Candidate gene analysis for drought tolerant tall fescue

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Candidate gene analysis for drought tolerant tall
fescue

• Emily Combs
• Saha Lab

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Why is drought resistance important?

• Drought is a major limiting factor in crop
  productivity drought can cause yield loss of up
  to 50 or more
• A large portion of arable land is arid (35) and
  the aridity and acreage is on the rise

Population PDF580 genotypes evaluated for drought
tolerance. Plants in left panel are well watered
and those in right panel have had water withheld
for seven days.
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Deciphering drought tolerance

• Conventional phenotypic selection
• Molecular mapping and QTL analysis
• Expression analysis of drought-stress genes
• Candidate gene analysis

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Candidate Gene Approach

• High gene conservation between plant species
• Genes that confer drought resistance in one
  species may do so in others
• Therefore, studies on drought resistance genes in
  other plants can be applied to tall fescue

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Objectives and Goals

• Find candidate genes for drought resistance
• Design primers for amplification of these genes
  in tall fescue
• Look for differences in the amplified regions of
  drought- resistant and susceptible tall fescue

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Genotype Selection
Materials and Methods

• Genotypes of tall fescue were screened for
  drought-stress resistance
• 4 drought- resistant and 4 susceptible genotypes
  selected
• DNA extracted using Qiagen extraction kit

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Database Search
Materials and Methods

• Searched literature for genes that confer drought
  resistance in different species
• Agris, Agricola, Bio Abstracts, CAB direct, and
  Google Scholar
• BLASTed drought resistance genes against tall
  fescue TCs to find orthologs
• Genes not found in tall fescue searched in wheat,
  rice, sorghum, and barley TCs
• Primers designed using Primer 3

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Electrophoresis
Materials and Methods

• Agarose
• 2 agarose gel
• 100 bp DNA ladder used
• Poly-acrylamide
• 6 PAG
• 10 bp DNA ladder used

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Sequencing
Materials and Methods

• Amplified products were extracted from
  poly-acrylamide gels
• Re-suspended products used for PCR
• Sea-Kem agarose gel-electrophoresis
• Bands extracted and purified using Montage kit
• DNA sent for sequencing

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Database Search
Results
192 drought resistance genes identified
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Agarose Gel
Results

• 60 primers run on agarose gel
• 24 primers failed to amplify

STS primers 106, 104, 101, 100, 97, and M
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Agarose Gel
Results

• 36 primers amplified
• 3 primers were polymorphic
• STS-112 (anti-oxidant defense)
• STS-114 (unknown)
• STS-115 (osmoprotection)

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Agarose Gel
Results

• 33 were monomorphic re-run on poly-acrylamide

200 bp
Primers 111, 110, 107, 103, 113 and M
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Poly-Acrylamide Gel
Results

• 7 primers polymorphic

39 592 914 947 279 366 704 348
Primer 120 agarose
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Polymorphic Primers
Results
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Sequencing
Results
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Alignment Report Primer 88
Results
T (39) T (366) T (348) S (592) S (947)
T (39) T (366) T (348) S (592) S (947)
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Conclusions

• 192 candidate genes identified 60 primer pairs
  developed 14 found polymorphic
• Tolerant genotypes had different alleles for
  resistance
• Polymorphic primers were mainly those associated
  with antioxidant defense

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Future Work

• Literature search and primer screening are
  continuing
• Screening of identified candidate genes with
  larger contrasting number of genotypes
• Verification of identified candidate genes using
  RNAi technology

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Acknowledgements

• Members of Saha Lab
• Steve Rhines
• Emily Edwards
• Noble Foundation for the scholarship

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Questions