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1 The wheat Sr50 gene reveals rich diversity at a cereal disease resistance locus Rohit Mago, Peng Zhang, Sonia Vautrin, Hana Šimková, Urmil Bansal, Ming-Cheng Luo, Matthew Rouse, Haydar Karaoglu, Sambasivam Periyannan, James Kolmer, Yue Jin, Michael A. Ayliffe, Harbans Bariana, Robert F. Park, Robert McIntosh, Jaroslav Doležel, Hélène Bergès, Wolfgang Spielmeyer, Evans S. Lagudah, Jeff G. Ellis, Peter N. Dodds * NATURE PLANTS 1

2 Supplementary Fig. 1. Isolation of phage lambda DNA clones from the Sr50 locus. Autoradiograph showing hybridisation of P 32 -labelled Mla-LRR probe B76 to DraI digested lambda clones 4, 5, 8, 11, 12, and 18 and genomic DNA of Gabo 1BL.1RS. The gel-purified region used to make the lambda genomic DNA library is indicated on the Gabo1BL.1RS lane in a dotted box with the fragments missing in deletion mutants indicated by arrows. Sequence analysis showed that all of the clones encoded Mla-related sequences but three contained various truncations or frameshifts and could not encode full length proteins and two only contained partial gene sequences corresponding to LRR fragments. Only clone 5 contained a nearly full length gene with no internal interruptions of the reading frame, but was truncated at a DraI site in the LRR encoding region and was missing the 3 end of the predicted gene. 2 NATURE PLANTS

3 SUPPLEMENTARY INFORMATION Supplementary Fig. 2 BAC contig containing Sr50. The contig contains 72 overlapping BAC clones that were ordered by high-information content BAC fingerprinting 17. BAC clones making the Minimum Tiling Path are shown in green. BAC clones positive for the NB-LRR PCR screen are indicated by a. NATURE PLANTS 3

4 Supplementary Fig. 3 Predicted exon-intron structures of ScRGA1 genes at the Sr50 locus. 4 NATURE PLANTS

5 SUPPLEMENTARY INFORMATION Supplementary Fig. 4 Analysis of transgenic plants. A Autoradiograph showing hybridization of a probe from the Sr50 gene to genomic DNA of pvecbar-sr50 T0 lines digested with NotI. The arrow shows the Sr50-specific band. Controls 1 and 2 are lines selected from tissue culture that did not show the selectable marker. Lines NATURE PLANTS 5

6 tested for stem rust resistance in figures 2A and S4B are indicated by a asterisks (** and *, respectively). B Stem rust phenotypes of selected pvecbar-sr50 T0 transgenic lines. Control 1 is a non-transgenic line selected from tissue culture. Infection types are shown at the bottom C Stem rust phenotypes of selected pvecneo-sr50 T0 transgenic lines. The non-transgenic Fielder parent, a nontransgenic line from tissue culture (control) and cv. Gabo are included as negative controls and the Sr50 resistance line Gabo1DL.1RS-DR.A1 is a positive control. Infection types are shown at the bottom. Lower panel shows gel image of PCR amplification of a 469bp (primers Sr50-5p-F3/R2) Sr50-diagnostic product showing the presence of the transgene only in lines showing resistance. 6 NATURE PLANTS

7 SUPPLEMENTARY INFORMATION Supplementary Fig. 5 Phylogenetic relationship of Sr50 and related Mla family NB- LRR proteins. A neighbor-joining tree of derived from predicted protein sequences of NATURE PLANTS 7

8 ScRGA1 genes, barley Mla genes (HvMla) 9,11,21-26, TmMlaA 12, Sr33 13, Sr35 27, Lr1 28, Lr10 29,30 and Lr21 31 is shown. Protein accession numbers are shown in brackets. 8 NATURE PLANTS

9 SUPPLEMENTARY INFORMATION Supplementary Fig. 6 Partial amino acid sequence alignment of Sr50 variants identified in S. cereale lines Frotier, Svalofs Otello and Dwarf Petkus R1. Amino acid differences from Sr50 are indicated in red. NATURE PLANTS 9

10 Supplementary Fig. 7 Distribution of Mla homologs in S. cereale accessions. DraI digested rye DNA was hybridised with probe B76 from barley Mla-LRR. The red box indicates those lines carrying identical Sr50 gene sequences. Also included are a Chinese Spring wheat addition line carrying rye chromosome 1 with Sr50 (CS+1R), Gabo1DL.1RS-DR.A1 and mutant line which contains a whole arm deletion of 1RS. 10 NATURE PLANTS

