Systematically integrating DNA information into breeding: The MAB Pipeline, case studies in and cherry Amy Iezzoni January 31, 2013 Cornell MSU Susan Brown Amy Iezzoni (PD) Kenong Xu Jim Hancock Dechun Wang Clemson Cholani Weebadde Ksenija Gasic Gregory Reighard Univ. of Arkansas John Clark WSU Texas A&M USDA-ARS Dave Byrne Cameron Peace Nahla Bassil Dorrie Main Univ. of Minnesota Gennaro Fazio Univ. of CA-Davis Kate Evans Chad Finn Karina Gallardo Jim Luby Tom Gradziel Vicki McCracken Chengyan Yue Plant Research Intl, Carlos Crisosto Nnadozie Oraguzie Netherlands Oregon State Univ. Eric van de Weg Univ. of New Hamp. Alexandra Stone Marco Bink Tom Davis Outline of Presentation

 The MAB Pipeline

 Apple skin color

 Cherry flesh color The MAB Pipeline “Jewels in the Genome” - discovering, polishing, applying

QTL MAB Pipelining Breeding (looks promising...) (polishing...) (assembling into masterpieces) Socio-Economics Surveys (example for apple) Washington Michigan Market Breeders Producers Producers Intermediaries

Fruit flavor 43 41 23 Fruit crispness 15 23 10 Exterior color 26 Fruit firmness 6 7 5 Shelf life at retail 7 7 3 Sweetness/soluble solids 6 7 3 Sugar/acid balance 9 7 External appearance 13 No storage disorders 7 4 Disease resistance 2 5 Storage life 5 Other fruit quality…2 3 Size 3 Juiciness 2 Tartness Shape Phytonutrient Aroma % of respondents020406080100 Reference Germplasm

