Comparative Transcriptional Profiling Of Berry Development Between Early-ripening Mutant And Its Parent

The variation characters of grape are mainly concentrated on peel color, berry size, mature period, rachis, etc. ‘Fengzao’ is the bud sport of ‘Kyoho’. It matures earlier nearly 30 d than ‘Kyoho’. In this study, the transcriptional profiles of these two grape cultivars were investigated in berry peel and flesh by RNA-Seq analysis from fruit set to full ripening to understand the dynamics and differential expression patterns of genes expressed between them during fruit development and ripening.The gene expression profiles at different developmental stages from fruit set to ripe berries（EL 27, EL 29, EL 31, EL 33, EL 34, EL 35, EL 37 and EL 38） were compared between ‘Fengzao’ and ‘Kyoho’ using RNA-Seq. A total of 40 libraries were constructed and 447,300,854 successful sequences were obtained. Annotation based on grape 12× V2.1 gene predictions, the expression profiles of 23,178 and 22,982 genes were generated for the flesh and peel, respectively. The results are as follow:（1） A total of 447,300,854 successful sequences were obtained. The expression profiles of 23,178 and 22,982 genes were generated for the flesh and peel, respectively. 20,944 were found to be present in at least one of the developmental stages for the fleshes of both WT and MT. 20,804 genes commonly expressed in the peels of both WT and MT, 905 genes（4.35%） were shown to be differentially expressed with the p value below 0.05.（2） The differential expression genes were assigned to GO categories. After the functional annotations, the greatest differential expression was found for two genes, VIT₂00s0238g00060（tmv resistance protein n-like） and VIT₂14s0030g00950（superoxide dismutase）. Considering the GO（gene ontology） annotation, the GO category of locomotion has obvious difference both in the flesh and peel between ‘Kyoho’ and ‘Fengzao’. The GO category of viral reproduction has difference in the peel between ‘Kyoho’ and ‘Fengzao’. The most common significant differentially expressed genes detected both in the peel and flesh between ‘Kyoho’ and ‘Fengzao’ are three genes, VIT₂14s0030g00950（superoxide dismutase）, VIT₂00s0238g00060（tmv resistance protein n-like） and VIT₂13s0067g01100（disease resistance protein at3g14460-like）. They are all down-regulated whether in the peel or flesh.（3） The unique expressed genes were classified by（expressed only in ‘Kyoho’ or ‘Fengzao’） the Gene Ontology（GO） database, rhythmic process and protin tag are only presented in the peel of ‘Fengzao’. Metallo chaperone is just only in the peel of ‘Kyoho’ and the flesh of ‘Fengzao’.（4） The K-means results showed no great difference in the gene expression profiles between ‘Kyoho’ and ‘Fengzao’ both in the flesh and peel. Most of the gene expression patterns between ‘Kyoho’ and ‘Fengzao’ are similar. The genes of this group expressed in the flesh nearly all expressed in Cluster 9 of the peel. While for that of Cluster 11 of flesh and Cluster 1 of peel, both of them showed a downward trend. Cluster 7 in MT flesh and Cluster 14 in MT peel represent genes showing the lowest level of expression at véraison. While in the corresponding clusters in WT flesh and peel, the expression level have no great changes and were steady.（5） To validate our RNA-seq results, we performed quantitative real time PCR（qRT-PCR） to determine the levels of expression from the list of genes differentially expressed between WT and MT.（6） The results indicated that the ROS level in ‘Fengzao’ is higher than that in ‘Kyoho’. Higher level of ROS maintains the production of ROS below the damaging levels and further motivates the ripening process earlier in ‘Fengzao’. Transcriptome results were validated by examining the expression of selected genes. These results provide new information regarding the network of events leading to grape ripening.In this research, three significant different genes were found, superoxide dismutase, resistance protein n-like and disease resistance protein at3g14460-like, it turned out to be the reason ‘Fengzao’ ripening earlier than ‘Kyoho’. The data reveal new candidate genes potentially responsible for the early ripening mutant of grape and will provide an invaluable resource for the on-going molecular investigation of grape berry ripening. These findings may help to a better understanding of the progress of berry ripening and encourage further studies to identify downstream elements of this cascade.