PCR-based Species Identification and Gene Tagging

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"PCR-based Species Identification and Gene
Tagging"

• Clint Magill, Ramasamy Perumal, C-L Yao
• Department of Plant Pathology Microbiology
• TAMU

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The Species to ID

• Downy Mildews Primary Host
• Peronosclerospora sorghi Sorghum
• P. maydis Maize
• P. sacchari Sugarcane
• P. philippinensis all the above
• Sclerospora graminicola pearl millet
• Peronospora sparsa rose

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Common Characteristics of these Pathogens

• Oomycetes-not true fungi
• Asexual conidia if dew
• Oospores from sexual reproduction in crop residue
• Obligate parasites
• Cant grow in pure culture
• Cant cross on demand
• Species differ in host preference and size of
  asexual conidia
• Unknown genome size
• No marker mutants or gene maps

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P. sorghi

• conidia-asexual spores
• antheridum and oogonium forming in leaf tissue

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• Chenglin Yao, PhD
• China quarantine service
• Protect against P. philippinensis
• Classify via average spore size in grain samples?
• We have P. sorghi

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Can we use pathogen DNA to detect the pathogen ?

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Detection of infected progeny from a systemically
infected maize plant via dot-blot hybridization
using genomic P. sorghi gDNA
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Can we use DNA to differentiate closely related
species?
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Peronosclerosporsa DNA PvuI digests
sacchari/cane leaf
sorghi/maize leaf
sacchari-G-maize leaf
sacchari-Y-maize leaf
sorghi-Thailand
philip/cane leaf
sorghi-Thailand
sorghi pt1/leaf
sorghi-3 leaf
srghi pt-1
srghi pt-3
maydis
Probe P. maydis gDNA
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Probe Development- P. sorghi
Repeated Sequence Clones, Hybridized to P. sorghi
gDNA
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Probe Development- P. sorghi
Repeated Sequence Clones, Hybridized to P. maydis
gDNA
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Peronosclerosporsa DNA PvuI digests
sacchari/cane leaf
sorghi/maize leaf
philip/cane leaf
sacchari-G-maize leaf
sacchari-Y-maize leaf
sorghi-Thailand
sorghi-Thailand
sorghi pt1/leaf
sorghi-3 leaf
srghi pt-3
srghi pt-1
maydis
Probe pMLY987
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Peronosclerosporsa DNA PvuI digests
sacchari/cane leaf
sorghi/maize leaf
philip/cane leaf
sacchari-Y-maize leaf
sacchari-G-maize leaf
sorghi-Thailand
sorghi-Thailand
sorghi pt1/leaf
sorghi-3 leaf
srghi pt-3
srghi pt-1
maydis
Probe pCLY83
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Dot-blot sensitivity test-probe pMLY12
ng of DNA
P. sorghi pathotype 1
P. sorghi pathotype 3
pt1-Infected sorghum leaf
pt3-Infected sorghum leaf
Botswana-Infected maize leaf
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Dot-Blot Hybridizations proble pMLY12
Colletotrichm graminicola
P. sorghi
Infected seed, with glume
Acremonium strictum
Healthy seed, with glume
Fusarium moniliforme
Infected seed, no glume
Infected seed, glumes 40d
Healthy seed, no glume
Infected seed, no glumes 40d
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How about converting clones to sequences for PCR?
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P. sacchari/maize leaf
P. sacchari/maize leaf
P. sacchari/cane leaf
P. phillip.//cane leaf
P sorghi/Thailand-1
P sorghi/Thailand-2
infected leaf pt1
infected leaf pt3
maize leaf _Bots
P sorghi pt1
P sorghi pt3
P. maydis
Marker
1 kb
PCR using pCLY83 based primers
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P. sacchari/maize leaf
P. sacchari/maize leaf
P. sacchari/cane leaf
P. phillip.//cane leaf
P sorghi/Thailand-1
P sorghi/Thailand-2
infected leaf pt1
infected leaf pt3
maize leaf _Bots
P sorghi pt1
P sorghi pt3
P. maydis
Marker
1 kb
PCR using pMLY12 based primers
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This sequence has been used to develop Real Time
PCR primers probe for rapid detection of SDM
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Are other regions useful for species comparisons?
How about rDNA? -present in many copies per
genome -extremely conserved in LS and SS
regions -sequences maintained by concerted
evolution -nontranscribed spacers between
units -internal spacers cut out after
transcription -data from other species suggest
these represent species-specific sequences
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16s rDNA
5
1
26s rDNA
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ITS1
ITS2
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2
5.8s
Primer sites for conserved fungal ITS primers as
described by White, Lee, Bruns and Taylor
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Marker
P. sorghi Thai1
P. sorghi Thai2
P. sorghi Thai1
P. sorghi Thai2
P. sorghi Thai1
P. sorghi Thai2
P. sorghi Thai1
P. sorghi Thai2
P. sorghi Thai2
P. sorghi Thai1
P. sorghi pt1
P. sorghi pt1
P. sorghi pt1
P. sorghi pt1
P. sacchari
P. sorghi pt1
P. sacchari
P. sacchari
P. sacchari
P. sacchari
P. maydis
P. maydis
P. maydis
P. maydis
P. maydis
flanking PCR
Cfo I
Hpa II
Sau 3AI
MspI
Fragments detected with P. sorghi rRNA probe
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P. sorghi Thai1
P. sorghi Thai2
P. sorghi Thai1
P. sorghi Thai2
P. sorghi pt1
P. sorghi pt1
P. sacchari
P. sacchari
P. maydis
P. maydis
M
M
ITS 1
ITS 2 5.8s
PCR using conserved ITS primers
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Peronosopora sparsa

