*4.8. Analysis of Alpha- and Beta-Carotenes by HPLC*

Avocado mesocarp and seed extracts were prepared as previously described [58], with minor modifications. Briefly, fresh avocado mesocarp and seed samples were separately ground in a mortar containing liquid nitrogen. Samples (approximately 2 g) were added to centrifuge tubes, after which they were treated with 4 mL acetone and homogenized (intermediate speed) for 1.5 min at 4 ◦C. The supernatant was then transferred to a new centrifuge tube, and the extraction of the residue was repeated twice. The extracts were mixed with 5 mL methanolic potassium hydroxide (15%, *w*/*v*), and then saponified for 2 h in the presence of nitrogen. A 3-mL aliquot of the mixture was diluted with 1 mL 10% sodium chloride and then added to a 2-mL solution comprising methylene chloride and water. The supernatant was washed three times with water, evaporated to dryness in the presence of nitrogen, and reconstituted in methanol/methyl tert-butyl ether (85:15). The subsequent HPLC analysis of carotenoids was completed with the 1290 HPLC system (Agilent, Santa Clara, CA, USA) and a YMC carotenoid C30 column (250 × 4.6 mm, 5 µm; Waters, Santa Clara, CA, USA) analyzed at 445 nm. The HPLC mobile phase consisted of methanol/water (96:4, *v*/*v*) and tert-butyl ether at a flow rate of 1.0 mL/min and the column temperature was maintained at 30 ◦C. The alpha- and beta-carotenes were identified by comparing the retention times of the peaks with those of commercial standards purchased from Sigma-Aldrich (Shanghai, China). Carotenoid contents were quantified based on external calibration curves (*R* <sup>2</sup> <sup>≥</sup> 0.999). Alpha- and beta-carotene contents were expressed as microgram per gram of fresh weight (µg/g FW). Samples were analyzed with three biological replicates and two technical replicates.

## **5. Conclusions**

This study provides a comprehensive overview of the NGST transcriptomes of the avocado mesocarp and seed at five developmental stages. NGST and SMRT transcriptomes results implied that the gene dosage variation and the associated changes in gene expression of most carotenoid biosynthetic-related genes might contribute to the higher carotenoid pathway flux in the avocado mesocarp than in the seed, and accelerate the carotenoid accumulation. The metabolite (alpha- and beta-carotene) profiling via HPLC in the avocado mesocarp and seed during five developmental stages in this study validated the results of our NGST and SMRT transcriptome profiling. Our study results provide new insights into the carotenoid contents and the molecular mechanisms underlying carotenoid accumulation in avocado.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/20/17/4117/s1. Table S1. Phenotypes of avocado cultivar 'Hass' at 75, 110, 145, 180, and 215 days after full bloom during the fruit developmental stage; Table S2. Transcriptomic data for 30 avocado samples; Table S3. Gene ontology annotations for the assembled avocado unigenes; Table S4. Enriched Kyoto Encyclopedia of Genes and Genomes pathways among the assembled avocado unigenes; Table S5. Data for the differentially expressed genes in various cDNA libraries for developing mesocarps and seeds; Table S6. Analysis of differential expression and annotation of unigenes related to the carotenoid biosynthetic pathway; Table S7. PacBio library and sequencing results in avocado mesocarp and seed; Table S8. Details regarding qRT-PCR primers; Figure S1. The photos of the tested avcoado samples per fruit developmental stage. Figure S2. Transcript and unigene length distributions; Figure S3. Chromatogram of α-carotene and β-carotene extracted from 215 DAFB mesocarp and seed of avocado 'Hass'.

**Author Contributions:** Y.G., X.D. and R.Z. conceived and designed the experiments; X.S., L.T. performed the experiments; Z.C., B.W. and N.W. analyzed the data; Z.X., Z.Z. and X.L. helped complete the experiments; W.M. and X.Z. contributed materials; and Y.G. wrote the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (grant number 31701883) and the Central Public-interest Scientific Institution Basal Research Fund for the Chinese Academy of Tropical Agricultural Sciences (grant number 1630092018003).

**Acknowledgments:** We gratefully acknowledge Pingzhen Lin from the Haikou Experimental Station of the Chinese Academy of Tropical Agricultural Sciences for their valuable support related to the avocado resource collection. We gratefully acknowledge three anonymous reviewers for their constructive comments. We thank Yajima for editing the English text of a draft of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
