Hui Li

In grapes ( Vitis L.), the transcription factors VvMYBA1 and VvMYBA2 , located on chromosome 2 (Chr2), are the primary genetic determinants of fruit skin color. However, the specific loci responsible for anthocyanin accumulation in the flesh remain unidentified. This study utilized ZhongShan‐HongYu (ZSHY, named by Tao Jianmin, Vitis vinifera L.) red‐fleshed grape germplasm, which accumulates anthocyanins in both skin and flesh, along with its selfed and hybrid progeny as experimental materials. Through bulked segregant analysis and RNA sequencing, LOC100233098 ( VvMYBA1 ), located on Chr2, was identified within the mapping region. The research found established that the red flesh phenotype is controlled by the dominant gene VvMYBA1 , with the color of leaves, skin, and flesh inherited together. Through fluorescence in situ hybridization, transient transformation of grape, and stable transformation of tobacco, the study determined that the insertion of a 408‐bp tandem repeat sequence within the VvMYBA1 promoter is the principal factor contributing to the significant increase in VvMYBA1 expression, ultimately leading to the red flesh phenotype in ZSHY. This mutation discovery will aid in the cultivation of new teinturier varieties, guide molecular breeding of grapes, and provide valuable resources for improving grape cross‐breeding.

Key message In our study, we discovered a fragment duplication autoregulation mechanism in ‘ZS-HY’, which may be the reason for the phenotype of red foliage and red flesh in grapes. Abstract In grapes, MYBA1 and MYBA2 are the main genetic factors responsible for skin coloration which are located at the color loci on chromosome 2, but the exact genes responsible for color have not been identified in the flesh. We used a new teinturier grape germplasm ‘ZhongShan-HongYu’ (ZS-HY) which accumulate anthocyanin both in skin and flesh as experimental materials. All tissues of ‘ZS-HY’ contained cyanidin 3-O-(6″-p-coumaroyl glucoside), and pelargonidins were detected in skin, flesh, and tendril. Through gene expression analysis at different stage of flesh, significant differences in the expression levels of VvMYBA1 were found. Gene amplification analysis showed that the VvMYBA1 promoter is composed of two alleles, VvMYBA1a and ‘VvMYBA1c-like’. An insertion of a 408 bp repetitive fragment was detected in the allele ‘VvMYBA1c-like’. In this process, we found the 408 bp repetitive fragment was co-segregated with red flesh and foliage phenotype. Our results revealed that the 408 bp fragment replication insertion in promoter of ‘VvMYBA1c-like’ was the target of its protein, and the number of repeat fragments was related to the increase of trans-activation of VvMYBA1 protein. The activation of promoter by VvMYBA1 was enhanced by the addition of VvMYC1. In addition, VvMYBA1 interacted with VvMYC1 to promote the expression of VvGT1 and VvGST4 genes in ‘ZS-HY’. The discovery of this mutation event provides new insights into the regulation of VvMYBA1 on anthocyanin accumulation in red-fleshed grape, which is of great significance for molecular breeding of red-fleshed table grapes.

ATP-binding cassette (ABC) transporters are a large class of superfamily involved in a variety of biological processes with multiple functions, including phytohormone transport, heavy metal ion detoxification, and so on. Anthocyanin pigmentation in grapes is a commercially important feature of this superfamily. To elucidate the mechanisms of the VvABC gene at different stages in grape berries, we analyzed and characterized the ABC family in ‘ZhongShan-HongYu’ (ZS-HY) berries using RNA-seq analysis. In this study, a total of 146 VvABC genes were identified by comprehensive bioinformatics analysis, which outlined their gene structure, chromosomal location, conserved domains, phylogenetic relationships, and collinearity analysis. The VvABC family could be divided into eight subfamilies based on the phylogenetic analysis. Fifty-eight VvABC genes were identified from the RNA-seq data, of which 31 belong to the ABCG subfamily, 15 belong to the ABCC subfamily, 8 belong to the ABCB subfamily, 2 belong to the ABCF subfamily, and only 1 belongs to each of the ABCA and ABCD subfamilies. We used qRT-PCR to detect the expression of VvABC genes in different organs and found that it changed significantly in different organs. Phylogenetic analysis showed that genes involved in anthocyanin transport in other species were closely related to members of the ABCC subfamily. Subsequently, analysis of the promoter elements and the protein interactions of the VvABCC genes using RNA-seq was performed. This study has improved our understanding of the functions of the ABC gene family and provided a basis for the role of ABC genes in grape anthocyanin transport.

Anthocyanins are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Glutathione S-transferases (GSTs) are thought to play a key role in anthocyanin transport. To clarify the mechanism of GST genes in the accumulation and transport of anthocyanin in the early fruit stage, we analyzed and characterized the GST family in the flesh of ‘Zhongshan-HongYu’ (ZS-HY) based on the transcriptome. In this study, the 92 GST genes identified through a comprehensive bioinformatics analysis were unevenly present in all chromosomes of grapes, except chromosomes 3, 9 and 10. Through the analysis of the chromosomal location, gene structure, conserved domains, phylogenetic relationships and cis-acting elements of GST family genes, the phylogenetic tree divided the GST genes into 9 subfamilies. Eighteen GST genes were screened and identified from grape berries via a transcriptome sequencing analysis, of which 4 belonged to the phi subfamily and 14 to the tau subfamily, and the expression levels of these GST genes were not tissue-specific. The phylogenetic analysis indicated that VvGST4 was closely related to PhAN9 and AtTT19. This study provides a foundation for the analysis of the GST gene family and insight into the roles of GSTs in grape anthocyanin transport.