Advances in Alien Gene Transfer in Wheat George Fedak

Transcription

1 Advances in Alien Gene Transfer in Wheat George Fedak Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, Canada K1A 0C6. 1

2 Milestones in Interspecific / Intergeneric Hybridization (1) First intergeneric hybrids - Wheat x rye, Wilson 1876 Colchicine - Blakeslee Wheat x barley, Farrer Wheat x Aegilops, Kihara 1937 Embryo rescue and in vitro culture - Kruse, 1973, Murashige and Skoog,1962 Pioneers Tsitsin, Ceoloni; Islam, Jauhar; Sears Mujeeb-Kazi; Friebe-Gill; Molnar-Lang; P. Chen; S. Xu Screening alien species for variability - Oliver et al Ph1 mutants - Riley and Chapman, 1958 Cytogenetic stocks - additions, substitutions Sears PBI, KSU, Dvorak, Joppa, P. Chen, Molnar-Lang Wheat and Maize pollination Laurie and Bennett,

3 Milestones in Interspecific / Intergeneric Hybridization (2) Chromosome - genome identification - C-banding, Lukaszewski & Gustafson 1983 Molecular markers - RFLP, RAPD, EST-SSR, DART Maps - T. monococcum - Dubcovsky et al T. dicoccoides - Peng et al 2000 Draft Genome Sequences - T. urartu - Ling et al FISH - Rayburn & Gill, 1985; Danilova et al (cdna) - GISH - Shwarzacher et al. 1989; Kruppa & Molnar-Lang, Ae.tauschii - You et al Luo et al Jia et al Next Generation Sequencing - RNAseg. - Yr15 T. dicoccoides - Ramirez-Gonzalez, Transcriptome analysis - Pm -Th. intermedium - Li et al Exome capture Sm1, Pozniak et al SNP - Sr Cad - Kassa et al KASP Sr Cad - Kassa et al. 2016; Neelam et al GBS - Sr24 - Gao et al STARP - Long et al Flow sorting - Dolezel et al

4 Estimates of Available Disease Resistance Genes Trait Leaf rust FHB Powdery mildew Stem rust Stripe rust Number of Loci 80 QTL in last 15 years 200 QTL 119 genes 141 loci ( 63 numerically designated genes) 67 genes 4

5 Traits Lacking in Primary Gene Pool BYDV resistance Leaf spotting resistance New disease threats ( e.g.ug99) 5

6 Examples of Recent Introgressions from Secondary and Tertiary Gene Pools Biotic Stresses Trait Source Reference Stem rust (Ug99) Current status eg. Sr43, Ae markgraffii Niu et al. 2014; 2017 Sr26 L. ponticum Dundas et al Additional Screening Zheng et al FHB E. repens, T. monococcum, T. timopheevii Fedak et al T. miguschovae, Ae. cylindrica, Th. elongatum Mildew A. cristatum Li et al D. villossum Xing et al. 2017; Zhang et al L. mollis Yang et al Th. intermedium Li et al Stripe rust 1B / 1R Qi el at S. africanum Lei et al L. mollis Yang et al WSMV Numerous species Ali et al BYDV Th. intermedium Ma & Tomita, 2013 Head and leaf blast Ae. ventricosa Cruz et al DON content Numerous species Fedak et al Resistance introgressed into wheat from 52 species from 13 genera ( 400,000 gene bank accessions) 6

7 Examples of Recent Introgressions from Secondary and Tertiary Gene Pools Physiolodical Traits Trait Source Reference Heat stress tolerance Synthetic hexaploids Cossani et al Wheat blast Ae. ventricosa Cruz et al Salt tolerance Th. distichum Marais et al Stress tolerance Ae. biuncialis Dulai et al Enhanced B glucan Wheat - barley translocations Cseh et al Improved protein and gluten Th. intermedium Li et al Grain yield, biomass, PS Emmer Merchuk -Ornat et al Enhanced kernels / spike A. cristatum Zhang et al Early maturity Ps. huashanica Wang et al Micronutrient content Ae. biunciales Farkas et al

