Transcript Slide 1

QTL and QTL allele
validation in cherry
Amy Iezzoni
Cameron Peace, Audrey Sebolt, Nnadozie Oraguzie,
Umesh Rosyara, Travis Stegmeir
25 July 2013
ASHS Palm Desert, CA
Outline of Presentation
 What is QTL Validation?
What is QTL allele Validation?
 QTL Validation: FW_G2 for Fruit Size
 QTL Allele Validation for FW_G2
 Other Jewels for Cherry
What is QTL Validation?
What is QTL Allele Validation?
Definitions
• QTL validation is confirming that the QTL
really exists in breeding germplasm using
breeding-friendly DNA tests.
• QTL allele validation is detecting and
determining the relative values of the
alleles present in breeding germplasm
detected by the breeding-friendly DNA
tests.
What is Required for QTL Validation?
• Segregating germplasm derived from
important breeding parents, representative
of a program
• Breeding-relevant phenotypic data
• Genotypic data for breeding-friendly
marker(s) at the QTL region
• QTL characterization software for multiple
generations & various family sizes (FlexQTL™)
Pedigree linked germplasm
Fruit weight
Schneiders
Rube
Regina
Empress Eugenie
Unknown
Van
Stella
Lambert
EF
Lapins
Data not
available
JI2420
Napoleon
Namati
Summit
Regina x Lapins
Bopparder Kracher
Unknown
(n= 101)
Sam
Namati x Summit
V-160140
Unknown
Krupno.
NY
(n= 76)
(n= 80)
Drogana Zholtaya
Valeriy Chkalov
Namati x Krupno.
NY x EF
(n= 167)
Pedimap software
Standardized Phenotyping
• Reference Germplasm Sets
• Standardized phenotyping
at multiple locations,
esp. for fruit quality
(www.rosbreed.org)
• Evaluations done for 2 – 3
years
• Available at Genome
Database for Rosaceae
(www.rosaceae.org/breeders_toolbox)
Genome Wide SNP arrays
• Genome-scanning SNP arrays developed and utilized for apple (9K), peach (9K)
and cherry (6K) by international RosBREED-led efforts
The statistical analyses must take advantage of
the family structure in the breeding program.
FlexQTL™ Statistical Software:
HiDRAS: European
Apple Project
Capabilities: Identify and quantify QTLs in different genetic backgrounds
(~allele mining)
Strategy: Ties together many segregating crosses through the common
ancestors in the pedigree utilizing the Identity by Descent concept.
What’s Required for QTL Allele Validation?
• A closer examination of the validated QTL
in the breeding germplasm, to quantify,
describe, and visualize:
- number of alleles present
- effect of each allele (or genotype, preferably)
- frequency of alleles
- origins of alleles
- distribution of alleles in important breeding
parents and other potential parents
Allele Validation
Mendelian/major
trait loci (MTLs)
Genes with allelic variation in
available germplasm
that explain/predict most or all
of the phenotypic variation
Quantitative
trait loci (QTLs)
…that explain/predict some
of the phenotypic variation
Useful for enriching for
superior alleles
Still valuable!!
mm
trait level
MM,
Mm
frequency
frequency
Definitive
qq
trait level
QQ,
Qq
QTL Validation
- confirming the FW_G2 QTL really exists
in cherry breeding germplasm
Example of a Valuable QTL Discovered:
FW_G2 for fruit size (Zhang et al. 2010)
• Trait has value to stakeholders
• QTL explains a significant amount of
trait variation
• Achieving the desired phenotype with
