Establishment Of High-quality Mitochondrial DNA Extraction System And Mitochondrial Genome Sequencing In Prunus Persica

The energy for life activities in plants is provided by mitochondria,and some important enzymes and proteins in plants are encoded by mitochondria DNA.Extraction of high-quality mitochondrial DNA is a necessary prerequisite for mitochondrial genome sequencing.Using leaves as materials,callus of six peach varieties ‘21st century',‘Hunchun',‘Zaoxianlu',‘Zhongshoutao',‘Mafirm',‘Ruiguang 21' were induced under completely dark conditions at 24?.Mitochondria were extracted and purified from‘21st century' peach callus by homogenization,differential centrifugation and sucrose shelf methods.Mitochondria DNA were extracted and purified by a series of methods such as SDS purification method.Finally,mtDNA of peach in ‘21st century' was extracted,which meets the requirements of sequencing.Rapid and efficient extraction technology system suitable for peach mitochondrial DNA was established,which enriched the related researches of mtDNA extraction in fruit trees belonging to Rosaceae.The mitochondrial genome of ‘21st century' peach was sequenced and the characteristics and functions of peach mitochondrial genome were analyzed,which laid the foundation for further researches.The main results are as follows:1.The callus induction rate of six peach varieties was significantly increased when 6-BA and 2,4-D were added to MS,and the medium induction time was also shortened.When the concentration of 6-BA reached 3 mg/L,the induction rate could reach 100%,and the callus grew well.The best medium for peach callus induction was MS+3.0 mg/L6-BA+1.0 mg/L2,4-D.2.Fluorescence microscopy test proved that the mitochondria extracted were complete and active.Electrophoresis analysis showed that the size of mitochondrial DNA bands in peach was about 23 kb.Nucleic acid purity analysis showed that the extracted mtDNA was free from RNA and protein contamination.Specific PCR detection of sfb,rbd1,nad4,coxI and atp4 genes indicated that the extracted mtDNA was not contaminated by nuclear DNA and chloroplast DNA,which could meet the requirements of high-throughput genome sequencing.3.The results of mitochondrial genome sequencing showed that the average GC content of mitochondrial genome in ‘21st century' peach was 59.33%.40 genes were detected in ‘21st century' mitochondrial genome.GO functional significance enrichment analysis showed that 40 genes in the mitochondrial genome were compared with 22 subgroups under three biological functions,including 5 molecular functional subgroups,7 cell component subgroups and 10 biological process subgroups.The main biological functions involved were cellular processes,metabolic processes,binding functions and catalytic activities.