8 Sr gene Origin and Chromosome Location of Stem Rust Resistance Genes Origin Chromosome Location Sources Sr 33 T. tauschii 1DL Sambasivam et al Sr 35 T. monococcum 3AL Zhang et al Sr 36 T. timopheevii 2B Tsilo et al Sr 39 Ae. speltoides 2B Liu et al Sr 40 T. araraticum 2B Wu et al Sr Cad Norin 40 6DS Heibert et al Sr 43 L. ponticum 7DL Lui et al

9 Disease Resistance Gene Combinations Parental hybrid combination AC Cadillac(SrCad-Bt10,Lr34)/Carberry(Fhb1,Lr34) // RL5405(Sr33)/ Carberry Doubled haploids 68 DH lines produced 9

10 Stem rust infection type of various gene stacks Order Gene type Stem rust infection type 1 Lr34-Sr42-Sr33-Fhb1 1-2 Lr34 Sr33-Fhb Lr34-Sr42-Fhb Lr34-Sr42-Sr Lr34-Sr42-Fhb1 1 6 Lr34-Sr Lr34-Sr42-Fhb1 1 8 Lr34-Sr Lr34-Sr Lr34-Sr Ac Cadillac Lr34-Sr RL5045 Lr Carberry Shaw - 3- Little club Note: Sr42 is SrCad; tightly linked to Bt10 10

11 Pyramids with combination of Sr33, Sr36, Sr42, (SrCad) Sr43 Hybrid combination AC Cadillac (SrCad ) / Lang (Sr36) // RWG 34 (Sr43)/ RL 5405 (Sr33) Progeny 78 F 1 plants screened 7 F 1 plants contained all four genes 54 DH plants produced Gene combinations No. of DH lines Sr33 Sr36 SrCad Sr43 7 Sr33 Sr36 SrCad _ 4 Sr33 Sr36 _ Sr43 2 Sr33 _ SrCad Sr43 1 _ Sr36 SrCad Sr43 4 Sr33 Sr36 2 Sr33 Sr43 2 _ Sr36 SrCad _ 3 _ Sr36 _ Sr

12 Crosses of Sr genes onto Canadian spring wheat cultivars Cultivars Carberry Hoffman Fusion Scotia FL62R1 Gene combinations Sr33 SrCad Lr34; Sr33 Sr39; Sr33 Sr43; Sr39 SrCad; Sr39 Sr40 Sr33 SrCad Lr34; Lr34 Fhb1; Sr39 Sr40 Sr33 SrCad Lr34; Lr34 Fhb1; Sr39 Sr40 Sr33 SrCad Lr34; Lr34 Fhb1; Sr33 Sr39; Sr40 SrCad; Sr39 Sr40 Sr33 SrCad Lr34; Lr34 Fhb1 ; Sr33 Sr39 ; Sr39 Sr40 BC2 produced in several combinations plus another 48 combinations 12

13 Search for new Sr Genes (Ug99) T. monococcum (;-1) Mapping population BC1 with AC Superb T. miguschovae (1-2) Mapping population Ae. cylindrica (1) Mapping population Triticale USDA (1) BC1 produced Rye USDA (1) Amphiploids with Rusty, Brigade, Langdon, Cocorit Primary triticale- ABIP (;) Top crossed, F2 produced Elymus repens (1) Crosses to separate 2 translocations Synthetic hexaploids (; 1) F2 produced Translocation lines 7E-7D (; -1) BC3F3 produced Lianquan Zhang - (A. Xue ) Ug99 funding 13

14 Amphiploids 8801 (ABE genomes) (;) Ma X H. villosa (ABV) (1-1) Zhong 4, Zhong 6, Zhong 7 (ABDE) (0-1) OK (ABDE) (;) (ABE) (1) (ABE) (;) PWM206 (ABDJ) (1) Ag-wheat hybrid (ABDJ) (;) Primary triticales -6X & 8X (0-1) 20 triticales-usda (1) 14

15 Translocation Lines KS-10 (7E-7D) (1) KS-24 (7E-7D) (1+) K (7E-7D) (;) 14/21 Knott translocation lines (; - 1) 15

16 Screening of 31 Synthetic hexaploids for Resistance to Ug99, Leaf Rust and Stripe Rust Disease package Number of lines *Lr + Sr + Yr 2 Lr + Sr 2 Lr + Yr 3 Sr + Lr 2 Sr + Yr 4 *Langdon x AS2399 *PI94655 x AS