McIntosh LivelRasp Jolana Williams F_Spartan PRI14-126 Starr OR38T610 F_Williams NJ53 PRI14-226 Minnesota Delicious KidsOrRed PRI14-644 Cox Sonora NJ8 GoldenDel F1_9433-2-2 PRI612-1 NJ24 RomBeauty NJ67634 F1_9433-2-8 F_Ill_#2 Mflor821 P21R4A30 F2_26829-2-2 NY65707-19 Keepsake Ill_#2 F_P21R4A30 M_MN1764 Prima NJ123249 BP Set Frostbite F_Honeycrisp NJ27 NJ110037 NorthSpy Sonya JerseyBla PRI668-100 Macoun Julyred F_Gingergold sibLiberty NY752 F_JamesGr PRI54-12 Jerseymac GingerGold MN1691 (n=160) NY913 F_IngMarie JamesGr F_Delfloki NJ37848 Tetofsky NJ130o PRI47-147 YelTransp IngMarie NJ117637 Longfield PRI610-2 NJ31 NJ88 Raritan DuchOlden NJ12 Monroe Rubin F_PattenGr Mantet Edgewood PRI1661-1 Vanda PRI855-102 WorcPearm Oriole RallsJan Sharon NY88 WSU2 Mavis Fireside PRI1661-2 PattenGr StateFair Chinook NY241 BenDavis PRI1018-101 F_Goodland PacQueen Goodland Close M_MolliesDel PacRose PRI643-11 NJ14 AurGoldGa NJ440249 Zestar Jongrimes NJ19237 BC-8S-27-2 Crandall Coop-17 Redfree Wagener M2439 BC-8S-31-56 FrancRus NJ35437 BC-SPA493 PCFW2-134 LadyWill CrownPR Lambourne P7R25A27 NJ136055 BlushiGol F_Undine NJ15 Suncrisp PRI669-205 PRI2482-100 Grifer Malinda NJ303955 CROP Glockenapfel Zari MolliesDel DrOldenbu Bellida PRI1235-100 F_Helios Any breeder’s NJ35934 XIIA277 F_EdGoulGol Civni BREEDING F_NJ15 Washington F_Sandow KAZ95-17-14 Ecolette Coop-15 Lundbytrop NJ109 F_MolliesDel Milwa A28/39 KAZ95-18-07 A849-5 PRI1469-100 BeautBath Delcorf KAZ96-05-04 Nicoter Rubinola VKrakers REFERENCE Goldrush Galmac NZ_RGxBB additional BelHerm Pilot Maigold Priscilla PEDIGREE PRI2059-101 BP SetLaFlam Rubinstep F_Brina Nevson Sundowner Scarlet CrippsPink Senshu Sansa Rucla Coop-10 NJ381049 Delblush StarkEarl Scifresh Undine PitmPine Linda (n=160)Scilate Delfloki germplasm X4598 Orin MN1764 SETS Shizuka WSU24 X3174 Toko SET AnanasRei MN1702 WSU37 F_MN1764 Camuzat AutGold GMAL4591 WSU17 BaumanRei Monark BernRos Telamon GMAL4593 M_Sunrise WSU36 Boskoop Delorina GMAL4595 with data BrixPlatt PCF-3-120 Sunrise Calamari Hatsuaki CIVG198 Jonafree WSU38 Calvilla Pop_BBxPR Arlet Dalitron CrimsSnow M_NY632 Croncels Gloster Ped-PinxCam NY632 n=480 DallaRosa PRI69-52 ShinGold 4-27-0019 EarlyGold Pop_GaxC15 Edelb WSU34 Ped-SonxNY241 Helios 5-87-0019 Fiamma 52-05-26 Apollo 25-120-9928 Freedom 5-90-0019 GelEdel Zonga Pop_HCxCP Gewurz Akagi Ped-HCxPit(692) 6-73-0019 Hampshire EdGoulGol Piros Santana HarbRei PRI1956-6 Ped_HCx1764 MN1914 Limoncini Fortune MPop_HCx1702 4A-100-0013 Magre SchonNord Shinsekai MN1628 MPop_HCxMon Ped-HCxZest GranSmith 4A-38-0013 Napoleone Silken Delgollune GoldenOr Ped-GoldrxNY913 Ontario PermainDor Dolgo Merlijn Pop_AurxHC Pristine RedFiled Angold NY75414-1 New York Sabina RenCanada Ariwa Tunda RenChamp CrimsCrisp Discovery WSU46 Robusta5 Belgica WSU35 WSU22 RosBian B_TxBB Rafzubin 4-159-9926 RosdiCal GoldPink RosDop Nicogreen Pop_ArlxCP Ped_HCxJonf RotStett WSU30 BP Set N_PxF Saturn SanLugano Pop_GDxSc Delcoros Sila E_FxGD Delearly Snowsweet NJ90 E_FxGlo TavBian Ligol TirSpitz Kerr Murray Dalinette (n=160) WeisWint SNP Genome Scans

Illumina Infinium® II SNP arrays: developed 2010, used 2011

9K 8K 6K

+1K Verde et al. 2012 Chagne et al. 2012 Peace et al. 2012 PLoS One PLoS One PLoS One Standardized Phenotyping

• Reference Germplasm Sets Public: 480 individuals Additional: +480 individuals

• Standardized phenotyping at multiple locations, esp. for fruit quality (www.rosbreed.org)

• Evaluations 2010 - 2012

• Available at Genome Database for Rosaceae (www.rosaceae.org/breeders_toolbox) Pedigree-Based Analysis

Pedimap FlexQTLTM for visualizing pedigrees for QTLdiscovery, (with alleles, traits, validation, characterization Identity-By-Descent)

RallsJan Delicious W inesap RomBeauty Jonathan M_PRI668-100 GoldenDel F2_26829-2-2

Fuji Crandall PRI14-126

PRI668-100

X-2771

X-3143

X-3318

Flow of a specific linkage block Fruit firmness in apple (HiDRAS data) Jewel Discovery

SNP genome scans Pedigree‐ Based QTL discovery Analysis Standardized phenotypic data Systematic QTL Validation – The MAB Pipeline

