Transcriptome analysis based on the characteristic difference between Shatang tangerine and Shatang Lantern tangerine

Abstract：【Objective】In order to investigate the reason for characteristic differences between Shatang tangerine (Citrus reticulat) and Shatang Lantern tangerine, we compared the size of plants, waxy arrangement and transcriptome information. By using transcriptome sequencing data, we screened out the differential genes among the leaves, pulp, tangerine pith and peel of Shatang tangerine and Shatang Lantern tangerine, and explored the significant differential genes related to wax biosynthesis in the peel and leaves.【Methods】Four types of tissue samples including leaf, pulp, tangerine pith and peel were taken at the same growth period and frozen in liquid nitrogen immediately for transcriptome sequencing. Three biological replicates were set up for every experiment. Characteristic analysis and toluidine blue staining experiment were observed and recorded by naked eyes. The transcriptome sequencing results between the two samples were analyzed to find out the key differential genes. The change threshold set as the absolute value of log2FC (fold change) was greater than or equal to 1 (FC greater than or equal to 2) and q value was less than 0.05. Besides, we used https://www.omicstudio.cn/index as a tool to analyze difference genes, like GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis.【Results】Compared with Shatang tangerine, the leaves of Shatang Lantern tangerine were larger and curly, and had irregular edge. The fruit was larger and had a coarse and roughly textured rind. The peel in Shatang Lantern tangerine showed obvious gully, and the surface of the peel wasfull of dots, like a“lantern”. After toluidine blue staining, the peel and leave of Shatang Lantern tangerine was covered with dye, indicating that the waxy composition in these parts was loosely distributed. After filtering raw sequencing data of transcriptome, the effective data size was 6.32-8.00 G, respectively. Besides, the effective data accounted for more than 96% of the original data, among which the bases with mass value ≥30 accounted for more than 97% (Q 30%), indicating that the quality of the measured transcriptomic data was good and the results were reliable. When compared with the reference genome of Citrus clementina, the matching rate was 93.32%-95.17%, which could meet the conditions of subsequent bioinformatics analysis. Through comparative analysis of transcriptome data, there were more differential genes in the leave of Shatang Lantern tangerine, followed by pulp, peel and tangerine pith. The main functions of differential genes were protein binding, molecular binding and ATP binding. The number of differential genes ranked in descending order was leaf, pulp, pericarp and tangerine pith, the number of up-regulated genes ranked in descending order was leaf, pulp, pericarp and tangerine pith, which was the same as the number of down-regulated genes. The number of differential genes in leaves were much higher than that of other tissue parts, and the number of down- regulated genes was the largest (3239), followed by the number of up-regulated genes (2443). Tangerine pith had the lowest number of differential genes.【Conclusion】These results indicated that the differences between Shatang tangerine and Shatang Lantern tangerine were mainly reflected in leaves, pulp and peel, which was consistent with the results observed by naked eyes. According to the differences in wax distribution of the two cultivars, the genes involved in the keratin, suberin and waxy biosynthesis pathway (map00073) were analyzed. There were 12 and 9 different genes related to wax biosynthesis in the leaf and peel of Shatang Lantern tangerine, respectively, including 5 identical down- regulated genes, named as CER1, CER3, HHT, cytochrome P45086A8 and cytochrome P45086A22. These genes were highly likely to be the key genes causing uneven waxy distribution on the surface of Shatang Lantern tangerine. Most of the differential genes were down- regulated except FACR2 in pericarp and cytochrome P45086B1 and cytochrome P45096A1 in leaf. Therefore, the characteristics of Shatang tangerine and Shatang Lantern tangerine were significantly different including morphology and waxy distribution. The transcriptome data showed that the number of differentially expressed genes was largest in the leaves, and there were five identical wax synthesis genes with the same expression trend in the middle leaves and the peel, which may be the key genes leading to the loss of wax on the surface of Shatang Lantern tangerine.