Dissection of the Pearl of Csaba pedigree identifies key genomic segments related to early ripening in grape.

Pearl of Csaba (PC) is a valuable backbone parent for early-ripening grapevine (Vitis vinifera) breeding, from which many excellent early ripening varieties have been bred. However, the genetic basis of the stable inheritance of its early ripening trait remains largely unknown. Here, the pedigree, consisting of 40 varieties derived from PC, was re-sequenced for an average depth of ∼30×. Combined with the resequencing data of 24 other late-ripening varieties, 5,795,881 high-quality single nucleotide polymorphisms (SNPs) were identified following a strict filtering pipeline. The population genetic analysis showed that these varieties could be distinguished clearly, and the pedigree was characterized by lower nucleotide diversity and stronger linkage disequilibrium than the non-pedigree varieties. The conserved haplotypes (CHs) transmitted in the pedigree were obtained via identity-by-descent analysis. Subsequently, the key genomic segments were identified based on the combination analysis of haplotypes, selective signatures, known ripening-related quantitative trait loci (QTLs), and transcriptomic data. The results demonstrated that varieties with a superior haplotype, H1, significantly (one-way ANOVA, P < 0.001) exhibited early grapevine berry development. Further analyses indicated that H1 encompassed VIT_16s0039g00720 encoding a folate/biopterin transporter protein (VvFBT) with a missense mutation. VvFBT was specifically and highly expressed during grapevine berry development, particularly at veraison. Exogenous folate treatment advanced the veraison of "Kyoho". This work uncovered core haplotypes and genomic segments related to the early ripening trait of PC and provided an important reference for the molecular breeding of early-ripening grapevine varieties.

Genome-wide characterization of long terminal repeat retrotransposons provides insights into trait evolution of four cucurbit species.

Cucurbits are a diverse plant family that includes economically important crops, such as cucumber, watermelon, melon, and pumpkin. Knowledge of the roles that long terminal repeat retrotransposons (LTR-RTs) have played in diversification of cucurbit species is limited; to add to understanding of the roles of LTR-RTs, we assessed their distributions in four cucurbit species. We identified 381, 578, 1086, and 623 intact LTR-RTs in cucumber (Cucumis sativus L. var. sativus cv. Chinese Long), watermelon (Citrullus lanatus subsp. vulgaris cv. 97103), melon (Cucumis melo cv. DHL92), and Cucurbita (Cucurbita moschata var. Rifu), respectively. Among these LTR-RTs, the Ale clade of the Copia superfamily was the most abundant in all the four cucurbit species. Insertion time and copy number analysis revealed that an LTR-RT burst occurred approximately 2 million years ago in cucumber, watermelon, melon, and Cucurbita, and may have contributed to their genome size variation. Phylogenetic and nucleotide polymorphism analyses suggested that most LTR-RTs were formed after species diversification. Analysis of gene insertions by LTR-RTs revealed that the most frequent insertions were of Ale and Tekay and that genes related to dietary fiber synthesis were the most commonly affected by LTR-RTs in Cucurbita. These results increase our understanding of LTR-RTs and their roles in genome evolution and trait characterization in cucurbits.

Identification of grape H3K4 genes and their expression profiles during grape fruit ripening and postharvest ROS treatment.

Fruit development is modified by different types of epigenetics. Histone methylation is an important way of epigenetic modification. Eight genes related to H3K4 methyltransferase, named VvH3K4s, were identified and isolated from the grape genome based on conserved domain analysis, which could be divided into 3 categories by the phylogenetic relationship. Transcriptome data showed that VvH3K4-5 was obviously up-regulated during fruit ripe, and its expression level was significantly different between 'Kyoho' and 'Fengzao'. The VvH3K4s promoters contains cis-acting elements of in response to stress, indicating that they may be involved in the metabolic pathways regulated by ROS signaling. The subcellular localization experiment and promoter activity analysis experiment on VvH3K4-5 showed that VvH3K4s may be regulated by H2O2. With H2O2 and Hypotaurine treatment, it was found that the expression pattern of most genes was opposite, and the expression level showed different expression trend with the extension of treatment time.

