Analysis of fruit quality and genetic difference of Orah mutant Yuwowuhe derived from radiation

Abstract:【Objective】Fruit quality analysis and whole genome resequencing as well as RNA-seq were conducted to reveal the difference of fruit quality and genetic background of Orah (Citrus reticulata Blanco) and its radiation mutagenesis line Yuwowuhe.【Methods】The fruit quality of orange varieties Orah and Yuwowuhe, the mutant of Orah derived from radiation as materials were evaluated. The leaves of Orah and Yuwowuhe were used for the whole genome resequencing, assembly and annotation. The ovaries of Orah and Yuwowuhe at flowering periods were used to extract RNA and sequenced by RNA-seq technology. The focus was on GO functional enrichment and KEGG pathway enrichment. Meanwhile, some differential expression genes (DEGs) were validated with qRT-PCR.【Results】The seeds number of Yuwowuhe were less than that of Orah. Compared with Orah, the fruits of Yuwowuhe matured earlier, had thicker pericarp and were harder to peel, and had bigger penetration and adherence. The fruits of Yuwowuhe had higher total soluble solids (13.17% ), ascorbic acid (31.56 mg · 100 mL-1) and solidity-acid ratio (29.70), low titratable acid (0.42 g·mL-1) and invert sugar (8.69 g·100 mL-1) in later fruit developmental stages. From the whole genome resequencing, assembly and annotation, we found that there were little differences in gene structure, function and non-coding RNA between Yuwowuhe and Orah, but a significant difference in the proportion of tandem repeats in genome (3.44% and 7.46%). Therefore, the genes located in or around of these tandem repeat sequences were conducted to do the functional enrichment analysis. GO functional enrichment demonstrated that the divergent genes related to small molecule binding and RNA binding were only involved in Orah, while Yu- wowuhe only present RNA polymerase activity and ATPase activity associated with transmembrane transport of substances. KEGG pathway enrichment showed that the divergent genes related to endocy- tosis and homologous recombination were only involved in Orah, while Yuwowuhe only involved galac-tose metabolism and peroxisome. At the same time, most genes were primarily involved in metabolic and biosynthetic processes. In addition, the genome of Yuwowuhe became bigger, while the number of genes decreased. The comparison of ovaries transcriptome sequencing between Yuwowuhe and Orah showed that a total of 4140 DEGs existed. GO enrichment analysis indicated that DEGs mainly in- volved in carbohydrate metabolism, oxidation-reduction process, β-galactosidase complex. The enrich- ment of KEGG pathway showed that DEGs mainly involved in phenylpropanoid biosynthesis, endoplasmic reticulum protein processing and secondary metabolite synthesis. Among them, most DEGs associ- ated with β-glucosidase, peroxidase, heat shock protein (HSPs), flavonol synthase and auxin response in the early growth stage (SAURs) were down-regulated in Yuwowuhe, while the expression of pollen-related G-receptor kinase genes were up-regulated. The qRT-PCR results showed that the relative expres- sion levels were basically consistent with the transcriptome variation trend, that is most DEGs were down-regulated in Yuwowuhe, which proved that the transcriptome data of ovary was reliable.【Conclusion】In summary, Yuwowuhe was an early ripenning mutant variety with better fruit quality compared with Orah. Analysing Yuwowuhe at the molecular level, on the one hand, the whole genome resequencing suggests that the tandem repeats in the genomes of Yuwowuhe are lower than that of Orah, the enrichment analysis of corresponding genes in or flanking the tandem repeats were mainly related to me- tabolism and secondary metabolites synthesis. On the other hand, according to ovary RNA-seq at flowering stage, DEGs related to phenylpropanol biosynthesis pathway, endoplasmic reticulum protein processing pathway, secondary metabolite synthesis pathway, glucose metabolism pathway, plant hormone pathway was significantly different between Yuwowuhe and Orah, suggesting that the lower fruit setting rate and seedless traits of Yuwowuhe might be regulated by these DEGs.