Salah F. Abou-Elwafa, Ke Xiao, Christian Jung Plant Breeding Institute Faculty of Agricultural and Nutritional Sciences Candidate Genes for Root Lesion Nematode Resistance in Barley PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

2 Production areas in the world where root lesion nematodes cause major problems Introduction PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014 Pratylenchus spp. P. penetrans P. neglectus

Objectives Genetic map construction of the Beysehir×Valentina (BV) DH population Mapping of RLN resistance QTLs Development of diagnostic markers for major QTLs associated with resistance to RLN Ultra fine mapping of major RLN resistance QTLs and identification of candidate genes 3 Objectives PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

4 Results P. neglectus resistance test with the BV population PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014 Number of nematodes Doubled haploids

Mapping of RLN resistance QTLs in the BV population 5 M34P42_B M31P44_V M34P45_B M35P36_V M35P40_V M32P40_B M32P40_B M33P40_V M31P42_B M35P40_V M35P36_V M36P37_B M32P41_B M32P44_V M32P40_B M31P35_V M33P45_V M33P40_B M36P40_V M35P40_B M31P40_V M35P34_B M36P34_B M35P35_V M34P46_V228 M34P46_B M34P41_B M36P33_B M35P33_B EBmatc M36P40_B M31P44_V M31P42_V M34P36_V GBMS M34P43_B M35P44_V M35P40_B M35P34_V M35P44_V M31P44_B M33P43_V M31P33_V M34P35_V M31P33_V M32P40_B M36P43_V M32P41_V M34P42_V M34P40_B M34P40_B M34P43_B M31P42_V M33P45_B M35P33_B M36P33_V M36P33_B M36P36_B M35P44_B M35P40_B M35P36_B M35P33_B M36P46_B M31P42_V M35P33_V M36P34_V M34P46_V M33P37_V M31P42_V M33P33_B M34P42_V M34P41_V M31P33_V M34P34_V M36P36_B M34P46_B M34P46_V M31P33_B M36P37_B M35P40_B M31P33_V M36P43_B M35P43_V M34P42_B M34P42_B M32P40_V M34P41_V M35P35_B M35P35_V M32P46_B M31P44_V M36P43_V M33P42_B M33P42_V M33P43_V M31P33_V M31P33_B M34P34_V M33P33_B M35P34_B M35P40_V339 M35P40_B M32P44_V M32P41_B M34P34_V bpb M32P43_B Bmag M34P37_B M34P37_V GBM M35P37_B M31P44_V483 M31P44_B M32P44_V M35P40_V M34P41_V M33P40_V M33P43_V M31P35_V M32P46_B M34P34_B M35P44_V Bmac M34P41_V234 M34P41_V M32P41_B M31P35_V M32P43_B M34P41_B M34P45_V M31P42_B Doubled haploids were used 388 AFLPs, SSRs and CAPSs markers were used Markers covered 1051 cM. The sizes of linkage groups ranged from 99 (LG 3H) to 206 cM (LG 2H) with an average marker density of 2.7 cM/ marker P. neglectus resistance QTL Ch. 1H Ch. 2H Ch. 3H Ch. 4H Ch. 5H Ch. 6H Ch. 7H Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

6 Comparative QTL analysis to detect common QTLs Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014 Galal et al. (in revision); Abou-Elwafa et al. (in prep.)

