Identification Of Shortened Fruit Mutant And Analysis Of Differentially Expressed Genes In Woodland Strawberry

Fragaria vesca, the woodland strawberry, is emerging as one of the important genetic resources improved modern cultivated strawberries and model for the cultivated octoploid strawberry as well as the Rosaceae family and other fruit tree genetic, cellular, developmental, molecular biological research due to its small and sequenced genome, diploidy (2n= 14,240 Mb genome), self-pollination, small stature, ease of growth, short life cycle, and facile transformation. Fruit shape index is a primary aspect of the fruit exterior quality. There are many types of fruit shapes in strawberries. The most common is a conical shape. In addition, there are other fruit shapes such as long-wedged shape, long conical shape and globose shape. In order to study formation mechanism of the strawberry fruit shape. The woodland strawberry’Yellow Wonder’and its shortened fruit mutant are used as materials. Phenotypic characterization of YW and sf plants and fruits, genetic development and the correlation with gibberellin were analyzed. Differentially expressed genes are analyzed using RNA-Seq. We have cloned gene associated with gibberellin synthesis. The conclusions are as follows.1. Wild-type and mutant were analyzed based on phenotypic characterization, cell analysis and the correlation with gibberellins.The leaf length and fruit length of mutant were significant shorter than those of wild-type. In addition, the mutant has dark green leaves compared to wild-type.The rate of pollen germination of mutant was less than wild-type, the mutant expressed a certain degree of fertility decline. Mutant receptacle was much shorter than that of wild-type receptacle, short fruit shape of mutant has been formed during the period of flower bud. Gibberellin level of wild-type obviously higher than that of mutant, short fruit mutant fruit shape can restore to the wild type fruit shape after exogenous application of GAs. The mutant was a GA-deficient mutant and a GA-sensitive mutant. Genetic analysis concluded that mutant belong to recessive inheritance controlled by single gene.2. Differentially expressed GA pathway genes were identified by comparisons between wild-type and mutant in fruit bud stage and small green fruit stage. Some GA pathway gene expressed differently in two stages. A total of 29 down-regulated GA genes were differentially expressed between wild-type and mutant in fruit bud stage transcriptomes including key genes of gibberellin synthesis:GA3ox3(gene10124), GA3ox4 (gene02231), GA20ox1(gene01062) and CYP450714C2-like (gene16769). A total of 28 genes were down-regulated including key genes of gibberellin synthesis in small fruit stage:GA3ox3(gene02611), GA20ox1(gene31924), CYP450 734A1-like (gene01176) and CYP450 714C2-like (gene16769). CYP450714C2-like (gene16769) differentially expressed in two stages.45 DEGs were selected for validation using qRT-PCR. The data revealed that the expression patterns of these genes measured by qRT-PCR were highly consistent with the expression patterns of the RNA-Seq data, suggesting that the transcriptome analysis was accurate and reliable.3. Using PCR technology, FvCYP714C2 gene were cloned from the wild-type and mutant strawberry.The full-length of DNA is 1940 bp. FvCYP714C2 gene sequence of wild-type and mutant were identical and has 100% homology with the CYP714C2 of Hawaii 4 which posted on the NCBI.There are five exons and four introns in full-length of DNA.The length of intron is 80 bp,92 bp,134 bp and 95 bp, respectively. The CDS length sequence of FvCYP714C2 is 1539 bp, encodes 512 amino acids and contains the main features of the cytochrome P450 conservative amino acid sequence FxxGxRxCxG. Contains the main features of the cytochrome P450 FxxGxRxCxG conservative amino acid sequence. Physical and chemical propertie suggesting that the gene encoding protein is hydrophobic unstable protein.4. FvCYP714C2 genes are expressed on root, stem, leaf, pedicel, flower bud, frower and the small green fruit of wild type and mutant. The express level was no difference in the stem, however, FvCYP714C2 gene expression in the wild-type were significantly higher than that in the expression of mutant on the root, leaf, pedicel, bud, flower and small green fruit. In terms of the expression of the same strain, FvCYP714C2 expression level in leaves were the highest, followed by in pedicel and the least in the roots and fruits.5. The plant overexpression vector pRI 101-ANFvCYP714C2 had been built. Using agrobacterium mediated infection,5 Arabidopsis thaliana plants that have lodge FvCYP714C2 gene have been obtained. GA1+3 level of FvCYP714C2 transgenic Arabidopsis thaliana plants were significantly higher than that of wild type plant. FvCYP714C2 have have promoted obviously vegetative growth of transgenic Arabidopsis thaliana. The shortened fruit mutant may be formed because the down regulation of FvCYP714C2.