus
Md-MYB1 gene
for red skin vs
non-red skin
Md-MYB10 gene
for red flesh
Vf locus – scab
resistance
Md-ACS1 gene
for storability
Second major
acidity locus
Md-ACO1 gene
for storability
Firmness QTL –
Md-Exp7 gene
Some Apple QTLs Known Previously
Ma locus – “high” vs
“medium” acidity, also
major QTL for crispness,
juiciness, consumer
liking, others
The Ma Locus
Ma locus – “high” vs
“medium” acidity, also
major QTL for crispness,
juiciness, consumer
liking, others
Ma = Malic acid
• Monogenic inheritance of apple acidity (Wellington 1924),
designated as Ma locus (Visser & Verhaeg 1978)
• Detected as a QTL (~40% phen variance) in single F1
populations (King et al. 2001, Leibhard et al. 2003),
also QTL for crispness, juiciness, and others
• More recently, detected as a QTL for acidity via
association mapping with SNPs (Kumar et al. 2012)
• = Malic acid transporter gene (Xu et al. 2012, Khan et al. 2013)
The Ma Locus
Ma locus – “high” vs
“medium” acidity, also
major QTL for crispness,
juiciness, consumer
liking, others
Ma = Malic acid
Is this QTL, LG16Ma, detectable
in U.S. breeding germplasm?
Is it associated with other socio-economically
valuable traits in addition to acidity?
If so, how can breeders exploit it?
QTL Validation
- confirming the LG16Ma QTL really exists
in apple breeding germplasm
QTL Validation Dataset
WA
• Germplasm from 3 major
U.S. breeding programs
- total n = 960
- all pedigree-linked
- multiple families & ancestors
- chosen to represent alleles of
57 important breeding parents
• Fruit quality standardized
phenotyping for 3 years
(at harvest and after 10 & 20 wks storage)
• Genome scanning with 8K
SNP array and a nearby SSR
MN
NY
QTL Validation Analysis
• Use Pedigree-Based Analysis software
McIntosh
Melba
W illiams
LivelRasp
F_Spartan
Jolana
Spartan
PRI14-126
Starr
OR38T610
Delicious
KidsOrRed
NJ53
F_W illiams
PRI14-226
PRI14-644
Cox
Gala
Sonora
NJ8
W inesap
GoldenDel
PRI612-1
F1_9433-2-2
RomBeauty
Mflor821
NJ24
NJ67634
F1_9433-2-8
RallsJan
Delicious
Winesap
RomBeauty
Jonathan
F_Ill_#2
M_PRI668-100
GoldenDel
F2_26829-2-2
P21R4A30
F2_26829-2-2
NY65707-19
Jonathan
Keepsake
Ill_#2
F_P21R4A30
M_MN1764
NJ123249
Prima
Frostbite
F_Honeycrisp
NJ110037
NJ27
NorthSpy
Honeycrisp
Melrose
Sonya
JerseyBla
PRI668-100
Julyred
Macoun
F_Gingergold
sibLiberty
NY752
PRI54-12
F_JamesGr
Liberty
W ealthy
GingerGold
F_IngMarie
Jam esGr
Tetofsky
NJ130o
Fuji
Crandall
MN1691
PRI14-126
Jerseymac
NY913
Florina
NJ37848
F_Delfloki
PRI47-147
YelTransp
IngMarie
NJ117637
Longfield
NJ88
NJ31
DuchOlden
PRI610-2
Raritan
NJ12
Monroe
Rubin
F_PattenGr
Mantet
Edgewood
W orcPearm
RallsJan
Mavis
PRI1661-1
Elstar
Vanda
PRI855-102
Oriole
Sharon
NY88
PRI668-100
Nicola
W SU2
Fireside
PRI1661-2
Chinook
StateFair
BenDavis
PattenGr
Gravenstein
F_Goodland
Close
M_MolliesDel
PacRose
PRI643-11
NJ14
AurGoldGa
NY241
PRI1018-101
PacQueen