breeding is very difficult
Breeding-Friendly DNA Markers Used
2 SSRs
• CPSCT038
• BPPCT034
These 2 SSRs defined 3 functional alleles for
FW_G2 in the bi-parental cross where the
QTL was discovered
Validation: Sweet Cherry Example
Utility assessment on
fruiting seedlings
EmpressEugenie
F_Van
Van
EmperiorFrancis
Lambert
Stella
Newstar
Sweetheart
PopSw-17
Pop-SwxSw-4
22 populations (219 seedlings), 0 cultivars
J12420
Beaulieu
Lapins
PopSwxAm-11
F_Bing
Kordia
Bing
Selah
PopSwxCh-3
PopSwxPM-4
EarlyBurlat
PMR-1
PopLaxAm-26
PopSwxRe-51
Regina
PopLaxCh-41
PopSwxTi-5
Dzherlo
Linda
PopGoxDz-10
Rube
Chelan
PopLaxLa-4
Gold
PopKo-8
Schneiders
Ambrunes
Kiona
Napoleon
PopSwxKi-1
PopLi-3
PopLaxRe-4
Pop-SexSe-7
PopKa-2
Rainier
Tieton
PopBi-2
Cashmere
Pop-LaxTi-11
PopSexPM-1
Katalin
PopRaxLa-1
PopSexAm-1
P8-79
PopLaxPM-3
PopSexVa-6
PopSe-16
Simple Validation and Functional Genotype Effects
for the Sweet Cherry Success Story
Phenotypic data collection
www.rosbreed.org/resources/fruit-evaluation
Simple Validation and Functional Genotype Effects
for the Sweet Cherry Success Story
Ran the DNA tests (2 flanking SSRs)
The previously identified G2 fruit
weight QTL was significant
Zhang G, Sebolt AM, Sooriyapathirana SS, Wang D, Bink MCAM, Olmstead JW, Iezzoni AF (2009). Tree Genetics & Genomes 6:1614-1642
Marker polymorphism
Sweet Cherry FW_G2 Example
QTL
FW_G2
Markers
CPSCT038 (SSR)
BPPCT034 (SSR)
No. Alleles
7
4
Alleles in important breeding parents
BPPCT034
223
common
Lambert
225 235
rare
NY54
Cristobalina
Windsor
common
Bing
237
250
rare
Glacier
Tieton
Kiona
Burlat
rare
Schmidt
Ulster
255
common
Napoleon
257
rare
Schneiders
QTL Validation - Summary
• The G2 QTL for fruit size was identified in
sweet cherry breeding germplasm
• The DNA test using the flanking SSR
markers was still associated with the trait
• Origin and distribution of alleles in
important breeding parents were
determined
QTL Allele Validation
- detecting and valuing FW_G2 alleles
in cherry breeding germplasm
Inheritance of functional alleles in pedigreelinked germplasm
Regina × Lapins
progeny classes
Mean fruit size for each
progeny class (g)
Simple Validation and Functional Genotype Effects
for the Sweet Cherry Success Story
Haplotyped and diplotyped every individual
Validation: Sweet Cherry Example
Alleles
Genotypes
Mean fruit wieght (g)
10
9
a
a
8
ab
b
bc
b
b
abc
b abc
34
25
abc ab
a
c
7
bc
6
5
4
77
92 108 11
2
41
15
4
5
2
6
39
4
225 235 223 255 237
BPPCT034 allele
225 223 223 223 235 235 235 225 237 255
235 235 223 255 255 235 237 255 255 255
BPPCT034 genotype
Number of
seedlings
Simple Validation and Functional Genotype Effects
for the Sweet Cherry Success Story
Calculated functional genotype effects
probability
BL
BN
AB
AA
AL
fruit size
probability
0
g
BN
AA
AB
AL
BL
firmness
g/mm
Functional Allele Distribution
AB
Empress
Eugenie
Napoleon
AC
Lambert
BO
AB
Emperor
Francis
JI2420
AH
?
AB
Van
Schmidt
Black Republican
?
AL
AO
Amim
Regina
Windsor
MIM17
AA
AB
AC
Sam
Bing
Gil-Peck
BN
NY54
AE
AB
AH
Early
Burlat
Stella
AB
Ulster
Beaulieu
AA
Rainier
Summit
?
Glacier
AE
Tieton
AC
PC7147-4
PC7146-11
Vic
Chinook
Kiona
PMR-1
P8-79
Brooks
AC
AA
AC
Selah
GG
AA AB’ AF AA
DD
Newstar
AE
AF
Benton
Chelan
Cashmere
AB
What crosses
to make?