Transcriptome analysis reveals mechanism of early ripening in Kyoho grape with hydrogen peroxide treatment.

BACKGROUND: In a previous study, the early ripening of Kyoho grape following H2O2 treatment was explored at the physiological level, but the mechanism by which H2O2 promotes ripening at the molecular level is unclear. To reveal the molecular mechanism, RNA-sequencing analysis was conducted on the different developmental stages of Kyoho berry treated with H2O2. RESULTS: In the comparison of treatment and control groups, 406 genes were up-regulated and 683 were down-regulated. Time course sequencing (TCseq) analysis showed that the expression patterns of most of the genes were similar between the treatment and control, except for some genes related to chlorophyll binding and photosynthesis. Differential expression analysis and the weighted gene co-expression network were used to screen significantly differentially expressed genes and hub genes associated with oxidative stress (heat shock protein, HSP), cell wall deacetylation (GDSL esterase/lipase, GDSL), cell wall degradation (xyloglucan endotransglucosylase/ hydrolase, XTH), and photosynthesis (chlorophyll a-b binding protein, CAB1). Gene expression was verified with RT-qPCR, and the results were largely consistent with those of RNA sequencing. CONCLUSIONS: The RNA-sequencing analysis indicated that H2O2 treatment promoted the early ripening of Kyoho berry by affecting the expression levels of HSP, GDSL, XTH, and CAB1 and- photosynthesis- pathways.

Transcriptome profiling of 'Kyoho' grape at different stages of berry development following 5-azaC treatment.

BACKGROUND: 5-Azacytidine (5-azaC) promotes the development of 'Kyoho' grape berry but the associated changes in gene expression have not been reported. In this study, we performed transcriptome analysis of grape berry at five developmental stages after 5-azaC treatment to elucidate the gene expression networks controlling berry ripening. RESULTS: The expression patterns of most genes across the time series were similar between the 5-azaC treatment and control groups. The number of differentially expressed genes (DEGs) at a given developmental stage ranged from 9 (A3_C3) to 690 (A5_C5). The results indicated that 5-azaC treatment had not very great influences on the expressions of most genes. Functional annotation of the DEGs revealed that they were mainly related to fruit softening, photosynthesis, protein phosphorylation, and heat stress. Eight modules showed high correlation with specific developmental stages and hub genes such as PEROXIDASE 4, CAFFEIC ACID 3-O-METHYLTRANSFERASE 1, and HISTONE-LYSINE N-METHYLTRANSFERASE EZA1 were identified by weighted gene correlation network analysis. CONCLUSIONS: 5-AzaC treatment alters the transcriptional profile of grape berry at different stages of development, which may involve changes in DNA methylation.

Genome-wide identification of small heat-shock protein (HSP20) gene family in grape and expression profile during berry development.

BACKGROUND: Studies have shown that HSP20 (heat-shock protein 20) genes play important roles in regulating plant growth, development, and stress response. However, the grape HSP20 gene family has not been well studied. RESULTS: A total of 48 VvHSP20 genes were identified from the grape genome, which were divided into 11 subfamilies (CI, CII, CIII, CV, CVI, CVII, MI, MII, ER, CP and PX/Po) based on a phylogenetic analysis and subcellular localization. Further structural analysis showed that most of the VvHSP20 genes (93.8%) had no intron or only one intron, while genes that clustered together based on a phylogenetic tree had similar motifs and evolutionarily conserved structures. The HSP20s share a conservedα-crystalline domain (ACD) and the different components of the ACD domain suggest the functional diversity of VvHSP20s. In addition, the 48 VvHSP20 genes were distributed on 12 grape chromosomes and the majority of VvHSP20 genes were located at the proximal or distal ends of chromosomes. Chromosome mapping indicated that four groups of VvHSP20 genes were identified as tandem duplication genes. Phytohormone responsive, abiotic and biotic stress-responsive, and plant development-related cis-elements were identified from the cis-regulatory elements analysis of VvHSP20s. The expression profiles of VvHSP20s genes (VvHSP20-1, 11, 14, 17, 18, 19, 20, 24, 25, 28, 31, 39, 42, and 43) were largely similar between RNA-Seq and qRT-PCR analysis after hydrogen peroxide (H2O2) treatment. The results showed that most VvHSP20s were down-regulated by H2O2 treatment during fruit development. VvHSP20s genes were indeed found to be involved in the grape berry development and differences in their transcriptional levels may be the result of functional differentiation during evolution. CONCLUSIONS: Our results provide valuable information on the evolutionary relationship of genes in the VvHSP20 family, which is useful for future studies on the functional characteristics of VvHSP20 genes in grape.

