Using the somatic cybrid Satsuma mandarin + Taoye sweet orange (G1+TYC), generated by protoplast fusion, and its mesophyll parent Taoye sweet orange (TYC) as materials, this study aimed to elucidate the physiological mechanism underlying fruit enlargement in G1+TYC and to identify candidate genes associated with the increased fruit size observed in this somatic cybrid.

Ovaries and pulp tissues of G1+TYC and TYC at different developmental stages were used as materials. Dynamic monitoring of fruit size traits, determination of sugar and acid contents, and measurement of endogenous hormone levels were performed across various fruit developmental stages including the mature stage. Combined with transcriptome and bioinformatics analysis, this study was conducted to clarify the physiological causes of fruit enlargement in G1+TYC and to identify key candidate genes controlling fruit size.

Dynamic monitoring of fruit development in three consecutive years showed that the transverse diameter of G1+TYC fruits was significantly larger than that of TYC starting from 75 days after flower (DAF), and the longitudinal diameter exhibited a significant superiority from 105 DAF onwards, with these differences persisting until 240 DAF. Taking the 2022 data as an example, the transverse and longitudinal diameters of the fruits from G1+TYC reached 79.14 and 66.75 mm at 240 DAF, respectively, while those from TYC were only 56.02 and 53.20 mm during the same period. This directly reflects the significant enlargement of fruit diameter of G1+TYC compared with TYC. The mature fruits of G1+TYC also had significantly higher fruit weight, segment width, single juice sac weight and area than TYC, indicating that the enlargement of juice sac volume contributed to the increase in segment width, thereby promoting the overall fruit size expansion of G1+TYC. Sugar and acid content determination revealed that the contents of fructose, glucose and sucrose in G1+TYC pulp were significantly higher than those in TYC during 90-240 DAF, whereas the contents of citric acid and total acids were significantly lower in G1+TYC. In terms of other organic acids, the difference in quinic acid and malic acid content between the two genotypes showed no obvious regularity. Endogenous hormone analysis demonstrated that the IAA content in G1+TYC pulp was significantly higher than that in TYC during 30-60 DAF. The GA₃ content of G1+TYC was significantly higher only at 0 DAF, while the ZT content showed poor repeatability across the two years of measurement. The ABA content increased rapidly after 90 DAF, and the ABA content in G1+TYC pulp was significantly higher than that in TYC at 180 DAF. Combined with transcriptome sequencing and GO enrichment analysis, five genes related to IAA metabolism were identified to be differentially expressed between G1+TYC and TYC during 30-60 DAF, which might serve as key candidate genes determining the fruit enlargement of G1+TYC.

This study revealed that enlargement of juice sac volume and elevated IAA content in pulp at early stage of fruit development were closely associated with fruit enlargement of G1+TYC. Moreover, candidate genes related to fruit enlargement in G1+TYC were identified based on transcriptome analysis, providing valuable genetic resources for citrus fruit size regulating mechanism research and further genetic improvement.