Transcriptome sequencing analysis of differentially- expressed genes involved in the spongy tissue of Olecranon peach (Prunus persica L.)

Abstract：【Objective】Spongy tissue is a serious physiological disorder in Olecranon peach (Prunus persica L.). The symptom occurs about 10 days before fruit ripening, and the pulp becomes spongy in texture and brown in colour, causing significant economic losses in peach production. However, little has been known about the underlying mechanism causing spongy tissue up to now. Here, the comparative transcriptomics was used to explore the molecular mechanism of spongy tissue formation.【Methods】Samples from spongy tissue (BGHM) and non-spongy tissue (BGFB) in unhealthy flesh, and tissue in healthy fruit flesh (JKG) of Olecranon peach were collected and used for total RNA extraction. The high-throughput sequencing (HTS) data of transcriptome was generated with HiSeq 6000 platform. The published genome of P. persica (GenBank accession: GCF_000346465.2) was used as a reference. The HTS reads were mapped to the reference genome and the expression level of each transcript was determined by calculating transcript per million (TPM) with FADU. Differentially- expressed genes (DEGs) were identified using DESeq with the screening criteria of p＜0.01 and |log2FC|＞1.0. For func-tional analyses, GO and KEGG enrichment analyses were performed to investigate the major pathways of DEGs.【Results】Clean reads per sample generated by RNA- seq ranged from 18.4 to 30.5 million reads, and the mapping rate ranged from 95.08% to 95.67%. A total of 4557 DEGs were identified between spongy tissue and healthy fruit flesh (BGHM vs JKG), 2410 genes were up-regulated and 2127 genes were down- regulated. 672 DEGs were identified between spongy tissue and non- spongy tissue (BGHM vs BGFB), including 539 up-regulated genes and 133 down-regulated genes. 4446 DEGs were identified between non- spongy tissue and healthy fruit flesh (BGFB vs JKG), with 2121 up- regulated and 2323 down-regulated genes. The GO terms enriched for DEGs of BGHM vs JKG were 190. In molecular function, ion binding, oxidoreductase activity, and inorganic molecular entity transmembrane transporter activity were significantly enriched. In biological process, the responses to stimulus, chemicals and organic substance were significantly enriched. In cellular component, cytoplasm, obsolete cytoplasmic part and membrane were significantly enriched. The 4557 DEGs were significantly enriched in the 11 pathways through the KEGG analysis. Most of the DEGs were significantly enriched in metabolism, carbohydrate metabolism, and energy metabolism. In the comparison between spongy tissue and non-spongy tissue (BGHM vs BGFB), 21 GO terms were enriched from 672 DEGs. The top three GO terms of molecular function were glycosyltransferase activity, hexosyltransferase activity and glucosyltransferase activity. In biological process, most of the DEGs were classified into the response to stimulus, organic substance and oxygen-containing compound. In cellular component, the DEGs were mainly annotated into cell periphery, endoplasmic reticulum and external encapsulating structure. The KEGG results revealed that most of the DEGs were significantly enriched in metabolism, biosynthesis of other secondary metabolites and phenylpropanoid biosynthesis. In this study, 33 DEGs related to cell wall metabolism were identified in BGHM vs JKG, of which 25 genes were up- regulated and 8 genes were down- regulated. 17 DEGs related to cell wall metabolism were found in BGHM vs BGFB, with 17 genes up-regulated and 1 gene down-regulated. These genes included polygalacturonase, pectin methylesterase, β- galactosidase, xyloglucan endotransglucosylase, β- D- xylosidase and expansin. Among them, 12 DEGs (PG-At1g48100, PG-QRT3, PG, 6 XET2, BXL7 and 2 EXP-A4) were found at a higher expression level in BGHM than BGFB or JKM. Furthermore, the expression level of genes associated with calcium transport showed that 5 DEGs were up- regulated in BGHM vs JKG, including calciumtransporting ATPase 1, 3 calcium-transporting ATPase 13 and cation/calcium exchanger 5 and 6 DEGs were down-regulated including 5 calcium-transporting ATPase and cation/calcium exchanger 2. Only 3 up-regulated DEGs were found in BGHM vs BGFB, and they belonged to calcium-transporting ATPase 13. In the transcriptome, genes involved in calcium sensors were detected in the DEGs: Calcineurin-Blike protein, Calmodulin protein and Calmodulin- like protein. Among them, 15 up- regulated and 13 down- regulated DEGs were found in BGHM vs JKG, while 13 up- regulated and 13 down- regulated DEGs were detected in BGHM vs BGFB.【Conclusion】In the present study, our data provided the most comprehensive transcriptomic resource of spongy tissue and non-spongy tissue in unhealthy flesh, and tissue in healthy fruit flesh of Olecranon peach. A set of DEGs were identified through comparative transcriptome analyses, which were potentially involved in the metabolism, carbohydrate metabolism and energy metabolism process. Furthermore, 12 genes associated with cell wall modifying enzymes were found up- regulated in the spongy tissue and the expression level of 3 genes associated with calcium transport and 23 genes associated with calcium sensor increased or decreased in the spongy tissue. It is speculated that the calcium metabolism disorder caused by the up- regulation and down- regulation of calcium transport and calcium sensor genes might result in the reduction of the stress resistance in Olec-ranon peach. The calcium metabolism disorder and accelerated degradation of the cell wall would lead to the occurrence of spongy tissue. The results provide a reference for the molecular mechanism of spongy tissue of Olecranon peach from the transcriptional level.