Molecular and genetic basis for quantitative partial resistance (QR) of western white pine against Cronartium ribicola

Transcription

1 Molecular and genetic basis for quantitative partial resistance (QR) of western white pine against Cronartium ribicola Jun-Jun Liu, Arezoo Zamany Pacific Forestry Centre, Canadian Forest Service Natural Resources Canada, Victoria, BC, Canada Richard Sniezko Dorena Genetic Resource Center, OR, USDA-FS Presentation at Fourth International Workshop on the Genetics of Host-Parasite Interaction in Forestry, Eugene, OR, July 31-Aug 5, 2011

2 Acknowledgement PFC-CFS-NRCan team: Arezoo Zamany (Lab technician), Holly Williams (Lab technician), Marie Girard-Martel (Ph. D student), Jordana Hutchinson (Co-op student), Craig Hammett (Intern student) USDA-FS team: Dr. Richard Sniezko, Dr. Jerry Hill, Angelia Kegley Supported by: CFS Genomics Fund CFS A-base Fund

3 Major gene resistance (MGR) to Cronartium ribicola (Kinloch et al., Phytopathol. 1999) Susceptible Reaction (Sus) Hypersensitive reaction (HR) -by single dominant gene Cr2

4 A saturated map of the Cr2 linkage ** RGA-AFLP markers (22) RAPD markers (8) AFLP markers (5) SCAR markers (2) (Liu et al., Phytopathol. 2006, 96: ; Liu et al., TGG, 2008, 4: ) On-going work: Fine genetic mapping of Cr2 using a full-sib family with ~3,600 seedlings.

5 Quantitative partial resistance (QR) on WWP stems NC + aecia A BR BR B C D

6 Research Objectives To find what genes/proteins contribute to quantitative partial resistance (QR) To determine defence mechanisms: all QR seed families have a same or different molecular mechanisms underlying their QR phenotypes? To find biomarkers/dna markers potentially for marker-assisted selection (MAS) in molecular breeding

7 Research methods 1. Phenotyping To select seed families To inoculate under controlled conditions To evaluate disease symptoms yearly from 2003 to 2010 To rank resistance levels for each infected seedlings 2. Genotyping - Candidate gene-based association study To select candidate genes/proteins To analyze functions of candidate genes/proteins for anti-fungal activity To find DNA variations (SNPs and indels) of candidate genes by sequencing PCR fragments or PCR clones To perform association genetics analysis for a linkage of genotypes with phenotypes To transfer some SNP/indel markers into PCR markers for MAS

8 Selection of WWP seed families: , 7 5 8~10 11~17 18~20

9 Phenotypic ranking of quantitative partial resistance (QR) Res/Sus Ranking Phenotypic traits Sus 0 Rust dead Sus 1 NC + aecia Sus 3 NC only; no aecia Sus 4 PBR, NC; no aecia Sus 5 BR, PBR, NC; no aecia Res 6 PBR only; no aecia Res 7 PBR & BR; no aecia Res 8 BR only; no aecia Res 10 SS-free (clean stem)

10 How to select candidate genes: Positional Candidates: QTL and physical mapping Functional Candidates: Genomics-based selection: Comparative transcriptomics-ngs (RNA seq) Proteomics-based selection: Comparative proteomics study Homology-based selection: WWP genes with similarities to known genes from other plants

11 Candidate genes we used currently: (1) PmAMP1: anti-microbial peptides (Zamanny et al., Can J Microbiol. in press) (2) PmCh4: class IV chitinase genes (Liu et al., Phytopathol. 2005, 95: ) (3) PmPR10: pathogenesis related protein family 10 (Liu et al., Physiol Mol Plant Pathol. 2006, 68:3-13) (4) PmTLP: thaumatin-like protein genes (Liu et al., Planta, 2009, 231: ; Liu et al., Plant Cell Rep. 2010, 29: )

12 Selection of candidates based on proteomic profiles Ch4s TLPs AMPs

13 PmAMP1 inhibits growth of C. ribicola and other fungal pathogens Recombinant Protein from E. coli A: C. ribicola B: Phellinus sulphurascens C: Ophiostoma montium; D: O. clavigerum

