Comparative Chloroplast Genomics And DNA Barcoding Of Fragariinae Torr.et A.Gray

The subtribe Fragariinae Torr.et A.Gray belongs to family Rosaceae Juss.,subfamily Rosoideae(Juss.)Arn.,tribe Potentilleae Sweet.Plants of this subtribe have high medicinal value,edible value and ornamental value.Chloroplast genomes of 12 populations of nine species including Chamaerhodos altaica,Comarum palustre,Dasiphora glabra,Dasiphora parvifolia,Farinopsis salesoviana,Fragaria nilgerrensis,Fragaria vesca subsp.vesca,Sibbaldia aphanopetala,and Sibbaldia cuneifolia were obtained in this study.Chloroplast genomes obtained in this study were combined with the chloroplast genome sequences of Fragariinae and related taxa downloaded from NCBI and the chloroplast genome sequences of Sibbaldianthe species obtained from previous study.Chloroplast genome sequences of a total of 31 species of 53 populations were processed using bioinformatics software,and Fragariinae were studied from the perspective of comparative chloroplast genomes and DNA barcoding.This study can provide ultrabarcoding for the identification of Fragariinae species and can provide a reference for the development and utilization of its germplasm resources;this study can provide basic data for further research on the evolution of the chloroplast genome of Fragariinae;this study lays the foundation for further exploration of the relationship among genera of Fragariinae;DNA barcoding evaluation of standard barcoding,developed hypervariable fragments and ultrabarcoding can provide molecular-level reference data for the identification of Fragariinae species.The main research contents and results are as follows:1.The chloroplast genome of Fragariinae was a tetrad structure consisting of a large single copy region(LSC),a small single copy region(SSC)and two inverted repeat regions(IRs),with a total length of 152,427bp-155,783 bp,and the total GC content was 37%-37.3%.The comparative analysis of chloroplast genome showed that the chloroplast genome of Fragariinae was highly conserved in terms of gene size,gene content,gene structure,gene order and quantity,and GC content.The chloroplast genome of Fragariinae contained 129 genes,including 84 protein-coding genes(PCGs),37 transfer RNA(t RNA)genes,and 8 ribosomal RNA(r RNA)genes.Gene boundary analysis showed there were subtle changes at the IR/SC boundary,and there were no obvious contraction and expansion.Genome Synteny analysis showed that the chloroplast genome sequences of Fragariinae had good collinearity without gene rearrangement and inversion.2.Based on the chloroplast genome sequence of Fragariinae,14 hotspot regions(trn R-UCU-atp A,trn H-GUG-psb A,trn G-UCC-trn R-UCU,trn GGCC-trnf M-CAU,5'-trn K-UUU-rps16,acc D-psa I,pet A-psb J,ycf4-cem A,rps15-ycf1,ndh G-ndh I,trn T-UGU-trn L-UAA,rps18-rpl20,trn L-UAA-trn FGAA,rpl32-trn L-UAG)can be used as candidate molecular markers,three of which are located in the SSC region and 11 are located in the LSC region.Those hotspot regions can provide specific barcoding for the identification of Fragariinae species.3.Based on Maximum Likelihood(ML)and Bayesian Inference(BI),phylogenetic trees of Fragariinae and related taxa were constructed.The results support Fragariinae was monophyletic,and Fragariinae was divided into two clades,of which Sibbaldianthe,Alchemilla,Farinopsis,Comarum and Sibbaldia were clustered together,and Dasiphora,Potaninia,Drymocallis,Chamaerhodos,and Fragaria were grouped together.Phylogenetic relationships among genera of Fragariinae were revealed.4.In the evaluation of the applicability of international common barcoding in Fragariinae,the performance of rbc L was the best,the performance of mat K was the second,and the performance of trn H-GUGpsb A with the shortest sequence length was the worst.Among the combined fragments of international common barcoding,the identification effect of mat K+rbc L was the best,the identification effect of rbc L+trn H-GUG-psb A and mat K+rbc L+ trn H-GUG-psb A was the second,and the identification effect of mat K+ trn H-GUG-psb A was the worst.The identification effect of combined fragments is generally better than that of single fragments,and its species clustering is more ideal.5.To evaluate the applicability of 14 hypervariable fragments as specific barcoding in Fragariinae,the identification of three fragments,namely trn RUCU-atp A,ycf4-cem A,and rps15-ycf1 was the most ideal.Among the combined fragments,the identification effect of trn R-UCU-atp A+ycf4-cem A,trn R-UCU-atp A+rps15-ycf1 and trn R-UCU-atp A+ycf4-cem A+rps15-ycf1 was better than that of ycf4-cem A+rps15-ycf1.Overall,the combined fragment identification effect of hypervariable fragments was better than that of single fragment identification,and the success rate and support rates of species identification were significantly improved.6.Compared with the common barcoding and its combined fragments and the screened hypervariable fragments and their combined fragments,the identification effect of the ultrabarcoding is better,and the identification success rate and support rates are high.