*2.1. Overview of the Morphology and NGST Profiling*

Morphological measurements, including mesocarp weight, seed weight, fruit length, and fruit width, gradually increased from 75 to 215 days after full bloom (DAFB), peaking at 114.74 g FW, 11.81 g FW, 83.07 mm, and 62.42 mm, respectively (Table S1). The photos of the tested avocado samples per fruit developmental stage are also presented in Figure S1. The RNA extracted from 15 mesocarp and seed samples were analyzed by RNA sequencing (RNA-seq), with three replicates per avocado fruit developmental stage. The sequencing of 30 cDNA libraries resulted in 20–26 million clean reads and 6.03–8.28 Gb of sequence data (Table S2). The generated avocado transcriptome data were deposited in the GenBank database (accession number PRJNA541745). The default parameters of the Trinity program were used to assemble the high-quality reads into 205,415 transcripts with a mean length of 1199.57 bp (N50 = 2063 bp) as well as 100,837 unigenes with a mean length of 847.40 bp (N50 = 1725 bp). Of these 100,837 unigenes, 59,969 (59.47%) were short (i.e., up to 500 bp), 16,511 (16.37%) were 501–1000 bp long, and 24,357 (24.16%) were longer than 1000 bp. The length distributions of all transcripts and unigenes are presented in Figure S2. These results demonstrated that the sequencing quality was sufficient for subsequent analyses.

## *2.2. Annotation and Identification of Unigenes*

Regarding the gene annotations, the BLASTX program revealed that 14,565 (37.05%), 20,712 (52.69%), 12,638 (32.15%), 19,065 (48.50%), 23,009 (58.54%), 22,403 (56.99%), 34,394 (87.50%), and 35,021 (89.09%) of the 39,309 avocado unigenes had significant matches with sequences in the Clusters of Orthologous Groups (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups (KOG), Pfam, Swiss-Prot, eggNOG, and the NCBI non-redundant protein sequence (Nr) databases, respectively. To further predict and classify functions, the annotated unigenes were analyzed according to GO assignments, COG classifications, and KEGG pathway assignments. A total of 20,712 unigenes were assigned to 49 sub-categories of the three main GO functional categories (cellular component, biological process, and molecular function) (Figure 1; Table S3). The molecular function category comprised the most unigenes, followed by the biological process and cellular component categories. The most common molecular function GO terms were catalytic activity (10,855 unigenes, GO: 0003824) and binding (10,148 unigenes, GO: 0005488). The top three biological process GO terms were metabolic process (10,654 unigenes, GO: 0008152), cellular process (9833 unigenes, GO: 0009987), and single-organism process (6516 unigenes, GO: 0044699). The most frequently observed cellular component GO terms were cell (8781 unigenes, GO: 0005623) and cell part (8769 unigenes, GO: 0044464) (Figure 1).
