Screening of the key early warning genes involved in superficial scald of Granny Smith apples

Abstract: 【Objective】Gene expression level usually changes before or with the ripening and senescence in fruit. To identify the early warning genes for the incidence of superficial scald, the Granny Smith apples during storage were subjected to transcriptome sequencing and qPCR analysis.【Methods】950 normal Granny Smith apples with even size and without mechanical damage were harvested at the commercial maturity stage from the Qianyang Apple Experimental Station. After harvest, the fruits were immediately transported to the laboratory and stored at room temperature for 12 hours. Then these fruits were stored at（0 ± 0.5）℃ with 90% relative humidity. These fruits were randomly divided into two groups, including a sampling group (150 fruits) and an investigation group (800 fruits). Then, the apples in the investigation group were further assigned to the cold storage investigation group (200 fruits) and a shelf-life investigation group (600 fruits). For the sampling group, 9 fruits were randomly selected at 0, 15, 30, 40, 50, 60, 70 and 80 d, and the ethylene production rate was measured at (0±0.5) ℃. After that the peel was immediately separated, frozen in liquid nitrogen and stored at −80 ℃ until 80 d when Granny Smith apples were seen scald (the healthy tissue and scalded tissue were sampled respectively). For the shelf-life investigation group, 40 fruits were randomly selected at 0, 15, 30, 40, 50, 60, 70 and 80 days, and their chromatic aberration was measured after transfer to room temperature [(25±0.5) ℃ with 85% relative humidity]. Meanwhile, the incidence of superficial scald was also evaluated. For the cold storage investigation group, the incidence of superficial scald was evaluated based onall 200 fruits at 60, 70, 73, 76, 79, 82 and 85 d, respectively.【Results】When Granny Smith apples were stored at a low temperature for 70 days, slight symptoms of superficial scald appeared on the fruit surface, and the incidence and disease index were 12.5% and 0.031, repectively; after 85 days of storage, the incidence increased to 93.5%, and the disease index increased to 0.439. The ethylene production rate increased slowly with the extension of storage time to 50 days and increased sharply after 50 days. The L* value was 78.46 after 15 days of storage and decreased significantly after 40 days. After 50 days of storage, the L* value showed a slight upward trend, and basically maintained at about 65. The change of a* value was relatively stable during storage. After 40 days of storage, the b* value was significantly higher than that after 30 days and remained stable at about 35 after 40 days. Based on the apple genome database, 247 genes related to superficial scald were extracted and identified. 28 genes with FPKM＞5 can be divided into 3 groups according to RNA-seq. For Group Ⅰ, the gene expression level in normal tissue decreased with the extension of storage time, and the gene expression level in room temperature storage was significantly higher than that in low-temperature storage. The expression level of MD10G1321200 in low-temperature storage for 30 days was more than 3 times higher than that for 50 days. For Group Ⅱ, the gene expression level in normal tissue was higher during low-temperature storage, and there was a significant difference between 30 days and 50 days after low-temperature storage. The gene expression level of Group Ⅲ in diseased tissue was significantly higher than that in normal tissue during low-temperature storage. The expression level of MD01G1106900 decreased gradually, and it was higher in normal tissue than in diseased tissue. The expression level of MD03G1059200 in low-temperature storage for 30 days was 44 times higher than that for 50 days. On this basis, the trend enrichment analysis and relative expression of the above candidate genes were analyzed by real-time fluorescence quantitative PCR in Granny Smith apples during low-temperature storage. The results showed that the expression level of MdPPO and MdPAL1 decreased at first and then increased, MdPPO began to increase after 60 days of storage, and MdPAL1 began to increase after 50 days. The expression level of MdPOD1 and MdPOD2 always decreased during low-temperature storage, especially in the later stage, but the difference was not obvious. On the 80^th day of storage, the expression levels of MdACO and MdPAL2 in diseased tissue were significantly higher than those in normal tissue. The expression level of MdCAT2 increased in the early stage of storage, reached the peak at 70 days of storage, and decreased at 80 days of storage. The expression level of MdCAT2 in normal tissue was higher. However, the expression levels of MdAFS and MdC3H increased significantly at 40 days and then decreased. The expression levels of MdAFS and MdC3H in 40 days were 1.94 times and 5.29 times higher than those in 30 days, respectively. The expression level of MdCAT1 increased sharply after 40 days, decreased sharply after 50 days, and then increased.【Conclusion】The superficial scald of Granny Smith apples mainly occurred in the middle and late periods of low- temperature storage. MdAFS, MdC3H and MdCAT1 can be used as early warning genes for superficial scald in Granny Smith apples during storage.