Xspecies analysis of Aloe vera

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Xspecies analysis of Aloe vera Oil Palm Zoe
Emmerson MRes in Advanced Genomic and Proteomic
Sciences 2009
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Aims

• Further proof of concept of the Xspecies method
  (Hammond et al., 2005).
• Two species, Aloe vera and Oil Palm which are
  distantly related to Arabidopsis will be
  hybridised to the ATH1 GeneChip and the results
  assessed for conserved genes.

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Introduction

• Still many commercially and scientifically
  interesting species have little or no genome
  sequence available in the public domain.
• Consequently, there are not many resources
  available for many important plant species which
  would aid, the understanding and conservation of
  the species.
• However, there is an Abundance of resources
  available for the study of Arabidopsis.
• Xspecies makes use of the already available
  technology for Arabidopsis and uses it to study
  other significant species.

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The Three Species

• Arabidopsis
• Model organism
• No economic value but many resources
• Aloe vera
• Native to sub-Saharan Africa, Arabian Peninsula
  and on some Indian Ocean islands
• Adaptations such as succulent
• Medicinal purposes for centuries
• Huge economic value, markets worth millions of
  dollars.
• Oil Palm
• Originates in Africa
• In-expensive food source and used in cosmetics
• Large market Oil Palm produces the highest yield
  per hectare
• Potential for use in biofuels

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Phylogenetic Tree
The use of phylogenies is important for this
project because of the need to see the
evolutionary distance between the study species
A. vera, Oil Palm and the GeneChip species A.
thaliana.
Phylogeny of flowering plants (adapted from
Fawcett et al., 2009)
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Introduction To Affymetrix Technology

• Transcriptomics is the study of gene expression
  at the RNA level from the individual cell to
  whole tissues.
• The transcriptome is dynamic and changes under
  the influence of the external environment and
  internal cues.
• Affymetrix GeneChip highly reproducible due to
  mass production.
• No need for technical replicates.
• Cost effective and produces accurate data.
• Each chip uses a probe set to represent each
  gene.
• Instead of having a single oligonucleotide to
  represent each gene, the GeneChip uses between
  11 and 20 probe pairs depending on the species.
• Each array contains multiple probe pairs in order
  to reduce the likelihood of random hybridisation
  and to increase the accuracy by using an average
  of the signal intensity across the probes.

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The ATH1 GeneChip

• The GeneChip that is to be used in this study is
  the Affymetrix Arabidopsis thaliana ATH1
• ATH1 has 11 probe pairs, containing a perfect
  match (PM) and a mismatch (MM) probe.
• The PM probes are precisely complementary to 11
  different 25 base pair sequences of the target
  gene.
• The MM probe is the same an the PM with the
  exception of the 13th nucleotide that is altered
  so that the sequence is no longer an exact match
  for the target sequence of the gene. This is
  intended to be a built internal control for
  background hybridisation.
• ATH1 GeneChip contains more than 22,500 probe
  sets which represent approximately 24,000 genes.
  The probe sets are scattered randomly across the
  surface of the chip to reduce the risk of losing
  entire probe sets in the case of uneven
  hybridisation, or damage to the surface of the
  chip.

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Xspecies Method

• The Xspecies method was developed by Hammond et
  al. (2005) as a response to the limited
  Affymetrix Genechip arrays available for a small
  number of plant and animal species.
• Xspecies allows for increase sensitivity when
  used with heterologous species.
• The specific Xspecies approach can greatly
  increase the number of potential genes that can
  be studied.
• Holds great potential to enable the study of
  agriculturally or ecologically important plant
  species for which there is no GeneChip array
  available.

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Materials Methods
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Results

• Shows that as the gDNA hybridisation intensity
  cut off increases the probe pairs and probe sets
  retained reduces.
• This tells us the hybridisation effectiveness of
  the gDNA from the heterologous species to the
  ATH1GeneChip array.