MicroRNA profiling analysis of developing berries for 'Kyoho' and its early-ripening mutant during berry ripening.

BACKGROUND: 'Fengzao' is an early-ripening bud mutant of 'Kyoho', which matures nearly 30 days earlier than 'Kyoho'. To gain a better understanding of the regulatory role of miRNAs in early-ripening of grape berry, high-throughput sequencing approach and quantitative RT-PCR validation were employed to identify miRNAs at the genome-wide level and profile the expression patterns of the miRNAs during berry development in 'Kyho' and 'Fengzao', respectively. RESULTS: Nine independent small RNA libraries were constructed and sequenced in two varieties from key berry development stages. A total of 108 known miRNAs and 61 novel miRNAs were identified. Among that, 159 miRNAs identified in 'Fengzao' all completely expressed in 'Kyoho' and there were 10 miRNAs specifically expressed in 'Kyoho'. The expression profiles of known and novel miRNAs were quite similar between two varieties. As the major differentially expressed miRNAs, novel_144, vvi-miR3626-3p and vvi-miR3626-5p only expressed in 'Kyoho', vvi-miR399b and vvi-miR399e were down-regulated in 'Fengzao', while vvi-miR477b-3p up-regulated in 'Fengzao'. According to the expression analysis and previous reports, miR169-NF-Y subunit, miR398-CSD, miR3626-RNA helicase, miR399- phosphate transporter and miR477-GRAS transcription factor were selected as the candidates for further investigations of miRNA regulation role in the early-ripening of grape. The qRT-PCR analyses validated the contrasting expression patterns for these miRNAs and their target genes. CONCLUSIONS: The miRNAome of the grape berry development of 'Kyoho', and its early-ripening bud mutant, 'Fengzao' were compared by high-throughput sequencing. The expression pattern of several key miRNAs and their target genes during grape berry development and ripening stages was examined. Our results provide valuable basis towards understanding the regulatory mechanisms of early-ripening of grape berry.

Comparative RNA-Seq profiling of berry development between table grape 'Kyoho' and its early-ripening mutant 'Fengzao'.

