Post on 22-Dec-2015
QTL and QTL allele validation in cherry
Amy Iezzoni Cameron Peace, Audrey Sebolt, Nnadozie Oraguzie,
Umesh Rosyara, Travis Stegmeir
25 July 2013ASHS Palm Desert, CA
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
Outline of Presentation
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
Schneiders
Rube
Empress Eugenie
Unknown
Lambert
EF
Napoleon
Bopparder Kracher
Unknown
V-160140
Unknown
Drogana Zholtaya
Valeriy Chkalov
NY
Regina
Van
JI2420
Namati
Sam
Krupno.
NY x EF
Stella
Summit
Namati x Krupno.
Lapins
Namati x Summit
Regina x Lapins
(n= 101)
(n= 76)
(n= 80)
(n= 167)
Fruit weight
Data not available
Pedimap software
RosBREED
Standardized Phenotyping
• Reference Germplasm Sets
• Standardized phenotypingat 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-scanning SNP arrays developed and utilized for apple (9K), peach (9K) and cherry (6K) by international RosBREED-led efforts
Genome Wide SNP arrays
The statistical analyses must take advantage of the family structure in the breeding program.
HiDRAS: European Apple Project
FlexQTL™ Statistical Software:
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 Quantitative trait loci (MTLs) trait loci (QTLs)
Genes with allelic variation in available germplasm
that explain/predict most or all of the phenotypic variation
…that explain/predict some of the phenotypic variation
frequ
ency
trait level
mmMM, Mm
frequ
ency
trait level
qqQQ, Qq
Useful for enriching for superior allelesStill valuable!!Definitive
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
Utility assessment on fruiting seedlings 22 populations (219 seedlings), 0 cultivars
Validation: Sweet Cherry Example
EmpressEugenie F_Van EmperiorFrancis Napoleon F_Bing Kordia Gold Dzherlo Linda Katalin
Van Lambert J12420 Bing PopKo-8 PopGoxDz-10 PopLi-3 PopKa-2
Stella Beaulieu Schneiders Rube EarlyBurlat Rainier PopBi-2
Newstar Lapins Ambrunes Chelan PMR-1 Regina Tieton Cashmere P8-79
Sweetheart Kiona Selah PopLaxLa-4 PopLaxAm-26 PopLaxCh-41 PopLaxRe-4 Pop-LaxTi-11 PopRaxLa-1 PopLaxPM-3
PopSw-17 Pop-SwxSw-4 PopSwxAm-11 PopSwxCh-3 PopSwxPM-4 PopSwxRe-51 PopSwxTi-5 PopSwxKi-1 Pop-SexSe-7 PopSexPM-1 PopSexAm-1 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)
Zhang G, Sebolt AM, Sooriyapathirana SS, Wang D, Bink MCAM, Olmstead JW, Iezzoni AF (2009). Tree Genetics & Genomes 6:1614-1642
The previously identified G2 fruit weight QTL was significant
Marker polymorphism Sweet Cherry FW_G2 Example
BPPCT034
CPSCT038FW_G2
47
QTL Markers No. Alleles
(SSR)
(SSR)
Alleles in important breeding parents
BPPCT034
223 225 235 237 255 257250commonLambert
rareNY54
Cristobalina
Windsor
commonBing
rareGlacierTietonKionaBurlat
commonNapoleon
rareSchneiders
rareSchmidtUlster
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 pedigree-linked 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
Genotypes
4
5
6
7
8
9
10
Mea
n fr
uit w
iegh
t(g)
225 223235 255 237
1110892774
aa
bbab
BPPCT034 allele
225235
223235
223223
223255
235255
235237235
235225255
237255
255255
396254253415412
aababc
abcabcbbbc
c
bc
BPPCT034 genotype
Number of seedlings
Alleles
Validation: Sweet Cherry Example
0
Simple Validation and Functional Genotype Effects
for the Sweet Cherry Success Story
Calculated functional genotype effects
fruit sizeBN AB AA ALBL
firmnessBNAA AB AL BL
prob
abili
typr
obab
ility
g
g/mm
Functional Allele DistributionEmpress Eugenie ?
