Association Mapping in European Winter Wheat

Wheat in Germany Wheat is the most important crop plant in Germany with more than 3 million hectares (mainly winter wheat) Yields have been stagnating and fluctuating at high levels over the last 10-15 years and wheat is getting less competitive compared to other crops Major factors for wheat production in Germany are diseases (e.g. Fusarium, Septoria) and environmental factors (such as drought, cold, etc.)

Association Mapping (AM) Improvement of varieties through breeding The WHEAT association study 1975 1976-1980 1981-1985 1986-1990 1991-1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 372 wheat varieties Phenotypic data   Genotypic data   Field evaluation of 372 wheat varieties for many traits ( 2 years x 4 locations x 2 replications), field evaluation of major diseases (Fusarium, Septoria, etc.) - Molecular markers (SSRs and SNPs) Candidate genes     Marker/Trait Association Mapping (AM)   Identification of molecular markers associated with traits that are important for wheat breeding Filed evaluation for F resistance were carried out for 372 wheat varieties, replicated in 2 years and 2 locations ； For genotyping part , 2 sets of molecular markers were used. One set is multi-alles systems , which includes 732 SSR makers cover the whole 21 wheat chromosomes and the other is a bi-allelic system, snp marker, which dereved from a novel illumina XXX chip. More details about the cihp were well described in a newly published paper “Title” in XX journal 2014. The AM statistical analysis part in this study we used the statistical software package Genstat. The 14th edition was used in this study. Through this study , we try to generate our knowledge about … ? 最后是 backcrossing   Improvement of varieties through breeding

Phenotyping Yield, yield components, France (SYNGENTA): quality parameters France (SYNGENTA): Saultain 2010 Janville 2010 Andelu 2009|2010 GERMANY (KWS LOCHOW): Wohlde 2009|2010 Seligenstadt 2009|2010 Disease resistance (Fusarium, Septoria, Drechslera) Traits measured in all eight environments Heading date - Specific weight Protein content - Thousand kernel weight Plant height - Zeleny volume Plot yield

Genotyping - 971 SSR markers for analysis of population structure - SNP arrays 12K wheat Infinium array 90K wheat Infinium array 35K wheat Affymetrix array >20 candidate genes (e.g.ppd, rht) SSR 12k 90k 35k sum number 971 9655 81587 35143 127356 polymorphic 781 5242 21200 20052 47275 mapped 412 3005 11676 13346 28439

Linkage disequilibrium LD in material Between one and several cM 90k data

Haplotype structure and SNPs Total number of haplotypes based on SNP marker analysis 16608 Arrays are in part complementary

SNPs and association studies Summary The data are complementary and the 90K and 35K arrays provide together a very useful marker set

Marker/trait associations for Fusarium resistance using molecular markers and candidate genes Rht-1D SSR system

The VALID study General In association studies, there are often many marker-trait associations (MTAs) identified which later cannot be confirmed and such MTAs are not useful for breeding Goals of the VALID project - Analysis of additional wheat material that is relevant for wheat breeding for the investigated traits in order to confirm the identified MTAs on other material - Validation of identified MTAs in specifically developed populations - Analysis of the wheat varieties for abiotic traits (e.g drought)

Validation of effects in more recent breeding material WHEAT Panel 372 unique genotypes European varieties mainly from 2000 to 2008 TROST Drought stress Panel 184 unique genotypes VALID Panel 133 unique genotypes European varieties mainly from 2007 to 2010 20 overlapping genotypes Field tests 2013/2014 Field tests 2009/2010 Field tests 2013/2014

Principal component analysis for all 518 wheat varieties (10,000 SNPs) * = WHEAT panel V = VALID panel Only very weak population structure in the entire material (breeder, type and origin) No significant subpopulation differences between the WHEAT and VALID panel Material is well-suited for association studies

Examples for specific traits: Fusarium (FHB) resistance plant height (PH) heading date HD

Phenotypic data for FHB, PH and HD Kosack Tuscan Phenotypic variation in the material Heritability is high in the material Candidate genes analyzed Kosack is the most resistant variety in all the 4 enviroments.

Candidate genes in the data sets HD and PH Heading Date in WHEAT Heading Date in TROST (13) Heading Date in VALID (13/14) Plant Height in WHEAT Plant Height in TROST (13) Plant Height in VALID (13/14)

MTA validation analysis Comparison MTAs for FHB between WHEAT and VALID Limited validation between both studies Loci  Validated loci Trait  VALID_BLUEs* MTA>=2.3  VALID_BLUEs MTA>=2.3 WHEAT >=3.0 PH 31 10(32%) HD 63 15(24%) Heritability WHEAT TRAIT h² PH 0,98 HD 0,98 Level of validation depends on heritability Preliminary data show the validation rate for yield is significantly lower (<10%)

Genetic validation approaches

Development of DH lines for yield 2 populations with fixed major genes (ppd, rht) were developed that were segregating for 2 or 3 identified QTLs each. Lines were identified by marker analysis with all possible QTL combinations and grown in the field.

Introgression of major MTAs into another line via MAS Goal: Pyramiding of additional QTLs in an already good line 6 QTLs were selected for backcrossing (4 crosses) Backcross of 1-2 QTLs into a high yield elite line Both lines are fixed for major genes (rht, ppd) and many other QTLs MAS with genome-wide marker set BC2S2 => more than 95% recurrent parent, trait fixed

Development of BC lines for Septoria and FHB Locus 1 Locus 2 Donor Donor Locus 1 Donor Donor

Summary and next steps A comprehensive phenotypic evaluation of European winter wheat material was performed over several years A detailed molecular marker analysis (SNPs, SSRs, candidate genes) of European wheat material was performed MTAs for important traits (disease resistance, yield and yield components, quality, drought) were identified Known candidate genes and first confirmed MTAs indicate that the approach produces useful data for breeding however the validation rate is relatively low (<30 to <10%) even for traits with high and intermediate heritability Mapping populations and introgression lines will further validate specific loci and open the door for using confirmed MTAs in wheat breeding Map-based cloning of confirmed and mendelized MTAs will be possible in the future

Acknowledgments Thank your for your attention! Erhard Ebmeyer Viktor Korzun Sonja Kollers Marion Röder Jie Ling Christine Zanke Gunther Stiewe Odile Argillier Maike Hinze Andrea Jansen Thomas Zschäckel Bernd Rodemann (BS) Christiane Balko (GLW) Frank Ordon (QLB) Martin Ganal Jörg Plieske Jana Lemm Thank your for your attention! ? Frank ordon