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 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

Bacterial Spot Control

Tomato Resistance Genes

• Shifts in bacterial populations and breakdown of avr genes

– Most tomato resistance is based off QTLs

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)

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 F2

populations with recombination

GBS

• GBS of ECW, 60R, and 93 F2 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

OBJECTIVE 1

Genotype and phenotype recombinants to fine map the region using markers associated with the bs6 locus

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 F3 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

Marker 1 - + + + + + + - - - - -550 Kb

Marker 2 - + + + + + + + - - - -21 Kb

Marker 3 - + + + + + + + + - - -666 Kb

Marker 4 - + - - + + + + + - - -560 Kb

Marker 5 - + - - - + + + + + - -757 Kb

Marker 6 - + + - - - + + + + + -

2.93 Mb

Marker 7 - + + - - - - + + + + +

Phenotype S R S S R R R R R S S S

OBJECTIVE 2

Identify and characterize annotated genes and further fine map the interval

Predicted Candidate Genes

• 13 annotated genes

– 1.22 Mb region

• C. annuumgenome release 1.55

Predicted Protein Family

Number of Genes

Kinase 6

Major Intrinsic Protein 1

Ubiquitin-Conjugating Enzyme

1

Lipid Binding Protein 1

Flavonol Synthase 1

Formyltetrahydrofolatedeformylase

1

Unknown 2

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 MappingDistance 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

CandidateGene

Mutation Effects

Gene 1 Single bp substitution nonsense mutation

Gene 2 Single bp insertion nonsense mutation

Gene 3 Single missense mutation

Gene 4 Three missense mutations

Gene 5 One silent mutation, three missensemutations

1.15 Mb

1.25 Mb

Tomato gene 17 (chromosome 10)

No hit No hit

Blast score ≥200Blast score <200

Syntenic Genes

Conclusions

• Pepper Genome

• ~3,400 Mb

• ~35,000 Genes

GBS

• 27 Mb Interval

Fine Mapping• 1.2 Mb Interval

• 13 Genes

Sequencing

• 620 Kb Interval

• 5 Genes

Gene Expression • Identifying bs6

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

Thank You

• 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

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. phytopathology47:265-284.

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 F2 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

Markers

Ma

rke

r E

Ma

rke

r F

Ma

rke

r G

Chromosome 6

Distance (Mbp)

Ma

rke

r C

Ma

rke

r B

Ma

rke

r A

Predicted Genes

Informative RILs

S

S

S

S

R

R

Phenotype

Gene 1

Gene 4

Gene 6Gene 2

Gene 3

Gene 7

Gene 8

Gene 9

Gene 10

Gene 11

Gene 12

Gene 13Gene 5

Ma

rke

r B

Ma

rke

r C

Ma

rke

r E

Ma

rke

r F

Ma

rke

r D

Ma

rke

r D

100,000 bp

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