Mitochondrial Genome Sequencing And Sequence Analysis Of Fragaria × Ananassa 'Benihoppe'

Strawberry(Fragaria × ananassa)is widely cultivated around the world.The fruit is widely praised for its unique aroma,bright red color,succulent texture and sweetness.It has rich vitamin content,high nutritional value,and it has wide range uses.It can be used as an auxiliary material and used to make strawberry flavoring.It is popular by people.Therefore,it is more practical to study its related physiological and molecular aspects.The genus of Fragaria L.is ploidy and diverse,and its mitochondrial genome composition and evolution-related information are beneficial to its genetic breeding and phylogenetic relationships.The mitochondria is an important organelle in eukaryotic cells and is a powerful tool for the study of species origin,genetic diversity,and phylogenetics.So far,nearly 127 plant mitochondrial genomes have been published in the NCBI database,most of which are angiosperms.However,no complete mitochondrial genome sequences have been reported for strawberry.In this experiment,the Fragariaxananassa 'Benihoppe' was used to sequencing its mitochondrial genomes by second-generation sequencing technology,and the assembled sequence was combined with the mitochondrial genome sequences of other 13 angiosperms to construct phylogenetic tree and the methylation analysis was performed on several of these mitochondrial genes.The main results of the study are as follows:1.The mitochondrial genome of Fragaria×ananassa 'Benihoppe' was sequenced,spliced and assembled by second-generation high-throughput sequencing technology.Finally,63 scaffolds were obtained with a total size of 109,851 bp and the GC content is 41.95%.13 protein-coding genes were found by gene annotation,5 of them namely NADH-dehydrogenase complex subunit(nadl,nad2,nad4,nad5,nad5-D2),1 of complex?(sdh4),cytochrome b gene(cob)respectively,2 of Cytochrome C oxidase subunit(cox1,cox3)and ATP synthase subunit(atp1,atp6)and cytochrome c biosynthetic genes(ccmB,ecmC)respectively;7 ribosomal protein genes(rp12,rpl16,rps4,rps 7,rps12,rps14,rps19);2 transfer RNA genes(tRNA);3 ribosomal RNA genes(rRNA)and 7 open reading frames(ORF).After analyzing the components of the assembled sequence on Fragaria×ananassa'Benihoppe',it contains a large number of gene spacers and repeats.2.The physicochemical properties,secondary structure prediction,phosphorylation site analysis and subcellular localization of 13 encoded proteins on Fragaria 'ananassa'Benihoppe' was analyzed by bioinformatics related software.In order to analyze the phylogenetic relationships of Fragaria×ananassa 'Benihoppe' with other plants,the SNPs were compared and analyzed with 13 kinds of plant mitochondrial genome sequences published in NCBI and the reference sequences obtained from the sequencing of Fragaria×ananassa 'Benihoppe',and based on the effective SNP to construct phylogenetic tree,and preliminary analysis of its evolutionary relationship:Fragaria×ananassa 'Benihoppe' with Fragaria vesca subsp.americana,apple(Malus×domestica)and Malus hupehensis var.mengshanensis are similt belong to Rosaceae is classified as a major branch,Ziziphus jujuba is located near their branch,indicating that their phylogenetic relationship is closer;Arabidopsis thaliana,grapevine(Vitis vinifera)and cucurbitaceae are located in an another large branch line indicates that they have a distant relationship with strawberries.3.The expression of five genes(nad4,nad5,nad5-D2,coxl,atp6)in Fragaria×ananassa 'Benihoppe' was analyzed by RT-qPCR.Three genes with large differences in expression(nad5,cox1,atp6)were selected and methylation analysis of gene fragments was performed using bisulfite sequencing PCR(BSP).The results showed methylation in the mitochondrial gene fragment of Fragaria xananassa 'Benihoppe',and the methylation level of the tested gene was negatively correlated with the gene expression level.