Studies On The Molecular Markers Related To Anthocyanins In Mulberry Plants

Anthocyanins are a kind of important secondary metabolites in plants, which are associated with the color of most flowers, fruits, vegetables, stems, and leaves. Anthocyanins play important roles in the physiological activities of plants. Anthocyanins in reproductive organs can attract insects to propagating pollon and herbivores to dispersing seeds through exhibiting colorful color. In addition, anthocyanins in vegetative organs can protect the plant. A large number of studies about anthocyanins have been reported so far. Screening molecular markers linked to anthocyanins related traits and the studies on the molecular mechanism of the anthocyanin formation also have been reported in many plants.Molecular marker is a powerful tool for genetic research, which plays an important role in breeding and identification and evaluation of germplasm resources. Mulberry is a perennial woody plant that has medicinal and economic value. The application of molecular markers in mulberry is mainly used in the analysis of germplasm’s genetic relationship and genetic diversity. Screening out the markers linked to important agronomic traits has not been reported yet. Therefore, in the present study, we tried to obtain the molecular markers linked to important traits of mulberry, which are essential for breeding of mulberry. Mulberry fruits are rich in anthocyanins and fruit color is an important fruit quality. So, we aimed to screen out the markers linked to the biosynthesis of anthocyanins in mulberry plants. Applicating these markers in the selection of target traits in early phase will improve the selection efficiency.In this research, we identified anthocyanin biosynthetic genes in M. notabilis using bioinformatic methods. SSR markers at the loci near these genes were designed and used to investigate the relationship of leave colour with the markers. According to intron-length polymorphisms of these genes, we further developed SCAR markers to explore the relationship between anthocyanins related markers and fruit color using the mulberry cultivars with different fruit colors. The main results are as follows:1.Screening markers linked to young leaves color of mulberryWe identified fifteen anthocyanin biosynthetic genes in M. notabilis using bioinformatic methods including four CHS genes, two CHI genes, two F3H genes, two F3’H genes, two OMT genes, one DFR gene, one ANS gene, and one UFGT gene. A total of196markers were developed in a70kb region according to the scaffold information. These196markers and other318SSR markers which were randomly distributed in mulberry genome were used in the screening and we tried to find those linked to young leaves color. The results showed that no such marker was found because the color of young leaves was susceptible to environmental conditions.To study the relationship between lavender color of young leaves and anthocyanin biosynthetic genes, we analyzed the transcriptional levels of these genes by qRT-PCR. The results indicated that most of transcriptional levels of anthocyanin biosynthetic genes in lavender young leaves were higher than those with light green. Only the F3’H2showed similar expression level in the two kind leaves. In the young leaf which anthocyanins coloration is the strongest, the transcriptional levels of anthocyanin biosynthetic downstream gene DFR and ANS were the highest, in particular, the transcriptional level of ANS corresponded exactly with anthocyanins coloration in young leaves. These results illustrated that anthocyanins coloration in young leaves was closely associated with the expression levels of anthocyanin biosynthetic genes. The downstream genes might have a direct impact on the synthesis of anthocyanins in young leaves of mulberry.2. Development of markers based on the intron-length polymorphisms of anthocyanin biosynthetic genesWe focused on the structure of anthocyanin biosynthetic genes and then developed five SCAR markers according to their intron-length polymorphisms in different mulberry cultivars, which have distinct fruit color. The markers developed here including CHS2-SCAR, CHI1-SCAR, DFR-SCAR, ANS-SCAR, and OMT1-SCAR. These markers will be used for the following investigation of the relationship between anthocyanin biosynthetic genes and fruit coloration. 3.The detection of SCAR markers in mulberry germplasm resources with different fruit colorWe selected eleven mulberry germplasm resources with three different fruit color as the experimental materials. Five SCAR markers were used to detect their allelic variation. The results showed that the band pattern of the five SCAR markers in mulberry cultivars or varieties was different with each other. The characteristic of each of eleven mulberry cultivars or varieties can be discriminated using these five markers. Meanwhile, we found that CHS2-SCAR, DFR-SCAR, and OMT1-SCAR were the specific markers of varieties with yellow-green fruit, and OMT1-SCAR amplified the specific band of Morns macroura. The fact that there wasn’t specific markers for mulberry cultivars with violet black fruit and white fruit suggested that CHS2, CHI1, DFR, ANS, and OMT1, may not be the key genes controling the violet black coloration in fruit.