Removal of a 10-kb Gret1 transposon from VvMybA1 of Vitis vinifera cv. Chardonnay

Authors: Yang, Yingzhen; KE, JOHN - Former ARS Employee; HAN, XIAOYAN - Michigan State University; Wuddineh, Wegi; SONG, GUO-QING - Michigan State University; Zhong, Gan-Yuan

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/31/2022
Publication Date: 9/6/2022
Citation: Yang, Y., Ke, J., Han, X., Wuddineh, W., Song, G., Zhong, G. 2022. Removal of a 10-kb Gret1 transposon from VvMybA1 of Vitis vinifera cv. Chardonnay. Horticulture Research. https://doi.org/10.1093/hr/uhac201.
DOI: https://doi.org/10.1093/hr/uhac201

Interpretive Summary: There are many economically important white grape cultivars and restoration of berry color in these cultivars may provide additional choices for grape industry and consumers. Berry color is an important fruit quality trait in grapes (Vitis) and largely influenced by the content of anthocyanins in berry skin. Anthocyanin content in berry skin is controlled by a cluster of genes named as VvMybA1-VvMybA4. Lack of anthocyanin pigments in most white grape cultivars is attributed to the loss of VvMybA1 gene expression due to insertion of a ~10-kb transposable DNA fragment, called Gret1, in the promoter of the VvMybA1 gene. In this study, we report the successful removal of the 10-kb Gret1 transposable DNA fragment from VvMybA1 in the white grape 'Chardonnay' through CRISPR/Cas9 editing which is a new genome technology recently developed and widely used by others. While restoration of berry color in the modified 'Chardonnay' is yet to be confirmed, this work demonstrates the technical potential of using the gene editing technology in modifying a critical color gene in grapes.

Technical Abstract: Many white grape cultivars have a nonfunctional VvMybA1 gene due to the presence of a 10-kb Gret1 transposon in the promoter sequences of VvMybA1. In this study, we successfully removed the 10-kb Gret1 transposon from a VvMybA1 allele in Vitis vinifera cv. Chardonnay through transgenic expression of Cas9 and two gRNAs simultaneously targeting two junction sequences between Gret1 LTRs and VvMybA1 with the aim of restoring VvMybA1 function. We generated 80 Agrobacterium-transformed and 106 bombardment-transformed transgenic vines and conducted molecular analyses of editing efficiencies in these vines and their progenitor calli. While the target editing efficiencies were as high as 17% for the 5’ target site and 65% for the 3’ target site, simultaneous editing of both 5’ and 3’ target sites (double editing) resulting in the removal of Gret1 transposon from the VvMybA1 promoter was 0.5% or less in most transgenic calli and vines, suggesting that these calli and vines had very few cells with their VvMybA1 alleles successfully double-edited at both target sites. Nevertheless, two bombardment-transformed vines were found to have the Gret1 successfully edited out from one of the two VvMybA1 alleles. Precisely removing more than a 10-kb DNA fragment from a grape gene broadens the possibilities of using gene editing technologies in modifying traits in grapes and other plants. Detailed molecular and sequencing analyses of the edited events in transgenic calli and vines revealed many interesting features of gene-editing, including large structural changes likely resulting from illegitimate recombination of highly homologous VvMybA genes in the VvMybA complex loci.