11 SUPPLEMENTARY INFORMATION Supplementary Table 1. Infection types a of genotypes carrying resistance genes Sr31, Sr50 and SrR Amigo tested with various P. graminis f. sp. tritici races. Pgt race (country of origin) Line 98-1,2,3,5,6 98-1,2,3,5,6 +Sr50 QFCSC TPMKC TTKSK TTKST (781219) b (130176) b (06ND76C) b (74MN1409) b (04KEN156/04) b (06KEN19v3) b (Australia) c (Australia) c (USA) c (USA) c (Kenya) c (Kenya) Federation Federation*4/Kavkaz (Sr31) 12= 2= Federation*4/Kavkaz sr31 ( ) d Gabo e ; Gabo 1DL.1RS-DR.A1 (Sr50) 12-4 ;1- ;1 1 1 Gabo 1BL.1RS (Sr50) 12-4 ; Gabo 1BL.1RS sr50 (M7) f 4 4 0; CSID5405 (Sr33) , a Infection types : ; to 1=R, 2=MR, 3=MS, 4=S b Isolate numbers of different Pgt races c Country of origin d sr31 mutant (Mago et al. 2005) 8 e The Gabo line contains genes conferring partial resistance to Ug99 derivatives ( f sr50 mutant (Mago et al. 2004) 5 NATURE PLANTS 11

12 Supplementary Table 2 Infection types a of genotypes carrying resistance genes Sr31, Sr50 and SrR Amigo tested with various Pgt races. Pgt race (country of origin) Line TTKSK TRTTF TKKTP QCMJC (04KEN156/04) b (06YEM34-1) b (13GER16-1) b (07WA ) b (Kenya) c (Yemen) c (Germany) c (USA) c LMPG*6/Sr31 (Sr31) 3 to 4 2- ; to 2- ; TAM107-1 (SrR Amigo ) 2- to 2 X to to 3 ; Federation*5/Gabo1BL.1RS-1-1 (Sr50) 2- ;1 0 to ; 4 a Infection types : ; to 1=R, 2=MR, 3=MS, 4=S b Isolate numbers of different Pgt races c Country of origin 12 NATURE PLANTS

13 SUPPLEMENTARY INFORMATION Supplementary Table 3. Infection types of the Australian stem rust differential set inoculated with putative Sr50-virulent Pgt mutant and its progenitor. The critical difference is shown in bold No Wheat lines Resistance Genes 98-1,2,3,5,6 98-1,2,3,5,6 +Sr50 1 Reliance Sr c 2 Marquis Sr7b Acme (durum) Sr9g, X Vernal Emmer Sr9e ; ;1-5 Einkorn (T. monococcum) Sr21 ;1- ;1 6 Line S Sr13, Sr17 2= 12= 7 McMurachy Sr Yalta Sr W2402 Sr7b, Sr9b TD Sr36 ;X= ; 11 Renown Sr7b, Sr Mentana Sr8a n/a n/a 13 Norka Sr15 X= ;X= 14 Festiguay Sr TAF 2 Sr44 ;1 2= Unknown Barleta Benvenuto Sr8b X X 18 Coorong triticale Sr27 ;1 ; 19 Satu triticale Sr Satu ; ; 20 SrNin triticale Sr Nin 12= ;1-21 Gatcher Sr2, Sr5, Sr6, Sr8a, Sr12 X c 22 Combination X Sr5b, Sr7b, Sr9b Kite Sr26 ; 12= 24 Agent Sr24 2= 2 25 Norin 40 Sr42 2= 2 26 Cook Sr5, Sr6, Sr8a, Sr36 0;= 0; 27 Banks Sr5, Sr8a, Sr9b, Sr12, Egret Sr5, Sr8a, Sr9b, Sr12 3= 3= 29 Mendos Sr11, Sr17, Sr36 ; 0; 30 Mildress Sr31 12= 2= 31 Mokoan Sr9b W3534 Sr C82.2 Sr32 2= 2 34 DK46 Sr35 ; ;1-35 Trident Sr38 X X 36 Gabo 1DL.1RS Sr50 ;12= Morocco Gabo1DL.1RS-DR.A1 Sr50 ;12= Gabo NATURE PLANTS 13

14 Supplementary Table 4. Segregation of rust phenotypes (resistant/susceptible) in VecBar T1 families Line Stem rust Leaf rust Stripe rust 2 12/3 0/12 0/15 10(1) 11/4 0/13 0/ /3 0/13 0/12 19d 10/4 0/15 0/15 14 NATURE PLANTS

