An approach to gene discovery in ALS crops

1
An approach to gene discovery in ALS crops

• Kevin Holland, Patrick Thimote, Ernesto Almira,
  Li Liu, and William Farmerie
• Interdisciplinary Center for Biotechnology
  Research
• University of Florida

2
ALS Crops

• Compact, high yield cultivars of crop plants
  developed for food production in space
• Significant challenges for ALS crops -- coping
  with non-terrestrial conditions
• CO2 concentration
• Light quantity and quality
• Temperature
• Relative humidity
• Microgravity

3
USU-Apogee Wheat

• Utah State University -- Bruce Bugbee
• Extremely short, high yield cultivar
• Full-dwarf hard red spring wheat (Triticum
  aestivum L.)
• High yields in controlled environments
• Higher yielding alternative to 'Yecora Rojo' and
  Veery-10

4
Characterization of ALS Crops

• Measuring interpreting cellular responses
• Quantitative gene expression monitoring
• Microarray
• preferred method for global gene expression
  analysis
• Commercial gene chips unavailable for ALS crops
• Species specificity of gene probes likely
  precludes using commercial chips

5
DYI gene chip construction

• Custom gene chip development technology rapidly
  evolving
• No longer cost prohibitive
• Need probe nucleotide sequences
• Public availability of a catalog of wheat gene
  cDNA sequences
• GenBank Unigene Database
• ? Applicability of public wheat catalog to dwarf
  cultivars

6
Why not sequence Apogee wheat?

• Comparison with existing publicly-available wheat
  sequences
• Identify possible unique genes
• Establish nucleotide sequence database for Apogee
  wheat
• Facilitate construction of Apogee-specific gene
  chips
• Relatively cheap insurance that probe sequences
  match targets

7
Apogee wheat cDNA library construction

• 6 g leaf tissue harvested for RNA prep
• Invitrogen cDNA library construction service
• 3 million primary clones
• 1.6 kb average insert

8
Naïve Cluster Assemble
10,842 Apogee EST Sequences
3,740 Singlet ESTs
7,095 ESTs in clusters
clustering
1,321 Contigs
6,011 unique assembly elements
assembly
950 Contig Singlets
9
NCBI Triticum aestivum UniGene Build 33

• 1,594 mRNAs 
• 56,513 EST, 3 reads 
• 156,352 EST, 5 reads 
• 158,789 EST, other/unknown 
• 373,248 total sequences in clusters
• 22,841 cluster sets total

10
Seed Cluster
10,842 Apogee EST Sequences
6,195 Apogee ESTs
20,865 unclaimed Unigenes
22,841 Unigene Sequences from GenBank
4,647 Apogee ESTs 1976 Unigenes
11
Seed Assembly
6,195 Apogee ESTs
2,953 singlets
770 contigs
509 contig singlets
assembly
4,647 Apogee ESTs 1976 Unigenes
5,839 unique assembly elements
1,607 Seed contigs
12
Assembly Statistics
13
EST Characterization

• What do our ESTs do?
• Gene Ontology (GO) categories
• Biological process
• Cellular component
• Molecular function
• Known biochemical pathways (KEGG)
• Transcription and Translation
• Replication, regulation, and repair

14
GO Classification

• Swissprot Database
• Most carefully annotated protein database
• Each Swissprot entry contains a list of GO
  associations
• Blastx search our Apogee assembly-set against the
  Swissprot DB
• Associates Apogee wheat ESTs with Swissprot
  entries and hence with GO terms
• Rank associations on the basis of Blast bit score
  and e-value

15
GO classifications for Apogee EST Assembly-Set

• 3445 of 5839 unique assembly elements classified
• Have at least 1 associated GO Term
• Most have multiple GO associations
• 2776 biological process
• 3062 molecular function
• 2058 cellular component

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Tae.116.C1

• Nucleolar RNA helicase II (DEAD-box protein 21)
• Associated GO Terms (6 of 9)
• Nucleic acid binding (mf)
• RNA binding (mf)
• Adenosinetriphosphatase (mf)
• ATP dependent RNA helicase (mf)
• Nucleus (cc)
• Nucleolus (cc)

17
KEGG Classification

• Kyoto Encyclopedia of Genes and Genomes
• Wiring diagrams of life
• KEGG Protein Networks
• Metabolic pathways
• Regulatory pathways
• Molecular complexes
• Network-network relations
• Network-environment relations

18
KEGG Summary

• Apogee EST assembly elements associated with KEGG
  ortholog database by Blastx search
• 3190 of 5839 assembly elements at e lt 10-3
• 2724 of 5839 assembly elements at e lt 10-10

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KEGG Pathways
20
Unique to non-Unigene
Common to both
Unique to Unigene
21
Unique to non-Unigene
Common to both
22
Summary

• 10,842 Apogee wheat EST sequences
• 43 have significant homology with domestic wheat
  Unigene ESTs (co-cluster)
• 57 are significantly different from Unigenes at
  the nucleotide sequence level
• gt 50 of the assembled Apogee ESTs are classified
  by GO and or KEGG associations