Study On Germplasm Innovation And Molecular Mechanism Of Flower Color Formation In Red-flowered Strawberry

The red-flowered strawberry, known as an ornamental strawberry, is an intergeneric hybrid derived from the cross of cultivated pineapple strawberry (Fragaria×ananassa Duch.) and a purple-colored marsh cinquefoil relative(Potentilla palustis (L.) Scop.). It is increasingly valued due to its pretty red flowers, prolonged flowering period and edible fruits. The red-flowered strawberry could be used as ground cover or pot flower now. In this study, the red-flowered strawberry cultivars ’Pink Beauty’,’Pretty Beauty’ and ’Pink Princess’, white-flowered strawberry cultivar ’Honeoye’ and the interspecies hybrid ’YH15-10’ with abundant white flowers, aromatic fruits, strong runners and high level tolerance to cold were used as materials to improve the flower color, flower diameter, fruit quality and cold resistance, and to create novel red-flowered strawberry germplasms. The genetic analysis of flower color and pigment content, qualitative and quantitative analysis of pigments using HPLC-MS method, the transcriptome sequencing and qRT-PCR expression analysis method were carried out to explore the molecular mechanism of flower color in red-flowered strawberry. This research was of important theoretical and practical significance to artificially control the flower color of red-flowered strawberry and breed novel flower color cultivars. The mainly conclusions were as follows:The backcrosses of red-flowered strawberry cultivars’Pink Princess’and’Pretty Beauty’ and white-flowered strawberry cultivar’Honeoye’were performed to improve the fruit quality and weight, and lay a foundation for breeding big red-flowered strawberry fruit cultivars. Most of the morphological characters were widely separated in backcross population hybrids with high broad sense heritability, which was mainly affected by the genetic effect. The flower color among backcross population showed different degree of separation, and was mainly between the flower colors of parents. The fruit shape of backcross population was mainly conical, and most of the fruits were significantly better than the red-flowered parent. 50.00% individuals in the backcross of Pink Princess × Honeoye exhibited higher fruit weight than that of the red-flowered parent at P<0.05, and 6.67% individuals showed significantly higher fruit weight than the high-fruit weight-parent ’Honyone’. Some red-flowered strawberry strains with big fruit and excellent fruit quality were selected in backcross population. These results showed that it could be effectively improved the fruit characteristics via the backcross between red-flowered strawberry cultivars and modern strawberry cultivars.The reciprocal crosses of red-flowered strawberry cultivars ’Pink Princess’, ’Pretty Beauty’ and ’Pink Beauty’ were performed to improve the flower color of red-flowered strawberry, and create novel germplasm with new flower color. These results showed that the flower color of hybrid population was widely separated, and ranged from white to red, and mainly concentrated in pink. Some strains showed darker colors at the basal part of petals, and a few strains showed double blossoms. Other morphological characters were also widely separated with high broad sense heritability, which were mainly affected by genetic effect. The hybrids bore short conical, abnormal, wedge, round or conical fruit and some individuals were sterility. Meanwhile, some hybrids were selected for the bright flower color and excellent fruit quality. These results showed that selecting bright flower color of red-flowered strawberry as parents could improve the flower color by the conventional cross.The reciprocal crosses of octaploid red-flowered strawberry cultivars’Pink Princess’ (2n=8x=56),’Pretty Beauty’(2n=8x=56) and synthetic white-flowered dodecaploid individual ’YH15-10’(2n=12x=84) were performed to create high polyploidy red-flowered strawberries with improved cold tolerance. The results showed that the fruit-setting rates between these reciprocal crosses were significantly different. When the low-ploidy parent was used as a female, it was more successfully to obtain hybrids. Most hybrids were confirmed to be decaploids (2n=10x=70) based on cytological study, and several ployploids were also observed, such as octaploid (2n=8x=56), enneaploid (2n=9x=63), undecaploid (2n=11x=77), dodecaploid (2n=12x=84), and 13-ploid (2n=13x=91). Big flower, excellent fruit quality and strong musky flavor were found in some hybrids. The LT50 of hybrids and parents was measured to analyze the cold tolerance. The cold tolerance of about 76.4% hybrids was significantly superior to that of ’Pink Princess’ at P<0.05. These novel high polyploidy red-flowered strawberry hybrids with improved cold tolerance could be obtained using interploidy cross and used in further red-flowered strawberry breeding and cultivar improvement.The fresh petal colors of hybrids and parents were evaluated based on the visual color measurement, Royal Horticultural Society Color Chart and Chroma Meter using the CIELAB system, and divided into white, light pink, pink, deep pink and red five color groups. There were anthocyanins and flavones but no carotenoids in petals of red-flowered strawberry based on color reaction and UV-visible spectra analysis. Hybrid seedlings from crosses of Pink Princess x Honeoye, Pretty Beauty x Honeoye and Pink Princess x Pretty Beauty were investigated to determine the inheritance pattern of flower color and pigment contents. The results showed that the flower color of red-flowered strawberry was inheritance of a quality trait controlled by oligogenes, and the pigment content was a quantitative trait controlled by polygenes. Seven kinds of anthocyanins were detected from the petals of red-flowered strawberry hybrids by the high performance liquid chromatography-mass spectrometry (HPLC-MS), and among all anthocyanins, cyanidin 3-O-glucoside (Cy3G) was most abundant. While there were no anthocyanins detected in white-flowered hybrids. The putative flavonoid metabolic process in red-flowered strawberry hybrids and in white-flowered strawberry hybrids was drawn, according to the metabolic profiling of coloration materials. The pattern of flavonoid metabolic flux flow and productivity of different branches were evaluated in red-flowered strawberry to determine core status of cyanidins in the process of flower color formation in red-flowered strawberry.The red-flowered petals and white-flowered petals in hybrids were used for transcriptome sequencing based on Illumina HiSeq 2500.91 835 unigenes with an average length of 717 bp were obtained after the de novo assembly of short reads by Trinity software, in which 47 058 unigenes were annotated. Therefore, a high-quality and comprehensive transcriptome database of red-flowered strawberry was constructed successfully, which almost penetrated into all aspects of the whole life activities.38 288 unigenes were annotated using blastx with NCBI non-redundant (Nr) database, in which unigenes assembled showed about 66.5% similarity with F. vesca. In addition,12 832 unigenes were assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and most of unigenes were annotated in the metabolic pathways.5000 DEGs were found between the red-flowered library and white-flowered library, in which 3 204 DEGs were up, and 2 375 DEGs were enriched 283 metabolic processes. Biological pathway analysis revealed that 120 unigenes were involed in structural genes of metabolic process related to flower color formation, and 165 transcription factors were related to flower color formation.The expression patterns analyses of 17 genes in petals of different flower colors in red-flowered hybrids were performed from the transcriptome database by qPCR method. An integrative approach to investigate the flower-color variation mechanisms was set up through a combination of transcriptome sequencing and metabolite analysis. The high expression of F3’H gene and DFR gene and ANS gene substrate specificity in red-flowered strawberry led to flow to the Cy branch. That was why Cy derivatives were the main anthocyandins in red-flowered strawberry. The effect of competition of the FLS gene leads to the different proportions of flavonoids and anthocyanin synthesis, and affects the different color formation in red-flowered strawberry hybrids. Meanwhile, ANS gene, UFGT1 gene and UFGT4 gene could be the main reason for the no accumulation of anthocyanidin in white petals of red-flowered strawberry hybrids. The competition alteration of the substrate between FLS gene and DFR gene seems to be anther reason for no accumulation of anthocyanidin in white petals.