Goodland
NJ440249
Jongrimes
W agener
NJ19237
Zestar
BC-8S-27-2
Crandall
Coop-17
BC-8S-31-56
M2439
Redfree
NJ35437
BC-SPA493
FrancRus
LadyW ill
CrownPR
X-2771
PCFW 2-134
Lambourne
P7R25A27
NJ136055
BlushiGol
Clivia
F_Undine
Splendour
Indo
NJ15
Suncrisp
PRI669-205
Braeburn
PRI2482-100
Grifer
NJ303955
Malinda
Ambrosia
Glockenapfel
Zari
Cameo
MolliesDel
DrOldenbu
Fuji
Bellida
PRI1235-100
F_Helios
Enterprise
NJ35934
XIIA277
Civni
F_EdGoulGol
X-3143
F_NJ15
F_Sandow
Ecolette
KAZ95-17-14
Cortland
A28/39
Coop-15
NJ109
Lundbytrop
Milwa
F_MolliesDel
KAZ95-18-07
A849-5
PRI1469-100
BeautBath
Delcorf
Kanzi
VKrakers
KAZ96-05-04
Nicoter
Idared
Rubinola
Goldrush
NZ_RGxBB
Ariane
Pilot
BelHerm
Priscilla
Maigold
LaFlam
PRI2059-101
Akane
Pinova
F_Brina
X-3318
Rubinstep
Nevson
Sundowner
Scarlet
CrippsPink
Senshu
Sansa
Rucla
Coop-10
NJ381049
Delblush
StarkEarl
Scifresh
Undine
PitmPine
Linda
X4598
Delfloki
Scilate
Orin
Topaz
MN1764
Shizuka
W SU24
X3174
Toko
AnanasRei
MN1702
W SU37
Camuzat
F_MN1764
AutGold
BaumanRei
BernRos
GMAL4591
Haralson
W SU17
Monark
Telam on
GMAL4593
W SU36
M_Sunrise
Boskoop
Delorina
Honeygold
GMAL4595
BrixPlatt
Calamari
Calvilla
PCF-3-120
Hatsuaki
Sunrise
CIVG198
Jonagold
CrimsSnow
M_NY632
Croncels
Gloster
W SU38
Jonafree
Pop_BBxPR
Arlet
Dalitron
Ped-PinxCam
NY632
DallaRosa
PRI69-52
ShinGold
4-27-0019
Pop_GaxC15
EarlyGold
Alkmene
Fiesta
W SU34
Edelb
5-87-0019
Helios
Fiamma
52-05-26
Apollo
5-90-0019
Empire
GelEdel
Gewurz
Zonga
Pop_HCxCP
Akagi
Ped-HCxPit(692)
Hampshire
EdGoulGol
HarbRei
PRI1956-6
Limoncini
Ped-SonxNY241
25-120-9928
Freedom
6-73-0019
Piros
Ped_HCx1764
MN1914
Santana
Fortune
Sandow
MPop_HCx1702
4A-100-0013
Magre
SchonNord
Shinsekai
MN1628
MPop_HCxMon
Ped-HCxZest
4A-38-0013
GranSmith
Napoleone
Silken
Delgollune
GoldenOr
Dolgo
Summerfree
Ped-GoldrxNY913
Ontario
PermainDor
RedFiled
Merlijn
Pristine
Pop_AurxHC
Angold
NY75414-1
Sabina
RenCanada
Ariwa
Tunda
RenChamp
CrimsCrisp
Discovery
W SU46
Robusta5
W SU35
Belgica
W SU22
RosBian
Rafzubin
B_TxBB
4-159-9926
RosdiCal
GoldPink
RosDop
Nicogreen
Pop_ArlxCP
Brina
Ped_HCxJonf
W SU30
RotStett
SanLugano
Pop_GDxSc
Sila
Creston
Stayman
Delearly
N_PxF
E_FxGD
Snowsweet
NJ90
E_FxGlo
TavBian
Ligol
Kerr
TirSpitz
Dalinette
W eisW int
Saturn
Delcoros
Murray
Galmac
• Validated position of LG16Ma for acidity!
malic acid
transporter gene
1
0.9
0.8
0.7
0.6 •
0.5
•
0.4
Posterior Intensity
Expected
LG16Ma locus
position
Sensory acidity atHharvest
Sensory acidity after
10wk
10 wks storage
Sensory acidity after
20wk
20 wks storage
Ma locus (L16)
locus (L08)
Evidence forAbitter
pit QTL there too
Some, but less, evidence for crispness/juiciness
S
terior Intensity
1
S
Sensory acidity HS
0.9
1
Sensory acidity 10w
0.8
Ma
locus
(L16)
Sensory acidity 20w
0.9
0.7
0.8
Ma locus (L16)
0.6
A locus (L08)
0.7
0.5
0.6
A locus (L08)
0.4
0.5
0.3
0.4
0.2
0.3
0.1
0.2
0
0.1
0 5 10 15 20 etc.
