Microarray Analyses

1
Microarray Analyses

• Microarrays completed
• Ploidy study 1x-2x-4x leaf tissue RNA
  comparisons 2 bioreps (8 slides total)
• In vitro tuberization 5 developmental stage
  comparisons 2 bioreps (10 slides total)
• In vivo tuberization 6 developmental stage
  comparisons (includes stolons) 2 bioreps (12
  slides total)
• Microarrays planned
• Ploidy study 1x-2x-4x root meristem RNA
  comparisons 2 bioreps (8 slides total)

2
Polyploidy Project ReviewSynthetic autopolyploid
series (P77)
Biorep 1 Biorep 2
1x
2x
4x
1x 4x
1x 4x
2xR3 2xR5
2xR3 2xR5
3
Ploidy Leaf Array Comparisons
Green 2x (R3) Red 4x Green 2x (R5) Red 1x
Green 1x Red 2x (R3) Green 4x Red 2x (R5)
4
Distribution of gene q-values for different leaf
ploidy comparisons
2x (R3)-2x (R5) Comparison 1x-4x Comparison
(Control experiment)
5
cDNA-AFLP

• cDNA-AFLP technique has been developed and use
  for gene expression profiling on the same
  materials used for microarray analysis
• Amplified double stranded cDNAs from two
  different leaf bioreps (Rep 1 Rep 2) and one
  root biorep (Rep 3) were used in analysis
• cDNAs are digested with Taq 1 and Ase 1 16
  primer combinations are used following the
  modified protocol (Bachem et al. 1996)

6
1X
2X
2X
4X
2X
4X
1X
2X
1
1
2
2
3
4
cDNA-AFLP patterns detected in various ploids
7
(No Transcript)
8
Candidate Ploidy Genes

• Gene lists are being compiled for genes that show
  significant expression differences in 1x-2x,
  2x-4x, and 1x-4x comparisons
• The microarray and cDNA-AFLP data sets will both
  be used to compile the gene lists
• Candidate ploidy genes from these lists will be
  verified by RT-PCR and Northern analyses
• RNA from both the synthetic autopolyploid series
  and wild Solanum species of varying ploidys will
  be used for gene verification analyses

9
Transformations and Late Blight Screening At
UW-Madison

• Repeated transformation of Katahdin and also
  transformed Russet Burbank, Dark Red Norland and
  Superior with RB
• Analyzed over 300 Kan-resistant plants for N and
  C terminus of coding region of RB
• Plants with both ends intact were tested for late
  blight resistance
• Late blight resistant transformants recovered for
  all cultivars, Dark Red Norland and Burbank sent
  to Chuck Brown and Jim Bradeen

10
Some General Observations

• Majority of kan-resistant plants tested did not
  contain RB gene or had truncated forms of RB.
  Suggest using another vector and reducing size of
  LR-PCR product.
• For each cultivar tested, especially Superior,
  there was a high percentage of plants that showed
  no resistance even though RB was present. This
  may be due to copy number/positional effects or
  truncation of the presumed native promoter. This
  warrants further investigation.
• Important to field test these lines next season.
  Lines are available to all collaborators.