17 Th. elongatum at 21 DAI 17

18 FHB symptoms on 7E lines following point inoculation Aneuploid Lines Severity % infected florets 7EL disomic addition 2n = 42 +2t 9.6 7ES disomic addition 2n = 42+2t E disomic addition 2n = E(7A) disomic substitution 2n = E(7B) disomic substitution 2n = E(7D) disomic substitution 2n = Chinese Spring parent 57.5 Sumai Dvorak and Knott,

19 Spikes of CS (a) and CS-7EL(b) 4 days after inoculation. Arrows indicate inoculated spikelets. c,d) Rachis with florets removed above and below point of inoculation, 21 days after inoculation. c) CS, d) CS-7EL. Miller et al Botany 89:

20 Wuhan/NuyBay Mapping Population 91 DH lines Blue: Type 1 resistance (initial infection) Red: Type 2 resistance (infection spread) Green: Type 3 resistance (toxin) Somers et al Genome 46:

21 GISH of 7E(7D) substitution line (M. Molnar) 21

22 Hybridization Procedure CS ph1bph1b fhbfhb X 7E substit. line Ph1bPh1b FHBFHB anthocyanin pigments CS ph1bph1b fhbfhb X F 1 7E Ph1bph1b FHBfhb (556) BC 1 F 1 ( Select with ph1bph1b marker PSR574 Select for FHB resistance in progeny) (124/ %) Meiotic analysis BC 1 F 2 Segregates ph1bph1b - FHB resistance - anthocyanin pigments - markers(7e SSR) 22

23 MI Ph1b x 7E(7D) 23

24 SSR markers specific for chromosome 7E Lingrang Kong (P.C.) Hu et al. Genes Genome 34:67-75, 2012 Gou Ouellet, Crop Sci. 56: ,

25 Line Primer Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu Lu short arm barc cfd cfd long arm cfd cfd barc FHB rating (%) band - no band Segregation of 7E-specific SSR markers on BC1F5 segregates from Ph1b x 7E(7D) 25

26 Line Primer 7D (long arm) Li Lii Liii Liv Lv Lvi Lvii Lviii Lix Lx 7ES 7EL 7E (7D) Ph1b CS wmc wmc wmc wmc barc wmc wmc barc barc gwm gpw wmc barc cfd wmc cfd TO group 2096-A1-7DL 2096-A3-7E 1480-B1-7E A2-7Dl A1-7E - - p7es Lu Lu A2-7DL A3-7E A1-7DL 1262-A3-7EL 3571-A2-7DL A3-7E A3-7DL A2-7E A1-7E - - p7es Lu Segregation of 7E-specific SSR markers on BC1F5 segregates from Ph1b x 7E(7D) 26

27 Line Primer p l m r e 7A gwm gwm barc wmc cfa gwm wmc B wmc gwm wmc wmc D wmc gdm wmc wmc barc barc gwm gpw wmc barc cfd gdm wmc wmc FHB Infection (%) BC1F5 segregates probed with primers from chromosomes 7A, 7B, and 7D of wheat 27

28 Green: Th. elongatum; Red: 6C6 GISH preparation of segregant (F. P. Han) 28

29 Linkage map of wheat chromosome 7A, 7B and 7D of wheat ( Somers et al. 2004) 29

30 Introgression of resistance to Fusarium head bright, stem rust and stripe rust from Triticum timopheevii (AG genome) 30

31 - Accession PI crossed & backcrossed to Crocus plants advanced to F7 by SSD. - TC67 selected. - Mapping population of 230 F7 RIL lines produced from TC67 X Brio cross - SSR markers - SNP - DH Malihipour et al Spikes of a T. timopheevii derivative at 21 days after point inoculation 31

32 32

33 Segregation of Fusarium head blight, stem rust and stripe rust in TC67(T. timopheevii) x Odessa129-2 (Ae. cylindrica) DH population (154 lines) Line FHB Sr- original Yr - Original QTL Resistance G03*E G03*B G03*E Resistant - Susceptible FHB: 5A Sr : 3B FHB: 5A Yr : 1B Yr : 1B Sr : 3B FHB + Sr FHB + Yr FHB + Sr Donor Trait Chromosome TC67 FHB 5A TC67 Sr 3B TC67 Yr 1B 33