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Aloe Results

• Volcano Plots
• Aloe RNA hybridisation was filtered using the
  optimum .cdf 200 on a 1.5 fold change and with a
  P value of lt0.05 in order to pick out the
  statistically significantly altered genes and to
  reduce the gene set to a more manageable size.
• This gave a result of 874 genes which were
  differentially regulated between leaf and root.
• All further analysis was carried out on this
  smaller gene set.

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Aloe Results

• Principle Component Analysis (PCA)
• 3 root replicates (blue) and 3 leaf replicates
  (pink) clustering at opposite ends of the graph.
• This demonstrates that the 3 replicates of the 2
  tissue types have given similar results.

• Simplified PCA, which shows a more definite
  separation of the two tissue types, root (blue)
  leaf (pink).

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Aloe Oil Palm Results
Pink is up-regulated in Leaf, blue is
up-regulated in Root.
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Results

• Of the total of 13 genes involved in this pathway
  there are 6 found in Aloe and 12 in Oil Palm.
• The 6 genes found in Aloe are common to all three
  species.

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Results

• There are 18 genes in this pathway which are
  found in Oil Palm.
• Could be due to the large unfiltered gene set.

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Results Venn Diagrams
Common to all 3 species 26 leaf genes involved
in photosynthesis.
6 root genes - metabolism processes
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Gene Functions

• The two most highly expressed genes in Aloe leaf
  have AGI codes
• At4g26710 has the description of expressed
  protein and is a V-type H-ATPase, which is a
  response to salt stress gene. (Ratajczak, 1999).
• At2g05070 is a light-harvesting chlorophyll a/b
  binding protein, which is integral to
  photosynthesis
• The two most highly expressed genes in the Aloe
  root have AGI codes
• At4g37370 is a cytochrome P450-like protein.
  Cytochrome P450 monooxygenases are a group of
  haem-containing proteins which catalyze various
  oxidative reactions (Naruskaka et al., 2004) and
  are involved in many metabolism processes.
• At1g04820 is a Tubulin alpha-2/alpha-4 chain
  (TUA4) which is a general house keeping gene.
  Microtubules are essential in plants and play
  vital roles alpha tubulin is one of the building
  blocks of the microtubules in many plant
  developmental processes. Tubulins are found in
  all tissue types but are mainly found in root
  tissue (Dhonukshe, 2006).

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C3'2009 2009 International Summer School on
Chips, Computers and Crops September 18th to
27th, 2009 Zhejiang University, Hangzhou, China

• The summer school on Chips, computers and crops
  aims to teach best practice in the application of
  state-of-the-art computational techniques and
  genomic technologies to crop research.
• The summer school covers interdisciplinary
  science, in a rapidly evolving field, bringing
  together ideas from the fields of mathematics,
  bioinformatics, genomics and post-genomic
  experiments on model organisms.

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Some Findings From China

• Several 6 base sequences occur in both
  Arabidopsis leaf and Aloe leaf, these have been
  identified to be involved in light
  responsiveness.
• Of overlapping genes between Arabidopsis and Aloe
  some of the most significant are chloroplastic
  glutamine synthetase, and glutamate decarboxylase
  which is involved in glutamate metabolic
  activity. One paper suggests this is primarily
  expressed in the leaves and is important for
  assimilating ammonia released from
  photorespiration.
• Of the genes more highly expressed in the roots
  of Aloe GO analysis found GO0000287 a magnesium
  ion binding gene.

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Conclusions Further Work
This project has tested the Xspecies method, and
has shown that it is able to

• Identify transcriptomic differences between leaf
  and root material.
• Pick up genes highly up regulated in each
  material and allow further analysis e.g., GO
  ontologies and pathway searches.
• Identifies genes common between target species.
• Further Work
• 2 more biological replicates for the Oil Palm
  leaf and root.
• Include other species of plant to further
  validate the methods.
• Hybridise both Aloe and Oil Palm to another
  monocot GeneChip such as the rice genome array.
• Validation experiments would have to be carried
  out such as qPCR.

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References

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