Development Of Strawberry EST-SSR Primers And Preliminary Study On Flowering Mechanism Of Everbearing Strawberry

Strawberry(Fragaria ananassa Duchesne)an important economic fruit tree,has a strong fragrance,sweet and sour taste,and is loved by consumers.Due to the wide variety and similar phenotypes of strawberry,it is difficult to distinguish by naked eye,which makes it more difficult to cultivate high-quality strawberries varieties without molecular assistant identification.The EST-SSR molecular marker has been favored by domestic and foreign researchers for its high reproducibility and easy operation.Strawberry EST-SSR primers have also been developed,but compared to other fruit trees,the number of effective EST-SSR marks in strawberry is still lower.Therefore,in this study,strawberry EST sequences published in the public database were used to search for and screen suitable SSR sites,design and synthesize strawberry EST-SSR primers,by which analyze the genetic diversity of 25 strawberry varieties.Eventually,they will be identified and distinguished in terms of molecular genetics.In addition,according to their flowering habits,strawberries can be roughly divided into two groups,seasonal flowering(SF)and everbearing(EB).Studying the annual flowering mechanism of strawberries to promote strawberry flower bud differentiation has certain market economic effects.Therefore,in this study,the differences of carbon-nitrogen ratio and the expression patterns of Fa LFY,Fa SOC1,Fa AP1,Fa FT,miR156,and miR172 were compared between EB cultivars ’Monterrey’ and SF cultivars ’Zhangji’ which were used as material in different growth stages,in order to preliminary explore the flowering mechanism of the EB cultivars,and provide theoretical basis for other crops to cultivate four season varieties.The specific research results are as follows:1.59795 EST sequences of strawberry were searched and downloaded from NCBI,redundant sequences were eliminated online by CD hit,and vector sequences were screened by seqman software.2992 of 24776 EST sequences were SSR-EST sequences,including 3516 SSRs.According to statistical analysis,dinucleotide and trinucleotide repeats were the main types of EST-SSR of strawberry.Based on the principle of specific primer design,SSR-EST sequence primers were designed anddetected.It was found that the number of polymorphic primers accounted for 52.5%of the total number of designed primers.2.Seven pairs of polymorphic primers were selected as core primers for genetic diversity analysis of 25 strawberry varieties.As a result,they were successfully distinguished completely,and their genetic relationships were identified.3.By measuring the content of soluble sugar and soluble protein to compare the changes in the carbon-nitrogen ratio of ’Monterey’ and ’Zhang Ji’ at different growth and development stages,it was found that high C/N is beneficial to the flowering of EB cultivars;There was no flowering signal appeared even if ’Zhang Ji’ had a high C/N stage.It is speculated that the "carbon-to-nitrogen ratio hypothesis" applies only to EB cultivars.4.The spatio-temporal specific expression of LFY,AP1,SOC1,FT,miR156 and miR172 genes in ’Monterey’ and ’Zhang Ji’ was detected by q PCR,and the differential expression was analyzed.Results found(1)There was a mutual promoting relationship between LFY and AP1 genes,and there seems to be periodic fluctuations in ’Monterey’,which may have deeper meaning for the ’Monterey’ anniversary flowering;(2)The expression trend of SOC1 gene was very similar in ’Monterey’ and ’Zhang Ji’,and the expression of ’Monterey’ was more stable than that of ’Zhang Ji’,indicating that SOC1 is an important gene to regulate the flowering transformation of EB cultivars;(3)The FT gene has a promoting effect on the SOC1 gene.The expression trend is stable and similar in ’Monterey’ and ’Zhang Ji’,but the expression of FT gene is lower in ’Monterey’.It can be speculated that the effect of FT gene on flower formation of EB cultivars is regulated by multiple genes and multiple pathways,and its expression abundance is not significant;(4)The expression level of miR156 in strawberry would decrease with the increase of seedling age,and miR172 would show the opposite expression pattern.In addition,the miRNA expression trend of the EB cultivars ’Monterey’ during the period from petal exposure to petal opening was completely opposite to the SF cultivars ’Zhangji’and other plants.During this period,the expression of miR156 increased and miR172 decreased,indicating that there are specific regulatory pathways and networks in’Monterey’.