Classical and Molecular Breeding for Resistance to Grape Pests & Diseases. Breeding / Genetics Projects

Transcription

1 Classical and Molecular Breeding for Resistance to Grape Pests & Diseases Andrew Walker Geneticist and Breeder Louis P. Martini Endowed Chair in Viticulture Breeding / Genetics Projects Broad and durable nematode resistance: Howard Ferris, Liang Zheng, Kristin Lowe, Britta Hult, Samuel Barros, Chin-Feng Hwang Fanleaf degeneration and rotundifolia: Yuri Takahashi, Brady Smith, Jeff Wheeler, Kenong Xu, Kirsten Skogerson Phylloxera: Jeffrey Granett, Tammy Roush, Hong Lin PD Breeding: Alan Tenscher, David Ramming, Alan Krivanek, Samuel Barros, Kurt Kabica Mapping and Gene Identification Efforts: Summaira Riaz, Alan Krivanek, Kenong Xu, Eileen Sweeney, Rong Hu, Hong Lin

2 Breeding and Genetic Strategies Evaluate and characterize germplasm Screening techniques, selection of parents, taxonomic investigations Crosses to develop new varieties and for genetic characterization Evaluation of selections in greenhouse and field Genetic mapping and genomics efforts to allow markeraided selection and characterization and location of genes Eventually genetic engineering with grape genes Genetic Transformation Characterize the gene of interest Extract DNA - identify and clone a single gene Design a gene construct Transform a plant cell Regenerate the transformed cell Select transformed cells Select transformed plants Release, or further hybridize

3 Why worry about phylloxera? Is resistance durable? How does it function? Phylloxera Resistance American Vitis species range from very high resistance to moderate resistance cinerea to champinii and labrusca none form tuberosities (?) Tissue culture test found similar results, identified selections of resistant species that are fed upon riparia, berlandieri, and rupestris, but

4 Species Total Phylloxera Root tip Feeding Root Base Feeding Eggs/Day V. berlandieri c9017 V. berlandieri c9019 V. berlandieri c9031 V. berlandieri c ab 6.6 def 2.2 bcd 3.5 de 167 bc 5.8 cde 3.6 cde 4.7 f 0 a 0.0 a 0.0 a 0.0 a 878 g 6.0 cde 6.8 gh 7.2 ij St. George 789 efg 8.0 ef 4.6 ef 6.0 g Cab Sauv 1081 h 7.0 def 9.6 i 8.3 k Species Total Phylloxera Root tip Feeding Root Base Feeding Eggs/Day V. riparia h 23.4 g 5.8 fg 7.0 hi V. riparia a 0.0 a 0.0 a 0.0 a V. riparia a 0.0 a 0.0 a 0.0 a V. riparia a 0.0 a 0.0 a 0.0 a St. George 789 efg 8.0 ef 4.6 ef 6.0 g Cab Sauv 1081 h 7.0 def 9.6 i 8.3 k

5 Genetics of Phylloxera Resistance Tammy Roush (with J. Granett), mapping resistance and susceptibility in V. vinifera Aramon x V. rupestris Ganzin population. Population consists of 100 individuals with about 200 SSR markers on map. Develop genetic markers for resistance and susceptibility-tuberosity and nodosity development are genetically independent. Tuberosity QTL Location VMC6e VMCNg1e2-2 Tuberosity 20.6 VMC5c VMC4h6

6 Nodosity QTL Locations VMCNg1e1 0.0 CTG Scu15 VVC VMC3d VVIQ Scu7 VMC4d CTG4952 VMC9h4-2 VMCNg4e10-1 CTG7356 VVMD CTG VMC2a VVMD CTG5983 Fanleaf Degeneration Disease complex caused by grapevine fanleaf virus (GFLV) and vectored by the dagger nematode, Xiphinema index One rootstock alternative exists - O vinifera x rotundifolia 89 Series and others in testing, but not the correct parentage

7 Muscadinia rotundifolia Genetically and morphologically different from Vitis 38 vs 40 chromosomes Hybridization produces very few and sterile offspring Non-host to most Vitis pests and diseases Will not root from dormant cuttings Fruit flavor very unusual Holy grail of grape breeding 89 Populations Olmo directed crosses in 1988 Two V. rupestris females A. de Serres and Wichita Refuge Six M. rotundifolia males Carlos, Cowart, Dixie, Magnolia, Southland and Trayshed Pollen from greenhouse-forced potted rotundifolia plants