34 Introgression of Fusarium head blight resistance from Elymus repens (StStH genome) BC1 advanced to F7 by SSD 16 lines selected in FHB nursery SSR Markers 34

35 Spike symptoms of: 1) Roblin, 2) P1131-5, 3) P , 4) P , 5) P , 6) P , 7) P B, 8) P E, 9) P at 21 days after inoculation with Fusarium graminearum spores 35

36 MI in Crocus x Elymus repens progenies ( Zeng et al.) 36

37 P (2n = 42) with two terminal translocation chromosomes (white arrows). 37

38 Banding patterns of 3D markers on telomeric translocation derived from Elymus repens (F2) 38

39 Fusarium resistance of telomeric translocations derived from Elymus repens Line 3DS 3DL PLr P1 P2 Crocus Sumai3 Roblin Average FHB resistance (% infected florets) Fedak et al

40 Imtrogression of Fusurium head bright from Triticum monococcum 40

41 200 accessions screened 2 lines selected - Line1 - see poster 54 by Ordon et al. - Line2 - Advanced to BC1F5 - Line M321 x Superb - DH population 70 lines - SNP-KASP markers T. monococcum at 21 DAI 41

42 Agronomic characteristics of FHB resistant lines obtained from wide crosses Genotypes Yield (kg/ha) Days to heading TSTWT (kg/hl) TKW (g) HT (cm) Protein (%) Flour (%) DON (ppm) Sumai M 321 (T. m.) S 184 (A. s.) AC Barrie Hoffman Roblin

43 M

44 AC Barrie 44

45 M

46 5AS FHB resistance QTL in M321 x Superb DH population FHB_5AS wsnp_ex_c6209_ Kukri_rep_c95804_119 Excalibur_c12644_58 RAC875_c2061_292 Excalibur_c47920_249 Kukri_c15450_476 BS _51 BS _51 RAC875_c68530_59 Excalibur_c6714_246 BS _51 BS _51 wsnp_ex_c25707_ Kukri_c29420_310 BS _51 BobWhite_c6468_104 wmc805 gwm293 BobWhite_c10901_240 BobWhite_c6759_365 wmc705 gwm304 barc186 cfa2250 BS _51 BS _51 BobWhite_c15454_63 BobWhite_c12392_149 Tdurum_contig17500_876 RAC875_c13164_695 BobWhite_c43981_90 Excalibur_c45894_552 wsnp_be399939a_ta_2_1 RAC875_rep_c91682_90 RAC875_c23340_2243 Excalibur_c37642_493 BobWhite_c46489_189 Excalibur_rep_c71254_415 BS _51 wsnp_ra_c29586_ IAAV1311 Excalibur_c26778_81 Tdurum_contig71499_211 BS _51 Ex_c4208_839 BS _51 BobWhite_c658_377 BobWhite_c15758_79 Excalibur_c49550_97 IAAV4799 CAP11_c1685_149 wsnp_ex_c40019_ Ex_c6479_750 cfa2141 D_contig04922_546 BobWhite_c15476_88 BS _51 IAAV7827 Kukri_c86812_193 wsnp_ra_c11532_ BS _51 RAC875_c47328_483 BS _51 BobWhite_rep_c63332_67 BS _51 wsnp_ex_c3136_ BobWhite_s66943_270 Excalibur_c32414_861 wsnp_ex_c2505_ BS _51 BS _51 BobWhite_c11539_336 GENE-2673_1457 JD_c48945_490 Chr5A

