Polymorphism Research Of Chloroplast Genome DNA In Brassica Juncea

Brassica juncea is one versatile Brassica allotetraploid species with the characteristics of resistances to drought,cold and barren soil.It is mainly distributed in the western region of China,has a long history of cultivation and shows rich resources.The chloroplast genome is of great significance in revealing the evolution of species origin and expounding its biological diversity because of its small molecular weight,noncategorization,high conservation and single parent inheritance.Based on the highly conserved nature of the chloroplast genome,the chlorogenic genome can be sequenced using Illumina sequencing technology under the premise of a valid reference sequence.In this study,we used Illumina sequencing technology to perform high-throughput sequencing of B.juncea accessions with wide range of diversitiers,to verify the reliability of mutation frequency by Sanger sequencing,and screen some genes for the polymorphism.The results are as follows:1.The optimization of chloroplast separation methods and conditions for large-scale extraction of chloroplast DNA.The following operations were optimized: an increase of 200 g centrifugation for 2 min during the chloroplast separation process,to remove cell wall fragments and nucleus particles,to eliminate the need for centrifugation with percoll gradient.In addition,this experiment used DTT instead of ?-mercaptoethanol with similar effect,for the irritation smell was smaller and the toxicity was lower.The method was simple in operation,but the experimental cost reduced and the purity and yield improved.2.Illumina sequencing technique to explore the polymorphism of chloroplast genomic DNA in Brassica juncea.The chloroplast genomes of 1549 Brassica juncea were mixed and sequenced(every 50 for a mixed pool,a total of 31),At above 2% of the variation frequency,A total of 573 variants were found,there are 199 located in the gene interval,and 374 located in the gene,And these 374 mutations were concentrated in 31 chloroplast genes.3.The verification of the reliability of Illumina sequencing results by using Sanger sequencing.The Sanger sequencing was performed by screening the appropriate sections for different variation frequencies of 2%-3%,6%-7%,20%,4%-23%,8%,10%,12%-15%.The results showed that the reliability of Illumina sequencing was more than 12%.The variation sites of many unreliable variational frequencies were eliminated,which laid the foundation for the follow-up study,and provided the reference for later research.4.Polymorphic analysis of a chloroplast gene rps19 and a gene spacer by Sanger sequencing.Among 96 materials from different provinces,In the rps19 gene found only four mutations in the material 1490,This material comes from the UK,which may have evolved over long periods of geographical isolation.In the gene spacer,52 material found two identical mutations,at site 64298,A change to T,and at site 64767,A change to C,respectively;but one material 887 from Sweden gave up to 30 variation sites.So the evolution rate of gene spacer was relatively faster relative to functional gene,This is also consistent with previous studies,in addition,geographical differences may also be one of the causes of DNA polymorphism.