8 89 Populations Segregate for and have strong resistance to: Root-knot nematodes Xiphinema index (Xi -dagger nematode vector of fanleaf virus) Phylloxera Pierce s disease (PD), Xylella fastidiosa (Xf) Must be V. rupestris x M. rotundifolia but SSR markers discovered the true parentage Xf & Xi Resistance Mapping x mapping population (9621) Michaeleen Doucleff: completed the 9621 map with AFLP markers and help from Alan and Summaira Summaira Riaz and Eileen Sweeney: completed a map of the 9621 with SSR, AFLP and ESTP markers and increased mapping population size to 182 SSR analysis discovered a parentage mix-up

9 89 Populations ( rupestris x rotundifolia = rupestris x arizonica) Back to the scene of the crime identified all the parents - most from Olmo s 1960 Mexico collection 4 true rup x rot; most were rup x rup (resemble true rup x rot) Have tested all of Olmo s Mexico collection for PD and X. index resistance S A.de Serres Pillans b43-56 b43-36 b b43-12 b arizonica x cordifolia 25 arizonica x vulpina N 96

10 M23 V.rup. Wichita Refuge female plants 55 S V.rup. A. de Serres 18 E b b M v M M W M28 M29 M30 M31 N PD/X. index Resistance of Olmo s Mexico Collections 4R/4S 8R,0I,0S 3R 4S 5R 4I 1S 3R2S 4R,2I,1S 9R 7I 8S 7R 20S

11 Taxonomic Investigations in Vitaceae and Vitis -Eric Wada / D. Potter Eric Wada focusing on arizonica / candicans group with high Xi and Xf resistance Nei s genetic distance b47-06 adeser b43-14 b43-15 b43-36 b b43-56 b47-32 b47-28 b47-27 b47-05 b46-43 b46-48 b42-26 b40-13 b40-14 b40-29 b40-50 b40-59 b40-61 b40-34 b41-13 barret27 b40-51 b45-05 outfh outsg b44-21 b42-33 b44-52 b41-23 b42-55 b42-51 b45-02 b45-26 b45-28 b44-11 b b b b b b b b b46-2 2

12 Xf & Xi Resistance Mapping x mapping population (9621) Michaeleen Doucleff: completed the 9621 map with AFLP markers and help from Alan and Summaira Summaira Riaz and Eileen Sweeney: completed a map of the 9621 with SSR, AFLP and ESTP markers and increased mapping population size to 182 SSR analysis discovered a parentage mix-up Important 9621 ( x ) Linkage Groups fm-vmc7g3 fm-vviu20 f-vmc5g7 fm-vmc6a11 f-vrip93 m-vvio55 fm-vmc3a10 m-vvia23 Sex fm-vvmd34 f-vvia f-vest-83 fm-ctg0193 f-vvin70 fm-vvin94 fm-vng1g1.1 fm-vvis70 fm-vvip26 fm-vmc6e1 fm-ctg fm-vvin64 PdR1 fm-vng3h8 f-ctg fm-vviv69 fm-vmc2a5 f-vvmd24 fm-vmc5a3 m-vmc6c10 m-vrip112 fm-vvip22 f-vmc2c3 m-vmc9f4 fm-vvc34 fm-ctg fm-vmc1e12 fm-vmc9c1 f-vvip05 m-vviq32 f-vvc62 fm-ng1e1 m-scu fm-vvip17 f-vmc9a2.1 f-vmc5h11 f-cd m-vmc5e9 fm-vmc5d11 fm-vviv70 fm-vmc3a7.2 fm-vvip31 fm-vvim03 fm-vviv33 fm-vng3a10 fm-west-4 XiR1a fm-vmc5a10 fm-cgf0660a m-vmc4h9 XiR1

13 89 Conclusions Very strong X. index AND PD resistance from V. arizonica types. We are using markers for Xi resistance and PD resistance. But need rotundifolia to tolerate GFLV infection as O39-16 does. Many more Vitis x Muscadinia hybrids have been made to address fanleaf (rup x rot) looks very promising - resists phylloxera, 3 strains of RKN, X. index, lesion, citrus AND ring nematodes. Vitis x Muscadinia Hybrids 4 rupestris x rotundifolia hybrids including longii / solonis x rotundifolia 1 doaniana (candicans x acerifolia) x rotundifolia 1 andersonii (aestivalis x riparia) x rotundifolia 2 monticola x rotundifolia Several others possible and testing continues Many other crosses have been made in 2004 May have fanleaf tolerance will go into replanted Clos du Bois trial

