Rootstock-mediated transcriptional changes associated with cold tolerance in Prunus mume leaves
The RNA was extracted by the Foregene Nucleic Acid Extraction Kit (Shenzhen, China) following the manufacturer standard protocol and purified using RNase-free DNase I (TaKaRa, Japan). The RNA concentration was checked through using 2200 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), and quality was determined through gel electrophoresis. Then, the RNA was treated with oligo (dT) and combined with fragment buffer to synthesize cDNA. After purification, the fragment was isolated in EB end repair and 1-A nucleotide addition. Then, ligation product was amplified through PCR and Illumina sequencing was performed by BGI, China. The raw reads (FASTQ) were filtered to remove the low quality reads and unknown base N content using the SOAPnuke (v 1.4.0) software. After getting clean reads, HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) (v 2.1.0) was used to compare the clean reads to the Prunus mume genome available at (https://www.ncbi.nlm.nih.gov/genome/?term=prunus%20mume). Bowtie2 (v 2.2.5) software was used to compare the clean reads to genomic sequences and the gene expression level was calculated by RSEM (v 1.2.8). After sequencing, the expression level of each sequence library was standardized as FPKM, and the most differentially expressed genes (DEGs) were selected for further differential analysis. The significance level of the transcripts was measured using FDR (False Discovery Rate) control method to rationalize p-value. The regulation of the genes was determined through an absolute log2fold change method with an FDR significance level of < 0.01. To find out the putative transcription factor (TF) related to these DEGs, Getorf (EMBOSS: 6.5.7.0) was used to identify ORF, the hmmsearch (v 3.0) was used to align ORF to TF protein domain and then the unigenes were identified according to TF family characterized by PlantTFDB 4.0. To further characterized the function of DEGs, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis were performed. The enrichment analysis of both GO and KEGG was performed using phyper function in R software with p-value < 0.13