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High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome.

Novaes E, Drost DR, Farmerie WG, Pappas GJ, Grattapaglia D, Sederoff RR, Kirst M - BMC Genomics (2008)

Bottom Line: However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation.In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this work created part of the foundation for the annotation of the E. grandis genome that is being sequenced by the US Department of Energy.In addition we demonstrated that SNPs sampled in large-scale with 454 pyrosequencing can be used to detect evolutionary signatures among genes, providing one of the first genome-wide assessments of nucleotide diversity and Ka/Ks for a non-model plant species.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Forest Resources and Conservation, University of Florida, PO Box 110410, Gainesville, USA. evandro@ufl.edu

ABSTRACT

Background: Benefits from high-throughput sequencing using 454 pyrosequencing technology may be most apparent for species with high societal or economic value but few genomic resources. Rapid means of gene sequence and SNP discovery using this novel sequencing technology provide a set of baseline tools for genome-level research. However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation.

Results: With the purpose of generating the first broad survey of gene sequences in Eucalyptus grandis, the most widely planted hardwood tree species, we used 454 technology to sequence and assemble 148 Mbp of expressed sequences (EST). EST sequences were generated from a normalized cDNA pool comprised of multiple tissues and genotypes, promoting discovery of homologues to almost half of Arabidopsis genes, and a comprehensive survey of allelic variation in the transcriptome. By aligning the sequencing reads from multiple genotypes we detected 23,742 SNPs, 83% of which were validated in a sample. Genome-wide nucleotide diversity was estimated for 2,392 contigs using a modified theta (theta) parameter, adapted for measuring genetic diversity from polymorphisms detected by randomly sequencing a multi-genotype cDNA pool. Diversity estimates in non-synonymous nucleotides were on average 4x smaller than in synonymous, suggesting purifying selection. Non-synonymous to synonymous substitutions (Ka/Ks) among 2,001 contigs averaged 0.30 and was skewed to the right, further supporting that most genes are under purifying selection. Comparison of these estimates among contigs identified major functional classes of genes under purifying and diversifying selection in agreement with previous researches.

Conclusion: In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this work created part of the foundation for the annotation of the E. grandis genome that is being sequenced by the US Department of Energy. In addition we demonstrated that SNPs sampled in large-scale with 454 pyrosequencing can be used to detect evolutionary signatures among genes, providing one of the first genome-wide assessments of nucleotide diversity and Ka/Ks for a non-model plant species.

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Proportion of GO categories found in the E. grandis unigenes. Proportion of categories of each Gene Ontology (GO) sampled by the E. grandis unigene sequences compared with the proportions found in the Arabidopsis genome annotation.
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Figure 2: Proportion of GO categories found in the E. grandis unigenes. Proportion of categories of each Gene Ontology (GO) sampled by the E. grandis unigene sequences compared with the proportions found in the Arabidopsis genome annotation.

Mentions: Next, we determined the proportion of annotated gene models for which homology was detected among E. grandis unigenes measuring over 100 bp. Homology was detected to 45% of Arabidopsis, 39% of Populus, and 22% of Oryza gene models (E value 10-5). The higher proportion of Arabidopsis genes that are apparent homologues to Eucalyptus is expected as the two species are more phylogenetically related than to Populus or Oryza [14]. Arabidopsis gene models are also more refined than those from the other plant species. Analyzing only the 22,032 Arabidopsis gene models for which there is any detected transcript evidence (TAIR v. 7.0) leads to a higher proportion of homologies: 58% with E value 10-5 and 39% with E value 10-20 (Table 3). Finally, we utilized the Gene Ontology (GO) classifications from the Arabidopsis best-hit gene models (E value 10-5). Proportions of best hits in each GO category were generally similar to those found in the Arabidopsis genome annotation (Figure 2). The GO annotation analysis reinforces the inference that a broad diversity of genes was sampled in our multi-tissue normalized cDNA pool.


High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome.