14 PmAMP1 inhibits spore germination of fungal pathogens

15 Effects of rust genotypes on protein levels of PmAMP1 in QR seed families Mean PmAMP1 levels (OD x mm) * * vcr2 AVcr2 * * F10: Sus Others: QR Type 1: F1, 2, 7, 8 0 F1 F2 F5 F6 F7 F8 F10 Seed Families Type 2: F5, 6, 10 PmAMP1 level was measured by Western blot analysis using its specific antibodies

16 PmAMP1 as biomarker to show different defence patterns among QR seed 3 families Pm-AMP1 (ODxmm) Families 1, 2 & 5: Full-sib Bark Rxn Families 6 & 7: Half-sib Bark Rxn Family 8: Half-sib Mechanism X Family 10: Full-sib Susceptible Family 1 Family 8 Family 5 Family 2 Family 6 Family 10 Family 7 Pattern 1: F1 Pattern 2: F5, 8 Pattern 3: F2, 6, 7 Pattern 4: F10 0 Healthy Moderate Severe Dead Scale of Infection Mean PmAMP1 levels in phenotypic categories of QR seed families show different dosage effects of rust infection

17 Genetic association of PmAMP1 SNPs with QR phenotypes Locus df_marker F_Marker p_marker df_model df_error MS_Error Rsq_model Rsq_marker ** ** * ** * ** PmAMP1 SNPs (6/15) contributes 4~7% of QR phenotypic variation Association analysis using MLM (y = marker + Q + K + e) in software TASSEL

18 Association of chitinase isoform with slow canker growth (SCG) Current Yr 1 Yr 2 Yr Current Yr 1 Yr 2Yr R-PmCh4A S-PmCh4A Chitinase protein was detected by Western blot analysis using anti-bodies

19 Genetic association of PmCh4B SNPs with quantitative partial resistance Variation name (a) Location p-marker (b) R 2 -marker A84G exon A112G exon T180C exon T218C intron * A227G intron T247C intron * G252T intron C282A intron * C397T intron * G412T intron * A428C intron ** A467T exon A468C exon T573C intron * C610T intron G691C intron * A695G intron ** G787C exon C806G exon * Indel200 intron * Indel613 intron ** L/S intron *** PmCh4B SNPs (12/21) contributes 8~12% of QR phenotypic variation

20 Transfer of a PmCh4B indel marker into PCR marker M L S PCR Genotyping: fast, easy, efficient. Three genotypes: L/L, L/S, S/S

21 PCR genotyping of chitinase PmCH4B gene to predict QR levels Average Resistance Levels a a ab b ab b a ab b 0 Hap-f Hap-e Hap-d Hap-c Hap-b Hap-a L/L S/S L/S (A) Haplotypes (B) Genotypes (Liu et al., Phytopathol. 2011, 101: )

22 DNA variation of PmTLP- L6 Locus df_marker F_Marker p_marker df_model df_error MS_Error Rsq_model Rsq_marker C69G A757C A838C A888T Inde Inde No association of PmTLP-L6 variations with QR phenotypes

23 Genetic association of PmPR10-2 SNPs with QR phenotypes Locus df_marker F_Marker p_marker df_model df_error MS_Error Rsq_model Rsq_marker E E E E Ind E PmPR10-2 SNPs (21/23) contributes 11~23% of QR phenotypic variation

24 PCR genotyping of PmPR10-2 gene Haplotypes (4): a b c d Genotypes (7): a/b; b/b; c/d; a/a; c/c; b/d; b/c

25 Genetic association of PmPR10-3 SNPs with QR phenotypes Locus F_Marker p_marker df_model df_error MS_Error Rsq_model Rsq_marker ** E * * E E * * * E ** PmPR10-3 SNPs (7/21) contributes 2~5% of QR phenotypic variation

26 Conclusion: Association genetics study identified four pathogenesis-related (PR) genes (PmAMP1, PmPR10-2, PmPR10-3, and PmCh4B) that contribute to quantitative partial resistance (QR) in western white pine. PmAMP1, as a biomarker, shows different defense mechanisms underlying QR in WWP seed families. Cross-pollination of QR seed families with different defense mechanisms would produce progenies with more durable or much broader resistance to WPBR genotypes Western white pine QR phenotypes are potentially predictable by biomarkers and DNA markers for MAS in molecular breeding.

27 Thank you! Any question