Socio-economics information Breeding markers DNA

information PBA: crop-wide

PBA: breeding Efficient, program precise, Supported by creative RosBREED’s in-development breeding Breeding Information Management System MAB Successes So Far Two jewels polished in RosBREED, put to routine use in demo breeding programs

marker‐assisted parent selection

for favorable alleles marker‐assisted seedling selection New generations enriched Routine MAB with QTLs

Improved efficiency in seedling evaluation since fall 2010 in RosBREED breeding programs

Thousands of inferior seedlings culled - est. $180K saved! Predicted to be (WSU’s apple and sweet cherry breeding programs) large-fruited (& self-fertile) Routine Use of QTLs in Breeding

allele 1 allele 2 carriers carriers  frequency

trait level 

Use of QTLs: Selecting for favorable QTL alleles enriches for favorable alleles and therefore results in a shift in progeny mean. RosBREED: Filling the Treasure Chest for Rosaceae Breeders Skin Color in Apple: Rf Rf

• Alleles = MYB1 promoter 0.00 Mb variations, simple PCR

• Alleles 2,3 = non-blushed

• Alleles 1, 4 = blushed

• Allele 5 = red-fleshed

29.48 Mb Rf = Md-MYB1/MYB10 33.72 Mb can be deduced from published information Rf

• Alleles = SNP haplotypes 0.00 Mb from 8K genome scan

• Allele “rf” = non-blushed

• Allele “Rf” = blushed, dominant

29.48 Mb Rf = Md-MYB1/MYB10 33.72 Mb no. individuals Rf - Predictions

“ PercOvrClr” = “% red / overcolor color of skin”

Rf

rfrf Rfrf RfRf

1 2 3 4 5 no <25% 25-50% 50-75% >75% Rf – Functional Genotypes

“ PercOvrClr” = “% red / overcolor color of skin” Rf

rfrf , ,

Honeycrisp, Gala, Zestar, Pacific Rose, Braeburn, Pinova, Rfrf McIntosh, , Splendour, Cameo, RfRfJonathan, Jonamac, Empire, Scired Apple: Skin Coloration Allele & Genotype frequencies in 192 cvs

Cover amount over rf rfrf apple skin surface Rf RfRf

Rfrf probability 1000 seedlings – how many with score >4? Rfrf x RfRf ---> 647 RfRf x RfRf ---> 818

Marker(s)? 1-3 SNPs from 8K array genome scan Application? Parents: crossing Selections: distinguish Rfrf / RfRf Peach: Maturity Date One trait locus, two ways of genotyping

Three 2-SNP haplotypes Two SSR alleles (AA,GA,AG) in four combinations (168,156) in two combinations GA:AA AA:AA AA:AG GA:AG 168:168 156:168

-40 -20 20 +20 +40 -40 -20 20 +20 +40 July days July days

Marker(s)? 2 SNPs from 9K array ...or ... 1 SSR Application? Parents: crossing Seedlings: culling Selections: advance What does the raw SNP data look like?

(haplotypes = linked alleles that are used to distinguish the different chromosome segments of interest) Ma locus: Most Probable Haplotype Alleles by IBD

Courtesy of Sujeet Verma

Sujeet Verma @ DC Workshop, 20 Flesh Color in Tart Cherry Flesh color in tetraploid tart cherry

Travis Stegmeir & Umesh Rosyara

• BACKGROUND – the Myb10 locus controls flesh color in sweet cherry.

• Objectives – Determine if the Myb10 locus controls flesh color in tetraploid tart cherry. If so, can DNA information be used to predict color? Flesh Color (visual rating)