14 Characterizing Fanleaf Resistance / Tolerance In the process of defining fanleaf tolerance: phytohormones, phytohormone balance, and analytical techniques - Kirsten Skogerson and Brady Smith. Creating new mapping populations with Vitis x Muscadinia crosses - Brady Smith Testing and creating alternatives to O39-16 Half V. vinifera and root-knot nematode susceptible 12 Vitis x Muscadinia selections under test Broad Nematode Resistance Crosses made in 1993 and 1994, screened for ease of rooting Selections were then tested against four Meloidogyne nematodes (R3, HarmA, HarmC and X. index) singly and in combination

15 Broad Nematode Resistance Tested against citrus (Tylenchulus semipenetrans), lesion (Pratylenchus vulnus) and ring (Mesocriconema xenoplax) nematodes Finally, they were tested for root-knot nematode resistance at different temperatures (22, 26, 30 and 32 C) Field trials in Santa Barbara (Chardonnay), Fresno (Fiesta) and Kern (top-worked) Advanced Nematode Resistant Selections (rufo x (rip x DR)) x rip (rufo x (rip x DR)) x champ c (rufo x (rip x DR)) x champ c (rip x Ram) x champ c (rufo x cinerea c9008)

16 (rupestris x rotundifolia Cowart ) Excellent combined resistance Resists ring nematode 71% dormant bench graft success Scheduled for Clos du Bois testing Green-grafting and GFLV inoculation underway Search for Root-knot Nematode Resistance Genes Past work found resistance to Meloidogyne incognita Race 3 to be inherited as a single dominant gene. Created V. champinii Ramsey (R) x V. riparia Riparia Gloire (S). Should also segregate for tolerance to salinity and lime, and segregates for sex. Kristin Lowe is mapping these traits.

17 Range of Parental Characteristics Ramsey (V. champinii) Riparia Gloire (V. riparia) RKN resistant Salt tolerant lime tolerant drought tolerant Deep- rooted high vigor rootstock RKN susceptible? No salinity tolerance? no lime tolerance? no drought tolerance Shallow-rooted low vigor rootstock Nematode Screening Protocol Inoculate with 1,500 J2 nematodes Allow feeding for 6-8 weeks Stain roots with Eosin Y dye- egg masses stained pink Count egg mass number per root mass to determine resistance

18 Ramsey x Riparia Gloire 200 SSR markers run on 186 genotypes Total distance = 1622cM; average distance between markers = 8.3cM LOD 4.0, map coverage 65% High conservation with other maps Riesling x Cab Sav, Syrah x Grenache, and 9621 MappingRKN resistance, sex, salinity tolerance, lime tolerance and rooting angle Ramsey x Riparia Gloire

19 Mapping Rooting Angle Determine genetic component (Nancy Sweet - MS project) of rooting angles in 9715 Compare herbaceous cuttings under greenhouse and tissue culture conditions (CRD 5 reps) Correlate with field grown dormant cuttings Green cutting data maps as a quantitative trait as three QTLs on 9715 map

20 Dormant cuttings in nursery rows Dormant cutting screen Trim all secondary roots

21 Dormant Cutting Screen Measured all primary root angles to nearest 5th degree Grouped roots into two categories: > 3mm, < 3mm Look for quantitative trait loci Rooting angle (whole system vs larger roots) Rooting number (ease of rooting) Dormant Cutting Data Parental controls from different rows Riparia Gloire Ramsey

22 QTLs identified for rooting angle of dormant cuttings VVIU20 VMC6b11 VVIO55 VrZAG93 VVIB23 SEX VVMD34 ] LOD % var VMCNG1g4 VMC5c6 CB CTG VMC3b8 VMC2h4 ] SCU5 VMCNG2h7 CTG LOD % var SCU15 CTG VMCNG1e1 VVIP05 VMC9c1 ] LOD % var 54.7 VMC1e VVC34 CTG VVIP22 VrZAG112 VMC6c10 UDV33 VMC2a VVIN64 VVIP26 CTG VVIN94 CTG VVIN70 QTLs identified for rooting angle of green cuttings VMC1c CTG VMCNG3f11 VMC6d12 ] LOD % var VVIB09 VVC82 AF ] LOD % var CTG VMCNG1b2.2a VMC2d9 VMC3h5 VMC6e VMC3a9 VVIS63 VMCNG1a2 SCU6 VMC2h3 CTG VMC6f5b