BACKGROUND: Early ripening is an important desirable attribute for fruit crops. 'Kyoho' is a popular table grape cultivar in many Asian countries. 'Fengzao' is a bud mutant of 'Kyoho' and ripens nearly 30 days earlier than 'Kyoho'. To identify genes controlling early fruit development and ripening in 'Fengzao', RNA-Seq profiles of the two cultivars were compared at 8 different berry developmental stages in both berry peel and flesh tissues. METHODS: RNA-Seq profiling of berry development between 'Kyoho' and 'Fenzhao' were obtained using the Illumina HiSeq system and analyzed using various statistical methods. Expression patterns of several selected genes were validated using qRT-PCR. RESULTS: About 447 millions of RNA-Seq sequences were generated from 40 RNA libraries covering various different berry developmental stages of 'Fengzao' and 'Kyoho'. These sequences were mapped to 23,178 and 22,982 genes in the flesh and peel tissues, respectively. While most genes in 'Fengzao' and 'Kyoho' shared similar expression patterns over different berry developmental stages, there were many genes whose expression were detected only in 'Fengzao' or 'Kyoho'. We observed 10 genes in flesh tissue and 22 genes in peel tissue were differentially expressed at FDR ≤ 0.05 when the mean expression of 'Fengzao' and 'Kyoho' were compared. The most noticeable one was VIT_214s0030g00950 (a superoxide dismutase gene). This ROS related gene showed lower expression levels in 'Fengzao' than 'Kyoho' in both peel and flesh tissues across various berry developmental stages with the only exception at véraison. VIT_200s0238g00060 (TMV resistance protein n-like) and VIT_213s0067g01100 (disease resistance protein at3g14460-like) were the two other noticeable genes which were found differentially expressed between the two cultivars in both peel and flesh tissues. GO functional category and KEGG enrichment analysis of DEGs indicated that gene activities related to stress and ROS were altered between the two cultivars in both flesh and peel tissues. Several differentially expressed genes of interest were successfully validated using qRT-PCR. CONCLUSIONS: Comparative profiling analysis revealed a few dozens of genes which were differentially expressed in the developing berries of 'Kyoho' and its early ripening mutant 'Fengzao'. Further analysis of these differentially expressed genes suggested that gene activities related to ROS and pathogenesis were likely involved in contributing to the early ripening in 'Fengzao'.

Comparative microbiome analysis reveals the variation in microbial communities between 'Kyoho' grape and its bud mutant variety.

Microbes are an important part of the vineyard ecosystem, which significantly influence the quality of grapes. Previously, we identified a bud mutant variety (named 'Fengzao') from 'Kyoho' grapes. The variation of microbial communities in grape and its bud mutant variety has not been studied yet. So, in this study, with the samples of both 'Fengzao' and 'Kyoho', we conducted high-throughput microbiome sequencing and investigated their microbial communities in different tissues. Obvious differences were observed in the microbial communities between 'Fengzao' and 'Kyoho'. The fruit and the stem are the tissues with relatively higher abundance of microbes, while the leaves contained less microbes. The fruit and the stem of 'Kyoho' and the stem of 'Fengzao' had relatively higher species diversity based on the alpha diversity analysis. Proteobacteria, Enterobacteriaceae and Rhodobacteraceae had significantly high abundance in 'Fengzao'. Firmicutes and Pseudomonas were highly abundant in the stems of 'Kyoho', and family of Spirochaetaceae, Anaplasmataceae, Chlorobiaceae, and Sphingomonadaceae, and genera of Spirochaeta, Sphingomonas, Chlorobaculum and Wolbachia were abundant in the fruits of 'Kyoho'. These identified microbes are main components of the microbial communities, and could be important regulators of grapevine growth and development. This study revealed the differences in the microbial compositions between 'Kyoho' and its bud mutant, and these identified microbes will be significant resources for the future researches on the quality regulation and disease control of grapevines.

A simple and efficient protocol for transient transformation of sliced grape berries.

Grape is an economically important crop but recalcitrant to Agrobacterium-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of "Shine-Muscat" (Vitis labrusca × Vitis vinifera) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using ß-glucuronidase (GUS) reporter system. Pre-culture increased the susceptibility of explants to the agrobacteria infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate agrobacteria entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of GUS transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene VvHPCA3, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop.

Large-scale discovery of non-conventional peptides in grape (Vitis vinifera L.) through peptidogenomics.