• Using color cards, an 1 overall flesh color is given. 2

3

4

5

WSU flesh color index card bcde f ghj kl nopu RB_S_3_09729116 BBBBBBBBBBABBB 14 Haplotypes RB_T_3_09782875 A A B A A B B A A B A B A A RB_S_3_10022424 B B B B B A A B B B B B A B RB_S_3_10105783 A B B A A B B B A B A B B B RB_S_3_10162979 BAAAAAAAAAAAAA identified in the RB_S_3_10264563 A A B A A B B A A B A B B A RB_S_3_10573974 ABBAAABBABABBB RB_T_3_10590166 A B B A A B B B A B A B B B RB_S_3_10626205 A B B A A B B B A B B B B B RB_S_3_10675150 A B B A A B B B A B A B A B RosBREED tart RB_S_3_10822211 A B B A A B B B A B A B A B RB_T_3_10908880 A B B A A B B B A B A B B B RB_T_3_12115409 A B B A A B B B A B A A B B RB_S_3_12383977 B A A B B A A A B A B A A B RB_S_3_12474678 BBBBBBBBBBABBB cherry germplasm RB_S_3_12500413 A B B A A B B B A B B B B B RB_T_3_12503462 A B B A A B B B A B A B B B RB_T_3_12539794 B B B A A B B B A B B B B B LG3_12.71Mb 3125512121221 RB_S_3_12944437 A B B A A B B B A B A B B B RB_S_3_12987920 B A B B B A A A B B B A A A RB_S_3_13025963 AAAAABBAABABAB RB_T_3_13063792 A B B A A B B B A B A B B B RB_S_3_13144730 BBBBBAABBBBABA RB_S_3_13208005 B B A B B A A B B A B A B A RB_T_3_13369328 AAAAAAAAAABAAA RB_S_3_13406263 ABBABAABBABABA RB_S_3_13433848 BABBBAAABABABA Region used to make RB_S_3_13466702 B B B B B A A B B A B A A A RB_S_3_13520194 BABBBAAABABBBA RB_S_3_13563908 A B B A A B B B A B A B B B haplotype call when RB_S_3_13567593 A B B A A B B B A B A A B B RB_S_3_13724726 A B B A A B B B A B A B B B RB_S_3_13754793 B A A B B A A A B A B A B A crossover took place. RB_S_3_13795019 B A A B B A A A B A B A A A RC3766‐391_3_13878008 AABAAABAAAAAAB RB_T_3_13881088 AABAAABAAAAAAB RB_S_3_14024780 A B A A A A A B B B B A A A It spans the predicted RB_S_3_14146853 BBBBBBBBABABBB RB_S_3_14316165 B A A B B A A A B A B B A A location of Myb10 RB_T_3_14442011 BABBBAAABABAAA RB_S_3_14521488 B A A B B A A A B A B A A A RB_S_3_14599590 BABBBAAABABBAA RB_T_3_15171728 A B B A A B B B B B B B B B RB_T_3_15305145 B A A B B A A A B A B A A A RB_S_3_15309954 A B B A A B A B A B A B B A RB_S_3_15357433 BAABBBAABABBAA RB_S_3_15455662 A B B A A B A B A B A A B A 4 haplotypes confer darker flesh color, 10 confer lighter color

• “Dark flesh” haplotypes: d, p, l, e • “Light flesh” haplotypes: b, c, f, g, h, j, k, n, o, u

Color differences among haplotypes were tested using ANOVA (Thanks Umesh!) “Dark flesh” haplotypes not created equal • d and p haplotypes tend to give rise to darker purple flesh (~4-5 on the color scale) • e and l tend to give a more moderate flesh color (~3 on the color scale)

d p + ‐ + ‐ +4.77 a3.00 b +4.22 a3.18 b e l ‐ 4.54 a1.91 c ‐ 3.61 c1.32 d Balaton x Surefire M172 x 25-02-29 Balaton x Surefire flesh distributions

B x S no "d" or "e" B x S only "e" (no "d") 10 7 9 6 8 7 5 6 4 5 4 3 3 2 2 1 1 0 0 12345 12345 B x S only "d" (no "e") B x S both "d" and "e" 10 12 9 10 8 7 8 6 5 6 4 4 3 2 2 1 0 0 12345 12345 M172 x 25-02-29 flesh distribution M172 x 25 no "p" or "l" M172 x 25 only "l" (no "p") 18 7

