De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology.

Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

Canales J ，Bautista R ，Label P ，Gómez-Maldonado J ，Lesur I ，Fernández-Pozo N ，Rueda-López M ，Guerrero-Fernández D ，Castro-Rodríguez V ，Benzekri H ，Cañas RA ，Guevara MA ，Rodrigues A ，Seoane P ，Teyssier C ，Morel A ，Ehrenmann F ，Le Provost G ，Lalanne C ，Noirot C ，Klopp C ，Reymond I ，García-Gutiérrez A ，Trontin JF ，Lelu-Walter MA ，Miguel C ，Cervera MT ，Cantón FR ，Plomion C ，Harvengt L ，Avila C ，Gonzalo Claros M ，Cánovas FM ... -  《-》

Transcriptome analysis in maritime pine using laser capture microdissection and 454 pyrosequencing.

Maritime pine (Pinus pinaster Aiton) is one of the most advanced conifer models for genomics research. Conifer genomes are extremely large and major advances have recently been made in the characterization of transcriptomes. The combination of laser capture microdissection (LCM) and next-generation sequencing is a powerful tool with which to resolve the entire transcriptome of specific cell types and tissues. In the current work, we have developed a protocol for transcriptomic analyses of conifer tissue types using LCM and 454 pyrosequencing. Tissue sections were isolated using non-fixed flash-frozen samples processed by LCM. Complementary DNA synthesis and amplification from tiny amounts of total RNA from LCM samples was performed using an adapted protocol for C: onifer R: NA A: mplification (CRA+). The cDNA amplification yield and cDNA quality provided by CRA+ were adequate for 454 pyrosequencing. Furthermore, read length and quality results of the 454 runs were near the optimal parameters considered by Roche for transcriptome sequencing. Using the CRA+ protocol, non-specific amplifications were prevented, problems derived from poly(A:T) tails in the 454 sequencing technology were reduced, and read length and read number considerably enhanced. This technical approach will facilitate global gene expression analysis in individual tissues of conifers and may also be applied to other plant species.

Cañas RA ，Canales J ，Gómez-Maldonado J ，Ávila C ，Cánovas FM ... -  《-》

Western white pine SNP discovery and high-throughput genotyping for breeding and conservation applications.

Western white pine (WWP, Pinus monticola Douglas ex D. Don) is of high interest in forest breeding and conservation because of its high susceptibility to the invasive disease white pine blister rust (WPBR, caused by the fungus Cronartium ribicola J. C. Fisch). However, WWP lacks genomic resource development and is evolutionarily far away from plants with available draft genome sequences. Here we report a single nucleotide polymorphism (SNP) study by bulked segregation-based RNA-Seq analysis. A collection of resistance germplasm was used for construction of cDNA libraries and SNP genotyping. Approximately 36-89 million 2 × 100-bp reads were obtained per library and de-novo assembly generated the first shoot-tip reference transcriptome containing a total of 54,661 unique transcripts. Bioinformatic SNP detection identified >100,000 high quality SNPs in three expressed candidate gene groups: Pinus highly conserved genes (HCGs), differential expressed genes (DEGs) in plant defense response, and resistance gene analogs (RGAs). To estimate efficiency of in-silico SNP discovery, genotyping assay was developed by using Sequenom iPlex and it unveiled SNP success rates from 40.1% to 61.1%. SNP clustering analyses consistently revealed distinct populations, each composed of multiple full-sib seed families by parentage assignment in the WWP germplasm collection. Linkage disequilibrium (LD) analysis identified six genes in significant association with major gene (Cr2) resistance, including three RGAs (two NBS-LRR genes and one receptor-like protein kinase -RLK gene), two HCGs, and one DEG. At least one SNP locus provided an excellent marker for Cr2 selection across P. monticola populations. The WWP shoot tip transcriptome and those validated SNP markers provide novel genomic resources for genetic, evolutionary and ecological studies. SNP loci of those candidate genes associated with resistant phenotypes can be used as positional and functional variation sites for further characterization of WWP major gene resistance against C. ribicola. Our results demonstrate that integration of RNA-seq-based transcriptome analysis and high-throughput genotyping is an effective approach for discovery of a large number of nucleotide variations and for identification of functional gene variants associated with adaptive traits in a non-model species.

Liu JJ ，Sniezko RA ，Sturrock RN ，Chen H ... -  《BMC PLANT BIOLOGY》

Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery.

Parchman TL ，Geist KS ，Grahnen JA ，Benkman CW ，Buerkle CA ... -  《BMC GENOMICS》

Combined de novo and genome guided assembly and annotation of the Pinus patula juvenile shoot transcriptome.

Visser EA ，Wegrzyn JL ，Steenkmap ET ，Myburg AA ，Naidoo S ... -  《BMC GENOMICS》