Novaes E, Drost DR, Farmerie WG, Pappas GJ, Grattapaglia D, Sederoff RR, Kirst M - BMC Genomics (2008)

Proportion of GO categories found in the E. grandis unigenes. Proportion of categories of each Gene Ontology (GO) sampled by the E. grandis unigene sequences compared with the proportions found in the Arabidopsis genome annotation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483731&req=5

Figure 2: Proportion of GO categories found in the E. grandis unigenes. Proportion of categories of each Gene Ontology (GO) sampled by the E. grandis unigene sequences compared with the proportions found in the Arabidopsis genome annotation.
Mentions: Next, we determined the proportion of annotated gene models for which homology was detected among E. grandis unigenes measuring over 100 bp. Homology was detected to 45% of Arabidopsis, 39% of Populus, and 22% of Oryza gene models (E value 10-5). The higher proportion of Arabidopsis genes that are apparent homologues to Eucalyptus is expected as the two species are more phylogenetically related than to Populus or Oryza [14]. Arabidopsis gene models are also more refined than those from the other plant species. Analyzing only the 22,032 Arabidopsis gene models for which there is any detected transcript evidence (TAIR v. 7.0) leads to a higher proportion of homologies: 58% with E value 10-5 and 39% with E value 10-20 (Table 3). Finally, we utilized the Gene Ontology (GO) classifications from the Arabidopsis best-hit gene models (E value 10-5). Proportions of best hits in each GO category were generally similar to those found in the Arabidopsis genome annotation (Figure 2). The GO annotation analysis reinforces the inference that a broad diversity of genes was sampled in our multi-tissue normalized cDNA pool.

Bottom Line: However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation.In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this work created part of the foundation for the annotation of the E. grandis genome that is being sequenced by the US Department of Energy.In addition we demonstrated that SNPs sampled in large-scale with 454 pyrosequencing can be used to detect evolutionary signatures among genes, providing one of the first genome-wide assessments of nucleotide diversity and Ka/Ks for a non-model plant species.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Forest Resources and Conservation, University of Florida, PO Box 110410, Gainesville, USA. evandro@ufl.edu

ABSTRACT

Background: Benefits from high-throughput sequencing using 454 pyrosequencing technology may be most apparent for species with high societal or economic value but few genomic resources. Rapid means of gene sequence and SNP discovery using this novel sequencing technology provide a set of baseline tools for genome-level research. However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation.

Results: With the purpose of generating the first broad survey of gene sequences in Eucalyptus grandis, the most widely planted hardwood tree species, we used 454 technology to sequence and assemble 148 Mbp of expressed sequences (EST). EST sequences were generated from a normalized cDNA pool comprised of multiple tissues and genotypes, promoting discovery of homologues to almost half of Arabidopsis genes, and a comprehensive survey of allelic variation in the transcriptome. By aligning the sequencing reads from multiple genotypes we detected 23,742 SNPs, 83% of which were validated in a sample. Genome-wide nucleotide diversity was estimated for 2,392 contigs using a modified theta (theta) parameter, adapted for measuring genetic diversity from polymorphisms detected by randomly sequencing a multi-genotype cDNA pool. Diversity estimates in non-synonymous nucleotides were on average 4x smaller than in synonymous, suggesting purifying selection. Non-synonymous to synonymous substitutions (Ka/Ks) among 2,001 contigs averaged 0.30 and was skewed to the right, further supporting that most genes are under purifying selection. Comparison of these estimates among contigs identified major functional classes of genes under purifying and diversifying selection in agreement with previous researches.

Conclusion: In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this work created part of the foundation for the annotation of the E. grandis genome that is being sequenced by the US Department of Energy. In addition we demonstrated that SNPs sampled in large-scale with 454 pyrosequencing can be used to detect evolutionary signatures among genes, providing one of the first genome-wide assessments of nucleotide diversity and Ka/Ks for a non-model plant species.

Show MeSH