Non-conventional peptides (NCPs), which are peptides derived from previously unannotated coding sequences, play important biological roles in plants. In this study, we used peptidogenomic methods that integrated mass spectrometry (MS) peptidomics and a six-frame translation database to extensively identify NCPs in grape. In total, 188 and 2021 non-redundant peptides from the Arabidopsis thaliana and Vitis vinifera L. protein database at Ensembl/URGI and an individualized peptidogenomic database were identified. Unlike conventional peptides, these NCPs derived mainly from intergenic, intronic, upstream ORF, 5'UTR, 3'UTR, and downstream ORF regions. These results show that unannotated regions are translated more broadly than we thought. We also found that most NCPs were derived from regions related to phenotypic variations, LTR retrotransposons, and domestication selection, indicating that the NCPs have an important function in complex biological processes. We also found that the NCPs were developmentally specific and had transient and specific functions in grape berry development. In summary, our study is the first to extensively identify NCPs in grape. It demonstrated that there was a large amount of translation in the genome. These results lay a foundation for studying the functions of NCPs and also provide a reference for the discovery of new functional genes in grape.

Overexpression of VvPPR1, a DYW-type PPR protein in grape, affects the phenotype of Arabidopsis thaliana leaves.

Pentatricopeptide repeat (PPR) proteins play important roles in plant growth and development. However, little is known about their functions in the leaf morphogenesis of Jingxiu grape (Vitis vinifera L.). Here, we explored the function of VvPPR1, which encodes a DYW-type PPR protein in grape. We showed that VvPPR1 is involved in the regulation of leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis thaliana. Analysis of structural characteristics showed that VvPPR1 is a DYW-type PPR gene in the PLS subfamily consisting of 15 PPR motifs. The N-terminal had a targeted chloroplast site, and the C-terminal had a DYW domain. Quantitative PCR analysis revealed that the expression level of VvPPR1 was highest in grape leaves. Subcellular localization revealed that VvPPR1 is localized in the cytoplasm and chloroplast. VvPPR1-overexpressing plants had rolled leaves, high degrees of anthocyanin accumulation, and longer trichomes. The expression levels of genes related to these phenotypes were either significantly up-regulated or down-regulated. These results demonstrate that VvPPR1 is involved in leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis; more generally, our findings indicate that VvPPR1 could be a target for improving the cultivation of horticultural crops.

Grape (Vitis vinifera) VvDOF3 functions as a transcription activator and enhances powdery mildew resistance.

DOF proteins are plant-specific transcription factors that play vital roles in plant development and defense responses. However, DOFs have primarily been investigated in model plants, and fairly limited research has been performed on grape (Vitis vinifera). In this study, we isolated and characterized a C2-C2 zinc finger structural DOF gene, VvDOF3, from the grape cultivar Jingxiu. The VvDOF3 protein showed nuclear localization and transcriptional activation ability, indicating that it functions as a transcription factor. The VvDOF3 gene was rapidly induced by exogenous salicylic acid (SA), jasmonic acid (JA), and powdery mildew infection. Overexpression of VvDOF3 in Arabidopsis thaliana enhanced resistance to Golovinomyces cichoracearum. Expression of the SA-responsive defense-related gene PR1 and the concentration of SA were up-regulated in transgenic Arabidopsis plants overexpressing VvDOF3. Together, these data suggest that VvDOF3 functions as a transcription factor in grape and enhances powdery mildew resistance through the SA signaling pathway.

Transcriptional Analysis of the Early Ripening of 'Kyoho' Grape in Response to the Treatment of Riboflavin.

Previous study has demonstrated that the riboflavin treatment promoted the early ripening of the 'Kyoho' grape berry. However, the molecular mechanism causing this was unclear. In order to reveal the regulation mechanism of riboflavin treatment on grape berry development and ripening, the different berry developmental stages of the 'Kyoho' berry treated with 0.5 mmol/L of riboflavin was sampled for transcriptome profiling. RNA-seq revealed that 1526 and 430 genes were up-regulated and down-regulated, respectively, for the comparisons of the treatment to the control. TCseq analysis showed that the expression patterns of most of the genes were similar between the treatment and the control, except for some genes that were related to the chlorophyll metabolism, photosynthesis-antenna proteins, and photosynthesis, which were revealed by the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The differentially expressed genes and weighted gene co-expression network analysis (WGCNA) analysis identified some significantly differentially expressed genes and some hub genes, including up-regulation of the photosynthesis-related ELIP1 and growth and development-related GDSL; and down-regulation of the oxidative stress-related ATHSP22 and berry softening-related XTH32 and GH9B15. The results suggested that the riboflavin treatment resulted in the variations of the expression levels of these genes, and then led to the early ripening of the 'Kyoho' berry.

