Identification and expression analysis of the anthocyanin synthesis-related R2R3-MYB gene family in Colocasia esculenta （L.） Schott

Objective This study aimed to identify and analyze the members of the R2R3-MYB gene family in taro Colocasia esculenta （L.） Schott， and to reveal the molecular mechanisms of the R2R3-MYB gene family in anthocyanin biosynthesis pathway in taro corm， thereby providing a theoretical foundation for breeding new taro varieties with high anthocyanin content.Method Ganyu 2 （white-fleshed） and Ganyu 3 （red-fleshed） were taken as experimental materials. The total anthocyanin content in flesh and cortex tissues of the two taro varieties during three corm development stages （early enlargement stage， middle enlargement stage， and late enlargement stage） were determined using the pH differential method. The members of taro R2R3-MYB gene family were identified using bioinformatic methods from the whole genome database of taro. Protein physicochemical properties， conserved domains， chromosomal localization information， and gene structural characteristics were analyzed and predicted. A phylogenetic tree of the R2R3-MYB gene fa-mily was constructed using MEGA 7.0. Anthocyanin biosynthesis-related genes were screened according to expression of R2R3-MYB family genes at the late corm enlargement stage. Key anthocyanin synthesis-related genes were further identified using correlation analysis and real-time fluorescence quantitative PCR.Result At the three corm development stages， the total anthocyanin content in the same tissue of Ganyu 3 （red-fleshed） was significantly higher than that in Ganyu 2 （white-fleshed） （P<0.05， the same below）. The highest total anthocyanin content among all samples was observed in the cortex tissue of Ganyu 3 （red-fleshed） at the late enlargement stage （6.23 mg/100 g）. A total of 97 members of the taro R2R3-MYB gene family were screened from the whole genome， which were all localized in the nucleus and unevenly distributed on 13 chromosomes and 9 contigs. Most proteins encoded 200~400 amino acid residues， which were acidic， and exhibited poor stability. Although the R2 and R3 repeat sequences of the MYB domain showed different cha-racteristic amino acid patterns， they were highly conserved between taro and Arabidopsis thaliana. All 96 taro R2R3-MYB family genes contained Motif 1， Motif 2， and Motif 3， and 1-5 introns. Phylogenetic analysis of different R2R3-MYB family members showed that taro R2R3-MYB proteins could be divided into 29 subfamilies， among which eight taro R2R3-MYB family proteins were closely related to Arabidopsis thaliana proteins involving in anthocyanin synthesis. Transcriptome sequencing and correlation analysis indicated that CeMYB63， CeMYB85， and CeMYB89 exhibited high expression in Ganyu 3 （red-fleshed） at the late corm enlargement stage but showed low expression in Ganyu 2 （white-fleshed） corms. Transcriptome sequencing and correlation analysis showed that genes CeMYB63， CeMYB85， and CeMYB89 in Ganyu 3 （red-fleshed） exhibited high expression at late corm enlargement stage， yet the three genes exhibited low expression in Ganyu 2 （white-fleshed）； the three genes were positively or highly positively correlated （P<0.01） with total anthocyanin content. Relative expression and transcriptome sequencing were consistent for anthocyanin synthesis-related genes CeMYB63， CeMYB85， and CeMYB89 in taro.Conclusion A total of 97 R2R3-MYB gene family members are identified in taro， and these family genes show conservation and diversity during evolution. Three key genes regulating anthocyanin synthesis in red-fleshed taro are identified as CeMYB63， CeMYB85， and CeMYB89.