Study on factors affecting fruit development of seedless ponkan cultivar Huagan No. 4

Abstract:【Objective】Ponkan (Citrus reticulata Blanco.) as the main Mandarins cultivar. Huagan No.4 is a new citrus cultivar selected from a bud sport of Jing’an ponkan, and its fruit quality is significantly improved. However, the cultivar has the problems of large flowering but low fruiting rate and slow fruit development, which is not conducive to the promotion of new cultivars and industrial deployment. Therefore, it is important to clarify the key factors affecting the fruit size of ponkan to provide a theoretical basis for the production of high-quality ponkan.【Method】Huagan No.4 (HG4) and Huagan No.2 (HG2) were used as test materials to observe the key factors affecting the fruit development. The main methods used in this study were making paraffin sections, measuring the transverse and longitudinal diameters of the fruits, sequencing transcriptome and analyzing the bearing base shoots with a diameter and fruiting site.【Result】Observation of the equatorial surface of young fruits from 5 to 45 days after flowering (DAF) revealed that cell layers in the white pericarp of HG4 was greater than HG2 at 5 DAF, but cell layers of HG4 was consistently smaller than HG2 from 15 to 45 DAF. The morphology of HG4 and HG2 was also observed during the cell division stage, and the white pericarp cell density of both continued to decrease from 5 to 45 DAF. The cell density of HG4 was consistently greater than HG2 from 15 to 45 DAF, and the significant difference at 35 DAF. Fruit transverse longitudinal diameters were measured at 30 day intervals starting at 80 DAF and were found to be consistently and significantly smaller in HG4 than HG2. The plant endogenous hormone IAA, GA₃ and Zeatin contents in young fruits of HG2 and HG4 showed an overall increase followed by a decrease from 15 days before flowering (DBF) to 45 DAF. The IAA contents of HG4 and HG2 were 117.08 ng/g and 290.21 ng/g, respectively, at 45 DAF, which were significantly different. The GA₃ content of HG2 peaked at 2.27 ng/g at 25 DAF, while HG4 was only 0.90 ng/g, with significant differences. The GA₃ and Zeatin contents of HG2 were higher than HG4 at 25 and 45 DAF. In order to further study the factors of HG4 fruit size, we sequenced the transcriptomes of HG4 and HG2 young fruits at 25 and 45 DAF. The study screened the differentially expressed genes using the Citrus Clementina genome with the criteria of p-value ＜ 0.05, log2FoldChange ＞ 1, and functionally analyzed the genes. Among the 173 differential genes identified, the transcription factors encoding the genes were screened. Three ERF transcription factors (Ciclev10005820m, Ciclev10005863m, Ciclev10021265m), two MADS (Ciclev10016394m, Ciclev10032489m), one GRF (Ciclev10025855m), and an ADAP  (Ciclev10025985m) by homologous sequence analysis with Arabidopsis transcription factors. These seven may be involved in the regulation of HG4 fruit size. Meanwhile, the homologous genes of YUC on the auxin synthesis pathway of HG4 (CmYUC10, CmYUC11), genes of GA20ox on the gibberellin(CmGA20ox1, CmGA20ox5), and genes of the cytokinin CmIPT2 were found to have an overall low level of expression in comparison to HG2, which is in accordance with the results of the plant endogenous hormone content measurement. CmGA20ox5 was consistent with the relative expression levels of GA20ox1 at 25 and 45 DAF. The expression were significantly lower in HG4 than HG2, suggesting that gibberellin has a greater possible influence on HG4 fruit development. In addition, the statistical analysis in the field revealed that the average thickness of bearing base shoots with a diameter corresponding to HG4 fruit transverse diameters ＜65 mm, 65 mm-70 mm, and ＞70 mm were 2.15 mm, 2.59 mm, and 2.97 mm, respectively. It found that the thickness was positively correlated with the size of the fruits. The proportion of large fruits with a transverse diameter ＞70 mm was as great as 40% for fruits siting on the tree upper, whereas only 4% for lower. 【Conclusion】Statistical analysis of data, measurement of plant endogenous hormone content and transcriptome analysis revealed that low expression of hormone metabolic pathway genes affects hormone content in HG4 fruits during early development period, which may lead to the inherent causes of smaller fruits. It found that the thickness of HG4 bearing base shoots with a diameter and fruiting site are also important factors affecting its fruit dimensions. Although the present study analyzed the content of endogenous hormone and expression of related genes to HG4 fruits development, hormones and signaling pathways were complicated and staggered. We can further combine the means of multi-omics such as genomes, transcriptomes and metabolomes to explore the mechanism of citrus fruit development in the future.

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