*4.5. Validation of Transcripts by Quantitative Real-Time PCR*

The expression levels of 10 unigenes related to carotenoid biosynthesis in the avocado mesocarp and seed were validated by a qRT-PCR assay, which was completed with a 96-well plate and the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Details regarding the qRT-PCR primers are presented in Table S8. Total RNA was extracted from the mesocarp and seed at the five developmental stages using RNAiso Plus Reagent (TaKaRa Bio Inc., Kusatsu, Japan) based on the manufacturer's protocol, then treated with RNase-free DNase I (New England Biolabs, Ipswich, MA, USA) to eliminate all contaminating DNA. The resulting RNA was applied for first strand synthesis by the PrimeScriptRT reagent Kit with gDNA Eraser (TaKaRa Bio Inc.). The concentration of cDNA was determined and diluted to 12.5 ng/µL. PCR was performed using QuantStudio7 Flex Real Time PCR System (Applied Biosystems, Foster City, CA, USA).The 20-µL reaction volumes comprised 2 µL cDNA, 10 µL SYBR *Premix Ex* Taq™ II (TliRNaseH Plus) (TaKaRa Bio Inc.), 1.0 µL each 10 µM primer, and 6 µL distilled water. The PCR program was as follows: 95 ◦C for 30 s; 40 cycles of 95 ◦C for 5 s, melting temperature of each primer for 30 s. The *PaActin7* gene was used as an endogenous control for normalizing data and 2−∆CT method was used for PCR data analysis. For each sample, the qRT-PCR analysis involved three biological replicates and two technical replicates.

## *4.6. SMRT Sequencing*

Poly-T oligo-attached magnetic beads were used to purify mRNA from the total RNA extracted from mesocarp and seed samples collected at each analyzed developmental stage. The mRNA from all five developmental stages was combined to serve as the template to synthesize cDNA with the SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). After a PCR amplification, quality control check, and purification, full-length cDNA fragments were acquired according to the BluePippin Size Selection System protocol, ultimately resulting in the construction of a cDNA library (1–6 kb). Selected full-length cDNA sequences were ligated to the SMRT bell hairpin loop. The concentration of the cDNA library was then determined with the Qubit 2.0 fluorometer, whereas the quality of the cDNA library was assessed with the 2100 Bioanalyzer (Agilent). Finally, one SMRT cell each was sequenced respectively with the PacBio RSII system (Pacific Biosciences, Menlo Park, CA, USA) for avocado mesocarp and seed.

## *4.7. Quality Filtering and Correction of PacBio Long-Reads*

Raw reads were processed into error-corrected reads of insert (ROIs) using an isoform sequencing pipeline, with minimum full pass = 0.00 and minimum predicted accuracy = 0.80. Next, full-length, non-chimeric transcripts were detected by searching for the poly-A tail signal and the 50 and 30 cDNA primer sequences in the ROIs. Iterative clustering for error correction was used to obtain high-quality consensus isoforms, which were then polished with Quiver. The low-quality full-length transcript

isoforms were corrected based on Illumina short-reads with the default setting of the Proovread program. High-quality and corrected low-quality transcript isoforms were confirmed as nonredundant with the CD-HIT (version 1) (http://weizhongli-lab.org/cd-hit/).
