Comparative Analyses Of Plastomes And Phylogeny Among Sedum

The genus Sedum,a large genus with about 400 recognized species,was classified in the family Crassulaceae of the order Saxifragales,occupying a quarter of the family.Sedum is the most taxonomically complex genus.Up to now,phylogenetic relationships within the genus remain unresolved.Owing to its maternal inheritance and conservative structure,chloroplast genome is frequently used in phylogenetic studies of species.Nuclear gene(ITS)data have been frequently used to phylogenetic relationships in Crassulaceae.Therefore,in this study,high-throughput second-generation sequencing technology was used to sequence 10 species of Sedum,including Sedum alfredii,S.burrito and S.dasyphyllum.We assembled the chloroplast genome and nuclear r RNA genes(nr DNA).Combining with the public released data,we discussed the following scientific problems:(1)chloroplast genomic characterization of Sedum;(2)the taxonomic status of Sedum;(3)the phylogenetic relationships between Crassulaceae and other families of Saxifragales.The main findings are as follows:1.Chloroplast genomic characteristics analysis of SedumIn this study,we used the CTAB to extracting DNA from Sedum,and genomes were sequenced by high-throughput sequencing technology.After the assembly and analysis of the Sedum chloroplast genome,we were successfully assembled complete chloroplast genomes of 9 Sedum species,except S.mocinianum.The complete chloroplast genomes of Sedum range from148,618 bp to 150,714 bp in size,with a typical circular and quadripartite structure,including a large single-copy region(LSC),a small single-copy region(SSC),and two reverse repeats(IRa and IRb).It contains 133 genes(85 proteincoding genes,36 t RNA genes,8 r RNA genes,and four pseudogenes).In the chloroplast genomes of Sedum,the AT content(62.04%~62.29%)is significantly higher than the GC content(37.71%~37.96%),and T base was more preferred between A and T base,C base was more preferred between C and G base.A total of 1268 SSR loci were detected,of which mononucletotide repeats were the highest.Except for Met,all of the types of biased codons ended with A or T in the chloroplast genomes of Sedum.2.Comparative analysis of the Sedum chloroplast genome sequencesIn order to investigate the differences of the Sedum chloroplast genome sequences,the m VISTA was used to analysed the consistency of the 9 complete chloroplast genome sequences.The results show that the coding region is more conservative than the non-coding region,and the IR regions is more conservative than the SC regions.This fact that the two IR regions were less variable was attributed to the conservation of the ribosomal RNA genes.Comparison of the inverted repeat-single copy(IR-SC)boundary regions of Sedum species showed a high degree of consistency.3.The analysis of genetic evolutionary selection patternsIn general,the ratio of nonsynonymous(d N)to synonymous(d S)substitution measures the levels of selective pressure operating in a protein coding gene.Combining with the public released data of the Saxifragales chloroplast genomes,we analyzed selection pressure of 76 protein-coding genes(PCGs)among 67 species.The result showed that the d N/d S ratios of chloroplast PCGs were all less than 1,indicating that purifying selection played an important role in the evolutionary selection patterns of PCGs.Ycf2 and rpl23 genes had a high evolution rate,which were 0.68 and 0.69,respectively.In addition,Three pairs of site models(M0 vs.M3,M1 a vs.M2 a,and M7 vs.M8)were used to test whether the evolution of plastid protein-coding genes was driven by positive selection.The result showed that 27 genes and 52 sites were detected under positive selection.There were 8 significant positive selection sites in rbc L.4.The analysis of phylogenyIn order to explore the phylogenetic relationship of the genus of Sedum,we used Get Organelle to acquire 10 species of Sedum chloroplast genomes sequences(mat K,rps16,trn L-trn F),nuclear ribosome sequences and internal transcriptional spacers(18S-ITS1-5.8S-ITS2-26S)from genomic data.Combining the data of 433 species from the NCBI public database to study the phylogenetic relationships of Sedum.RAXML(Bootstrap)was used to construct the maximum likelihood tree for 443 species.The results were as follows:(1)Sedum belongs to Sempervivoideae,and its species are distributed in all five clades of the Sempervivoideae.Sedum is proposed to be a polyphyletic group.(2)S.burrito,S.clavatum,S.plumbizincicola,S.hernandezii and S.palmeri are in one clade.S.furfuraceum is sister to S.pacense.S.mocinianum is sister to S.hintonii.The clade of S.alfredii and S.makinoi are located at the basal position of Acre clade.(3)S.dasyphyllum is located in the Leucosedum Clade and is sister to S.mucizonia.In order to reveal the phylogenetic relationship between Saxifragales and Rosids,79 protein-coding genes of 10 Sedum species were extracted by DAMBE,and combined with 164 species released from the NCBI database,maximum likelihood trees and Bayesian trees were constructed.The results showed that:(1)S.hernandezii,S.mocinianum,and S.furfuraceum are in one clade.S.plumbizincicola,S.alfredii and S.makinoi are in one clade.(2)Saxifragales had a closer relationship with Rosids.This incongruence between chloroplast gene and combined trees(ITS and chloroplast sequences)of Sedum hernandezii and Sedum plumbizincicola.This study laid a foundation for further study on the phylogeny of Sedum.