Anatomical structure, hormone change, and transcriptome analysis of the large-fruit mutant of Jinhong Bingtang Orange

Abstract: 【Objective】Fruit size is a major agronomic trait for evaluating fruit appearance quality. Therefore, it is important to analyze the molecular mechanism of fruit size for high-quality citrus breeding. Bud sport is one of the sources of fruit size mutation. A large-fruit mutant of Jinhong was found previously. In this research, we explored the mechanism of the mutation in order to provide target genes for citrus molecular design breeding.【Methods】Jinhong Bingtang orange and its large-fruit mutant as experimental material were sampled at 30, 50, 70, 90, 120, 150, 170, 190 and 250 days after bloom (DAB). Juice sacs at 70, 120 and 170 d were collected for RNA-seq. Young leaves of large-fruit mutant were collected for ploidy testing. Volume of juice sacs was determined using water replacement method. Paraffin section was used for cytological observation of juice sacs. The plant hormones were determined by liquid chromatography- mass spectrometry. Transcriptome analysis were performed on theabove samples. Differentially expressed genes (DEGs) between the wild type and the mutant at same developmental stage were screened on the standard of FDR＜0.05. Common DEGs of those three developmental stages were obtained using Venn analysis. GO and KEGG enrichments were mapped using TBtools, and GO and KEGG were used to analyze the DEGs between Jinhong and its large- fruit mutant. Genes related to plant hormones were shown with the heat maps, which were drawn based on FPKM of the DEGs.【Results】Flow cytometry analysis showed that the large-fruit mutant was a diploid. Jinhong and large- fruit mutant had the same number (11) of carpels. Compared with Jinhong, the number of juice sacs of the large-fruit mutant increased, and volume of juice sacs enlarged. Results of paraffin section showed that the cell number of the large-fruit mutant was larger than that of Jinhong. IAA content of the mutant was always higher than that of Jinhong at 70 and 120 DAB, but it was lower at 170 DAB. The GA3 content in the large-fruit mutant was always greater. ZT content was low, and the ZT content of the large-fruit mutant was always lower than that of Jinhong at the three developmental stages. Except at 70 DAB, JA content of the large-fruit mutant was lower than that of Jinhong. At 70 DAB, there were 3118 DEGs between the two materials, including 1549 up- regulated and 1569 down- regulated genes, which were enriched in plant hormone signal transduction, MAPK signaling pathway, plantpathogen, etc. At 120 DAB, there were 1952 DEGs, with 377 up- regulated and 1575 down- regulated, which were enriched in photosynthesis - antenna proteins, plant-pathogen interaction and MAPK signaling pathway, etc. At 170 DAB, there were 611 DEGs, with 372 up-regulated and 239 down-regulated, enriched in photosynthesis - antenna proteins, phenylpropanoid biosynthesis, etc. Besides, 88 DEGs were identified in the three stages and KEGG analysis showed that the top five most significantly enriched pathway were fatty acid elongation, photosynthesis- antenna proteins, cutin, suberine and wax biosynthesis, phenylpropanoid biosynthesis and plant hormone signal transduction. Furthermore, DEGs encoding hormone signaling were further analyzed. 24 DEGs were found at least two periods, including 4 genes related to ABA, 6 related to IAA, 3 related to BR, 2 related to CTK, and other hormone-related genes. Among them, GH3.6 was up-regulated in the large-fruit mutant at 70 and 120 DAB. IAA27 was up- regulated in the large- fruit mutant at 120 and 170 DAB, and the difference gradually increased. GH3.1, SAUR36 and auxin-induced protein 22D were up-regulated in the large-fruit mutant at 70 DAB. ARR2, TIFY10A and TGA9 were down-regulated in large-fruit mutant at 70 and 120 DAB, and was upregulated at 170 DAB.【Conclusion】the development of juice sacs was possibly the cause of large-fruit phenotype in the mutant, and hormone levels may influence fruit size. The transcriptome analysis provided a relatively complete molecular platform for future studies on the difference of citrus fruit size.