Stduy On The Evolution Of Chloroplast Genomes In Chrysosplenium L. Of China

The species of Chrysosplenium L.is a member of the Saxifragaceae family.The basal leaves are alternate or opposite.China is the core area of global Chrysosplenium L.distribution.The first large-scale molecular phylogeny of the Chrysosplenium L.was completed in 1997.After that,scientists have conducted in-depth research on this species.As core area of global Chrysosplenium distribution,few samples about Chrysosplenium had been collection in China.The comprehensive analysis of the Chrysosplenium L.chloroplast genome has not been reported.This research optimized the technology of isolation of chloroplast DNA,and then obtained high quality DNA of chloroplast.Using the next-generation sequencing technology,Illumina,we sequenced the chloroplast genome of 30 Chrysosplenium in 27 species.We analysed evolution of chloroplast DNA at level of sequence and structure.Reconstructed the phylogenetic relationship in Chrysosplenium L.,providing molecular evidence for the identification of new species of Chrysosplenium L..Determine the developmental position of Chrysosplenium L.and Saxifragales in the APG classification system.The results are as follows:1.This study uses wild Chrysosplenium L.as materials.The chloroplast genomic DNA was extracted by gradient centrifugation and high salt-low pH method respectively,and total DNA was extracted by modified CTAB method.Comparing two different chloroplast DNA extraction methods,all of which can obtain high quality chloroplast DNA.Both of the above chloroplast DNA extraction methods require a large amount of fresh leaves as a material.Due to the lack of samples,most wild Chrysosplenium L.still adopt the CTAB method to extract total DNA for sequencing.2.Assembly using the ABYSS.The final chloroplast genome size we obtained was 150,750-154,342 bp,of which SSC was 17,115-18,137 bp,LSC was 81,830-84,437 bp,and IR was 25,318-26,666 bp.The GC content of C.microspermum was 37.05%,which was much smaller than that of other species.In the case of not counting the C.microspermum,the average GC content of Subgen.Chrysosplenium was 37.38%,which was significantly lower than the average GC content of Subgen.Gamosplenium 37.62%.3.Most of the Chrysosplenium L.chloroplast genome contains 117 different genes,including for distinct rRNA,31 distinct tRNA and 82 distinct peptide-coding genes.In C.sinicum,C.nepalense,C.sangzhense,the position of a tRNA has changed,trnK-UUU has been transferred from the LSC region to the IRs region.InfA gene loss in C.gonggaense.There is only one ycf1 gene in the IR region of C.gonggaense,C.alternifolia,and C.lectus-cochleae,which is a single copy.The rps19 gene of C.badongense and C.griffithii is a single copy.Species in which IR region expansion/contraction events occur in Chrysosplenium L.are concentrated in high latitudes and high altitude regions.4.The phylogenetic relationship of species within Chrysosplenium L.was reconstructed using 79 protein coding sequences.The four phylogenetic trees of neighbor-joining(NJ),Maximum Likelihood(ML),Maximum parsimony(MP),and Bayesian represent the same topology.Except for the C.microspermus,Chrysosplenium L.can be divided into two large branches and nine small branches,which are basically consistent with the morphological description.5.It was confirmed that Chrysosplenium L.is more closely related to other species of the Saxifragaceae family and forms a good sister branch.Using molecular evidence to determine that Chrysosplenium L.belongs to Saxifragaceae.Reconstructed the position of Saxifragales in the APG system.Our phylogenetic analyses roughly confirmed that Rosales and Saxifragales formed a clade that was sister to Vitales.This studies have shown that there is expansion of single copy(SC)and IR regions of angiosperms cp genomes during evolution.