Fine Mapping and Candidate Gene Characterization of the Pepper Bacterial Spot Resistance Gene bs6 Rebecca L. Wente, Jian Li, Samuel F. Hutton, Upinder Gill, Jeffrey B. Jones, Gerald V. Minsavage, Robert E. Stall
INTRODUCTION
Bacterial Spot Disease of pepper and tomato Caused by multiple Xanthomonas species Symptoms include lesions on leaves and fruit Reduces marketable yield (Ritchie, 2000) Hot and humid environments (Stall and Civerolo, 1993) C. Hardin L. Kumar
Bacterial Spot Control Bacterial spot was controlled with copper and streptomycin Xanthomonas has developed resistance to both (Jones et al., 1991, Stall and Thayer, 1962) Cultural controls, bacteriophages, and SAR inducers (Obradovic et al., 2004) Bacterial spot resistance is a major objective for many breeding programs
Tomato Resistance Genes Gene Source Resistance Rx3 (Yang et al. 2005) Hawaii 7998 HR to Race T1 (Jones and Scott 1986) Xv3 (Rx-4) (Wang et al. 2011, Robbins et al. 2009, Zhao et al. 2015) Hawaii 7981 (PI128216) HR to race T3 (Scott et al. 1996, Scott et al. 1995) Xv4 (Astua-Monge et al. 2000) S. pennelli LA716 HR to race T3 and T4 (Jones et al. 1995) Shifts in bacterial populations and breakdown of avr genes Most tomato resistance is based off QTLs
Pepper Resistance Genes Bs1-4 are dominant resistance genes Elicit an HR Most breeding efforts use Bs1, Bs2, and/or Bs3 All are susceptible to race 6 Mutations in avr genes and shifting bacteria populations have broken resistance (Gassmann et al., 2000, Stall et al., 2009)
Pepper Resistance in Tomato Bs2 is a bacterial spot resistance gene from pepper AvrBs2 is highly conserved across Xanthomonads However, mutant strains have overcome resistance EFR from Arabidopsis provides broad spectrum resistance to pathogens Transgenic tomatoes with Bs2+EFR have lower disease ratings and increased yield (Horvath et al., 2012)
Non Transgenic Transgenic
bs5 and bs6 bs5 and bs6 are recessive, non-hypersensitive response resistance genes (Jones et al., 2002) Pyramiding bs5 and bs6 results in resistance to races 0-10 (Vallejos et al., 2010) Includes resistance to race 6 Syntenic tomato genes Potential for CRISPR/Cas9 genome editing The location of bs5 was recently discovered bs6 has not yet been located
Population Early Calwonder (ECW) is susceptible An ECW near-isogenic line containing bs6 was created (60R) (Vallejos et al. 2010) ECW and 60R were crossed to produce F 2 populations with recombination
GBS GBS of ECW, 60R, and 93 F 2 plants (Elshire et al., 2011) bs6 was mapped to a 27Mb region on chromosome 6 (p<0.0001) Between loci 53 and 54
Objectives Genotype and phenotype recombinants to fine map the region using markers associated with the bs6 locus Identify and characterize annotated genes in the fine mapped interval
Genotype and phenotype recombinants to fine map the region using markers associated with the bs6 locus OBJECTIVE 1
Disease Screens Infiltration of X. euvesicatoria Race 6 Maintained in greenhouse for 3 weeks Phenotyped as resistant or susceptible
Mapping and Fine Mapping Recombinants were selected in the 27 Mb interval identified by GBS Selfed to produce recombinant inbred lines (RILs) Developed CAPS and HRM markers RILs were genotyped and phenotyped for disease response Compared to fine map the bs6 region
Fine Mapping Selected 58 F 3 RILs CAPS markers 5.6 Mb region Between markers E and I
Fine Mapping No. RILs Per Recombinant Haplotype Distance Markers ECW 60R 1 1 1 5 1 1 1 2 19 3 550 Kb 21 Kb 666 Kb 560 Kb 757 Kb 2.93 Mb Marker 1 - + + + + + + - - - - - Marker 2 - + + + + + + + - - - - Marker 3 - + + + + + + + + - - - Marker 4 - + - - + + + + + - - - Marker 5 - + - - - + + + + + - - Marker 6 - + + - - - + + + + + - Marker 7 - + + - - - - + + + + + Phenotype S R S S R R R R R S S S
