1. 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. 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 http://nematode.unl.edu http://www.plantwise.org/KnowledgeBank/PWMap.aspx?speciesID=35060&dsID=43900

3. 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. 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

5. Mapping of RLN resistance QTLs in the BV population 5 M34P42_B416 0.00 M31P44_V154 17.59 M34P45_B266 21.68 M35P36_V615 23.70 M35P40_V420 25.56 M32P40_B400 27.38 M32P40_B344 28.42 M33P40_V427 29.51 M31P42_B467 29.82 M35P40_V267 31.71 M35P36_V264 33.94 M36P37_B169 35.93 M32P41_B109 37.30 M32P44_V66 37.42 M32P40_B62 42.57 M31P35_V309 43.77 M33P45_V133 45.77 M33P40_B453 49.52 M36P40_V501 50.31 M35P40_B299 52.57 M31P40_V137 53.72 M35P34_B151 55.23 M36P34_B333 59.95 M35P35_V85 62.22 M34P46_V228 M34P46_B229 66.69 M34P41_B254 69.25 M36P33_B409 78.62 M35P33_B383 82.69 EBmatc0003 84.15 M36P40_B116 84.77 M31P44_V213 87.85 M31P42_V188 88.54 M34P36_V167 93.24 GBMS119 93.97 M34P43_B370 96.03 M35P44_V489 98.28 M35P40_B116 104.06 M35P34_V73 106.59 M35P44_V658 108.73 M31P44_B93 114.14 M33P43_V185 115.80 M31P33_V70 118.26 M34P35_V119 119.49 M31P33_V309 122.97 M32P40_B269 123.97 M36P43_V137 126.06 M32P41_V429 126.91 M34P42_V174 126.96 M34P40_B305 128.98 M34P40_B504 130.54 M34P43_B292 133.23 M31P42_V560 135.82 M33P45_B128 139.35 M35P33_B286 142.50 M36P33_V386 149.67 M36P33_B375 153.90 M36P36_B63 0.00 M35P44_B102 3.94 M35P40_B108 8.55 M35P36_B100 12.05 M35P33_B486 19.68 M36P46_B208 22.28 M31P42_V114 22.37 M35P33_V491 23.61 M36P34_V213 26.80 M34P46_V135 28.85 M33P37_V130 33.11 M31P42_V325 41.76 M33P33_B291 45.66 M34P42_V419 51.11 M34P41_V81 51.58 M31P33_V359 55.69 M34P34_V257 55.89 M36P36_B154 59.38 M34P46_B166 61.01 M34P46_V110 61.65 M31P33_B350 62.04 M36P37_B279 63.89 M35P40_B167 67.50 M31P33_V200 69.96 M36P43_B91 71.97 M35P43_V276 77.04 M34P42_B101 82.16 M34P42_B548 84.67 M32P40_V439 85.30 M34P41_V106 87.87 M35P35_B195 89.49 M35P35_V57 94.23 M32P46_B159 95.21 M31P44_V164 98.68 M36P43_V451 102.10 M33P42_B225 103.97 M33P42_V222 106.04 M33P43_V243 110.11 M31P33_V286 116.15 M31P33_B293 120.50 M34P34_V239 133.06 M33P33_B441 135.98 M35P34_B58 144.39 M35P40_V339 M35P40_B336 145.82 M32P44_V80 147.06 M32P41_B251 150.94 M34P34_V253 156.62 bpb0037 160.30 M32P43_B70 163.08 Bmag206 164.10 M34P37_B321 166.51 M34P37_V247 168.13 GBM1174 169.94 M35P37_B255 170.61 M31P44_V483 M31P44_B306 171.33 M32P44_V86 171.36 M35P40_V351 171.43 M34P41_V466 172.07 M33P40_V551 172.12 M33P43_V159 173.99 M31P35_V71 175.54 M32P46_B133 175.56 M34P34_B103 176.18 M35P44_V177 177.36 Bmac186 177.85 M34P41_V234 M34P41_V101 180.98 M32P41_B145 180.99 M31P35_V364 182.33 M32P43_B281 183.32 M34P41_B153 186.79 M34P45_V331 195.19 M31P42_B380 196.76 226 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. 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. 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

8. 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

9. 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) 45.09939.62219.63342.43 1H251.620204.51163.59824.64 2H340.517278.92085.35024.44 3H302.678242.48272.36623.31 4H285.184237.92971.30624.36 5H309.084250.116101.10131.93 6H266.364216.68866.51224.33 7H335.274263.95481.07323.58 Total2,135.8181,934.222 (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. 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 251.620 2.895248,9660.00099 2H 340.517 3.857305,4000.00089 3H 302.678 3.245261,4200.00086 4H 285.184 3.312267,0000.00094 5H 309.084 3.434263,4170.00085 6H 266.364 3.284309,6100.00116 7H 335.274 3.540303,0220.00090 Results PLANT 2030 Status Seminar, Potsdam, March 31 - April 2, 2014

11. 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. 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, 2014 4 Megabases

13. 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

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

15. 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