*2.3. Screening of Di*ff*erentially Expressed Genes during Avocado Mesocarp and Seed Development*

An analysis of differentially expressed genes (DEGs) in the avocado mesocarp and seed during five fruit developmental stages revealed 16,903 DEGs (Table S5). There were 4013–4828 DEGs between the mesocarp and seed at five time-points, with some minor variability in the number of DEGs among the five fruit developmental stages. The number of DEGs increased considerably during mesocarp development. The largest number of DEGs (1516) was detected between 75 and 215 DAFB. Similarly, the number of DEGs sharply increased during the whole seed development stage, with a 4.40-fold increase from 75 vs. 110 DAFB to 75 vs. 215 DAFB. These results indicated that the fifth mesocarp and seed developmental stages may be associated with the most dramatic changes in enzyme contents and multiple metabolic pathways.

(8781 unigenes, GO: 0005623) and cell part (8769 unigenes, GO: 0044464) (Figure 1).

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

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

that the sequencing quality was sufficient for subsequent analyses.

*2.2. Annotation and Identification of Unigenes* 

**Figure 1.** Gene Ontology functional annotations of avocado unigenes. The unigenes were divided into three categories (biological process, cellular component, and molecular function). A total of 19,065 unigenes were functionally characterized based on 26 Eukaryotic Orthologous Groups (KOG) categories (Figure 2). Among the 26 categories, 'General function prediction only' represented the largest group (5557 unigenes, 26.38%), followed by 'Posttranslational modification, protein turnover, chaperones' (1959 unigenes, 9.30%), and 'Signal transduction mechanisms' (1729 unigenes, 8.19%). 'Extracellular structures' and 'Cell motility' were the two smallest groups. Additionally, 12,638 unigenes were assigned to 130 pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Table S4), with carbon metabolism (571 unigenes), biosynthesis of amino acids (516 unigenes), and ribosome (465 unigenes) representing the three most common pathways. **Figure 1.** Gene Ontology functional annotations of avocado unigenes. The unigenes were divided into three categories (biological process, cellular component, and molecular function). A total of 19,065 unigenes were functionally characterized based on 26 Eukaryotic Orthologous Groups (KOG) categories (Figure 2). Among the 26 categories, 'General function prediction only' represented the largest group (5557 unigenes, 26.38%), followed by 'Posttranslational modification, protein turnover, chaperones' (1959 unigenes, 9.30%), and 'Signal transduction mechanisms' (1729 unigenes, 8.19%). 'Extracellular structures' and 'Cell motility' were the two smallest groups. Additionally, 12,638 unigenes were assigned to 130 pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Table S4), with carbon metabolism (571 unigenes), biosynthesis of amino acids (516 unigenes), and ribosome (465 unigenes) representing the three most common pathways.

**Figure 2.** Eukaryotic Orthologous Groups classification of the assembled unigenes. **Figure 2.** Eukaryotic Orthologous Groups classification of the assembled unigenes.

An analysis of differentially expressed genes (DEGs) in the avocado mesocarp and seed during five fruit developmental stages revealed 16,903 DEGs (Table S5). There were 4013–4828 DEGs between the mesocarp and seed at five time-points, with some minor variability in the number of

*2.3. Screening of Differentially Expressed Genes during Avocado Mesocarp and Seed Development* 

enzyme contents and multiple metabolic pathways.

in the carotenoid biosynthetic pathway (Table 1).

with a 4.40-fold increase from 75 vs. 110 DAFB to 75 vs. 215 DAFB. These results indicated that the fifth mesocarp and seed developmental stages may be associated with the most dramatic changes in

A comparison of the avocado mesocarp and seed at five developmental stages based on the KEGG pathway enrichment among all DEGs resulted in the identification of the carotenoid biosynthetic pathway in four of the five developmental stages (Figure 3). The DEGs detected in the avocado mesocarp and seed transcriptomes included 17 unigenes that putatively encode 11 enzymes

*2.4. Differentially Expressed Carotenoid Biosynthetic Genes between the Avocado Mesocarp and Seed* 
