Transcriptome Analysis Provides Insights into Korean Pine Tree Aging and Response to Shading
We hypothesized that gene expression changes during tree aging, and age and shading effects on the development and physiology of Korean pine trees, can be detected at the transcriptional level. To prove this hypothesis, we investigated the transcriptomes of Korean pines of different ages and the effects of shading on them, providing a theoretical basis for Korean pine management.
2. Materials and Methods
2.1. Plant Material
2.2. CDNA Library Preparation and Transcriptome Sequencing
Total RNA extraction from each tree was performed using a plant RNA purification reagent, as per the manufacturer’s instructions (Invitrogen, San Diego, CA, USA). RNA was quantified using a Qubit RNA kit (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Five micrograms of total RNA from each tree was used to generate an RNA library for Illumina paired-end sequencing. In total, 27 libraries were constructed.
After isolation from 5 μg of total RNA, the mRNA was purified using mRNA capture beads and then broken into 300–500 bp short fragments using magnetic beads. Subsequently, these fragments were employed as templates for the synthesis of first-strand cDNA, using a random hexamer primer. Then, second-strand cDNA was synthesized and cleaned up to prepare for end repair and the addition of poly A. Thereafter, these strands were connected to sequencing adapters and the desired cDNA was isolated using Hieff NGS® DNA Selection Beads (YEASEN, Shanghai, China). To enrich the desired cDNA, PCR amplification was performed. PCR products were tested on 2% agarose gel. Finally, the libraries were sequenced using the Illumina HiSeqTM 2500 platform (Illumina, CA, USA).
2.3. Transcriptome Assembly and Annotation
To identify the Korean pine protein-coding genes, blastx was used to search our assembled sequences against the blastx_swissprot, protein, blastp_swissport, Pfam, SignalP, eggnog, blast2GO, Pfam2GO, and Kegg databases with an E value < 1 × 10−5. Gene Ontology (GO) terms associated with the top hits in the nine protein databases were assigned to the annotated transcripts. After sequence clustering with Trinity software, unigenes were obtained and used for further analysis.
2.4. Identification of Differentially Expressed Unigenes (DEUs)
To identify the DEUs, 27 sets of sequencing reads were mapped pairwise to the assembled reference transcripts, and 36 comparisons were produced. Then, the normalized expression value was calculated as the fragments per kilobase of transcript per million mapped reads (FPKM). A pairwise comparison between two samples was performed to identify the DEUs with a false discovery rate (FDR)-corrected p value cutoff of 0.05 and a minimum expression fold change of 2.
2.5. Statistical Analyses and Graphics
While this study focused on age and shading, other environmental factors, such as soil quality, water availability, and pest presence, may also significantly impact tree growth and physiology. In this work, the sampled trees grew closely, so other environmental factors had a lower impact on their growth and physiology. In the future, the influences of other environmental factors can be studied further. In conclusion, our findings offer valuable insights into how age and shading affect the development and physiological traits of Korean pine trees, providing a foundation for further studies and practical forest management strategies.
4.1. Transcriptomic Analysis Reveals the Age-Related Developmental and Physiological Changes in Korean Pine Trees at the Transcriptional Level
4.2. Shading Affects the Expression of Physiological Response-Related Genes in Korean Pine
4.3. The Expression Level of PkDAL1 Is Age-Dependent, Independent of the Reproductive State or Growth Conditions of Korean Pine Trees
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