Identify and characterize annotated genes and further fine map the interval OBJECTIVE 2
Predicted Candidate Genes 13 annotated genes 1.22 Mb region C. annuum genome release 1.55 Predicted Protein Family Number of Genes Kinase 6 Major Intrinsic Protein 1 Ubiquitin-Conjugating Enzyme Lipid Binding Protein 1 Flavonol Synthase 1 Formyltetrahydrofolate deformylase Unknown 2 1 1
Sequencing Sequenced 13 genes ECW and 60R Identified polymorphisms Designed new markers in 1.22 Mb Interval Further fine mapped the interval Sequenced candidate genes of 3 RILs recombinant in new interval Fine mapped based on polymorphisms
Further Fine Mapping Distance Marker Controls No. RILs Per Recombinant Haplotype ECW 60R 7 1 1 1 1 7 1 1 1 5 2 1 10 2 2 E - + + + + + + + + + + - - - - - - 4.33 Mb a - + - + + + + + + + + + - - - - - 92 Kb b - + - + + + + + + + + + - - - - - 404 Kb c - + - + + + + + + + + + + - - - - 162 Kb d - + - - + + + + + + + + + - - - - 46.7 Kb e + - - - + + + + + + + + - - - - 34.6 Kb f - + - - - - + + + + + + + - - - - 34.1 Kb g - + - - - - + + + + + + + - - - - 551 Kb h - + - - - - + + + + + + + + - - - 552 Kb i - + - - - - - + + + + + + + - - - 533 Kb j - + - - - - - - + + + + + + - - - 67.1 Kb k - + - - - - - - - + + + + + + - - 163 Kb l - + - - + - - - - - + + + + + + - 1.37 Mb m - + - - + - - - - - + + + + + + + 715 Kb n - + - - + - - - - - - + + + + + + 363 Kb I - + - - + - - - - - - + + + + + + S R S S S S R R R R R R R S S S S Resistance Phenotype
Sequencing Results Candidate Gene Gene 1 Gene 2 Mutation Effects Single bp substitution nonsense mutation Single bp insertion nonsense mutation Gene 3 Single missense mutation Gene 4 Three missense mutations Gene 5 One silent mutation, three missense mutations
Syntenic Genes 1.15 Mb Tomato gene 17 (chromosome 10) No hit No hit 1.25 Mb Blast score 200 Blast score <200
Conclusions Pepper Genome ~3,400 Mb ~35,000 Genes Fine Mapping 27 Mb Interval 1.2 Mb Interval 13 Genes Gene Expression 620 Kb Interval 5 Genes Identifying bs6 GBS Sequencing bs6 in Tomato bs6 has a weak effect and can be difficult to phenotype Developed tightly linked markers for MAS Syntenic genes in tomato could accelerate genetic engineering efforts Pyramiding bs5 and bs6 should achieve more durable resistance in peppers and tomatoes
2Blades Foundation Committee Samuel Hutton Hugh Smith Jeffrey Jones Tomato Breeding Lab Jessica Chitwood Dolly Cummings Tim Davis Nate Brown Reza Shekasteband Jose Diaz Kazuyo Ueda Keri Druffel Judith Lopez Edgar Sierra Jasmine Lopez John Smeda Bacterial Plant Pathology Lab Jeffrey Jones Jerry Minsavage Thank You
Questions
Works Cited Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S., and Mitchell, S.E., 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 6:e19379. Gassmann, W., Dahlbeck, D., Chesnokova, O., Minsavage, G.V., Jones, J.B., and Staskawicz, B.J., 2000. Molecular evolution of virulence in natural field strains of Xanthomonas campestris pv. vesicatoria. J. bacteriology 182:7053-7059. Horvath, D.M., Stall, R.E., Jones, J.B., Pauly, M.H., Vallad, G.E., Dahlbeck, D., Staskawicz, B.J., and Scott, J.W., 2012. Transgenic resistance confers effective field level control of bacterial spot disease in tomato. PLoS One 7:e42036. Jones J, Scott J (1986) Hypersensitive response in tomato toxanthomonas campestris pv. vesicatoria. Plant Dis Jones, J., Minsavage, G., Roberts, P., Johnson, R., Kousik, C., Subramanian, S., and Stall, R., 2002. A non-hypersensitive resistance in pepper to the bacterial spot pathogen is associated with two recessive genes. Phytopathology 92:273-277. Jones, J., Woltz, S., Jones, J., and Portier, K., 1991. Population dynamics of Xanthomonas campestris pv. vesicatoria on tomato leaflets treated with copper bactericides. Phytopathology 81:714-719. Lorieux, M., 2012. MapDisto: fast and efficient computation of genetic linkage maps. Molec. Breed. 