15 SUPPLEMENTARY INFORMATION Supplementary Table 5. List of rye accessions used No. AUS No. Name Putative origin Tetra-Petkus Kisvardai Hungry European rye collection USA King II Sweden Tetraploid rye from King II Sweden * Svalofs Otello Sweden Svalofs Varne Sweden Canadian rye Canada Adams USA Antelope Canada Balbo USA Elbon USA * Merced USA Pierre USA Prolific Canada Rosen USA Rymin USA Snoopy rye mixed Mexico Saskatoon Australia Everest France Wiloszanowskie Poland Wiloszanowskie N Poland Karlshudska Yugoslavia Dominat: 2793 Romania Petka South Africa Petkuser Somerroggen South Africa W2 Pakistan Wrens Abruzzi Spain Rahu New Zealand Derzavinskaja 29+2 Soviet Union Pervenec Shi Soviet Union Kormovaja 61 Seln. Soviet Union Zarecanskaja Zelenoukosnaja Soviet Union * Odesskaja mnogoletniaja Soviet Union Gazelle Canada SSR*1 South Africa Snoopy Seln. Mexico * Coloma USA South Australian Australia Caribou 6B Carsten Roggen Germany * Dwarf Petkus R1 United Kingdom Dwarf Petkus R2 United Kingdom Dwarf Petkus R3 United Kingdom Dwarf Petkus R4 United Kingdom Dwarf Petkus R5 United Kingdom Dwarf Petkus R6 United Kingdom NATURE PLANTS 15

16 Dwarf Petkus R6-2 United Kingdom Dwarf Petkus R6-3 United Kingdom Mikulikie Wazesne South Africa South Australian Australia * Castelo Branco Portugal Estacao Agraria de Visco 5035 Portugal * Frotier France Kartner 1 Germany Gaiton Germany Centero do Alto Portugal Purmitoi Germany Emory USA Dominant Ceha Burnburger Futterroggen M South Africa Ceske South Africa Rye impurity in W165 India Ratborske M Soviet Union Derzavinskaja 2942 Soviet Union Korovaja 6 1 Soviet Union Pamirskaja Soviet Union Pervenec Soviet Union Zarecianskaja Selenoukosnaja Soviet Union SV 6970 Sweden Svalofs Ponsi Sweden Petkus II Sweden AMC 242 Turkey * AMC 127 Iran Frontier Canada Puma Canada ND Seln no. 1 USA ND Seln no. 5 USA Dankouskie Nowe (rye) Poland Saratovskaja 5 Soviet Union Saratovskaja Kurpnozernaja Soviet Union * Zitomirsksja Tetra Soviet Union Rah 101 Poland Rah 1 Poland Dacold USA Centeio BR1 Brazil Bevy Australia * Imperial USA Elect Austria Eho Austria Westwood Australia Kenya rye Australia Gator rye USA Wrens rye USA Secale Resgene A Australia Secale Resgene B Australia 16 NATURE PLANTS

17 SUPPLEMENTARY INFORMATION Secale Resgene C Australia Secale Resgene D Australia Kenya -12 Australia Kenya -12 Australia Gator - 28 Australia Gator - 28 Australia * Wrens 5 Australia Wrens 5 Australia Elbon-Gator 17 Australia Elbon-Gator 17 Australia Elbon-Gator 17 Australia Wrens 5 Australia Kenya 12 Australia Kenya 12 Australia Kenya 12 Australia Elbon-Gator 17 Australia Wrens 5 Australia * Indicates accessions which amplified with Sr50 primers NATURE PLANTS 17

18 Supplementary Table 6. Stem rust infection types on S. cereale accessions. Rye Line ID Name Origin Infection type with Pgt race a 34-2,4,5,7, ,12, ,2,3,5,6 98-1,2,3,5,6 +Sr ,6,7,11 AUS b Odesskaja Mnogoletniaja Soviet Un 0 2= ;1 ;1 ;, AUS b Castelo Branco Portugal 0; ;1 ; ; 0; AUS b Coloma USA 0; ;1 ;, ;12= 0; AUS b AMC 127 Iran 0; 12= 2= 12- ;1 AUS b Zitomirskaja Tetra Soviet Un 0; ;12= ; ;12-0; AUS c Imperial USA ; AUS d Svalofs Otello Sweden 0; ;1 0; 0;1 0; AUS d Frotier France 0; ;= ; ;12= 0; AUS e Wrens 5 Australia ; 12-12= 12=, 3 ;1 AUS e Merced USA ; 0; 2+ ;1-, 2 ; AUS f Dwarf Petkus R1 UK =, 3 - Gabo 1DL.1RS-DR.A1 Australia ;1 12= 12= 3+ ; Gabo Australia ;1= Morocco a Culture accessions numbers: 34-2,4,5,7,11, ; 34-2,12,13, ; 98-1,2,3,5,6, ; 98-1,2,3,5,6 +Sr50, ; 126-5,6,7,11, 334 b Contains sequence identical to Sr50, c No amplification d Contains single amino acid change from Sr50 (Figure S6) e Contains inverted sequence in Sr50 coding region f Contains five amino acid substitution in Sr50 (Figure S6) 18 NATURE PLANTS