25 30 35 40 45
0
Genetic Distance (cM
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Genetic Distance (cM)
Intensity
Posterior
QTL
Validation
Results
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Ma locus (L16)
A locus (L08)
Posterior Intensity
Sensory acidity H
Sensory acidity 10wk
Sensory acidity 20wk
0
Ma locus
A locus
0
A locus
Results shown for the FlexQTL™
analysis of WSU RosBREED data
5 10 15 20 25 30 35 40 45 50 55 60 65
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Titratable acidity H
Titratableacidity 10wk
Titratable acidity 20wk
Ma locus
A locus
0
Sensory acidity H
Sensory acidity 10wk
Sensory acidity 20wk
Ma locus
A locus
0
5 10 15 20 25 30 35 40 45 50 55 60 65
Genetic Distance (cM)
Posterior Intensity
Posterior Intensity
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
5 10 15 20 25 30 35 40 45 50 55 60 65
Genetic Distance (cM)
Genetic Distance (cM)
(second acidity QTL
on LG8 also consistent)
Titratable acidity H
Titratableacidity 10wk
Titratable acidity 20wk
Ma locus
0
Sensory acidity H
Sensory acidity 10wk
Sensory acidity 20wk
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
5 10 15 20 25 30 35 40 45 50 55 60 65
Genetic Distance (cM)
Posterior Intensity
Posterior Intensity
• LG16Ma for acidity
consistent over:
- storage durations
- years
- families
- breeding programs
Posterior Intensity
QTL Validation Results
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
5 10 15 20 25 30 35 40 45 50 55 60 65
Genetic Distance (cM)
Titratable acidity H
Titratableacidity 10wk
Titratable acidity 20wk
Ma locus
A locus
0
5 10 15 20 25 30 35 40 45 50 55 60 65
Genetic Distance (cM)
QTL Validation Results
• So, the QTL is there, but...
o
What functional alleles are present
in breeding germplasm?
o
What are the effects and value of those
alleles? (alone or in combination with others)
o
What is their occurence?
-
frequencies
sources (founders, important parents)
• And besides with genome-scan SNPs, can
all this be captured with a simple DNA test?
QTL Allele Validation
- detecting and valuing LG16Ma alleles
in apple breeding germplasm
Functional Alleles Present
• With SNP and SSR haplotyping:
o
11 alleles in Washington State Univ.
apple breeding program germplasm
o
15 more in Univ. Minnesota and Cornell
Univ. breeding germplasm
• Are these all functionally unique?
Effects of Functional Alleles
Titratable Acidity
(TA, mEq/L)
above the
season average
of 0.70
(2011 at harvest,
WSU seedlings,
n=214)
High-acid alleles
Ma1 Ma2 Ma3 Ma4 Ma5
xx1 ma1 ma2 ma3 ma4 ma5
Low-acid alleles
• For acidity, can simplify to two types:
“Ma” (high acid) and “ma” (low acid)
Effects of Functional Alleles
• But various effects on other traits...
Crispness
rating (1-5)
above season
average of
2.90
(2011 at harvest,
WSU seedlings,
n=214)
Bitter pit
incidence
rating (1-5)
below season
average of
1.50
Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 ma2 ma3 ma4 ma5
(As in other graphs,
high here is good)
Values of Functional Alleles
• So, each of the 11 alleles can be +ve or -ve
for effect on acidity, crispness, bitter pit incid.
- often trade-offs
• Which ones are most desirable?
• Overall value of each allele can be
determined as socio-economic value
of its relative effect on each trait
(Example of Socio-Economics Results –
Market Intermediary $ Values)
Trait
Threshold
Value*
Shelf life
1 week
$0.134
Crispness
not  very
$0.129
Flavor
weak/mild
$0.128
Appearance
 full/intense
3% defects
Firmness
14 lb
$0.009
Size
100-count
$0.002
$0.123
* Willingness-to-pay of market intermediaries
per lb of a 42 lb box
Values of Functional Alleles
• Using $ values from RosBREED’s survey
results of market intermediaries...
