Changes in soluble sugar content and differential gene screening in Ziziphus jujuba Mill.‘Lingwuchangzao’ propagated by root tiller and grafting

【Objective】Ziziphus jujuba Mill.‘Lingwuchangzao’belongs to Rhamnaceae Jujube. Its fruits are rich in protein, sugar and other nutrients, and have high economic value and market development potential. It uasually propagated by root tillers and grafting. The study aimed to reveal the differences of soluble sugar metabolism in the fruits of Lingwuchangzao propagated by different propagaion methods through studying on the changes of soluble sugar content in different developmental stages of the fruits of Lingwuchangzao propagated by root tillers and grafting in order to provide a scientific basis for improving fruit quality.【Methods】The investigation was carried ou at the white ripening stage (NB vs JB), coloring stage (NZ vs JZ), and mature stage (NC vs JC) on the fruits of Lingwuchangzao propagated by root suckers and grafting as experimental materials, . The contents of glucose, fructose, sucrose and soluble sugar in the fruits of the three periods were determined respectively. Combined with transcriptome data analysis, significant differential genes were screened from four key sugar metabolism pathways, glycolysis/gluconeogenesis (ko00010), starch and sucrose metabolism (ko00500), fructose and mannose metabolism (ko00051), and amino sugar and nucleotide sugar metabolism (ko000520) with FDR＜0.05 and |log2FC|≥1 as the standard. By integrating phenotypic data with transcriptome data for WGCNA analysis, key differential genes regulating soluble sugar synthesis and metabolism at different developmental stages of Lingwuchangzao fruits were identified, followed by qRTPCR verification to ensure the reliability of the results.【Results】The soluble sugars in Lingwuchangzao included sucrose, fructose, glucose and others. The fructose was the main sugar accumulated at white ripe stage and coloring stage, while sucrose was the main sugar at mature stage. However, sugar accumulation exhibited significant developmental stage specificity in the fruits of Lingwuchangzao propagated with different methods. At the white ripe stage, the soluble sugar content of the fruits from Lingwuchangzao propagated by grafting was 17.55% higher than that from root tillers. At the coloring stage and maturity stage, the soluble sugar content of fruits from the Lingwuchangzao propagated by root tillers was 24.38% and 27.91% higher than that from grafted trees, respectively. At the coloring and mature stages, the sucrose content in the fruits fruits from Lingwuchangzao propagated by propagated by root tiller was 50.63% and 49.32% higher than that from grafted trees, respectively. At the coloring stage, the fructose content of in the fruits fruits from root tiller propagated trees was 20.25% higher than that from grafted trees. At the mature stage, the fructose content in the fruits from grafted trees was 13.32% higher than that from root tiller propagated trees. As the fruit grew and developed, the glucose content in the fruits from grafting propagated trees was 36.08%, 20.67%, and 26.85% higher than from the trees propagated by root tiller, respectively. 24 significantly differentially expressed genes (DEGs) were screened. Among them, the genes EP3 (ncbi_107406053), BAM1 (ncbi_107422617), BGLU11 (ncbi_107426268), PGMP (ncbi_107414395) showed higher expression level, and the difference of the gene expressions between the coloring period and the maturity period was significant, indicating the potential for further fluorescence quantitative analysis. Through GO functional enrichment analysis, it was found that there were significant differences in carbohydrate metabolism, hydrolytic enzyme activity and secondary metabolite synthesis between the trees propagated by two different methods. These differences might be closely related to the adaptability of trees and the fruit quality. The analysis of soluble sugar metabolism pathways and differential genes revealed that the gene PGMP (ncbi_107414395) appeared multiple times in the metabolic pathways, indicating that this gene might play an important role in regulating soluble sugars in Lingwuchangzao and could serve as a key candidate gene for further validation studies. Additionally, weighted gene co-expression network analysis (WGCNA) showed that the black, brown, and green modules were significantly associated with soluble sugar metabolism. Genes in the black module exhibited a strong positive correlation with fructose, with a correlation coefficient of 0.87; genes in the brown and green modules showed strong positive correlations with soluble sugar and sucrose, with correlation coefficients of 0.71, 0.86, 0.74, and 0.83, respectively. Finally, the genes ncbi_ 112492650 and HERC2 (ncbi_107420452), which exhibited the highest connectivity and high expression level in the black module, were selected as key candidate genes for subsequent validation. Through the fluorescence quantitative analysis (qRT-PCR), it was found that the trend of gene expression levels in the fruits from the trees grafted by root-tiller was basically consistent with the transcriptome sequenc-ing results at the white ripe stage, coloring stage and mature stage, indicating that the transcriptome data had high reliability.【Conclusion】This study revealed the difference of soluble sugar metabolism in the fruits of Lingwuchangzao propagated by two differnt methods , providing an important theoretical basis for optimizing the propagation mode of Lingwuchangzao. The genes ncbi_112492650, HERC2 (ncbi_ 107420452) and PGMP (ncbi_107414395) would play a key role in regulating soluble sugar metabolism of Lingwuchangzao, and could be used as subsequent key verification genes.