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RNA-seq analysis of Quercus pubescens Leaves: de novo transcriptome assembly, annotation and functional markers development.

Torre S, Tattini M, Brunetti C, Fineschi S, Fini A, Ferrini F, Sebastiani F - PLoS ONE (2014)

Bottom Line: These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance.We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq.Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

View Article: PubMed Central - PubMed

Affiliation: Institute for Plant Protection, Department of Biology, Agricultural and Food Sciences, The National Research Council of Italy (CNR), Sesto Fiorentino, Italy.

ABSTRACT
Quercus pubescens Willd., a species distributed from Spain to southwest Asia, ranks high for drought tolerance among European oaks. Q. pubescens performs a role of outstanding significance in most Mediterranean forest ecosystems, but few mechanistic studies have been conducted to explore its response to environmental constrains, due to the lack of genomic resources. In our study, we performed a deep transcriptomic sequencing in Q. pubescens leaves, including de novo assembly, functional annotation and the identification of new molecular markers. Our results are a pre-requisite for undertaking molecular functional studies, and may give support in population and association genetic studies. 254,265,700 clean reads were generated by the Illumina HiSeq 2000 platform, with an average length of 98 bp. De novo assembly, using CLC Genomics, produced 96,006 contigs, having a mean length of 618 bp. Sequence similarity analyses against seven public databases (Uniprot, NR, RefSeq and KOGs at NCBI, Pfam, InterPro and KEGG) resulted in 83,065 transcripts annotated with gene descriptions, conserved protein domains, or gene ontology terms. These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance. Finally, 14,202 microsatellite markers and 18,425 single nucleotide polymorphisms (SNPs) were, in silico, discovered in assembled and annotated sequences. We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq. The assembled and annotated sequences together with newly discovered molecular markers provide genomic information for functional genomic studies in Q. pubescens, with special emphasis to response mechanisms to severe constrain of the Mediterranean climate. Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

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Top 30 metabolic pathways in Q. pubescens.This table shows the KEGG metabolic pathways of plants that were well represented by unique sequences of Quercus pubescens. The number of sequences and enzymes involved are described
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pone-0112487-g005: Top 30 metabolic pathways in Q. pubescens.This table shows the KEGG metabolic pathways of plants that were well represented by unique sequences of Quercus pubescens. The number of sequences and enzymes involved are described

Mentions: In order to identify the biological pathways active in Q. pubescens, the assembled contigs were annotated with corresponding EC numbers against the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways database [31]. By mapping EC numbers to the reference canonical pathways, a total of 4,050 contigs (4,22%) were assigned to 138 KEGG biochemical pathways (Table 2). Figure 5 shows the 30 KEGG metabolic pathways mostly represented by unique sequences of Q. pubescens. These include “Purine metabolism” (548 members), a metabolic pathway of central significance in plant growth and development [49]. For instance, purine is involved in building blocks for nucleic acid synthesis and a well-known precursor for the synthesis of primary products and secondary products [50], [51]. This is consistent with enzymes specifically involved in phenylpropanoid metabolism (total 485 seqs, 41 enzymes) detected in our analysis. These include both phenylalanine metabolism, the entry point in phenylpropanoid biosynthetic pathway, as well as downstream enzymes involved in general and branch pathways of phenylpropanoid biosynthesis, such as flavonoid biosynthesis. Additionally, others highly represented pathways are “Starch and sucrose metabolism” (427 members) “T cell receptor signalling pathway” (302 members) and “Glycolysis/Gluconeogenesis” (221 members).


RNA-seq analysis of Quercus pubescens Leaves: de novo transcriptome assembly, annotation and functional markers development.

Torre S, Tattini M, Brunetti C, Fineschi S, Fini A, Ferrini F, Sebastiani F - PLoS ONE (2014)

Top 30 metabolic pathways in Q. pubescens.This table shows the KEGG metabolic pathways of plants that were well represented by unique sequences of Quercus pubescens. The number of sequences and enzymes involved are described
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231058&req=5

pone-0112487-g005: Top 30 metabolic pathways in Q. pubescens.This table shows the KEGG metabolic pathways of plants that were well represented by unique sequences of Quercus pubescens. The number of sequences and enzymes involved are described
Mentions: In order to identify the biological pathways active in Q. pubescens, the assembled contigs were annotated with corresponding EC numbers against the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways database [31]. By mapping EC numbers to the reference canonical pathways, a total of 4,050 contigs (4,22%) were assigned to 138 KEGG biochemical pathways (Table 2). Figure 5 shows the 30 KEGG metabolic pathways mostly represented by unique sequences of Q. pubescens. These include “Purine metabolism” (548 members), a metabolic pathway of central significance in plant growth and development [49]. For instance, purine is involved in building blocks for nucleic acid synthesis and a well-known precursor for the synthesis of primary products and secondary products [50], [51]. This is consistent with enzymes specifically involved in phenylpropanoid metabolism (total 485 seqs, 41 enzymes) detected in our analysis. These include both phenylalanine metabolism, the entry point in phenylpropanoid biosynthetic pathway, as well as downstream enzymes involved in general and branch pathways of phenylpropanoid biosynthesis, such as flavonoid biosynthesis. Additionally, others highly represented pathways are “Starch and sucrose metabolism” (427 members) “T cell receptor signalling pathway” (302 members) and “Glycolysis/Gluconeogenesis” (221 members).

Bottom Line: These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance.We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq.Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

View Article: PubMed Central - PubMed

Affiliation: Institute for Plant Protection, Department of Biology, Agricultural and Food Sciences, The National Research Council of Italy (CNR), Sesto Fiorentino, Italy.

ABSTRACT
Quercus pubescens Willd., a species distributed from Spain to southwest Asia, ranks high for drought tolerance among European oaks. Q. pubescens performs a role of outstanding significance in most Mediterranean forest ecosystems, but few mechanistic studies have been conducted to explore its response to environmental constrains, due to the lack of genomic resources. In our study, we performed a deep transcriptomic sequencing in Q. pubescens leaves, including de novo assembly, functional annotation and the identification of new molecular markers. Our results are a pre-requisite for undertaking molecular functional studies, and may give support in population and association genetic studies. 254,265,700 clean reads were generated by the Illumina HiSeq 2000 platform, with an average length of 98 bp. De novo assembly, using CLC Genomics, produced 96,006 contigs, having a mean length of 618 bp. Sequence similarity analyses against seven public databases (Uniprot, NR, RefSeq and KOGs at NCBI, Pfam, InterPro and KEGG) resulted in 83,065 transcripts annotated with gene descriptions, conserved protein domains, or gene ontology terms. These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance. Finally, 14,202 microsatellite markers and 18,425 single nucleotide polymorphisms (SNPs) were, in silico, discovered in assembled and annotated sequences. We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq. The assembled and annotated sequences together with newly discovered molecular markers provide genomic information for functional genomic studies in Q. pubescens, with special emphasis to response mechanisms to severe constrain of the Mediterranean climate. Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

Show MeSH
Related in: MedlinePlus