Analysis On The Complete Chloroplast Genome Of Cultivated Strawberry(Fragaria×ananassa)and Development Of Molecular Markers

The genus Fragaria belongs to subtribe Fragariinae within tribe Potentilleae of subfamily Rosoideae in family Rosaceae.The Fragaria includes about 24 species mainly distributed in Asia,Europe and America.Cultivated strawberry(F.×ananassa)is octoploid,also known as pineapple strawberry,exhibits the characteristics of bright color,high nutrition,rich flavor and high yield and is highly favored by growers and consumers.Chloroplast DNA is unaffected by such changes in ploidy and maternal inheritance,which can provides moleeular information for phylogenetic analyses.Thus,in this study,we report the first complete chloroplast genome of cultivated strawberry(F.×ananassa'Benihoppe\)based on high-throughput sequencing technology.In addition to mapping gene map of F.×ananassa chloroplast genome,we conduct comparative analysis against nine other Rosaceae species,including Fragaria species in particular.Based on the chloroplast genome sequences of Fragaria,cpDNA molecular markers are also developed.The major results were showed as following:1.The chloroplast of cultivated strawberry(F.×ananassa 'Benihoppe')was separated and purified by high salt-low pH buffer followed by Percoll density gradient centrifugation.The chloroplast DNA(cpDNA)was extracted by SDS-Protein K method.This method could obtain high integrity of chloroplast and high quality and purity of cpDNA,which could meet the basic requirements of subsequent genome sequencing experiments.2.The complete chloroplast genome of F.×ananassa 'Benihoppe' was sequenced using Illumina sequencing technology,and the map of chloroplast genome was also mapped.The F.xananassa 'Benihoppe' chloroplast genome is a typical circular double-stranded DNA molecule with a quadripartite structure;it is 155,549 bp in size and consists of IR(25,936 bp)regions separated by LSC(85,531 bp)and SSC(18,146 bp)regions.The genome encodes 130 genes,of which 18 genes are located in inverted repeats.There are 112 genes,including 78 protein-coding genes,30 tRNA genes and 4 rRNA genes.The gene content and gene order of F.×ananassa 'Benihoppe' are identical to those of the previously reported the genus Fragaria chloroplast genomes.A total of 39 repeat structures and 61 SSR loci were detected.Comparative analysis of the overall nucleotide sequence identity among ten complete chloroplast genomes confirmed that for both coding and non-coding regions in Rosaceae,single copy regions exhibit higher sequence variation than IRs.The Ka/Ks ratio of most genes was less than 1,suggesting that most genes are under purifying selection.Moreover,three octoploid strawberries which were(F.×ananassa 'Benihoppe',F.chiloensis(GP33),F.virginiana(0477))showed a high degree of conservation in genome,a number of SNPs and InDels were also revealed.3.Based on ten chloroplast genome sequences of Fragaria,six cpDNA markers(ndhE-ndhG,atpF-atpH,trnR-atpA,rps2-rpoC2,ndhJ,rbcL)were developed,and 19 Fragaria germplasm were used to verify their feasibility and comparative analysis.As a result,six markers were amplified in 19 sources,but the agarose gel could not distinguish different amplification products.3,5,13,7,3,21 variable sites were detected,respectively;the parsimony-informative sites were 2,3,9,6,2,20,showed high polymorphism.Thirteen haplotypes Were generated in the eombined sequences of six markers,haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.953 and 0.00514,respectively;the total number of mutation sites was 50,of which 41 were informative sites.UPGMA analysis showed the differences in the clustering results of each marker on the tested materials.Combining six marker sequences can not only distinguish the species of the tested Fragaria genus(bootstrap value greater than 80%),but also Duchesnea indica formed a single branch.The result showed that the six molecular markers have application value in identification and validity of species in the Fragaria genus.