Chloroplast Genome Analysis And Molecular Marker Development Of Helianthus Annuus L.

Helianthus annuus,in the genus Asteraceae,is an important oil crop with high economic value,and its germplasm resources are abundant,including local varieties and wild resources.Helianthus germplasm resources are the material basis for the selection and improvement of new varieties.Therefore,the effective identification of Helianthus germplasm resources is very important for Helianthus breeding.The chloroplast genome is the genetic material existing in the chloroplast.Its evolution is slow,its structure is conservative,and it contains a lot of genetic information.It is of great value for the study of the origin of species and the relationship between species.SSRs are evenly distributed and abundant in the genome,and have high polymorphisms.Genomic DNA barcoding and the development of SSR molecular markers based on the chloroplast genome are effective means for species identification.Based on this,this study carried out research on the chloroplast genome of H.annuus germplasm materials and screened candidate barcodes and SSR primers for interspecific identification of Helianthus,which supplemented the chloroplast genome database of Helianthus plants and provided a reference for the rapid identification of H.annuus species.In this study,bioinformatics methods were used to compare and analyze the genomic characteristics and differences of 10 cultivated H.annuus germplasm materials,and to construct the phylogenetic relationship of Helianthus.Applicability evaluation,design of cp SSR primers and verification of primer polymorphism,the research results are as follows:(1)The second-generation high-throughput sequencing technology was used to obtain the complete chloroplast genome sequences of 10 H.annuus materials.The genome sequences were conserved and ranged from 151,100 bp to 151,150 bp in length,with 84 protein-coding genes,8 r RNAs and 35 t RNAs.A total of 18 genes contained introns,two of which contained 2introns(ycf3,clp P).Codon usage is similar,and A/U is preferred in base selection.Arginine,tryptophan and leucine are the amino acids with the most synonymous codons,and methionine and tyrosine are the amino acids with the fewest synonymous codons.The repeat sequences and SSRs of the 10 H.annuus materials were mostly located in the non-coding intergenic regions,and a small part of the SSRs were distributed in the gene coding regions,including mat K,rpo B,rpo C1,rpo A,ycf1 regions.(2)The results of IR region boundary analysis,sequence difference alignment,and collinearity analysis all showed that the genome structure of the 10 H.annuus germplasm materials is conservative,the gene distribution at the boundary is highly similar,and there is no gene rearrangement or loss in the genome.The fragment consistency is high,the coding region is more stable than the non-coding region,and the IR region has no obvious amplification trend.Nucleotide polymorphism(Pi)value analysis obtained a total of 4 fragments with high variation,including 2 non-coding fragments and 2 coding region genes,of which the Pi value of psa J gene was 0.05599,which was the highest nucleotide polymorphism fragment.In the phylogenetic analysis of species,material J-04 is the most distantly related to other germplasm materials,and it is a separate branch with H.annuus cultivar HA89(PET2).The genetic relationship between J-07 and J-01 and other materials is slightly close to that of material J-04,and the genetic relationship of other germplasm materials is relatively close.(3)The applicability of universal barcodes(mat K,psb A-trn H,and rbc L fragment)was evaluated from the perspective of sequence variation and species identification.The results showed that the fragment with the highest proportion of variant sites was psb A-trn H,reaching13.04%.There are few mutation sites in mat K fragment and rbc L fragment.The phylogenetic tree shows that both psb A-trn H fragment and rbc L fragment can distinguish J-04 from other germplasm materials,but the bootstrap value is low,only 66%.The psb A-trn H fragment can distinguish different species of H.annuus and can be considered as a candidate barcode for interspecies identification of Helianthus.Through the whole fragment alignment of 10 H.annuus germplasm materials,5 highly variable fragments were screened from the ratio of variation sites,including 3 non-coding regions and 2 coding regions.The results showed that the identification results between the trn H-GUG-psb A fragment and the psa J fragment were confusing,the trn G-GCC-trn M-CAU fragment could not distinguish different species of Helianthus,and neither could be developed as candidate barcodes for the identification of Helianthus species.The phylogenetic tree gathers 10 H.annuus materials in one place and distinguishes them from other Helianthus species.The psb C-trn S-GCU fragment can only distinguish material J-04 from other germplasm materials,and the bootstrap support rate is higher than 85%.According to the 20 SSR sites,20 pairs of primers were designed.After preliminary screening,the primers Hacp SSR4,Hacp SSR13 and Hacp SSR19 can amplify the corresponding bands,which can be further explored for their potential value as molecular markers.