Isolation And Validation Of F Locus Responsible For Floral Anthocyanin Pigmentation In Potato

Anthocyanins in floral petals play an important role in attracting pollinators,promoting seed dispersal and enhancing tolerance to abiotic/biotic stress.It could also add ornamental value for plants.Although some structural genes（CHS、F3H、DFR）involved in potato anthocyanin biosynthesis have been identified,genes controlling anthocyanin production in floral petal is still not clear.In this study,PGSC003DMG400019217（StMYB19217）as F gene was identified by genetic analysis and functional verification.Main results are as follows:（1）Four polymorphism In Del markers were screened in the end of chromosome10,and the main QTL was located between markers ta CR10-223 and ta CR10-362（51.15 Mb-52.16 Mb）,with a size of 1.01 Mb.（2）According to the genomic annotation,five MYB genes in the mapping regions were selected as candidate genes.Cluster analysis and sequence alignment showed that the five MYB candidates were clustered into the same group and shared similar conserved domain with the MYB genes involving anthocyanin biosynthesis in the other species.Thus,we speculated that the five MYB candidates possibly play the similar roles in plant anthocyanin biosynthesis.（3）Genetic analysis showed that StMYB19217 was co-segregated with the phenotype of anthocyanin production on flower petals.Furtherly,we tested the expression level of StMYB19217 in F₂individuals.The results showed that StMYB19217 was specifically expressed in pigmented petals but can’t be detected in unpigmented petals.The other four MYB candidate genes had no expression in flower petals.Above results suggested that StMYB19217 could be the regulatory gene responsible for the F locus in potato.（4）To verify the function of the StMYB19217,overexpression vector was successfully constructed and transformed by tobacco transient transformation.The result clearly showed that StMYB19217 could induce anthocyanin accumulation in tobacco leaves.In order to clarify the function of StMYB19217 in anthocyanin biosynthesis of potato petals,we knocked out the target gene in a diploid potato CIP65 with purple petals by CRISPR/Cas9 and finally obtained 14 positive transgenic lines.Two transgenic plants（KO-3 and KO-16）were screened and used for further phenotype evaluation.Compared with the wild type,both KO-3 and KO-16 had white flowers and no anthocyanin accumulation on petals.These results demonstrated that StMYB19217 was the key regulatory gene controlling anthocyanin biosynthesis in potato petals.This study improved our understanding on the biosynthesis and regulatory mechanism of anthocyanin production in potato.