FR-H3: A New QTL to Assist in the Development of Fall-Sown Barley with Superior Low Temperature Tolerance

Outline Introduction Germplasm Linkage Maps Phenotyping & QTL Analysis Conclusions and Implications

Introduction

Fall-Sown Malting Barley New to the U.S. Two varieties approved by AMBA ◦Both two-row, lack LTT 49% malting barley planted has been six- row over the past 5 years Most selection has been done within a spring growth habit framework Advantages ◦Higher Yielding ◦Conserves Water

Adaptation to Target Regions Winter Hardiness Disease Resistance

Winter Hardiness Complex Trait Vernalization Sensitivity Photoperiod Sensitivity Low Temperature Tolerance

Vernalization Sensitivity The requirement of a period of low temperature in order to transition from the vegetative to reproductive state

Photoperiod Sensitivity Flowering is dependent on sufficient day length ◦Short Day ◦Long Day

Low Temperature Tolerance The capacity to survive low temperatures Induced response Reduced after reproductive transition

Defined by LTT, VS, & PS Winter ◦Varying LTT, VS, PS varies Facultative ◦Varying LTT, No VS, PS varies Spring ◦Lack LTT and VS, Short PS irrelevant Growth Habits

Winter Hardiness: VS Winter Genotype: Vrn-H2_/vrn-H1vrn-H1/vrn-H3vrn-H3

Winter Hardiness: PS PPD-H1 ◦Recessive Allele Insensitivity to long photoperiods PPD-H2 ◦Deletion Short PS

Winter Hardiness: LTT FR-H1 ◦VRN-H1? FR-H2 ◦≥11 C-repeat binding factor genes ◦Copy number variation

Low Temperature Tolerance Studies FR-H1 first reported Dicktoo x Morex (Hayes et al. 1993) FR-H2 discovered Nure x Tremois (Francia et al. 2004) FR-H1 and FR-H2 validated through GW- AM (von Zitzewitz et al. 2011) Galiba et al Skinner et al Francia et al Stockinger et al Galiba et al. 2009

Is LTT due only to FR-H1 and FR- H2 or are there other, undiscovered determinants of this critical trait?

Resistance to Biotic Stresses Barley Stripe Rust ◦Incited by Puccinia striiformis f. sp. hordei ◦Prominent in Pacific NW  First reported in Oregon in 1995 ◦Spread by wind QTL and Genes Identifies Chen et al Hayes et al Toojinda et al Castro et al Vales et al Rossi et al. 2006

Scald ◦Incited by Rhynchosporium secalis ◦Spread by splashing rain ◦Reduce functional green leaf tissue on plant Many Genes and QTL identified Fungicides effective but more effective to deploy resistance genes Pyramiding resistance genes ◦Effective and durable Resistance to Biotic Stresses Abbott et al Garvin et al Jensen et al Cheong et al Li and Zhou 2011

Overall Scheme Two doubled haploid populations Genotyping ◦Custom 384-OPA Linkage Maps (JoinMap 4) Phenotyping ◦LTT ◦VS (FT & FLN) ◦BSR ◦Scald QTL analysis (Windows QTL Cartographer) Pooled data when appropriate

Germplasm Two doubled haploid populations (half-sibs) ◦NB3437f/OR71 (N=111) and NB713/OR71 (N=124) OR71 ◦Facultative, Moderate LTT, BSR resistant, good malting quality NB3437f ◦Facultative, High LTT, BSR susceptible, poor malting quality NB713 ◦Winter, High LTT, BSR susceptible, poor malting quality

NB3437f NB3437 bulk seed lot grown <1% flowered without vernalization VRN-H2 deletion confirmed

Genotyping Parents previously genotyped with Barley OPA 1 & 2 (Barley CAP) ◦3,072 SNPs total Custom 384-OPA ◦Barley consensus map (Close et al. 2009) ◦Maximize polymorphism, genome coverage, focus on specific regions/genes of interest  Concurrent MAS study

Linkage Mapping NB3437f/OR71 (220 SNPs) NB713/OR71 (217 SNPs) Combined population (157 SNPs)

NB3437f/OR71 3_ _ _ _ _08391_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ H2H 3H4H5Ha6H7H 5Hb 5Hc 5Hd 5He

NB713/OR71 2_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _0510 2_0974 3_0385 1_1233 2_1210 3_0889 3_0006 3_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ H2H 3H4H5Ha6H7Ha 5Hb 7Hb

Combined Population 2_ _ _ _ _ _ _ _ _0293 1_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Hb 2_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ H2H 3H4Ha5Ha6H7H 5Hd 4Hb 3_ _ Hc

Phenotyping LTT ◦Controlled Environment  MRI ◦Field Conditions  SPMN, LNE, FCCO, FMT Final Leaf Number ◦GH: V+/V- Flowering Time ◦GH: V+/V- ◦Field: COR Disease Resistance ◦BSR and Scald  COR

