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

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 open-pollinated families from the Heilongjiang province of China. The research showed that (1) 21.3GB 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 open-pollinated 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.