23 What s next? Cuttings taken tocorrelate known rootstock vigor with rooting angles -dormant and green Rootstock selections being evaluated for rooting ability, nature of roots (size, diameter, nature of branching and rooting angle) Verify rooting angles are consistent and that green and dormant rootings are equivalent Continue mapping efforts Develop templates for measurements Develop screen for salt tolerance, lime tolerance? Progress Towards Breeding PD Resistant Wine Grapes Andrew Walker and Alan Tenscher S R

24 Pierce s Disease Native to the southern US, Mexico and South America Caused by a xylem-inhabiting bacterium - Xylella fastidiosa Vectored by xylem-feeding leafhoppers (sharpshooters) Kills grapevines within 2-4 years by clogging the xylem -vines desiccate Inoculation and Stem Symptoms

25 Defining Xf / PD Resistance PD resistant cultivars show symptoms in the greenhouse test system Leaf symptoms are far more variable and severe than cane symptoms, and complicated by water/root stresses Lake Emerald - no symptoms and very high Xf levels No cane symptoms and very low Xf levels PD Inoculation: Old vs. new system testing in half the time and half the space

26 Important 9621 ( x ) Linkage Groups fm-vmc7g3 fm-vviu20 f-vmc5g7 fm-vmc6a11 f-vrip93 m-vvio55 fm-vmc3a10 m-vvia23 Sex fm-vvmd34 f-vvia f-vest-83 fm-ctg0193 f-vvin70 fm-vvin94 fm-vng1g1.1 fm-vvis70 fm-vvip26 fm-vmc6e1 fm-ctg fm-vvin64 PdR1 fm-vng3h8 f-ctg fm-vviv69 fm-vmc2a5 f-vvmd24 fm-vmc5a3 m-vmc6c10 m-vrip112 fm-vvip22 f-vmc2c3 m-vmc9f4 fm-vvc34 fm-ctg fm-vmc1e12 fm-vmc9c1 f-vvip05 m-vviq32 f-vvc62 fm-ng1e1 m-scu15 PdR fm-vvip17 f-vmc9a2.1 f-vmc5h11 f-cd m-vmc5e9 fm-vmc5d11 fm-vviv70 fm-vmc3a7.2 fm-vvip31 fm-vvim03 fm-vviv33 fm-vng3a10 fm-west-4 XiR1a fm-vmc5a10 fm-cgf0660a m-vmc4h9 F & F Breeding Strategy D x F = 9621 mapping population Utilize PdR1 marker to screen progeny- Alan Krivanek Repeatedly backcross to noble wine varieties Currently have 3 year (seed to seed) generation time Progeny from 2005 crosses will have between 75% and 87% vinifera and PD resistance

27 PdR1 Marker Segregates as a single dominant gene Tested in A81 (75% vinifera) and 0062 (F2-7; 50% vinifera) families Used in the crosses of R selections as parents for 2005 crosses to wine grape types (F2-35; 50% vinifera) Could switch mapping populations and will have greenhouse data by Fall/Winter PD Resistant Table Grapes Collaborationwith David Ramming since 1999 Used his advanced vinifera table and raisin selections and crossed with multiple PD resistance sources Collaboration continues, but focus for our group shifted to wine grapes in 2004

28 Proven Potential of Classical Breeding A81-17 = A38-17 x % vinifera = B x Highly resistant to PD and has PdR1 marker From rupestris x arizonica to vinifera quality in6 years A81-17 Next Steps Characterizing Grape Resistance Genes Increase population sizes to allow for greater recombination and finer mapping resolution. Add AFLP markers to SSR-based framework maps. Utilize characterized EST sequences and Resistance Gene Analogs (RGAs) on the genetic map. Develop BAC libraries and physical mapping efforts. Utilize the sequenced grape genome!

29 Resistance Gene Utilization Splice out genes of interest. Utilize constitutive and tissue specific promoters to transform grapes. Confirm gene function in susceptible genotypes range of cultivars, environments and transformation events. Select for desired / consistent viticultural and enological trait expression. Keep breeding programs going! Thanks!