| Galla chinensis is a traditional Chinese medicine,which has important medicinal value and can be used as industrial raw materials.Galla chinensis is the gall of Rhus species induced by Rhus galla aphid stimulation.But whether there is gene difference between gall tissue and host plant has never been studied,and the taxonomy and identification of Rhus is also very little.DNA barcoding technology can quickly and accurately identify species.Chloroplast genome research can be applied to species identification,chloroplast genetic engineering,phylogenetic and taxonomic research.DNA barcoding technology and chloroplast genome have their own advantages,and both can be applied to species identification and taxonomic research.In this study,PCR amplification and Sanger sequencing were performed on host plant and Galla chinensis,and the number of variation sites of host plant and Galla chinensis were compared,so as to preliminarily explore the genetic differences between gall and host plant.PCR amplification,Sanger sequencing,Illumina high-throughput sequencing and were performed on host plant and Galla chinensis.The general DNA barcoding sequence PCR amplification,Sanger sequencing,Illumina high-throughput sequencing and sequence assembly were performed on Rhus species in order to evaluate the efficiency of general DNA barcoding sequence identification and obtain chloroplast genome.Chloroplast genome annotation,structure,comparison and phylogenetic analysis were performed on Rhus species,and develop molecular markers to identify these species.In order to provide important basis and reference for species identification,protection and utilization of of Rhus family,and providereference for phylogenetic research of Anacardiaceae.The main results of this study are as follows:1.Comparison of the general DNA barcoding sequence of Galla chinensis and host plants(1)The successful amplification rates of ITS2,psbA-trnH,rbcL and matK sequences of R.chinensis,R.punjabensis var.sinica,R.potaninii and their galls were 100%,91.7%,100%and 75.0%respectively.Comparison of the galls and host plants showed that ITS2,psbA-trnH and rbcL sequence had no variable sites.2.Identification of the general DNA barcoding sequence of Rhus(1)The ITS2 and psbA-trnH sequences of R.chinensis,R.punjabensis var.sinica,R.potaninii,R.wilsonii,R.hypoleuca and R.typhina were amplified and sequenced with 100%success rate.The number of variable sitesin ITS2 and psbA-trnH sequences were 28 and 69,and the percentage of variable sites was 12.3%and 9.6%,respectively.(2)Based on genetic distance analysis,the identification efficiency of ITS2 and psbA-trnH sequences were 60.0%and 40.0%,respectively.Based on NJ tree analysis,the identification efficiency of ITS2 and psbA-trnH sequences were 66.7%and 33.3%,respectively.3.Analysis of chloroplast genome of four Rhus species(1)The chloroplast genomes of R.punjabensis var.sinica,R.potaninii,R.hypoleuca and R.wilsonii spanned 159,617,159,620,159,472 and 159,814bp in size,respectively,and possessed a typical circular structure with GC content of 37.9%.(2)Eeach genome contains 133 genes except R.wilsonii which contains 132 genes,with a total of 19 genes repeated in the IR region.infA is a pseudogene shared by four species,and rps19 is only annotated as pseudogene in and R.punjabensis var.sinica and R.potaninii.(3)There were 50,50,45 and 54 long repeats,and 86,84,79 and 72 SSRs were identified in R.punjabensis var.sinica,R.potaninii,R.wilsonii and R.hypoleuca,respectively.(4)The comparative analysis showed that three highly variable regions,namely ccsA-ndhD,trnD-trnY and trnT-trnL,were identified.trnD-trnY combined with 1 single nuclear polymorphysm(SNP)loci could be used to identify these species.(5)The ML phylogenetic tree based on chloroplast genome showed that R.chinensis had the closest genetic relationship with R.hypoleuca,R.punjabensis var.sinica had the closest genetic relationship with R.potaninii,and R.typhina was closely related to R.wilsonii. |
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