| Xanthopappus subacaulis C.Winkl.is a perennial medicinal herb of the genus Xanthopappus(Asteraceae)that mainly grows in alpine meadows and dry slopes of the Qinghai-Tibet Plateau and its adjacent areas.It has multiple stress tolerance traits,such as cold,drought and salinity resistance,and medicinal values,such as cooling blood and stopping bleeding.Previous studies on X.subacaulis have focused on its biological characteristics,chemical constituents,pharmacological activities,stress physiology,phylogeny and demographic history,but no research has been reported on its genetic diversity based on SSR molecular markers.X.subacaulis is a promising medicinal plant with multiple stress resistance.Therefore,it is necessary to use various molecular markers to investigate the population genetic structure and genetic diversity of X.subacaulis.In this study,we first performed full length transcriptome sequencing of X.subacaulis using single-molecule real-time sequencing technology(SMRT)and annotated gene functions using multiple databases.We analyzed the distribution,types and features of SSR loci and screened SSR molecular markers with high polymorphism and stability.Based on this,we selected 200 individuals from 40 populations of X.subacaulis for genetic diversity and population genetic structure analysis,aiming to provide reference data for the development,utilization,genetic breeding and diversity conservation of X.subacaulis.The main results are as follows:⑴We performed full-length transcriptome sequencing of X.subacaulis using the PacBio Sequel sequencing platform.After removing redundancy from the raw data,we obtained 46 100 Unigenes.Nr database alignment showed that X.subacaulis had the highest homology with Cynara cardunculus.In GO database,14 735 Unigenes were successfully annotated to three primary categories and 92 secondary categories.KEGG metabolic pathway analysis showed that 16 699 Unigenes were successfully annotated to five primary pathways and 34 secondary pathways.Meanwhile,in KOG database,33 487 Unigenes were successfully annotated to 25 major classes.Meanwhile,we predicted 52 898 CDS sequences,1 791 transcription factors,966 transcription regulators and 179 long non-coding RNAs.⑵We used MISA software to detect SSR loci of X.subacaulis Unigenes and found that 12 303 Unigenes contained 16 441 SSR loci with a frequency of 26.69% and an average distribution distance of 7.35 kb.X.subacaulis SSR loci had most nucleotide repeat types,ranging from mononucleotides to hexanucleotides,among which trinucleotide repeat type was the most abundant(36.77%),followed by dinucleotide repeat type(30.36%).AG/CT and ATC/ATG were the dominant motifs for dinucleotide and trinucleotide repeat types,respectively.We designed 145 pairs of X.subacaulis SSR primers using Primer Premier 5 software and screened out 15 pairs of primers with high polymorphism and stability.Capillary electrophoresis detected that these primers contained 102 alleles with a polymorphism ratio of 100%,polymorphism information content(PIC)ranged from 0.381 to 0.839 with an average of 0.635.⑶The analysis of genetic diversity of 40 populations of X.subacaulis showed that the average values of observed alleles(Na)and effective alleles(Ne)were 1.64 and1.461,respectively,and the average value of Shannon’s information index(I)was 0.442;the average values of observed heterozygosity(Ho)and expected heterozygosity(He)were 0.544 and 0.467,respectively,among which 17 populations had He above than0.5,indicating that they had higher genetic diversity;the important genetic diversity indices of X.subacaulis populations from Qinghai Chaka Salt Lake to Gansu Er Long Mountain north were higher than those south;the average value of genetic differentiation coefficient(Fst)among populations was 0.642,gene flow(Nm)was0.167,molecular variance analysis(AMOVA)found that X.subacaulis genetic variation mainly came from among populations(60%),individual genetic variation was small(40%),indicating that X.subacaulis gene exchange among populations was infrequent,genetic differentiation was large.⑷According to the calculation of ΔK and ln P(D)values,K=2 is the most suitable number of taxa.By STRUCTURE analysis,the X.subacaulis populations could be divided into two genetic lineages when K=2.Group 1 included 22 populations from the Qilian Mountain and Qinghai Lake area,while Group 2 consisted of 18 populations from Aemye Ma-chhen Mountain and Hengduan Mountain.Populations from eastern Qilian Mountain,southern Qinghai Lake and northern Aemye Ma-chhen Mountain showed significant gene introgression.These results were also supported by UPGMA cluster analysis and PCo A analysis.Mantel test results showed that geographic distance significantly affected the genetic differentiation among X.subacaulis populations,and natural geographic barriers such as mountains and lakes severely hindered gene exchange among X.subacaulis populations. |
PDF Full Text Request