30:1231-1235. Lu, F., Lipka, A.E., Glaubitz, J., Elshire, R., Cherney, J.H., Casler, M.D., Buckler, E.S., and Costich, D.E., 2013. Switchgrass genomic diversity, ploidy, and evolution: novel insights from a network-based SNP discovery protocol. PLoS genet. 9:e1003215. Obradovic, A., Jones, J., Momol, M., Balogh, B., and Olson, S., 2004. Management of tomato bacterial spot in the field by foliar applications of bacteriophages and SAR inducers. Plant dis. 88:736-740. Ritchie, D., 2000. Bacterial spot of pepper and tomato. The Plant Health Instructor PHI-I-2000-1027-01/DOI: 10.1094. Robbins MD, Darrigues A, Sim S-C, Masud MAT, Francis DM (2009) Characterization of hypersensitive resistance to bacterial spot race T3 (Xanthomonas perforans) from tomato accession PI 128216. Phytopathology 99 (9):1037-1044 Stall, R. and Civerolo, E., 1993. Xanthomonas campestris pv. vesicatoria: cause of bacterial spot of tomato and pepper, p. 57-60. In: Swings, J. and Civerolo, E. (eds.), Xanthomonas. Chapman & Hall, London, UK. Stall, R. and Thayer, P., 1962. Streptomycin resistance of the bacterial spot pathogen and control with streptomycin. Plant Dis. Rep. 46:389-392. Stall, R.E., Jones, J.B., and Minsavage, G.V., 2009. Durability of resistance in tomato and pepper to Xanthomonads causing bacterial spot. Annual rev. phytopathology 47:265-284. Vallejos, C.E., Jones, V., Stall, R.E., Jones, J.B., Minsavage, G.V., Schultz, D.C., Rodrigues, R., Olsen, L.E., and Mazourek, M., 2010. Characterization of two recessive genes controlling resistance to all races of bacterial spot in peppers. Theoret. appl. Genet. 121:37-46. Wang H, Hutton SF, Robbins MD, Sim S-C, Scott JW, Yang W, Jones JB, Francis DM (2011) Molecular mapping of hypersensitive resistance from tomato Hawaii 7981 toxanthomonas perforans race T3. Phytopathology 101 (10):1217-1223 Yang W, Sacks EJ, Lewis Ivey ML, Miller SA, Francis DM (2005) Resistance in Lycopersicon esculentum intraspecific crosses to race T1 strains of Xanthomonas campestrispv. vesicatoria causing bacterial spot of tomato. Phytopathology 95 (5):519-527 Zhao B, Cao H, Duan J, Yang W (2015) Allelic Tests and Sequence Analysis of Three Genes for Resistance toxanthomonas perforans Race T3 in Tomato. Hortic Plant J 1 (1):41-47
Genotyping By Sequencing GBS of ECW, 60R, and 93 F 2 plants (Elshire et al., 2011) Illumina HiSeq 2500 system Raw reads were processed with the UNEAK pipeline (Lu et al,. 2013) Linkage groups for bs6 were constructed using MapDisto (Lorieux, et al., 2012) LOD min=5.0 r max=0.3 Classical option to estimate recombination fraction Kosambi mapping function Identified the polymorphic SNPs for genetic mapping
Distance (Mbp) 100,000 bp Chromosome 6 Markers Marker A Marker B Marker C Marker D Marker E Marker F Marker G Phenotype S S Informative RILs S R R S Predicted Genes Gene 1 Gene 5 Gene 7 Gene 11 Gene 13 Gene 2 Gene 6 Gene 8 Gene 12 Gene 3 Gene 9 Gene 4 Gene 10 Marker F Marker E Marker D Marker C Marker B
Gene Expression Experiments Gene expression experiments are ongoing ECW and 60R plants were inoculated with Xanthomonas, inoculated with water, and uninoculated Tissue Collected at 1, 24, and 48 hours RNA extraction DNase treatment cdna Synthesis qrt-pcr of each gene Reference Genes Complete for all but one gene Analysis using the ddct method