19 SUPPLEMENTARY INFORMATION Supplementary Table 7. Primers used for PCR amplification of Sr50, ScRGA1 candidates and BAC ends. Marker Forward primer Reverse primer Sr50-F1, R1 TAGCGCTGCTCACATCCACCTC GATCCGCCGTTGTCGGCATTTGT Sr50-F2, R2 ATTCATGCTTTTATACTCACTAATATC GGGCGTGACTGTGCTGCTT Sr50-5p-F3,R2 TTCAGTGAAGTTGCCGCTGT GCATGCTCTCAAGCTCCTTCT ScRGA1-B- F1, R1 ATCTTTGGGTCTCGGGTGCC GACGCACAAGGTGTTTATGCTGAT ScRGA1-B- F2, R2 TCAAGTCATTCTTTCGCGAAACT GCAGACGCACGCACACACGTTAT ScRGA1-C- F1, R1 TCTGCATGTAGATTAATGTACTAG GTAGCTGCAGCAGCTCTGAAA ScRGA1-C- F2, R2 ATGAATGCATAGTTTGTAGCATGGC GACTGCAAGTCTGCGACTGCGA ScRGA1-D- F1, R1 AGCTTGAAGAAATTAAAGCTTCAG GCACCCCTGAACCTGATGCAGT ScRGA1-D- F2, R2 AGCTCCCTTTAATTTGGCAGC GTCCTCGTCACTATCACACACCA ScRGA1-E- F1, R1 TGGAGGTATATTTCATATTTCCAGTC GTTGCACCGCTTATATCCGT ScRGA1-E-ex3- F1, R1 TATCTGTTGTACTCTTGCAACGG CAGATCTCACAGTGAAGCATAGTTGA ScRGA1-F- F1, R1 ATTCATCTATTTTGTAGGTTTCCGG CCATCTTAATTTGTTCGTTGCCT ScRGA1-F- F2, R2 AGGCAACGAACAAATTAAGATGG CATGCAAATATCTGCTACATACCTGA ScRGA1-G- F1, R1 ATCAAAGGTCCGGATCTCTG GCTGCTAGAGAAGCACAATTGA RACE 5p,3p GATTCCTGCCTTTCTTAAACAAGCCGA TCGGCATGATGTCTTTGTTCG p2d7-f-end GGCGGGCTGCTAGTATTTCC GCCATCGGATCTGGAGAGAA p2d7-r-end CGTTGCAATGATGTACCATACG ACCGAGCTCGTGTGCTCAA p2e7-f-end CAACAAGACGCACACCACCT GTGCAGTTGCAGAGGACCTG p2c2-f-end TTCGCAGGTTCATCATGGTC CTCCCGAATTGGAAAGTGGA p2c2-f-end CCTTGGCCTTTAGCTTGTGG TTGCCGGAAGCAAGAACTTT p1f7-f-end CGGAGTGTTTGGATGAAAGG CCGATCCAGGGGATATAGGT p1f7-r-end CTTCGTTAGGAATGGCAGGT CATGCCTGATTCAATGTTGC p2b8-f-end GCACGCATGCATGTAGTTGA GGGAAGCTCCTGGTTTGTTG p2b8-r-end ATCCGTGGGAGCTGTAGGTG AGATGGATTGGGCTGTGGAT p2c8-f-end CGCTCAGTTTGCCGAAAAG ATCGGAGTCGTCGGAGAGAG p2c8-r-end GGTCCCTTGCTCGTGAGTTC TGTGATGGTGATGCTTGTGC p2c7-f-end TCTGAAGCCGGTCGAGTCTTC GGGAGTACTAGTCTCGCATCA p2c7-r-end CATGGCTGCCACTCTCAAAG TCACGCACGTCAAGTCAAAA p2a3-f-end TGGTACTGTGAAAGCGATTCTTATC GACGGCAAGATGGAGCAAGGA pindigobac5 GGATGTGCTGCAAGGCGATTAAGTTGG CTCGTATGTTGTGTGGAATTGTGAGC NATURE PLANTS 19

20 Supplementary references 21. Zhou, F., et al., Plant Cell 13, (2001) 22. Halterman, D.A., Wei, F. & Wise, R.P. Plant Physiol. 131: (2003) 23. Halterman, D., Zhou, F., Wei, F., Wise, R.P. & Schulze-Lefert, P. Plant J. 25: (2001) 24. Halterman, D.A. & Wise, R.P. Plant J. 38: (2004) 25. Shen, Q.H., Zhou, F., et al. Plant Cell 15: (2003) 26. Wei, F., Wing, R.A. & Wise, R.P. Plant Cell 14: (2002) 27. Saintenac, C., et al. Science 341: (2013) 28. Cloutier, S., et al. Plant Mol. Biol. 65: (2007) 29. Loutre, C., et al. Plant J. 60: (2009) 30. Feuillet,C., et al. Proc. Natl. Acad. Sci. USA 100: (2003) 31. Huang, L., et al. Genetics 164: (2003) 20 NATURE PLANTS