Bitter pit incidence
Crispness
Total
socioeconomic
value of
allele
Acidity
High-acid alleles tend to
be most valuable overall
Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 ma2 ma3 ma4 ma5
Most valuable allele because of
In contrast, these only valuable
for bitter pit (low incidence)
valuable contribution to all 3 traits
Values of Functional Alleles
• Strategy: recombination to get even more
valuable alleles (assuming three different genes)
Bitter pit incidence
Crispness
Total
socioeconomic
value of
allele
Acidity
Ma1 Ma2 Ma3 Ma4 Ma5 xx1 ma1 ma2 ma3 ma4 ma5
Can we combine best of each
into a single allele?
Frequencies of Functional Alleles
Frequencies of Functional Alleles
Shown for parent pool (58 cultivars and selections)
of WSU apple breeding program
ma other
ma5
ma4
ma3
Ma1
ma2
Ma2
Ma3
ma1
xx1
Ma other
Ma4
Ma5
Sources of Functional Alleles
Parents
(founder sources)
(McIntosh)
Shown for parent pool (58 cultivars and selections)
of WSU apple breeding program
(Frostbite)
ma Honeycrisp other
Arlet, Aurora, Arlet, Cameo,
Enterprise
ma5
Enterprise, Fuji, Gala,
Splendour, WA 2
ma4
(Lady
ma3
Williams, Braeburn
Granny Smith)
Ma1
(Red Delicious,
Golden Delicious)
Aurora, Gala,ma2
Sansa, WA 2
Ma2
(Granny
Smith)
(Red Delicious)
Cripps Pink
Ma3 (Lady Williams)
Arlet, Cameo,
Cripps Pink, ma1
Splendour
(Golden Delicious)
xx1
Honeycrisp
Ma other
(Duchess of Oldenburg)
WA5
Ma4(Rome
Beauty)
Ma5 WA7 (NJ90)
Simple Locus-Specific DNA Test
• All previous effects, values, frequencies
were defined by 8K-array SNP haplotypes
• Converted to a simple PCR marker –
“Ma-indel” – captured most information
• Can now use in high-throughput MAB
• Already used to screen many parents and
>10,000 seedlings (WSU & UMN) in spring 2013
Ma-indel DNA Test:
a “Jewel in the Apple Genome”
Other Jewels for Apple
Other Apple Jewels Available Now
The LG8A Locus – as big as LG16Ma
for acidity
LG16Ma: additivity
9
6
Very
High
High
2011 at-harvest TA
All WSU populations
5
Cat TA ±sd
9 = 0.94 ±0.15
7 = 0.67 ±0.06
5 = 0.67 ±0.14
3 = 0.53 ±0.11
1 = 0.43 ±0.09
8
2
7
MedHigh
4
Med.
Med.
1
Very
Low
dominance
High
MedHigh
3
Low
LG8A: partial
Together:
some epistasis
Best to
consider together
The Bp13 Locus
– combines well with Bp16 (Ma)
Homozygous “high”
for both loci
Homozygous “high”
for Bp16 (Ma)
Homozygous
“high” for Bp13
Not homozygous
for either locus
The Rf locus
– skin color amount, type, hue
probability
Cover amount over
apple skin surface
Allele & Genotype
frequencies in 192 U.S. cvs
rf
Rf
rfrf
RfRf
Rfrf
Md-ACS1 & Md-ACO1
- the ethylene synthesis genes
from harvest to 20 weeks storage
plus 1 week at room temperature
(Newtons, M2 of Mohr Digi-Test)
Change in firmness
F=favorable
0
2
4
6
8
10
12
14
N=non-favorable
ACS1
4
some
softening
Both genes - additive
6
more
softening
8
10
FF
0
2
4
6
8
10
12
14
FN NN
ACO1
12
14
ACS: FF FF
ACO:NN FN
ACS1
FF
FN NN
FF FN FN FN
FF NN FN FF
ACO1
allele frequencies
in U.S. cultivars
No N/N N/N
U.S. cultivars
found
Md-ACS1 & Md-ACO1
www.rosbreed.org/breeding/jewel-use/apple
Md-ACS1 & Md-ACO1
Apple Jewels
= QTLs & Their Alleles Validated,
with DNA Tests Developed
...and Used in MAB
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