A Complete Transcriptional Landscape Analysis of Pinus elliottii Engelm. Using Third-Generation Sequencing and Comparative Analysis in the Pinus Phylogeny

Abstract

The planting of Pinus elliottii Engelm. has now reached close to three million ha in China. Molecular breeding as part of the improvement program for P. elliottii in southern China has been carried out in recent years. Third-generation sequencing (Pacbio sequencing technology, TGS) was used to obtain the exome of P. elliottii for molecular breeding. A total of 35.8 Gb clean reads were generated using TGS. After removing the redundant reads, we obtained 80,339 high-accuracy transcripts. Significantly, a total of 76,411 transcripts (95.1%) were blasted to public annotation databases. We predicted 65,062 intact coding sequences (CDSs), 8916 alternative splicing events, 1937 long non-coding RNAs, and 22,109 simple sequence repeats (SSRs) based on these obtained transcripts. Using the public databases and the data obtained above, 23 orthologous single-copy genes were identified to analyze the phylogenetic relationships for Pinus firstly including P. elliottii. Many positive selection genes involved in important biological processes and metabolism pathways were identified between P. elliottii and other pines. These positive selection genes could be candidate genes to be researched on the genetic basis of superior performance. Our study is the first to reveal the full-length and well-annotated transcripts of P. elliottii, which could provide reference for short transcriptome sequences in the research of genetics, phylogenetics, and genetic improvement for the non-reference genome species.Forests 2019, 10, 942 2 of 21 and are highly heterozygous. Like other conifers, the genome assembly of pines is also very difficult to determine because of our inability to obtain inbreeding lines [6]. The genome size of pines is about 17-35 gigabase pairs (Gb) [8]. The fist pine genome of Pinus taeda (22 Gb) was first published in 2014 [9,10]. Subsequently, the genome of Pinus lambertiana (31 Gb) was published [11]. The published pine genome contains highly abundant transposable elements. These published pine species genomes provide important genetic information for Pinus genetic study. However, interspecific divergences make it difficult for other pines to reference these published genomes. Whole genome sequencing is complex, time-consuming, and expensive. Thus, a better strategy is needed to obtain genome information for pines. Single-molecule, real-time (SMRT) sequencing developed by Pacific BioSciences (PacBio) is a third-generation sequencing (TGS) technology, more concisely referred to as "PacBio sequencing". PacBio sequencing has been widely used to sequence full-length transcripts and is well-suited for the non-reference species to obtain a complete transcriptional landscape [12]. TGS could generate full-length cDNA sequences, which would help provide a reference for second-generation sequencing (SGS) technologies in the assembling process and prediction of coding sequences (CDS), simple sequence repeat (SSR), long non-coding RNAs (long ncRNAs, lncRNAs), and alternative splicing (AS) events. Based on the full-length transcript ...

Full-length SMRT transcriptome sequencing and microsatellite characterization in Paulownia catalpifolia

“…Total RNAs extraction were performed using the EZ-10 DNAaway RNA mini-prep kit (Sangon Biotech Co., Shanghai, China) following the manufacturer’s instructions. The total RNAs of three samples above were mixed equally according to the method of Diao 53 to form the sample S for transcriptome sequencing. The degrees of RNA degradation and contamination were evaluated using 1% agarose gels 39 .…”

“…All non-redundant HQ transcripts were aligned using a previously described method 56 . Candidate AS events were identified using the selection criteria described by Diao et al 53 .…”

A transcriptome-based association study of growth, wood quality, and oleoresin traits in a slash pine breeding population

“…Clean reads were then mapped to the full-length transcript sequences developed in our former work [6] using the RNA-seq software STAR [24], and GATK [25] was adopted for SNP calling. Identified SNP sites were further annotated and analysed for their effect on the expression level of genes and associated protein products.…”

“…To implement MAB, increase the effectiveness of GS and explore the gene editing, detection of candidate genes for quantitative traits would be preferable through association study [5]. A transcriptome-referenced association study (TRAS, also called transcriptome wide association study (TWAS)), with a genome-wide association study (GWAS-like) approach was recently carried out as part of the improvement program for slash pine breeding populations in China [6].…”

“…Relationships between gene expression and trait performance can be investigated through association transcriptomics which could reduce the sequence need by the entire genome [12]. The transcriptome obtained by single molecule long-read sequencing was recently used as a reference sequence for scoring population variation in both sequence SNPs (SNP markers) and expression level variants (gene expression markers, GEMs); this method was termed a TRAS and holds promise as a GWAS-like approach in trees [6][7][8]. GWAS has been used in discovering the genetic basis of complex traits in many different plants, including Arabidopsis thaliana [13], Oryza sativa [14], and Populus trichocarpa [15], conifers [16,17].…”