Phylogenetic Relationship And Molecular Marker Development Of Prunus Species Based On Chloroplast Genome

Prunus mume,Prunus armeniaca and Prunus salicina are important fruit trees in China are important fruit trees in China.They can not only be used as fresh food,but also process fruit wine and candied fruit,which is of great economic and research value.Chloroplast genome has the characteristics of small size,cytoplasmic inheritance,low nucleotides replacement rate,haploid property and highly conserved genome structure,which is an ideal genome for thestudy of plant breeding,phylogeny and geographical domestication.In this study,chloroplast separation was compared by plant chloroplast DNA kit,high salt-low pH combined with Percoll density gradient and improved high salt-low pH method..The chloroplast integrity was detected by fluorescence microscope examination.The concentration and purity of cpDNA was detected by agarose gel electrophoresis and nucleic acid detector.Through complete chloroplas genome sequence alignment,gene rearrangement detection and gene enrichment analysis,the differences between direct sequencing and reference sequencing were analyzed to provide reference for the extraction and sequencing in Prunus Mume.Through chloroplast genome sequencing and systematic evolution tree construction,the evolutionary relationship of Prunus genus was explained from the perspective of chloroplast genome,and the simple repetitive sequence information of chloroplast genome was analyzed to distinguish Prunus mume,Prunus armeniaca and Prunus salicina.Furthermore,the development of SSR molecular markers provided a reference for the study of chloroplast genome and the phylogeny,germplasm identification and genetic population analysis of the Prunus genus.The main findings are as follows:1.The effect of chloroplast separation was compared by improved high salt-low pH method,plant chloroplast DNA kit,high salt-low pH combined with Percoll density gradient method,and chloroplast DNA of Prunus mume variety ’Longyan’ was extracted.The results showed that complete chloroplast and high concentration and high purity chloroplast DNA could be obtained by improved high salt-low pH method,which was the most suitable method for chloroplast separation of Prunus mume.Chloroplast genome sequence alignment,gene rearrangement detection and gene enrichment analysis showed that the significant difference between direct sequencing method and reference splicing sequencing method was a redundant sequence in IR region.The similarity of genome rearrangement was higher than 99%.2.The chloroplast genome sequences of Prunus mume variety’longyan’,Prunus armeniaca variety ’Jintaiyang’ and Prunus salicina variety ’Gaixian Dali’ were obtained by high throughput sequencing.Genome analysis showed that trnY-ATA was a specific gene of Prunus armeniaca,trnI-TAT was not arranged in the same order on Prunus mume and Prunus salicina and it was specific to the two species.Comparison of chloroplast complete gene sequence showed that 17 different regions were positively correlated with the distribution of SSR.From the point of view of chloroplast genome,systematic evolution analysis proved that Prunus armeniaca and Prunus salicina were more closely related to each other than Prunus mume.3..We designed 162 pairs of primers from the chloroplast fenome of three Prunus genus plant,the DNA amplification and polymorphism of 60 Prunus genus plants were detected by PAGE gel analysis.And two SSR primer pairs were screened out with clear polymorphic markers which could be used not only in the genetic population analysis but also in germplasm identification and a genetic diversity analysis.