Study On The Mitochondrial Genome Of Allotetraploid Cucumis×hytivus

Mitochondria.an important organelle of plants,are directly related to multiple traits such as resistance and fertility.Sequencing analysis of plant mitochondrial genome provides unique insight for revealing genome structure and evolutionary relationships.Allotetraploid Cucumis×hytivus Chen&Kirkbrid（2n=38,HHCC）is a heterotetraploid,which is synthesized by cross between wild cucumber C.hystrix and cultivated cucumber C.sativus,followed by chromosome doubling.The synthetic allotetraploid is resistant to high and low temperature and have a long growth cycle,which enriches cucumber genetic resources and broadens the channels for variety improvement.At present,the nuclear genome and chloroplast genome information of allotetraploid C.×hytivus have been published,but the relevant information surrounding the mitochondrial genome is very scarce.The allotetraploid C.×hytivus mitochondria originated from its paternal parent cucumber C.sativus,but it can’t be ignored that it will cause some genetic variation after allopolyploidy.Through sequencing and analysis,it is helpful to analyze all the genetic information of the allotetraploid C.×hytivus mitochondrial genome,and it is also the only way to discover useful genes for variety improvement and breeding.In the early stage of this experiment,cucumber C.sativus was used as the test material,and mitochondria were extracted by optimizing the composition of homogenization buffer,differential centrifugation and percoll gradient precipitation.The improved CTAB method is used to further extract and purify mitochondrial DNA,helping us to establish a mitochondrial DNA extraction system that is universally applicable to cucumber crops.In the later stage,we separately extracted allotetraploid C.×hytivus complete mitochondrial DNA for high-throughput sequencing of its mitochondrial genome,analyzed its structural features and genetic composition,and explored the lateral migration of information between it and chloroplast genome.At the mitochondrial genome level,the evolutionary relationship of related species of cucurbitaceae crops was interpreted,and the formation mechanism of the allotetraploid C.×hytivus mitochondrial genome was preliminarily explored.The main research contents and results are as follows:1.Research on extraction and purification methods of cucumber mitochondrial DNAFirstly,the composition of the tissue homogenate buffer and the differential centrifugal force combination were optimized by using cultivated cucumber C.sativus yellow leaves.In order to avoid excessive chloroplast genome interference,the yellow seedlings obtained by direct shading growth of seeds were used as the final material,combined with percoll density gradient centrifugation to obtain mitochondria on the optimal system,and 3%CTAB is used to lyse the mitochondria.Mitochondrial DNA（mt DNA）was refined repeatedly with chloroform-isoamyl Alcohol.The integrity of the mitochondria after extract using homogenization buffer A(EGTA 1 mmol·L^-1,Tricine 10 mmol·L^-1,Cysteine 8mmol·L^-1,BSA 0.1%,PVP 1%,MOPS-KOH 25 mmol·L^-1,mannitol 400 mmol·L^-1)is better.When the centrifugal force of separating cell-disrupted-tissue and purifng mitochondria were 2000×g and 17000×g respectively,the obvious mitochondrial aggregate was harvested.The mt DNA obtained by repeated experiments were with no other genome contamination.The purity and quality of allotetraploid C.×hytivus mitochondrial DNA extracted according to this system also meet the sequencing requirements.2.Sequencing and analysis of allotetraploid C.×hytivus mitochondrial genomeSequencing the complete mitochondrial genome of the allotetraploid C.×hytivus showed that the total length of the mitochondrial genome was 1,686,697 bp,composed of three circular DNAs.All genes were located on the largest main loop,which contained 37protein-coding genes,21 t RNAs and 6 r RNAs.There were a large number of repetitive sequence fragments in plant mitochondrial genome.After the sequence alignment of the main loop mitochondrial genome,a total of 1,121 pairs of repetitive sequences were detected.The sequences migration of chloroplast genome to the mitochondrial genome is common in cucurbitaceae crops.159 pairs of migration sequences occurred between the cytoplasmic genomes,accounting for 4.43%of the length of the mitochondrial genome.Compared with the mitochondrial genome of cucumber C.sativus in Gen Bank,9 amino acid variations were detected in 4 protein-coding genes,and other SNPs and Indels occurred in non-coding regions.It showed that the allotetraploid C.×hytivus mitochondrial genome undergoed hybridization and doubling to cause recombination and mutation.The comparison between species showed that the mitochondrial genome structure and gene composition of several cucurbitaceous crops in this study were similar,and the evolutionary relationship at the mitochondrial genome level was verified that the allotetraploid C.×hytivus is the closest to cucumber.