7 Results 7 PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014 Mapping crossover events within the critical interval R M3 M1 M2 M4 Large DH population Valentina Beysehir 1209 DH lines Phenotyping for RLN and Genotyping using NGS-based BSA 52 Recombinant DH lines

Phenotypic bulks were sequenced using NGS: –The R-bulk: 20 DHs into two bulks the „best“ (10 DHs) and the „good“ (10 DHs) –The S-bulk: 20 DHs into two bulks the „worst“ (10 DHs) and the „bad“ (10 DHs) Each bulk was sequenced in a separate lane (4 lanes) using Illumina HiSeq 2000 (paired-end sequences) 8 Sequencing distributional extremes for fine mapping and candidate gene identification The best The good The bad The worst Genotype Number of nematodes/genotype Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014 Number of nematodes Doubled haploids

Development of consensus reference sequence using the barley pseudo-reference genome 9 Chromosome Pseudo-reference length (million-bp) Consensus length (million-bp) Total read count (million) Average coverage 0H (1) H H H H H H H Total2, , (1) Sequences which are not assigned to any of the seven barley chromosomes Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

10 Identification of sequence variants between the two phenotypic bulks Reads from the R-bulk were mapped to the consensus reference sequence Sequence variants were identified (Probabilistic algorthim) Chromosome Length (million-bp) No. of variants (million) No. of variants (>90% frequency) Variants density 1H , H , H , H , H , H , H , Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

11 Analysis of sequence variants Applying high-intensity criteria –Frequency ≥90%, coverage ≥5 and a phred quality score ≥40 Window size: 1 Mb SNP number Ch. 1H SNP number Ch. 2H SNP number Ch. 6H Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

12 Co-localization of a major QTL to the most divergent region of the same chromosome SNP number Ch. 6H Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, Megabases

Candidate gene identification 13 Results Criteria; i) Located within or close to the most divergent region ii) Putative function of resistance genes to RLN iii) Exhibit differential expression pattern between genotypes possessing extreme phenotypes PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014 GeneGene full name Gene model/ EST E-value/ identity (%) HvSAM1S-adenosylmethionine synthase 1Yes 0.0/ 90 HvSAM3S-adenosylmethionine synthase 3Yes 0.0/ 93 TuSHV3Glycerophosphoryl diester phosphodiesterase 1No 2e-47/ 49 AtRPM1Disease resistance protein RPM1 No 4e-37/45 HvHDGHydroxysteroid dehydrogenaseYes 2e-44/ 88 HvALDHAldehyde dehydrogenaseYes 8e-133/ 58 HvPKProtein kinase superfamily proteinYes 3e-48/ 92 AtERF1Ethylene-responsive transcription factor 1No 3e-31/ 90 TuBGL5Glucan endo-1,3-beta-glucosidase 5No 1e-42/ 66 AtWAK3Wall-associated receptor kinase 3No 4e-18/ 48 AtWAK5Wall-associated receptor kinase 5No 2e-65/ 75 HvRGA2 No 9e-14/62 OsHRGPHydroxyproline-rich glycoprotein-likeNo 4e-74/ 40 HvCBF7C-repeat binding factor No 6e-86/ 100 ZmG3PpGlycerol 3-phosphate permeaseNo 1e-119/ 45 AtLRRReceptor-like serine/threonine-protein kinase No 8e-161/ 95 AtCRRK25Cysteine-rich receptor-like protein kinase 25No 4e-63/ 71 AtTMKL1Kinase-like protein No 2e-78/ 67 AtSUVH1 Histone-lysine N-methyltransferase, H3 lysine-9 specific No 4e-8/ 29 SiGTL1Trihelix transcription factorYes 8e-60/ 67 TuHSDL2 Short-chain dehydrogenase/reductase family 42E member 1-like No2e-65/ 91 TuGT-2Trihelix transcription factorNo 2e-113/ 79 TuDFRDihydroflavonol-4-reductaseNo 6e-8/ 71

Expression analysis of candidate genes 14 Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

15 Acknowledgements Federal Ministry for Education and Research (BMBF) Eberhard Laubach International Barley Genome Sequencing Consortium Institute of Clinical Molecular Biology (IKMB), CAU Kiel Institute of Phytopathology and Applied Zoology Karl-Heinz Kogel Division of Biochemistry Uwe Sonnewald Christian Jung Ke Xiao Ahmed Galal Smit Shah Bettina Rohardt Monika Bruisch Erwin Danklefsen