LTT: MRI and SPMN

LTT: LNE & FCCO

LTT QTL Results FR-H3 FR-H2 VRN-H2

5H and MRI QTL Scans

Final Leaf Number

FLN QTL Results VRN-H2 VRN-H3 VRN-H2

Flowering Time

FT QTL Results VRN-H3 VRN-H2 PPD-H2

Disease: BSR and Scald

BSR & Scald QTL Results Rps4 Rrs15 Rrs2

Combined Population QTL Analysis VRN -H3 Rps4 FR-H3 FR-H2

Conclusions and Implications

LTT: Challenges and Opportunities Assessing LTT is challenging Lowest Temperatures ◦SPMN: -27°C; LNE: -26°C; FCCO: -25°C; FMT: -32°C; MRI: -13.5°C  No Survival at FMT  Nearly complete survival FCCO Other factors Controlled Freeze Tests $$ SPMN has been informative

FR Loci Expectations FR-H1 ◦~24cM region between flanking markers  All parents monomorphic within 5 cM (n = 27 markers) FR-H2 ◦Expected QTL  Marker polymorpisms  Differences in LTT ◦Complicated by 5H fragmentation

5H Fragmentation Chromosome rearrangements ◦e.g. Inversion Copy Number ◦CBF genes related to degree of LTT (Stockinger et al. 2007) Heterozygosity in Parents ◦Genotyping of parents revealed no heterozygosity ◦Multiple plants used for crossing not all were genotyped ◦Retention of heterozygosity? High density mapping, cytogenetics

FR-H3 Discovery Genetic basis of superior LTT in NE unknown: necessary for molecular breeding Previous Reports on 1H (Hayes et al. 2003) ◦Dicktoo x Morex  Pleiotropic effect of PPD-H2  PPD-H2 distal to FR-H3 SPMN ◦Additive effect of 29% in combined population

Vernalization Sensitivity: validation and discovery Role of VRN-H2 Confirmed ◦NB3437f x OR71  All progeny flowered under V- conditions  VRN-H2 deletions ◦NB713 x OR71  Segregated for VS  VRN-H2 presence/absence

Unexpected role of VRN-H3 Variation in exotic germplasm Wong ◦Released: China 1941 ◦Pedigrees of NB3437f & NB713 NE contributed the early flowering allele VRN Model ◦Recessive vrn-H3 required for VS ◦Dominant Vrn-H3 flowers very early ◦NB713 has VS  alternative recessive allele ◦Recessive allele triggered only under long days Vernalization Sensitivity: validation and discovery

LTT and VS: uncoupled NB3437f x OR71 and NB713 x OR71 similar LTT values NB713 x OR71 ◦High survival with and without VS ◦VRN-H2 QTL was detected in MRI  VS may have delayed vegetative to reproductive transition through critical times

NB713 x OR71: Facultative Vs. Winter

BSR Resistance First report in fall-sown barley background Resistance donors ◦Kold or Strider  Winter six-rows released by OSU  Effective resistance since BSR first reported in the area in 1995 Large-effect QTL on 1H ◦58-88% variation Favorable 1H LTT and BSR alleles in repulsion ◦Progeny with high survival and BSR resistance ◦~20 cM apart in combined map

Combined Population QTL Scan

Scald Resistance All parents have some resistance ◦Selected for “field resistance” NB3437f x OR71 ◦Favorable alleles on 4H and 7H contributed by NB3437f NB713 x OR71 ◦Favorable alleles on 2H and 7HL contributed by OR71 Results confounded by year effect ≥4 loci segregating

Prospects for Disease Resistant, LT Tolerant Fall-Sown Barley Fall-sown barley advantages ◦e.g. Yield and water conservation Pre-requisites ◦Sufficient LTT, disease resistance, grain with a premium Assessment of malting quality not possible ◦BSR (COR) and LT injury (SPMN)

OrNe Germplasm Continuation Used in crossing ◦LTT and scald resistance Limited number incorporated in breeding program ◦OBELT Included in association mapping panel in the TCAP ( ◦Assessed for multiple phenotypes including malt quality

Final Thoughts Multitude of favorable alleles discovered for future MAS and GS schemes Newly discovered FR-H3 and superior Nebraska FR-H2 ◦Targets for LTT introgression Finding undiscovered QTL shows that the search for more LTT QTL should continue as well as identifying candidate genes and causal polymorphisms

Acknowledgements OSU Barley Project ◦Patrick Hayes ◦Alfonso Cuesta-Marcos ◦Ann Corey ◦Tanya Filichkin ◦Yada Chutimanitsakun ◦Natalie Graham Committee Members ◦Glenn Howe, Tony Chen, James Hermes Collaborators ◦CSIS: Luis Cistue ◦UNL: Stephen Baenziger ◦UM: Kevin Smith ◦Anheuser-Busch: G. Hanning, J. Heward ◦MRI: Ildikó Karsai, Zoltan Bedo ◦JHI: J. Russell, Robbie Waugh USDA/AFRI

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