AA
AE
AA
EE
BB
CC
AB
AP
Cowiche
Lapins
AB
Sweetheart
maternal parent
pollen parent
“How To” for Rosaceae Breeders
Cherry fruit
size
Functional Alleles for FW_G2 in the
Michigan Sour Cherry Breeding Program
Pandy38
UjfehertoiF
NY-6935
Montmorency
RhSchatten
ErdiBotermo
M172
EnglaiseTimp
Surefire
25-02-29
25-14-20
RSxET
UFxSurfire
25x25
M172x25
Montx25
N = 79
N = 36
N = 72
N = 57
N = 22
Phenotypic data –
standardized phenotyping
• Standardized phenotyping protocols can
be found at http://www.rosbreed.org/resources/fruit-evaluation
FW_G2 in tetraploid sour cherry
• 17 alleles for the G2 QTL
region were identified in sour
cherry using SNP markers
• 17 alleles likely an overestimation of the number of
functional SNP alleles in the
sour cherry breeding materials
Gbrowse view of the peach sequence for
the G2 fruit size QTL region
Tomato: fw2.2
A fruit size gene was
discovered in tomato that
is a regulator of cell
division
Tomato and cherry
fruit are both
enlarged ovaries
Breeding-Friendly DNA Markers Used
3 SSRs
• CPSCT038
• SSR linked to the Cell Number Regulator
candidate gene: PavCNR12 & PceCNR12
• BPPCT034
7 sour cherry G2 SSR-flanked QTL haplotypes
hypothesized instead of 17 based on new SSR data
RB_S_2_14926622
CPSCT038
RB_S_2_15084429
RB_S_2_15127760
RB_S_2_15129278
RB_S_2_15162260
RB_S_2_15172649
RB_S_2_15337787
RB_S_2_15372418
RB_S_2_15492297
RB_S_2_15598480
CNR
RB_S_2_15658996
LG2+18K_SSR
RB_S_2_15747822
RB_S_2_15778222
RB_2_15846482
RB_T_2_15863936
RB_S_2_15873315
RB_S_2_15873418
RB_S_2_15894385
RB_S_2_15894441
RB_T_2_16005866
RB_T_2_16111179
RB_S_2_16118423
RB_S_2_16142700
RB_S_2_16229065
BPPCT034
d
A
204
A
A
B
B
B
B
B
B
B
b
A
185
A
B
A
A
A
A
B
B
A
l
A
185
A
B
A
A
A
A
B
B
A
e
A
190
A
B
A
A
A
A
B
B
A
h
A
190
A
B
A
A
A
A
B
B
A
o
A
190
A
B
A
A
A
B
B
B
A
k
A
192
A
B
A
A
A
B
B
B
A
m
A
null
A
B
A
A
A
B
B
B
A
a
A
185
A
B
A
A
A
A
B
B
A
q
A
192
A
B
A
A
A
A
B
B
A
c
A
192
A
B
A
B
B
A
B
A
B
g
A
192
B
A
B
B
B
A
B
A
B
p
A
190
B
A
B
B
B
B
B
A
B
j
A
192
B
A
B
B
B
B
B
A
B
n
f
i
A B A
192 190 null
B B A
A A A
B B B
B B B
B B B
B B B
B A B
A B B
B B B
A
B
B
B
B
235
B
A
A
A
A
218
B A
A B
A B
A A
A B
241 228
A
A
A
A
A
206
A
A
A
A
A
206
A
A
A
A
A
206
A
A
A
A
A
206
B
A
A
A
A
210
A A A A A A A A
A B B B B B B B
A B B B B B A B
A A A A A A B A
A B B B B B B B
235 237 237 225 237 237 255 228
7 sour cherry
CNR alleles (bp)
– 2 (250)*
– 4 (210)
– 5 (212)
B A A B B B B B A A B B B B B B B
250 210 210 210 210 210 210 212 235 239 239 239 225 225 225 228 228
– 6 (235)
A A A A A A A A A A A A B B A B A
A B B A B B B B B B A A A A A A A
B B B B B B B B B B B B B B B B B
–
7
(239)
B A A B A A A A A A B B B B B B B
B A A B A A A A A A B B B B B B B
– 8 (225)
B A A B A A A A A A B B B A B B B
B A A A A A A A A A B B B A B B A
A B B A B B B B B B A A A A A A A
– 9 (228)
* Same as in sweet
cherry based on other
marker data
Sour cherry - putative PavCNR12 &
PcrCNR12 alleles
Mean fruit weights based on the presence
or absence of putative PavCNR12 &
PcrCNR12 alleles (n=274)
2/no 2
8/no 8
7+8/no 7 or 8
Number of progeny
128/146
139/135
56/56
Mean fruit weight (g)
5.64/5.30
5.11/5.81
4.66/5.74
0.0700
0.0002
0.0004
P-value
FW-G2 exists in sweet cherry and sour
cherry breeding germplasm!
• In sweet cherry, 9 ancestral haplotypes for
the G2 region were identified
• In sour cherry, 8 ancestral haplotypes for
the G2 region were identified
Other Jewels for Cherry
Other cherry “Jewels” available now!
JUN
Bloom Time
JUL
Maturity Date
Self-fertility
Flesh Color
Disease Resistance
Fruit Size
Firmness
Acidity
Conclusion
RosBREED has and continues to provide DNA tests
for valuable traits that have been challenging to
plant breeders’ efficiency
Sour cherry breeding program
• Prior to RosBREED, I had “no clue” about the
inheritance of any fruit quality or disease
resistance trait in sour cherry.
• Now I not only have an understanding of trait
inheritance, but I have DNA markers for parent
selection, cross design and seedling selection.
• The end result is increased breeding efficiency!
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