*4.4. Expression Analysis of Longan WRKY Genes in Various Tissues and Different Flowering Stages*

The RNA-seq data for analyzing the expression patterns of *WRKY* genes in different longan tissues were downloaded from the NCBI Sequence Read Archive (GSE84467). Three pairs of nine-year-old "SJ" and "SX" *D. longan* trees which displayed opposite flowering phenotype were used for comparative expression analysis of *DlWRKY* during floral induction. All those trees were grown at an experimental orchard in the South Subtropical Crops Research Institute of the Chinese Academy of Tropical Agricultural Science in Zhanjiang (110◦160 E, 21◦100 N), China. Three different kinds of apical buds, including the dormant stage (T1), the emergence of floral primordia stage (T2), and the floral organ formation stage (T3) of "SJ" and "SX", were used in this study. The samples obtained for the T1, T2, and T3 in "SJ" and "SX" were collected on 20 November 2016, 24 December 2016, and 1 January 2017, respectively. For each sample, we used three biological replicates from three different trees. Each biological replicate contained the mixed buds which were collected from the four cardinal directions of each tree. All samples were collected from 10:00 am to 12:00 am and were frozen immediately in liquid nitrogen and stored at −80 ◦C. According to the manufacturer's instructions, the total RNA was extracted by using the quick RNA Isolation Kit (Hua Yue Yang Bio Co., Ltd., Beijing, China) and the genomic DNA residues were removed during RNA extraction. We used an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) to test the RNA concentration and the quality of each sample. The RNA quality was also confirmed by RNase free agarose gel electrophoresis. The RNA-seq experiment was performed as described by our previous study [66]. The RNA-seq data were uploaded to the NCBI Sequence Read Archive (SRS2241241, SRS2241242, SRS2241243, SRS2241244, SRS2241245, SRS2241246, SRS2241247, SRS2241248, SRS2241249, SRS2241250, SRS2241251, SRS2241252, SRS2241253, SRS2241254, SRS2241255, SRS2241256, SRS2241257, and SRS2241258). The fragments per kilobase of the exon model per million mapped values (FPKM) were log10-transformed, and heat maps with hierarchical clustering were exhibited using the software Mev4.9.0 [67].
