Chloroplast Phylogenomics Of Rubus In China

Rubus Linn.belongs to subfamily Rosoideae of Rosaceae.It’s a species-rich genus with worldwide distribution.China is the diversity center of this genus,with 204 species and 100 varieties,which accounts for 97% of Asian species.Morphological mutations,hybridization,polyploidy,and apomixis frequently occured in Rubus,which make it difficult to study the taxonomy,phylogeny and evolution of Rubus.At present,phylogenetic study based on a few chloroplast DNA fragments could not resolve the interspecific relationship in Rubus with their fewer mutation sites and limited phylogenetic information.The chloroplast(cp)genomes of most angiosperms are mainly maternal inheritance.Simultaneously,they have moderate size,nucleotide substitution rate and high collinearity among different groups.Therefore,plastomes have been accidentally applied to phylogenomics research at different classification levels during recent years.However,the related research on Chinese Rubus is still lacking.In order to evaluate the application potential of cp genome in phylogenetic research in Rubus,and to investigate the phylogenetic species relationship within the genus,in this study,60 individuals representing 55 Chinese Rubus species(including variants,from 20 subsections of 3 sections)were sequenced by genome resequencing technology on the high-throughput sequencing platform of Illumina.Based on the assembly and annotation of cp genomes,plastid genome comparative analysis was carried out on 24 representative species of Rubus.Then phylogenetic analysis were carried out using Maximum Likelihood and Bayesian Inference based on 7 matrices.The main results are as follows:(1)The plastid genomes of 24 Rubus species remained highly conservative with the typical quartile structures formed by a large single copy region(LSC),a small single copy region(SSC),and a pair of inverted repeat regions(IRs).Plastome size,gene composition and structural characteristics within the genus were also highly conservative,with genome size ranging from 155,464 bp to 156,506 bp,and the length variation of LSC accounts for the main difference in genome size.The plastome had 112 unique genes,including 79 protein-coding genes,29 tRNAs,and 4 rRNAs.At the same time,the internal genome were highly collinear,and the IR boundaries have not changed significantly.A total of 2,245 repetitive sequences and 1,149 SSR sites were detected in 24 Rubus plastome sequences.The sequence variation of the whole genome were relatively conservative,and there was different degree of variation between different regions.Meanwhile,the IRs was the most conservative.According to the results of nucleotide polymorphism analysis,nine hypervariable regions were detected,i.e.trnH-psbA,trnK-rps16,rps16-trnQ-psbK,petN-psbM,trnT-trnL,petA-psbJ,rpl16 intron,ndhF-trnL and ycf1,which can be used for subsequent DNA barcode and population genetics research.(2)The result of phylogenetic analysis based on the IR matrix failed to solve the infrageneric relationship.While phylogenetic analysis based on the remaining six matrice(whole chloroplast genome,LSC,SSC,coding region,non-coding region and hypervariable region matrix)showed similar topology with minor differences,which greatly improved the resolution.Phylogenetic analysis based on the whole chloroplast genome showed that sect.Idaeobatus is a polyphyletic group with three strongly distant clades.Sect.Malachobatus,R.pectinaris of sect.Chamaebatus and R.pectinaris of sect.Idaeobatus form a monophyletic clade.Combined with previous cytology and morphological classification,it’s likely that Rubus spp.evolved from diploid(2n = 14)to polyploid,from compound leaf to simple leaf,the inflorescences evolved from corymbose,single flowers to panicles,racemes,fruit colour evolved from orange to red.It was found that partial branches in phylogenetic tree were inconsistent with morphological traits among subsections and interspecies,which is speculated that it is caused by the highly conserved Rubus cp genomes with limited phylogenetic information.It is also possibly related to chloroplast capture,convergent evolution,incomplete lineage sorting and so on.In this study,the basic characteristics of Rubus plastid genomes were summarized for the first time.Based on the complete chloroplast genome,a relatively reliable phylogeny framework of Rubus was preliminarily provided by phylogenomics research,which revealed the potential of plastid genome for resolving phylogenetic relationship in Rubus,and provided genomic resources for the study on the plastome evolution mechanism of Rubus.