Studies on cytology and related gene expression pattern of a large-fruited bud mutant from ‘Cuiguan' pear (Pyrus pyrifolia)

Abstract: 【Objective】Fruit size at harvest is determined by both cell number and cell size of flesh which result from cell division and cell expansion processes, respectively. Cell division is regulated by cell cycle. Some important regulatory proteins of cell cycle have been studied, such as cyclins (CYCs) and cyclin-dependent kinases (CDKs) . CDKs control the key transitions in the plant cell cycle. CDKA plays a pivotal role in both the G1-to-S and the G2-to-M transitions, while a reduction in CDKB1 activity results in a block at the G2-to-M transition. Plants contain much more CYCs than previously described in other organisms, some of them have been found to associate with CDKs. D-type cyclins (CYCD) are thought to regulate the G1-to-S transition and function at the G2-to-M transition. A-type cyclins regulate the S-to-M phase, and B-type cyclins control both the G2-to-M transition and the intra-M-phase.To investigate cellular and molecular mechanism related to fruit size in pear, ‘Cuiguan'pear (Pyrus pyrifolia Nakai.) and its spontaneous mutant with larger fruit size (named‘Panzhuang Dacuiguan') were used as materials in this study.【Methods】AFLP (amplified fragment length polymorphism) was used to analyze the genomic differences between ‘Cuiguan' and ‘Panzhuang Dacuiguan'. 128 Eco R I/Mse I selective primer pairs were adopted in AFLP analysis. To minimize the effect of low-intensity background peaks (noise) , threshold value for fragment selection were set towards average signal intensity and fragment fre-quency. FCM (flow cytometry) analysis was carried out to record the chromosome ploidy. Genome size wasmeasured using the Otto method. The filtered fluid was then centrifuged at a speed of 5 000 r·min^-1 for 2min. The system's light source of FCM was 488 nm argon lasers. Seasonal changes in longitudinal andtransverse diameters were measured at regular intervals during the course of fruit development. Freshweight, longitudinal diameter and transverse diameter of both‘Cuiguan'and‘Panzhuang Dacuiguan'fruits were measured at harvest. The flesh width was calculated from the difference between the largestwidth of the transverse section of the fruits and core diameter. Fruit (flower) samples were collected for cytol-ogy and gene expression analysis during 0-28 d after full bloom (DAFB) . Paraffin section was used for mi-croscopy observation of cell number and size of the fruits. Cell number was determined by counting the num-ber of cell layers. Cell size was determined by measuring the average diameter of seven contiguous cells.Ten observation zones per paraffin section were measured. Quantitative real-time PCR (Q-PCR) was per-formed to test the gene expression. Each Q-PCR reaction mixture contained SYBR Premix Ex TaqTM (10.0μL) , both primer (0.4 μL, 10 μmol·L^-1) , c DNA (2 μL) , and RNase-free H2O (7.2 μL) in a total volume of 20 μL. The reaction started with a preliminary step of 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The Q-PCR primers were designed using Primier 3 according to obtained sequences of c D-NA fragments.【Results】A total of 116 polymorphic primer pairs were chosen from 128 selective primerpairs for AFLP analysis which amplified 8 620 DNA fragments. The polymorphic rate and Neips association coefficient between them were 8.18% and 0.957 4, respectively, confirming that‘Panzhuang Dacui-guan'was a sport of‘Cuiguan'. FCM analysis showed that both‘Cuiguan'and‘Panzhuang Dacuiguan'were diploid. Therefor, the larger fruit size in‘Panzhuang Dacuiguan'was not the result of the chromosomedoubling. At maturity stage, ‘Panzhuang Dacuiguan'had a 17% larger fruit diameter and a 64% heavierfruit weight than those of‘Cuiguan', while no difference in internal quality was observed between two culti-vars. Cell division started at 0 DAFB (days after full bloom) and continued till 24 DAFB in‘Panzhuang Dacuiguan', longer than that of ‘Cuiguan' by 4 d. Cell number (cell layers) in the floral-tube tissue wasnine cell layers more in‘Panzhuang Dacuiguan'than in‘Cuiguan'at 28 DAFB. The average areas of thecell and cell nucleus at full bloom stage were larger in‘Panzhuang Dacuiguan'than those in‘Cuiguan'. Q-PCR analysis indicated CYCD3 expression of‘Panzhuang Dacuiguan'fruit during early fruit developmentwas 1.2 times as high as that of‘Cuiguan'. In other cyclin-related genes including CYCA2、CDKA1 and CDKB2, different expressions were also observed between‘Panzhuang Dacuiguan'and‘Cuiguan'.【Conclu-sion】The larger fruit size of‘Panzhuang Dacuiguan'pear, the spontaneous mutant of‘Cuiguan'pear, could be contributed to the increased cell number in the fruit flesh which was related to extended period ofcell division. Our work verified the important role of cell division in regulating pear fruit size.