| Xinjiang red-fleshed apple(Malus sieversii f.niedzwetzkyana(Dieck)Langenf) is the formae speciales of Malus sieversii. Different from domesticated apples, red-fleshed apples possess a dramatically high anthocyanin phenotype with highly pigmented foliage, floral and fruit tissues. M. sieversii f.neidzwetzkyana is an important wild plant resource with high ornamental and medical value for its red phenotype, but their flavor quality of fruits are mainly bad. In order to improve the fruit quality of the wild red-fleshed apple, we crossed M. sieversii f.neidzwetzkyana with cultivar apples in 2006 and obtained large cross population. Used the cross population as material, we studied the genetic basis of the red phenotype, the segregation law of red and green phenotype, the callus culture and somaclonal variation of red-fleshed apple. The purpose of this study is to uncover the genetic development of apple red character and provide theoretical support for red-fleshed apple breeding and utilization. The main results are shown as follows:1. MdMYB10 was cloned from the red plant in cross population. In nucleotide level, its coding domain sequence(CDS) had 100% identity to MdMYB10_Maypole(AB744002) and 99% identity to MdMYB1_Golden Delicious(DQ886415) and MdMYBA_Tsugaru(AB279598); its promoter sequence had 99% identity to MdMYB10_niedzwetzkyana(EU518250) and 98% identity to MdMYB1_Golden Delicious(DQ886415). Its promoter also contained six repeat sequences which was the character of MdMYB10.2. The MdMYB10 locus genetype of hybrid progeny was identified by PCR, including eight red plants and eight green plants in F1 population and ninety-five red plants and five green plants in BC1 population. The result indicated MdMYB10 was specific in red plants and not in green plants.3. The hybrid progeny of MdMYB10/MdMYB10×MdMYB1/MdMYB1 were almost all red plants, but still exsit 5% green plants. The average segregation ration of red and green phenotype of twelve MdMYB1/MdMYB10×MdMYB1/MdMYB1 hybridized combinations was 0.6:1, the highest segregation ration was 0.89:1, but the low segregation ration was only near 0.2:1. Nine green plants selected from the hybrid progeny of MdMYB10/MdMYB10× MdMYB1/MdMYB1 were identified by SSR and demonstrated six green plants among them were hybrid. This indicated the red phenotype was dominant and controlled by MdMYB10, and MdMYB10 locus might exsit mutation mechanisms.4. Red callus was induced from the leaf of red plant named ?Zihong 3‘. The anthocyanin composition of red callus was detected by HPLC, and found three anthocyanins: cyanidin-3-galactoside, cyanidin-3-arabinfuranoside, and cyanidin-3-xyloside. Among them, cyanidin-3-galactoside was the main anthocyanin composition account for over 94% of the total peak area. NAA 0.3 mg/L combined with 6-BA 1.0 mg/L was the best hormone combination for red callus anthocyanin accumulation. 3% sucrose was the best carbohydrate source for red callus culture. Low nitrogen concentration was favor to callus anthocyanin accumulation but inhibited callus growth. Under the condition of nitrogen deficiency anthocyanin content could up to 2 mg/g. Auxin could inhibit callus anthocyanin synthesis. Cytokinin could promote callus anthocyanin synthesis and might exsit interaction with auxin in anthocyanin synthesis regulation.5. The expression difference of anthocyanin synthesis-related genes between red callus and yellow callus was analysed. Except MdTTG1, other anthocyanin synthesis regulatory genes and structure genes were higher expressed in red callus than that in yellow callus. Further study indicated that the expression of anthocyanin synthesis-related genes could quickly be inhibited after 2 hours of 0.25 mg/L 2,4-D treatment.6. The expression of IAAs and ARFs family genes in red and yellow callus was analysed and obtained 12 differently expressed IAA genes and 13 differently expressed ARF genes. Further, Y2 H and Bi FC assay showed that MdARF3 and MdARF4 protein interacted with MdMYB10 protein, respectively.7. Global gene expression was analyzed in red-fleshed apple calli treated with NAA(0.3 and 10 mg/L) and 2,4-D(0.03 and 0.6 mg/L) using RNA-seq. In our results, a total of 3,070 and 2,533 genes were differently expressed(log2 ration≥2 at P<0.0001) in 2,4-D and NAA treatment, respectively. Genes involved in anthocyanin and flavonoid synthesis and transport into vacuole were widely down-regulated. In auxin signaling pathway, 9 Aux/IAA family genes and 7 ARF family genes were both up-regulated. Moreover, 298 transcription factors were differently expressed in NAA and 2,4-D treatment. Among them, some members of MYB, bHLH and WD40 family directly regulating anthocyanin and flavonoid synthesis were down-regulated by NAA and 2,4-D. Auxin also affected gene expression in other plant hormone signaling pathways, such as cytokinin, ethylene, and GA, which also influenced anthocyanin biosynthesis. This provides a valuable overview of the transcriptome changes and gives insight into the molecular mechanism that auxin inhibited anthocyanin biosynthesis in red-fleshed apple calli.8. A green mutation was found during leaf regeneration in red-fleshed apple tissue culture. The anthocyanin content and anthocyanin synthesis-related gene expression in green mutation was both declined. PCR and sequence analysis demonstrated the MdMYB10 locus in green mutant occurred deficiency mutation. |
