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, 16 (12), 29047-59

De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus Densiflora)

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De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus Densiflora)

Le Liu et al. Int J Mol Sci.

Abstract

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.

Keywords: EST-SSR marker discovery; Pinus densiflora; simple sequence repeats; transcriptome sequencing; unigene assembly.

Figures

Figure 1
Figure 1
Sequence length distribution of contigs (A) and unigenes (B).
Figure 2
Figure 2
e-Value (A); similarity (B); and species distributions (C) of the unigenes.
Figure 3
Figure 3
COG function classification of the unigenes.
Figure 4
Figure 4
GO functional distribution of sequences. Biological process (blue); cellular component (red); and molecular function (green).
Figure 5
Figure 5
Frequencies of different repeat motifs in the EST-SSRs.
Figure 6
Figure 6
PCR amplification using 20 primer pairs randomly selected in P. densiflora. ”×”: void amplification. M: DNA Marker DL2000.
Figure 7
Figure 7
PCR amplification using four primer pairs (AD) in six related species. 1: P. densiflora, 2: P. taeda, 3: Pi. sitchensis, 4: Pi. asperata, 5: Ab. Firma, and 6: Ab. Fabri. M: DNA Marker DL2000.

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