Study On The Effect Of McMYB10 Promoter On Anthocyanin Accumulation In Crabapples With Different Leaf Color Types

Malus crabapples are one of the most important ornamentaland economic germplasm resources, providing abundant plantlandscape species and favorable research material to exploit themechanism of color formation, which belongs to the Rosaceae, Malus Mill family. To understand the effect of difference and number distribution of McM YB 10 promoter on anthocyanin accumulation in crabapple leaves, we selected 15 crabapple cultivars:the purple-leaf color cultivars,’Royalty’; spring-red cultivars ’Radiant’Crabapples is an apple tree (Malus) plants with ornamental value and the value of economic function, which has the advantages of diverse varieties, beautiful tree form, richful leaf color type, and bright color. The difference about the McMYB10 promoter of the variable leaf type of ornamental can affect the plant color, which is a good material for studying the mechanism of leaf color. This paper studied 15 crabapple cultivars including often purple-leaf cultivar Malus cv.’Royalty’; spring-red cultivars Malus cv.’Radiant’, Malus cv.’Indian Magic’, Malus cv.’Prairifire’, Malus cv.’Red Begonia’, Malus cv.’Red Splender’, Malus cv.’Kelsy’ and Malus cv.’Strawberry Parfait’; green-leaf color cutivars Malus cv. "Spring Snow’, Malus cv.’Donald’, Malus cv.’Dolgo’, Malus cv.’Snowdrift’, Malus cv.’Red Jade’, Malus cv.’Flame’ and Malus cv.’Jewelberry’. The main results were as follows:1. We collected five phyllotaxies of the 15 ornamental crabapple cultivars and photographed the phenotype; HPLC analysis was performed to detect the content of different varieties of leaf flavonoids; qRT-PCR analysis was performed to analyse of the relative expression of McMYB10 in different varieties and different phyllotaxy. Our results indicated that the anthocyanin content in purple-leaf cultivar was higher than other cultivars, and there are no obvious anthocyanin content in green leaf cultivars. In spring-red cultivars, with the color changed from red to green, the anthocyanin content gradually decreased from 1 to 5 leaf phyllotaxies. The qRT-PCR results showed that the expression of McMYB10 in crabapple leaf was corresponded to leaf color and flavonoid content. The relative expression levels of McMYB10 in purple and spring-red cultivars were higher than green-leaf cultivars. Meanwhile, the first and second phyllotaxies had a relatve higher expression of McMYB10 than in other phyllotaxies.2. We extracted the genomic DNA and cloned McMYB10 promoter in different crabapple cultivars, and compared the difference of McMYB10 promoter by sequencing and electrophoresis. The results showed that there is only one R6 fragment in purple-leaf cultivar, and there is only one R1 fragment in green-leaf cultivar. However, the R1 and R6 fragments were detected in spring-red cultivars at the same time.3. We used flow cytometry to detect the ploidy of crabapple. Our results showed that Maluscv. ’Royalty’ and Malus cv.’Snowdrift’ were triploid and other varieties were diploid. We used real-time fluorescence quantitative PCR method to analyse and calculate the number of copies of McMYB10 promoter in different cultivars. The results showed that the number of copies of McMYB10 gene promoter is significantly different in different type of leaf color. It also showed that the number of R1 and R6 promoter determined the color depth of buds in spring-red cultivars.4. Sulfite hydrogen salt sequencing (BSP) analysis was used to analyse the methylation level of McMYB10 promoter. The results showed that there is a negetive relationship between the methylation level of McMYB10 promoter and the expression level of McMYB10. Therefore, the methylation level of McMYB10 promoter affected the expression of its own, thus affected the development of leaf colour and resulted in the change of leaf color in the late development stage.To sum up. there is a closely relationship between the difference and distribution of McMYB10 pomoter and the coloraion of crabapple leaves. The distribution of R1 and R6 determined the different color of crabapple leaves, and the different number of R1 and R6 determined the red color of new buds in spring-red crabapple cultivars. Meanwhile, the methylation level of McMYB10 promoter affects the expression level of McMYB10, and affects the color of leaves in the last development stages.