Complete Chloroplast Genome And Comparative Analyses Of Three Bramble Resources (Rubus)

Chloroplast is an important organelle for photosynthesis and energy conversion of green plants,and has autonomous genetic information.Compared with the nuclear genome,the chloroplast genome is smaller and maternally inherited,and its gene order,gene content and base composition are highly conserved.At the same time,the nucleotide evolution rate of the chloroplast genome is moderate,with significant differences between coding and non-coding regions.Therefore,the chloroplast genome is suitable for phylogenetic studies at different taxonomic levels.Rubus L.plants are an important source of small berry fruit trees.The edible cultivar of its fruit is often referred to as raspberry,They are widely distributed and diverse in the world,and their genetic relationships are complex.The resources of Rubus in China are extremely abundant,but most of them are in the wild without effective development and utilization.In this study,three representative species,R.coreanus,R.lambertianus var.glaber and blackberry cultivar ’Arapaho’ were identified by PCR combined with high-throughput genomic sequencing.The chloroplast genome sequence of genomic assembly and bioinformatics analysis provides more molecular basis for the difficult classification and evolutionary relationships of the genus Rubus.The main results were listed as follows.1.This study sequenced,assembled and annotated the complete chloroplast genome sequence of three Rubus species.Bioinformatics analysis showed that the sample chloroplast genome is similar to the chloroplast genome structure of most higher plants,and is a typical tetrad ring structure.It includes one large single copy area(LSC),one small single copy area(SSC)and two reverse repeat areas(IR).The complete chloroplast genome size of R.coreanus,R.lambertianus and blackberry ‘Arapaho’ is 155405 bp,156569 bp and 156621 bp,respectively.The difference in size of the chloroplast genomes of these three samples was mainly due to the variation of the intergenic regions.The chloroplast genomes of R.coreanus and R.lambertianus contain 111 different genes,including 80 protein-coding genes,27 t RNA genes and 4 r RNA genes,and 18 repeat genes in the IR region;the Black Berry ’Arapaho’ contains 112 different genes,including 80 protein-coding genes,28 t RNA genes and 4 r RNA genes,have 18 repeat genes in the IR region.The GC content of the chloroplast genome of the three Rubus samples is 37.32%,37.17% and 37.13% in R.coreanus,R.lambertianus,and ’Arapaho’,which was not significantly different from other higher plants.2.Combined with other reported genome-wide sequence analysis of Rubus,the expansion and contraction of the IR region is very common in evolution.The IR expansion and contraction observed at the IR/SSC/LSC boundary may be caused by molecular recombination of two short repeats(ycf1,ycf1-f),which are often located at the IR boundary.3.The number of SSRs in the three chloroplast genomes was 44(R.coreanus),54(R.lambertianus)and 40(blackberry ’Arapaho’),and the labeling densities were 0.283,0.345,and 0.255 SSR per kb,respectively.We define a single nucleotide more than 5 bp as a repeating single nucleotide,and the major single nucleotide repeats in the three chloroplast genomes are A or T,accounting for 27.27% to 40%,47.5% to 68.18%,respectively.The A and T contents in the SSR locus region are also relatively high.The data obtained in this study indicate that the SSR polymorphism of the chloroplast genome of Rubus is weak,mainly single nucleotide repeats,very few dinucleotide or trinucleotide repeats,no polynucleotide repeats.4.With reference to the chloroplast genome sequence of the blackberry ’Arapaho’,the number of In Del sites in the chloroplast genome sequences of Rubus coreanus and Rubus lambertianus var.glaber were compared to 388 and 361.respectively,the size of the inserted and deleted fragments ranged from 2-34 bp,mainly from the intergenic regions.There were 22 In Del sites that affected gene mutations in Rubus coreanus.The nucleotide insertion accounted for about 81.82%.The longest insertion fragment was 34 bp and the longest deletion fragment was 26 bp,all of which occurred in the rbc L gene.There are 13 In Del sites in Rubus lambertianus var.glaber that affect the gene mutation,the nucleotide insertion accounts for about 84.61%,the longest insertion fragment is 14 bp,occurs in the ndh F gene,and the longest deletion fragment is 30 bp,occurs in the rbc L gene.Based on the genomic sequence of the whole chloroplast genome of blackberry ’Arapaho’,a total of 19,042 SNP sites were screened in Rubus coreanus and Rubus lambertianus var.glaber,96.58% of which were located in the genetically modified region.SNP sites were detected in almost all introns.Based on the CDS sequence(protein coding sequence),the three chloroplast genomes were analyzed for codon bias.The results showed that the codon bias was highly consistent in the three samples and highly preferred codons ending in A/T.5.Using the chloroplast genome-wide sequence information,the reported phylogenetic gene tree of the genus Rubus was reconstructed by the maximum likelihood method.The results showed that the species relationship revealed by the chloroplast genome sequence obtained in this study was consistent with the previously reported systemic relationship: the hollow berry group in the genus Rubus was in the basal group,and the raspberry group had the recent systemic differentiation.There is a closer relationship between R.lambertianus and blackberry ‘Arapaho’.