Research On The Phylogenetics Of Aspidistra Based On Chloroplast Genomics And Transcriptomics

The plant genus Aspidistra is of certain ornamental and medicinal value.In recent years,there has been a continuous increase in the number of newly reported species,which has led to confusion in morphological classification and uncertainty about the phylogenetic relationships between classification groups.This study selected 29 representative Aspidistra species based on morphology,geographic distribution,and chromosome number,and used quantitative taxonomy methods to identify important classification traits for this genus.Through high-throughput sequencing technology,complete chloroplast genome sequences,and transcriptome data were obtained for comparison of genomic analysis.This provided a large number of candidate gene fragments and SSR molecular marker sites that could be used in DNA barcode research.Based on the chloroplast genes and nuclear genes,we constructed a phylogenetic tree of the Aspidistra genus,and combined evidence from morphology,cytology,and molecular systematics to explore the phylogenetic relationships among various taxonomic groups within the genus.The main results are as follows:(1)Quantitative classification results show that Aspidistra can be divided into four major groups and eight small groups based on the growth status of rhizomes,leaf length,perianth tube shape and length,whether there are appendages at the base of the lobes,whether there is a ridge at the base of the lobes,shape of pistil,diameter of stigma,and number of stamens.(2)Chloroplast genome comparative analysis results show that the chloroplast genome of the 29 representative Aspidistra plants is highly conserved,with good collinearity,consistent gene arrangement order,no rearrangement or inversion,more conserved coding regions than noncoding regions,and similar codon preferences.The inf A gene in Aspidistra oblanceifolia,Aspidistra sichuanensis,Aspidistra leshanensis,Aspidistra arnautovii,Aspidistra linearifolia,Aspidistra yingjiangensis,Aspidistra omeiensis,Aspidistra minutiflora,Aspidistra sutepensis,Aspidistra longipetala,and Aspidistra ovatifolia has a false gene due to the termination codon advanced mutation.Three polymorphic SSR loci were detected at atp F intron region,rps15-ycf1 intergenic spacer region,and atp I-rps2 intergenic spacer region.There is no obvious boundary contraction and expansion,and high consistency is also observed.However,some Aspidistra species have differences in the number of bases at the border gene distance region,including Aspidistra zhangii,Aspidistra fungilliformis,Aspidistra claviformis,Aspidistra linearifolia,Aspidistra guangxiensis,Aspidistra obliquipeltata,Aspidistra minutiflora,Aspidistra radiata,Aspidistra ovatifolia,and Aspidistra oblanceifolia.(3)Transcriptional assembly annotation of 29 Aspidistra species resulted in high annotation rates across six major databases,and annotations in the NR database were mostly related to the genes of Asparagus officinalis.A total of 463,312 SSR loci were detected,and 470 SSR loci were polymorphic.In polymorphic SSR loci,six SSR loci were found in all 29 Aspidistra species.A total of 430 single-copy orthologous gene groups were obtained,and four of them were under positive selection.They separately encoded the photosystem I assembly factor PSA3,uncharacterized protein,thioredoxin-like protein AAED1,and diphthamide biosynthesis protein 3-like.(4)Hotspots suitable as DNA barcode candidate genes for Aspidistra species identification were selected.These include the coding regions(inf A,ccs A,ndh D,atp F,ccs A,cem A,ndh B,rpl20,rps11,rps16,psb A,rpo C2,ycf1,and ycf2)and non-coding regions(atp F intron region,rpo C1 intron region,rps15-ycf1,trn E-trn T,atp I-rps2,ccs A-ndh D,pet A-psb J,rps16-trn Q,trn C-pet N,trn S-trn R,trn V-rps7,trn T-psb D)of the chloroplast genome,as well as genes coding for photosystem I assembly factor PSA3,uncharacterized protein,sulfiredoxin-like protein AAED1,and diphthamide biosynthesis protein 3 were identified in the transcriptome.(5)Based on the chloroplast genes and nuclear genes,we reconstructed a phylogenetic tree of the Aspidistra genus,and 29 species of Aspidistra were classified into six clades through comprehensive comparison.Aspidistra zhangii was the earliest divergence and had its own branch.Aspidistra subrotata,Aspidistra punctata,and Aspidistra punctatoides were closely related.Aspidistra ovatifolia,Aspidistra radiata,Aspidistra patentiloba,Aspidistra guangxiensis,and Aspidistra obliquipeltata were also closely related.Aspidistra dolichanthera,Aspidistra bicolor,Aspidistra formosa,and Aspidistra cavicola were closely related.Aspidistra minutiflora,Aspidistra arnautovii,Aspidistra linearifolia,Aspidistra yingjiangensis,and Aspidistra omeiensis were closely related.Aspidistra fimbriata,Aspidistra leshanensis,Aspidistra sichuanensis,and Aspidistra oblanceifolia were closely related.Differences in position exist for Aspidistra longipedunculata,Aspidistra clausa,Aspidistra longipetala,Aspidistra fungilliformis,Aspidistra claviformis,Aspidistra quadripartita,and Aspidistra sutepensis.This study preliminarily explored the phylogenetic relationships of the Aspidistra genus plants based on evidence from morphology,chloroplast genomes,and transcriptomes,and screened out a number of candidate genes that can be used as DNA barcodes for the Aspidistra genus.