16 6 14 5 12 10 4 8 3 6 2 4 2 1 0 0 12345 12345

M172 x 25 only "p" (no "l") M172 x 25 both "p" and "l" 7 12

6 10 5 8 4 6 3 4 2

1 2

0 0 12345 12345 MASS for flesh color

• If dark colored flesh is desired, select seedlings possessing the dark flesh haplotypes d, p, l and e • If light colored flesh is desired, select seedings not possessing the dark flesh haplotypes • If red flesh is desired, the more moderate e and l color haplotypes could be selected bcde f ghj kl nopu RB_S_3_09729116 BBBBBBBBBBABBB RB_T_3_09782875 A A B A A B B A A B A B A A RB_S_3_10022424 B B B B B A A B B B B B A B RB_S_3_10105783 A B B A A B B B A B A B B B RB_S_3_10162979 BAAAAAAAAAAAAA Designing an RB_S_3_10264563 A A B A A B B A A B A B B A RB_S_3_10573974 ABBAAABBABABBB RB_T_3_10590166 A B B A A B B B A B A B B B RB_S_3_10626205 A B B A A B B B A B B B B B efficient marker RB_S_3_10675150 A B B A A B B B A B A B A B RB_S_3_10822211 A B B A A B B B A B A B A B RB_T_3_10908880 A B B A A B B B A B A B B B RB_T_3_12115409 A B B A A B B B A B A A B B RB_S_3_12383977 B A A B B A A A B A B A A B system to distinguish RB_S_3_12474678 BBBBBBBBBBABBB RB_S_3_12500413 A B B A A B B B A B B B B B RB_T_3_12503462 A B B A A B B B A B A B B B RB_T_3_12539794 B B B A A B B B A B B B B B the functional LG3_12.71Mb 3125512121221 RB_S_3_12944437 A B B A A B B B A B A B B B RB_S_3_12987920 B A B B B A A A B B B A A A RB_S_3_13025963 AAAAABBAABABAB RB_T_3_13063792 A B B A A B B B A B A B B B haplotypes. RB_S_3_13144730 BBBBBAABBBBABA RB_S_3_13208005 B B A B B A A B B A B A B A RB_T_3_13369328 AAAAAAAAAABAAA RB_S_3_13406263 ABBABAABBABABA RB_S_3_13433848 BABBBAAABABABA RB_S_3_13466702 B B B B B A A B B A B A A A RB_S_3_13520194 BABBBAAABABBBA In tetraploid tart cherry we have found that RB_S_3_13563908 A B B A A B B B A B A B B B RB_S_3_13567593 A B B A A B B B A B A A B B the haplotypes are easier to genotype if an RB_S_3_13724726 A B B A A B B B A B A B B B RB_S_3_13754793 B A A B B A A A B A B A B A SSR is used as the SSRs allow the ability to RB_S_3_13795019 B A A B B A A A B A B A A A RC3766‐391_3_13878008 AABAAABAAAAAAB RB_T_3_13881088 AABAAABAAAAAAB distinguish multiple alleles with one marker. RB_S_3_14024780 A B A A A A A B B B B A A A RB_S_3_14146853 BBBBBBBBABABBB RB_S_3_14316165 B A A B B A A A B A B B A A RB_T_3_14442011 BABBBAAABABAAA RB_S_3_14521488 B A A B B A A A B A B A A A RB_S_3_14599590 BABBBAAABABBAA RB_T_3_15171728 A B B A A B B B B B B B B B RB_T_3_15305145 B A A B B A A A B A B A A A RB_S_3_15309954 A B B A A B A B A B A B B A RB_S_3_15357433 BAABBBAABABBAA RB_S_3_15455662 A B B A A B A B A B A A B A Go to G-browse in GDR • Identify your region of interest and then identify SSR containing transcripts Systematic QTL Validation – The MAB Pipeline

Socio-economics information Breeding markers DNA information PBA: crop-wide

PBA: breeding Efficient, program accurate, creative breeding Take Home Message MAB Pipelining can begin in strawberry!

• QTLs are available to validate • Phenotypic data is available for breeding germplasm • Genome wide genetic data will soon be available • Build haplotypes for QTL regions of interest and determine if the different haplotypes are associated with differences in phenotype Questions?