Genome-wide association study of berry-related traits in grape [Vitis vinifera L.] based on genotyping-by-sequencing markers.

Deciphering the genetic control of grape berry traits is crucial for optimizing yield, fruit quality, and consumer acceptability. In this study, an association panel of 179 grape genotypes comprising a mixture of ancient cultivars, landraces, and modern varieties collected worldwide were genotyped with genotyping-by-sequencing using a genome-wide association approach based on 32,311 single-nucleotide polymorphism (SNP) markers. Genome-wide efficient mixed-model association was selected as the optimal statistical model based on the results of known control loci of grape berry color traits. Many of the associated SNPs identified in this study were in accordance with the previous QTL analyses using biparental mapping. The grape skin color locus was found to be associated with a mybA transcription factor on chromosome 2. Two strong and distinct association signals associated with berry development periods were found on chromosome 16. Most candidate genes of the interval were highlighted as receptor-like protein kinase. For berry weight, significant association loci were identified on chromosome 18, as previously known, and on chromosome 19 and chromosome 17, as newly mapped. Berry flesh texture was newly located on chromosome 16; candidate genes in the interval were related to calcium. Berry flavor was determined on chromosome 5. Genomic regions were further investigated to reveal candidate genes. In this work, we identified interesting genetic determinants of grape berry-related traits. The identification of the markers closely associated with these berry traits may be useful for grape molecular breeding.

Comparison of reactive oxygen species metabolism during grape berry development between 'Kyoho' and its early ripening bud mutant 'Fengzao'.

Enzymes and non-enzyme elements related to the metabolism of reactive oxygen species (ROS), such as catalase (CAT), superoxide dismutase (SOD), ascorbic acid (AsA), glutathione (GSH), NADPH oxidase (NOX), hydrogen peroxide (H2O2), superoxide anion (O2-), lipoxygenase (LOX) and malondialdehyde (MDA), were measured in 'Kyoho' and its early ripening bud mutant 'Fengzao' to compare ROS level changes and investigate the potential roles of ROS in grape berry development and the ripening process. In addition, the anthocyanin and sugar contents as well as berry diameter were also investigated at different berry development stages. The results showed that the H2O2 content and LOX activity exhibited obviously different trends between 'Fengzao' and 'Kyoho' during the berry development stages. Before berry softening, the SOD activity, LOX activity and H2O2 content were significant lower in 'Fengzao' than in 'Kyoho', but there were no significant differences in the production rate of O2-, ROS scavengers (CAT, AsA, GSH) and MDA content between them, which indicated that the higher oxidation status in 'Fengzao'. It may promote the faster development of 'Fengzao' berry than 'Kyoho' before berry softening (EL31-33). The significant higher LOX and CAT activities at EL-34, as well as significant higher LOX activity and H2O2 content at EL-35 in 'Fengzao' than in 'Kyoho' indicated H2O2 was acted as the appropriate oxidative stress factor and the signal molecule to further accelerate the berry ripening of 'Fengzao'. The increasing O2- and H2O2 after EL-35 in 'Fengzao' further promoted the ripening process. Furthermore, after the spraying of 300 µmol/L H2O2 solution on 'Kyoho' at EL-31 stage, the berries matured 15 days earlier than the untreated. Evidence in this study indicated that the overall oxidation status (ROS levels) in 'Fengzao' is higher than in 'Kyoho' and H2O2 could promote the early ripening of 'Kyoho' berry.

Histological and Molecular Characterization of Grape Early Ripening Bud Mutant.

An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape.