Tissue sections, hormone changes, and transcriptomes analysis of the large-fruited bud mutation of Pyrus pyrifolia‘Cuiguan’

Abstract:【Objective】Fruit size is a very important agronomic trait, and large fruit cultivars are more appreciated in the market. Genetic background, cultivation measures, and chemicals could regulate fruit size. A large-fruited bud mutant of the Cuiguan pear (Pyrus pyrifolia) was found previously. The mechanism of mutation of this mutant were detected by studying the physiology, biochemistry and gene expression of the mutant.【Methods】A large- fruited bud mutant from Cuiguan pear (BCG) and normal Cuiguan pear (CG) were studied. All trees were grafted on seedling rootstocks. The fruit flesh of CG and BCG were sampled on 0, 10, 20, 30, 40 days after blossoming (DAB). Three independent biological replicates were analyzed per treatment (each replicate was composed of a pool of 5 fruits). The histological observation of the pulp tissue of the cross section was obtained from fruit samples. Three pears in each sample were used for slicing. The anatomical images were observed using a microscopic imaging system of Eclipse Ti-S (Nikon). The plant hormones were determined by liquid chromatography - mass spectrometry. A total of 100 mg of sample were extracted by ethyl acetate. The supernatants were evaporated to dryness. The dry matter at the bottom of the tube was dissolved with methanol. The super-natant was injected into a LC-MS (AB Qtrap5500). The content of trans-zeatin (tZT) (Parent ion: m/z = 218.0; quantitative ion: m/z =134.0) and gibberellin A3 (GA3) (Parent ion: m/z =345.2; quantitative ion: m/z =239.1) were analyzed. The standards were tZT (Z0876) and GA3 (48880) (Sigma-Aldrich). The calibration curves were plotted using 0, 2.5, 5.0, 10, 12.5, 25 and 50 ng·mL-1 of each standard. The total RNA was extracted using the CTAB method. The RNA purity and concentration were assessed using a NanoDrop 2000 (Thermo). The cDNA libraries were constructed using NEBNext Ultra RNA Library Prep Kit (NEB) according to the manufacturers instructions. The cDNA fragments of preferentially 250-300 bp in length were purified. Subsequently, USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA. Then PCR was performed, and the PCR products were purified and library quality was assessed by the Agilent Bioanalyzer 2100 system. Each library (approximately 10 ng) was used for Paired-End sequencing using Illumina HiSeq™ 4000 (Illumina). The raw sequence data were filtered to remove low- quality reads. The clean reads were mapped to the Pyrus reference genome (NCBI, GCF_000315295.1) using Hisat2 software. The unique mapped reads were used in subsequent analyses. The gene expression level was calculated using the method of fragments per kb per Million reads (FPKM) by RSEM. The raw counts were analyzed using DESeq2 software based on negative binomial distribution. The genes/transcripts with differential expression between groups were selected by p-adjust ≤ 0.05 and |Log2Foldchange| ≥ 1.【Results】The median value of mature fruit weight of BCG and CG was 425.8 g and 269.0 g respectively. The mature fruit of BCG was 59.4% larger than that of CG. The Paraffin section results showed that the cross-section area of cell of BCG was bigger than that of CG. The content of tZT decreasing in both of CG and BCG after blossoming. On 10 DAB, tZT content was of BCG higher than that of CG. At other time points, the difference of tZT content was not significant. The GA3 content exhibited the trend of down-up-down. On 20, 30 and 40 DAB, the content of GA3 of BCG was higher than that ofCG. The significant difference was only found on 20 DAB. BCG and CG fruits were sampled at five stages and subjected to RNA-Seq analysis. Between 42.7 and 61.1 million paired-end reads (raw data) were obtained from each library. After a stringent quality filtering process, the counts of clean reads per library ranged from 42.33 to 60.52 M, with a Q30 percentage ≥ 93.4%. The SRA data of RNA-seq was deposited in Genome Sequence Archive in the National Genomics Data Center (https://ngdc.cncb.ac.cn/) (accession number, CRR327161- CRR327190). The differentially expressed genes (DEGs) were identified by pairwise comparisons of the libraries from CG and BCG at each sample time. A high number of DEGs (264 upregulated and 345 downregulated DEGs) were found on 10 DAB, and a low number of DEGs (52 upregulated and 62 downregulated DEGs) were found on 20 DAB. 609 and 639 DEGs were found on 0 and 30 DAB respectively. After combining the DEGs in all five stages, 2015 DEGs were found in the young fruit development in pear. The Venn diagram showed that a large number of DEGs were specific at each stage. 302 DEGs were shared in more than two stages. It is generally believed that DEGs shared in different periods might be the key genes. Among the 302 DEGs, only one gene was found to be differentially expressed at all stages, which was a long non-coding RNA (lncRNA, LOC103927682). It was down-regulated in BCG and presumed to be a negative regulatory gene. Six genes were significantly differentially expressed at the four stages. A late embryo enrichment protein D-29 (LOC103944693) was significantly up- regulated in BCG at the last four stages. Three genes were related to metal elements. The Metallothionein 1 (LOC103944922), metal nicotinamide transporter YSL2 (LOC103951610) and iron superoxide dismutase (LOC103953698) were down- regulated in BCG at the last four stages. A gene for RNA- dependent RNA polymerase 1 (LOC103943088) was found to be up- regulated in BCG at most stages. An extensin gene (LOC103937946) was significantly up- regulated in BCG on 10 and 20 DAB. In addition to analyzing the differential genes shared by different periods, we analyzed the differential genes on 0 and 10 DAB. On 0 DAB, the expression of auxin-binding proteins ABP19a, KLUH and gibberellin regulatory protein 6 was significantly up-regulated in BCG. The expression of gibberellin 3-β-dioxygenase was down-regulated. On 10 DAB, the expression of the expansin was significantly up-regulated, and two members of gibberellin 2-beta-dioxygenase were significantly down-regulated in BCG. The three cytokinin-related genes (AHP1, ARR17 and ORR10) were significantly up-regulated in BCG.【Conclusion】This study revealed the possible reasons for the large fruit bud sport of Cuiguan pears in the physiological, biochemical and molecular levels. The cytokinins and gibberellins might be involved in the control of fruit size. The transcriptome analysis provided a relatively complete molecular platform for future studies on the difference of pear fruit size.