Optimization Of Chloroplast DNA Extraction And Comparative Genomics Study Of Four Allium Vegetables

In this study,four kinds of vegetables Allium fistulosum,Allium sativum,Allium cepa,and Allium tuberosum,were used as experimental materials,optimization of the extraction method of chloroplast DNA and comparative genomics research,the results as follow:（1）By using the leaves of Allium fistulosum as the experimental material,chloroplast DNA was extracted by High-salt Low-pH（HSLp）method and SucroseDNase（SucDNase）method.The results showed that HSLp method was superior to SucDNase method.These two methods acquired similar DNA purity,and the OD₂₆₀/OD₂₈₀ values were 1.984 and 1.977 respectively.But in DNA yields,HSLp method was 0.482μg/g（Fresh Weigh,FW）and ScuDNase method was 0.059μg/g·FW.HSLp method was 8.17 times than ScuDNase method.On the basis of the control of total DNA extracted by the kit,the primers of chloroplast MatK,mitochondrial Atp6 and nuclear LFY gene were designed to compare the copy number ratio of three organelles DNA.The results showed that the extraction effect of cpDNA by HSLp method was 130.56 times than by Kit method,and 5.57 times than by SucDNase method.HSLp method was 23.43 times of SucDNase method.The above results showed that the yield and content of DNA in chloroplasts extracted by HSLp method was better than that of ScuDNase method.Then chloroplast DNA was extracted from Allium cepa,Allium sativum and Allium tuberosum.by HSLP method,and high yield and purity were obtained.（2）The leaves of Allium fistulosum was used as the experimental material,the cpDNA purity optimization test of four vegetable crops of Allium was carried out.By using different concentrations of salt ion reagents（1.0 mol/L,1.5 mol/L,2.5mol/L,2.5 mol/L）extraction of cpDNA and analysis of purity.The results showed that 1.5 mol/L was the optimum concentration of salt ion.Percoll density gradient and centrifugal force screening result showed that 3220g was the optimum centrifugal force;When the percoll density gradient was 50%,the whole chloroplast would float between the Percoll and the water layer.Larger nuclear fragments would sink at the bottom of the Percoll layer;When the percoll density gradient were 30%,the whole chloroplasts could sink into the bottom of the Percoll layer,and lighter nuclear fragments and parts of mitochondria float in the water layer.Combination of two concentrations,they were better to separate chloroplast,mitochondria and nuclear fragmentsit.cpDNA of Allium fistulosum,Allium sativum,Allium cepa,and Allium tuberosum were obtained by using the optimized scheme of this experiment.（3）In this study,chloroplast DNA of four species of Allium vegetables were sequenced with high-throughput sequencing,the genome sequences of chloroplasts of Allium fistulosum,Allium sativum,Allium cepa,and Allium tuberosum were obtained,with the size of 153109bp,153189bp,153586bp,154057bp and the content of GC36.9%,36.7%,36.9%,36.9%.They had four obvious chloroplast structural characteristics,the length of which were LSC 82206bp,SSC 17906bp,IRA 26498bp,IRB 26498bp of Allium fistulosum;LSC 82024bp,SSC 17906bp,IRA26498bp,IRB26498bp of Allium sativum;LSC 82731bp,SSC 17932bp,IRA26461bp,IRB26461bp of Allium cepa;LSC 83081bp,SSC 17959bp,IRA 26508bp,IRB26508bp of Allium tuberosum.By gene annotation and phylogenetic analysis,there were 112 functional genes in chloroplast genome of Allium L.,however infA lost in Allium tuberosum;Allium tuberosum from their common ancestors and then Allium sativum became an independent branch.Allium fistulosum and onion had close relationship with each other,and their confidence level was 100%.In this study,the chloroplast DNA extraction methods of four Allium vegetable crops were optimized and their chloroplast genomic sequences were obtained,they filled in the blank of chloroplast genome sequence of Allium fistulosum and Allium tuberosum,and laid a good foundation for further research.