? Regina
?
Beaulieu
Emperor Francis
JI2420Schmidt
Early Burlat
Ulster
Lambert Sam
Stella PMR-1
GlacierTieton
Cashmere
PC7147-4 PC7146-11
Chelan
GG
KionaCowiche
NapoleonBlack Republican
VanBing
Rainier ChinookVic
Summit
Lapins
Newstar
Brooks
DD EE BB CC
Sweetheart
P8-79
Selah
Benton
Windsor MIM17
maternal parent pollen parent
ABAB
AB AB
AB
AA
ABAA AF
AA
AA AB’
AC
AA
AB
AA
AC
AB
AB
AH
AH
AC
AP
AE
AE AE
AE
AA
AC
AF
AC
BOAL AO Amim
Gil-Peck
NY54
BN
What crosses to make?
RosBREED
Cherry fruit size
“How To” for Rosaceae Breeders
Functional Alleles for FW_G2 in theMichigan Sour Cherry Breeding Program
Pandy38 Montmorency
UjfehertoiF NY-6935 RhSchatten ErdiBotermo M172 EnglaiseTimp
Surefire 25-02-29 25-14-20 RSxET
UFxSurfire 25x25 M172x25 Montx25
N = 72 N = 57 N = 79 N = 36
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 over-estimation 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
Tomato and cherry fruit are both
enlarged ovaries
A fruit size gene was discovered in tomato that
is a regulator of cell division
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
7 sour cherry CNR alleles (bp)
– 2 (250)*– 4 (210)– 5 (212)– 6 (235)– 7 (239)– 8 (225)– 9 (228)* Same as in sweet cherry based on other marker data
d b l e h o k m a q c g p j n f iRB_S_2_14926622 A A A A A A A A A A A A A A A B A
CPSCT038 204 185 185 190 190 190 192 null 185 192 192 192 190 192 192 190 nullRB_S_2_15084429 A A A A A A A A A A A B B B B B ARB_S_2_15127760 A B B B B B B B B B B A A A A A ARB_S_2_15129278 B A A A A A A A A A A B B B B B BRB_S_2_15162260 B A A A A A A A A A B B B B B B BRB_S_2_15172649 B A A A A A A A A A B B B B B B BRB_S_2_15337787 B A A A A B B B A A A A B B B B BRB_S_2_15372418 B B B B B B B B B B B B B B B A BRB_S_2_15492297 B B B B B B B B B B A A A A A B BRB_S_2_15598480 B A A A A A A A A A B B B B B B B
CNR RB_S_2_15658996 B A A B B B B B A A B B B B B B B
LG2+18K_SSR 250 210 210 210 210 210 210 212 235 239 239 239 225 225 225 228 228RB_S_2_15747822 A A A A A A A A A A A A B B A B ARB_S_2_15778222 A B B A B B B B B B A A A A A A A
RB_2_15846482 B B B B B B B B B B B B B B B B BRB_T_2_15863936 B A A B A A A A A A B B B B B B BRB_S_2_15873315 B A A B A A A A A A B B B B B B BRB_S_2_15873418 B A A B A A A A A A B B B A B B BRB_S_2_15894385 B A A A A A A A A A B B B A B B ARB_S_2_15894441 A B B A B B B B B B A A A A A A ARB_T_2_16005866 A B B A A A A A B A A A A A A A ARB_T_2_16111179 B A A B A A A A A A B B B B B B BRB_S_2_16118423 B A A B A A A A A A B B B B B A BRB_S_2_16142700 B A A A A A A A A A A A A A A B ARB_S_2_16229065 B A A B A A A A A A B B B B B B B
BPPCT034 235 218 241 228 206 206 206 206 210 235 237 237 225 237 237 255 228
Sour cherry - putative PavCNR12 & PcrCNR12 alleles
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
P-value 0.0700 0.0002 0.0004
Mean fruit weights based on the presence or absence of putative PavCNR12 &
PcrCNR12 alleles (n=274)
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 JUL
Bloom Time Maturity Date
Self-fertilityFlesh Color
Disease Resistance
Fruit Size Firmness Acidity
ConclusionRosBREED 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
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