Complete Genome Analysis And Phylogeography Of Chloroplast In Halenia Elliptica

Chloroplast genome research is of great significance to chloroplast genetic engineering,development of molecular markers,breeding of stress-resistant varieties,molecular phylogeography,conservation of germplasm resources and phylogenetic relationships analysis of species.In this paper,Halenia elliptica,which belongs to the genus of Gentianaceae,is through high-throughput sequencing,splicing and assembly of chloroplast genome,gene annotation,analysis of genome characteristics and phylogenetic analysis,trnH-psbA and rps2-rpoC2 non coding regions were screened out,and molecular markers were developed,which were applied to the study of phylogeography in different populations of H.elliptica.This study set a precedent for the whole chloroplast genome sequencing of H.elliptica,and laid a foundation for the development of molecular markers related to H.elliptica,the study of phylogeography,the cultivation of fine varieties and the protection of germplasm resources.The main conclusions are as follows:(1)The length of the complete chloroplast genome was 153 341 bp,which was a circular double-stranded and typical tetrad structure.The length of the large single copy region(LSC)was 82 811 bp,the length of the small single copy region(SSC)was 18 278 bp,and the length of the two reverse repeat regions(IR)was 26 126 bp.The complete cp genome of H.elliptica contains 135 genes,including 88protein-coding genes,37 t RNA genes,8 rRNA genes and two pseudogene.The overall GC content of the cp genome is 38.1%.By analyzing the chloroplast genomes of H.elliptica,the 88 protein genes encoded 25051 codons,the most of which was leucine with 2708 codons,and the least of which was Cysteine with 288 codons,there are 32 codons with preference,and it preferred use T and A with the codons ending;There are 173 SSR loci in the cp DNA of H.ellipitica.The number of mononucleotide,dinucleotide,trinucleotide,tetranucleotide and hexanucleotide are 128,34,4,6 and 1,but didn't found pentanucleotide;The expansion or contraction analysis of IR boundary showed that the main differences of chloroplast genome in different genera of Gentianaceae were the position of rps19 gene and the length of ycf1 gene;Through the analysis and comparison of chloroplast genome,it was found that genes were more conservative than spacer regions,and IR regions(83k-109 k,127k-153k)were more conservative than LSC and SSC;Phylogenetic analysis of 14 sequenced related species from Gentianaceae and two outgroups showed that the Halenia was closely related to the Swertia.(2)The chloroplast gene fragments psb A-trn H and trnS-rpoC2 of 283 individuals in 42 populations were amplified by PCR.A total of 27 haplotypes and 34 parsimony information loci were identified.The total genetic diversity Ht value is0.941,the average gene diversity Hs value is 0.545,the Gst and Nst values of genetic differentiation among different cohabitation groups were 0.421 and 0.680,After the u-statistical test,it is found that Nst is greater than Gst(P<0.01),and the level of genetic differentiation among populations was high,suggesting that there was a certain phylogenetic structure in the existing distribution area of H.elliptica.The AMOVA analysis(1000 Bootstraps)showed that the genetic variation among populations was 63.4%,and the genetic variation within populations was 36.6%,and the genetic differentiation coefficient Fst=0.63366(P<0.10),further revealing that the genetic variation of H.elliptica mainly existed among populations.Neutral test was performed on all individuals of H.elliptica with the cp DNA joint sequence,the results showed that Tajima's D(-0.33800,P>0.10),Fu & Li' D*(0.60727,P>0.10)and Fu &Li' F*(0.40097,P>0.10)were not significant,but Tajima's D value was negative.It is speculated that the H.elliptica has not experienced recent expansion on the whole level.The results of the analysis of the Mismatch distributions showed that in the whole range of H.elliptica distributed leaves,the mismatch distributions showed an "L-shaped" curve,indicating that the H.elliptica distributed leaves experienced population reduction at first,and then quickly reached individual balance.The populations with high genetic diversity were mainly distributed in the southeast of Qinghai Province,the northeast of Qinghai Tibet Plateau and the south of Qinghai Tibet Plateau,suggesting that these three areas might be the micro shelters of H.elliptica.The Maxent model was used to simulate the current and future ecological suitable areas of H.elliptica.The results show that the distribution area of H.elliptica will shrink slightly in the next 60-80 years.This trend is presumed to be due to global warming.