Elucidation of the Resistance

Mechanism in Sorghum bicolor cv.

Bk7 to Colletotrichum sublineola

Terry Felderhoff

University of Florida

Sorghum on the Move

2002

2012

Source: USDA Census of Agriculture

• Interest in sorghum for biofuels and chemicals • Production in new areas ▫ Increase of 75% in southeastern states ▫ Need for regional adaptation

• Existing sugarcane infrastructure in Florida ▫ Sweet sorghum and sugarcane are complementary crops

Anthracnose

• Colletotrichum

sublineola ▫ Thrives in warm, humid

conditions Major problem in

southeastern U.S.A.

▫ Diverse natural population Genetic variation found

between geographic areas

▫ Infects all aerial parts of

sorghum

▫ 70% yield reduction Source: http://www.plantwise.org /KnowledgeBank/Datasheet.aspx

Colletotrichum sublineola

• Anthracnose ▫ Aggressive fungal disease

Host species specificity

▫ Infects and kills living tissue

▫ Hemibiotrophic

Starts with biotrophic stage No cell death

Shifts to necrotrophic stage Cell invasion and death

Source: http://www.padil.gov.au/thai-bio/Pest/Main/140459/30916

Source: Crouch and Beirn, Fungal Diversity, 2009

Anthracnose Management

• Unmitigated disease damage is a challenge to high yield production in southeast

• Fungicide can control disease ▫ Can become expensive ▫ Health risks

• Resistant germplasm ▫ Genetic resistance exists ▫ Bk7

Anthracnose resistant Florida inbred line Mechanism unknown

▫ Transfer resistance into elite lines

Aided by knowledge of allelic location

Bk7 Early Hegari Source: Dr. Wilfred Vermerris, UF

Mapping Population

• Population of lines selected/created for mapping alleles of interest ▫ Two inbred parents crossed

Bk7 and Early Hegari

▫ Heterozygous F1 selfed ▫ Multiple F2 progeny grown ▫ Continued selfing within lines ▫ 135 F4 and F5 lines

• Increased recombination ▫ Genetic mosaic ▫ Disease resistance similar within

lines, but differing between lines

Genotyping by Sequencing

• Truncated genome sequencing

• Identification of Single Nucleotide Polymorphisms (SNPs)

• Relatively fast, inexpensive, and high data output

• GBS data generated at Cornell University

Source: http://cbsu.tc.cornell.edu/lab/doc/GBS_overview_20111028.pdf

Marker Data

• Markers filtered (removed) for ▫ <10% minor allele frequency (MAF)

SNPs of sequencing errors have low MAF

▫ Inbred heterozygosity (FIT) score of <0.8 Expected level of heterozygosity in inbred line Low quality SNPs have lower FIT

• 2012 ▫ 101,075 unfiltered markers ▫ 2,453 filtered markers

• 2013 ▫ 130,569 unfiltered markers ▫ 6,727 filtered markers

Disease Trial Designs

• Field trial in Quincy, FL

▫ Two replications

▫ Natural and manual inoculation

• Greenhouse trial at UF

▫ Three replications

▫ Manual inoculation

▫ Observed after 4 weeks

• Field trial in Live Oak, FL

▫ Two replications

▫ Natural inoculation

▫ Observed during soft dough stage

• Field trial in Citra, FL

▫ Three replications

▫ Natural inoculation

▫ Observed during soft dough stage

• Experiments performed during 2012 and 2013 2012 2013

Phenotype Analysis

• Scored on scale of 1-5

▫ Disease score non-linear

▫ Majority of variation derived from 1/2 vs all other scores

▫ QTL analysis uses linear models

▫ Data suggest dominant gene(s) Expected ratio for single gene:

F4 56.25 : 43.75

F5 53.125 : 46.875

Expected ratio for two genes: F4 80.86 : 19.14

F5 78.03 : 21.97

2013 Live Oak F4

Mean Freq Cum Pro

1 15 57.7

1.5 3 69.2

2 2 76.9

2.5 1 80.8

3 2 88.5

3.5 2 96.2

4 1 100

2013 Live Oak F5

Mean Freq Cum Pro

1 57 52.3

1.5 8 56.6

2 14 72.5

2.5 7 78.9

3 9 87.2

3.5 3 89.9

4 10 99.1

5 1 100

Phenotype-Genotype Analysis

• Determine distribution of marker alleles across infected and uninfected lines ▫ Fisher’s exact test, -log10 of p-value

▫ H0: no difference in frequency of allele between contrasting disease phenotypes

▫ Ha: high frequency of Bk7 alleles in asymptomatic plants

▫ False discovery rate (FDR)with α=0.95 used as threshold

Marker Allele Early Hegari Bk7

Disease Phenotype

Asymptomatic a b a+b

symptomatic c d c+d

a+c b+d

a+b+c+d=n

Methods for Phenotypic Grouping

• Four different methods to create groups of contrasting lines ▫ Original 5-point scale reduced for 2x2 Fisher’s test

• Extremely resistant method ▫ Average scores of 1 vs. 2/3/4/5

• Resistant method ▫ Average scores of 1/2 vs. 3/4/5

• Susceptibility method ▫ Average scores of 4/5 vs. 1/2/3

• Consistency method ▫ Scores that were always 1/2 across reps vs. scores that

were always 4/5 across reps

Agreement Among Disease Scores

• Cohen’s kappa agreement

• Comparison of consistency within phenotypic grouping ▫ Consistency method has greatest kappa scoring

▫ Susceptible method has lowest kappa scoring

▫ Resistant method > extremely resistant method

Susceptible Method Consistency Method

Citra Quincy Leaf Area Midrib Citra Quincy

Leaf Area Midrib

Live Oak 0.12* 0.17* -0.02 0.12 Live Oak 0.45* 0.19* 0.26* 0.17*

Citra 0.66* -0.01 -0.02 Citra 0.15* 0.31* 0.18*

Quincy -0.01 -0.02 Quincy -0.03 -0.06

Leaf Area -0.02 Leaf Area 0.20*

Chromosomal Rearrangement

• Marker order of GBS based on reference genome, but parental lines unrelated to BTx623 ▫ Identify chromosomal rearrangements in population

compared to reference genome

• Markers analyzed with JoinMap ▫ Linkage mapping orders markers based on

recombination frequencies

• Marker ordering by JoinMap compared with reference order ▫ Inversions, translocations, or deletions

Comparison of JoinMap to GBS Order Chromosome 7

Current Research

• Identification of candidate resistance genes in loci identified with mapping analysis • Narrowing down list of candidate genes ▫ Finer mapping of loci to narrow down area to

identify candidate genes ▫ UF cultivars derived from Bk7 can be used to narrow

down candidates

• Validate candidate genes ▫ qRT-PCR after inoculation with anthracnose to test

for increased gene production ▫ VIGS using Brome mosaic virus to reduce protein

product and test reaction to anthracnose inoculation

Current Research

• Microscopy of pathogen development ▫ Tissue sectioning of infected tissues at different

fungal developmental stages

▫ Staining to identify production of biochemicals

• Multiple fungal isolate test ▫ Using fungal isolates from different geographical

areas on most resistant and susceptible lines

▫ Narrow vs broad resistance

Thank you!

▫ Dr. Wilfred Vermerris

▫ Dr. Lauren McIntyre

▫ Dr. Jim Olmstead

▫ Dr. Jeff Rollins

▫ Dr. Ana Saballos

▫ Dr. Alisa Huffaker

▫ Jose Lopez

▫ John Bonkowski

▫ Undergraduate students

▫ UF Plant Disease Clinic

▫ SunGrant Initiative

▫ USDA-BRDI