EST–SSR marker development and transcriptome sequencing analysis of different tissues of Korean pine (Pinus koraiensis Sieb. et Zucc.)

Abstract

Korean pine is a gymnosperm, and gymnosperms have relatively large genome sequences and lack a model organism reference genome. Understanding the important gene expression in the tissue growth process of needles (T 1 ), stems (T 2 ), female flowers (T 3 ) and cones (T 4 ) of the Korean pine is necessary to develop and compound the enzyme genes related to secondary metabolite synthesis. Simple sequence repeat (SSR) markers were explored based on the expressed sequence tag (EST). High-throughput sequencing technology was used to compare and analyse transcriptomes of four different tissue parts of the Korean pine, yielding many differentially expressed unigene sequences. Fluorescently labelled SSR primers were designed to analyse the polymorphism level of 60 openpollinated families from the Heilongjiang province of China. The research showed that (1) 21.3 GB of data was obtained from the transcriptome sequencing, and 41,476 candidate unigenes were identified based on sequence annotation using various databases. Clusters from orthologous groups and gene ontology function classification tools were used to divide the annotated transcript sequences into 56 functional categories. (2) Cones had the highest number of expressed genes during puberty, with rich expression as they were being formed. (3) By pathway enrichment analysis, 16 key enzyme genes related to fatty acid synthesis in other homologous species were obtained. (4) Ten novel polymorphic fluorescence labelling were used to identify 60 openpollinated families with a medium polymorphism level. The research showed that high-throughput sequencing technology could analyse the transcriptome expression level between different organisms, and SSR markers were successfully developed.

Phenotypic Variability and Genetic Diversity in a Pinus koraiensis Clonal Trial in Northeastern China

“…The PIC, Shannon's Information Index (I), and Fixation Index (F) were 0.5736 ± 0.294, 1.15 ± 0.07, and −0.11 ± 0.03, respectively, and were comparable to the findings of [16] that analyzed SSR markers. The great genetic diversity observed in P. koraiensis is an essential factor for improving the efficient selection of genotypes that might resist different environmental hazards (climate, parasites, etc.)…”

“…The DNA concentration and quality were tested using 1% agarose gel electrophoresis and a gel imaging system (Tanon-2500R, Tanon Science & Technology Co., Ltd., Shanghai, China). Sixteen published Simple Sequence Repeats (SSR) primer pairs were selected from previous studies ( [16,37]) and used to perform PCR analysis (Table S1).…”

“…Several factors, including the mating system, affect the maintenance of high genetic diversity in tree populations to varying degrees [81][82][83]. One study [16] demonstrated that the pollen source of P. koraiensis was greatly affected by wind direction during the pollination season. The Fixation Index (F) of −0.11 ± 0.03 that we found in this orchard complex suggested strong inbreeding.…”

“…Although the selection of superior P. koraiensis trees and elite materials has traditionally been based on phenotypic characteristics, many genetic markers have been developed to characterize Korean pine genetic diversity [14,15] and assess clone fingerprints, paternity analysis, and pedigree reconstruction for breeding populations in tree improvement programs [16,17]. However, no study to date has linked the genetic variation in Korean pine with phenotypic characteristics and genetic markers.…”

Approaches to variant discovery for conifer transcriptome sequencing

“…Prior to the advent of Next Generation Sequencing platforms, Expressed Sequence Tag (EST) libraries, based on captured and sequenced cDNA have been a mainstay of gene discovery and functional genomics [ 2 , 3 ]. Expressed Sequence Tag (EST) libraries have long been a rich resource for markers such as microsatellites or Simple Sequence Repeats (SSRs) [ 4 , 5 ]. Indeed, the conserved nature of gene sequence across conifers has meant that frequently, EST based markers from one species can be studied in related species, giving insight to evolutionary processes and increasing the pool of available markers across a genus [ 6 – 8 ].…”