47 MBDS TJBJ TDBG Leaf rust QTL in M321 x Superb DH population Chr1B barc187 RAC875_c16352_594 BobWhite_c29613_126 BobWhite_c16816_284 BobWhite_c23972_382 BobWhite_c65478_119 RAC875_c864_88 BS _51 BobWhite_c11235_370 BobWhite_c28635_785 BS _51 BS _51 BS _51 wsnp_ex_c6611_ BS _51 BobWhite_c20015_225 RAC875_c7674_634 CAP7_c1625_317 BS _51 BobWhite_c33069_114 BobWhite_c41673_67 Tdurum_contig66511_957 BobWhite_c11044_322 Tdurum_contig85620_277 BS _51 BobWhite_c5812_440 Excalibur_c64439_87 BS _51 Excalibur_c27915_560 BobWhite_c15152_341 RAC875_c100059_123 Excalibur_c3557_66 IACX886 BS _51 RAC875_c16136_1597 wsnp_ex_c18384_ IACX1240 BS _51 BS _51 BS _51 BS _51 CAP12_c6260_64 Excalibur_rep_c68706_1084 RFL_Contig1823_1044 BobWhite_c23617_167 BobWhite_c294_450 CAP7_c199_62 BobWhite_c44460_821 BS _51 wmc44 BS _51 RAC875_c34361_709 BobWhite_c8293_236 Excalibur_c15389_501 CAP7_rep_c6352_375 GENE-0223_239 BobWhite_rep_c50057_164 BS _51 CAP7_c1788_146 BS _51 BS _51 BobWhite_c20073_382 Ex_c29452_302 BS _51 Tdurum_contig13879_919 Tdurum_contig42960_865 wsnp_ex_c750_ Kukri_c44587_130 Lr71 flanking marker BS LR QTLs for the three rust races tested Possibly Lr71? (originally identified from spelt wheat) 47

48 Introgression of Fusarium head blight resistance from Triticum miguschovae (AGD genome) 48

49 Roblin T. miguschovae at 21 DAI 49

50 Population advanced to F5 DH mapping population 137 lines Superb x MSB55 SNP markers Symptoms on BC2 spike of hybrid between Superb and T. miguschovae at 21 days after point inoculation 50

51 Fusarium incidence on DH population derived from Superb X MSB55 (137 lines) 51

52 QTL for Fusarium index derived from Superb X MSB55 DH population (137 lines) Genetic Map (cm) LG Chromosome Peak LOD* D A A 2.61 *LOD significance threshold (p<0.05) =

53 Introgression of Fusarium head blight resistance from Aegilops cylindrica (CD genome) Odessa homozygous line from AC Superb population DH mapping population lines Manica x Odessa129-1 SNP markers 53

54 QTL for FHB resistance in Manica3 (Maringa) x Odessa129-1(Ae. cylindrica) DH population (319 DH lines) Line FHB QTL Resistance G02*A FHB: 5A + 7D + 2D FHB G02*A FHB: 5A + 7D + 2D FHB G02*A FHB: 5A + 7D FHB G02*A FHB: 5A FHB + Resistant - Susceptible Donor Manica3 Manica3 Chromosome 5A 7D Odessa129 2D 54

55 New approach to Identify New Genes for Resistance to Leaf Rust, Stripe Rust, Powdery Mildew 300 genotypes - Triticum; Aegilops; Synthetic hexaploids Phenotyping - 2-3years Genotyping - SNP assays - Exome capture - sequencing, allelic diversity - KASP assays - discover new genes, assign markers 55

56 Acknowledgements Dr. F. P. Han Dr. B. Zhang Dr. M. Molnar Dr. C. Hiebert Dr. T. Fetch Dr. A. Xue Dr. Steven Xu Dr. A. Malihipour Dr. B. McCallum Dr. A. Rybalka Dr. T.Ternovska Dr. J. Zeng Dr. S. Miller Dr. W. Cao Dawn Chi Danielle Wolfe Kerry Boyle 56

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59 Thank you! 59

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61 Pyramids with combination of Sr33, Sr35, Sr43 Hybrid combination RWG 34 (Sr43) / RL 5405 (Sr33) // RL6099 (Sr35) Progeny A total of 82 DH plants were produced Gene combinations No. of DH lines Sr33 Sr35 Sr43 11 Sr33 _ Sr43 9 _ Sr35 Sr43 8 Sr33 Sr35 _ 3 61

62 Pyramids with combination of Sr35, Sr36, SrCad Hybrid combination AC Cadillac (SrCad) / Lang (Sr36) // RL6099 (Sr35) Progeny 180 F 2 plants screened Gene combinations No. of lines Sr35 Sr36 SrCad 16 Sr35 Sr36 _ 1 Sr35 _ SrCad 1 _ Sr36 SrCad 2 62