• downy mildew of roses
• Imported from California?
• MS Thesis, Sharon Ross

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Peronospora sparsa-specific ITS primers
F1 Peronospora sparsa TGGCTGGCTGCTACTGGGC Perono
spora tabacini ---------A--G---A-A Phytophthora
megakaya --- --T----G-----A Chladosporium
sp G--- --GG--- -C---T Botrytis sp
A-A-CTC- -CCT--T-T F7 Peronospora sparsa
TATCGCGAGCGTTTGGGCCC Peronospora
tabacini -----T-------CT-A-- Phytophthora
megakaya ------------------T Chladosporium sp -
--AAACT-T-GC-TAA- Botrytis sp -T-T-T T--T ---
--G
ATG
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Nested Primer PCR for Peronospara sparsa ITS
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Sclerospora graminicola

• pearl millet downy mildew
• Joint project with ICRISAT
• Aparna Viswinathan, MS
• Dale Hess, Bamako, Mali
• Sivi Siviramakrisnan, India

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PCR Amplification of ITS-2 Using Standard Primers
1
1.5
2.5
Magnesium concentration (mM)
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ITS Amplification Oomycete Primers
OLD
NEW
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other variable sequences potentially useful for
probe development

• COX spacer between mitochondrial cytochrome
  oxidase I and II subunits
• ?-tubulin (intron 3 region) (single copy)
• Translational elongation factor-1? (intron 4
  region)

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COX spacer Consensus (P. sorghi) (hits are all
Oomycetes)
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COX spacers
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?-tubulin sequence comparisons for maize vs
sugarcane isolates
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• EF-1? base sequence comparisons

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Tagging and mapping disease resistance genes in
sorghum -Ramasamy Perumal
-Seriba Katilé -Clint Magill
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Anthracnose resistance
Objective Identify DNA-based markers that
co-segregate with gene Cg1 that confers
resistance to Colletotrichum graminicola Tools
AFLP SSR markers (from BAC contig sequences)
Cross SC748-5 (resistant) by BTx 635
(susceptible)
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The AFLP Technique
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Segregation for anthracnose resistance and
susceptibility in F2 population from a cross
between BTx623 (susceptible parent) and SC748-5
(resistant parent)
Pedigree Year F1 reaction F2 population F2 population Ratio fit ?2-value
Pedigree Year F1 reaction Resistant Susceptible Ratio fit ?2-value
BTx623SC748-5 1999 Resistant 235 60 31 3.41
BTx623SC748-5 2000 Resistant 105 41 31 0.73
This is a subset of the population tested by
Mehta et al. (2005)
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F2 progeny selected for DNA analysis
Parents BTx 623 x SC
748-5 (Susceptible)
(Resistant)
F1 self pollinations
Homozygous Homozygous
Heterozygous Resistant Susceptible
Segregating (29) (29)
(13)
Disease genotypes of F2 individuals verified in
F3/F4 for use in gene tagging These must be
correct!!
Disease Scoring College Station 1999
2000 College Station 2003
Georgia 2003 College Station 2004 - 4
consecutive weeks data (Dr. L.
Prom, USDA)
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Cosegregation of AFLP marker Xtxa6227 and the Cg1
locus in F2-3 progeny derived from the cross of
BTx623 and SC748-5. AFLP templates from parental
inbreds BTx623 (cg1cg1) and SC748-5 (Cg1Cg1) and
IS3620C (mapping parent) were run as controls to
aid in the identification of polymorphic bands.
Co-segregation of dominant SSR marker SSR 1 and
the cgf1 locus in F2-3 progeny derived from the
cross of ATx623 and SC748-5. Genomic DNA from
parental inbreds BTx623 (cg1cg1) and SC748-5
(Cg1Cg1) were run to aid in the identification of
parental alleles for SSR 1. The amplified band
from the SSR 1 allele was 152 bp (BTx623) or 155
bp (SC748-5)
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Relative positions of amplified fragment length
polymorphism (AFLP) and Simple Sequence repeats
(SSRs) markers linked to gene cg1 in the off-end
of linkage group J in a segregating population
derived from the cross BTx623SC748-5
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Conclusions

• SC748-5 - major dominant gene
• AFLP - Xtxa6227 - linked in coupling phase 1.8
  cM from cg1 (LG 5)
• SSR1- (CT)8 repeat motif - 3.5 cM
• 5CCATGAATGGACTCGCTGT3 5CGGAACAGTAAAACCAACGA
  3 - BAC clone 117e5_04 that contains
  txa455(05)
• First report placing a gene for resistance to
  Colletotrichum graminicola on the sorghum
  molecular marker map

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Identification, chromosome location, and
diagnostic markers for a new gene (Shs1) for
resistance to sorghum head smut

• Objectives
• Determining the inheritance and chromosomal
  location of the of sorghum head smut (Sporisorium
  reilianum) resistance gene Shs1 from BTx635
• Identification of markers linked to the Shs1
  using high throughput AFLP technology
• Conversion of dominant AFLP marker into
  STSs/SCARs marker derived by cloning and
  sequencing specific AFLP marker

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Segregation ratio of the F 23 families (B1
BTx635)
Total F 23 families Resistant families Susceptible families Segregation ratio ?2 value
161 134 (85 Resistant 49 Heterozygous) 27 31 5.82
Markers linked to Shs locus in LG 03
AFLP marker Designation Size of the Marker (bp) Primer Combination Map Distance (cM)
Xtxa 3450 168.7 Ectg Mccc 5.8
Xtxa 2058 279.7 Egga Mccg 7.9
Xtxa 3823 293.9 Etac Mctc 9.6
http//sorgblast.tamu.edu
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QTL mapping of downy mildew resistance in maize