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