Transcriptome Sequencing And Primary Phylogenetic Analyses For Gynostemma Pentaphyllum And G.cardiospermum

The development of the high throughput sequencing technology accelerate the research on the transcriptome of the non model organisms.Gynostemma pentaphyllum,which belong to Gynostemma(Cucurbitaceae),is one of the most important Chinese herbal medicines.It contains many kinds of effective components,such as gypenosides,flavonoids and polysaccharides.The classification and phylogenetic relationship of the species in Gynostemma was blurred because of the cross and transition between morphological characters in this genus.Several previous studies analyse the phylogenetic relationship in Gynostemma but the species they collected were not enough which could not completely resove the phylogenetic relationship in Gynostemma.In this study,Illumina HiSeqTM 2000 sequencing technique was used to sequence the two species of G.pentaphyllum and G.cardiospermum,it produced 40257 and 44000 Unigene respectively after assembly the raw reads,then we obtained 71607 non reduandant unigenes after pooling unigenes from the two species and obtained 4517 differentially expressed genes(DEG)after comparing the unigenes of two species.The 71607 non reduandant unigenes and the 4517 DEG were annotated in Nr,Swissprot,GO,COG,and KEGG protein database respectively from which we obtained the relevant genes involved in the biosynthesis of Triterpenoid saponin.The development of molecular markers is essential in the analysis of the genetic structure of the population.In this study,we mined 3891 EST-SSR loci using MIS A software from the 71607 Unigenes,of which 2586 primer pairs were designed successfully.We randomly selected 360 markers in 6 G.pentaphyllum individuals and found that the 216 loci were polymorphic.30 polymorphic loci were selected from 216 loci to analyse the polymorphism in 12 species of Gynostemma,of which 15 pairs of primers can be successfully amplified in all species,The cross-species amplification of these 30 EST-SSRs was 92.33%.Among them,15 pairs of primers were amplified with an average of 6.73 alleles in the 26 individuals.The EST-SSR molecular markers screened in this study can be used to analyze the genetic diversity of Gynostemma and its related species.The molecular marker that used in previous phylogenetic research in Gynostemma were RFLP and ITS markers.In this research,we obtained 4566 pairs of orthologous genes from the transcriptome of G.pentaphyllum and G.cardiospermum,of which there were 533 pairs orthologous genes have high homology with APVO single copy gene database.In the light of the different stilus structure and fruit types between the two subgenera of Gynostemma,we screened 6 genes which associate with carpel development in flower from 4566 orthologous genes,one(G-AGL-29-like)of the six genes could amplified successfully in G.pntaphyllum,and homology search in the NCBI showed the successful amplified gene was homologous gene with AGL-29-like which belong to the MADS-BOX in the genome.The nrDNA G-AGL-29-like showed more mutation sites than cpDNA(rbcL,matK,trnH-psbA)in 12 species of Gynostemma.The phylogeny analysis showed that the 12 species of Gynostemma were divided into 2 categories,one category includes G.pentagynum,G.caulopterum,G.guangxiense,G.compressum,G.cardiospermum and G.laxiflorum.among which G.pentagynum was the original species because it was located in the phylogenetic tree base;the other category includes G.longipes,G.pubescens,G.pentaphyllum var.dasycarpum,G.burmanicum,G.laxum and G.pentaphyllum.The results also showed the Subgen.Trirostellum was more primitive than Subgen.Gynostemma.Beast analysis also showed the Subgen.Gynostemma was the evolved groups and the species in this group could not be distinguished clearly because of incomplete differentiation or the hybridization or introgression.G.guangxiense and G.compressum,which belong to Subgen.Gynostemma in the morphology plant taxonomy,were clustered with G.caulopterum in the MP,ML and Beast phylogenetic tree,we infer the G.guangxiense and G.compressum maybe the transitional group between the 2 subgenus in Gynostemma based on their fruit type;G.longipes and G.pentaphyllum may be same species based on morphology and molecular evidence.There are so many InDels which could not provide enough effective mutation information in trnH-psbA region in G.cardiospermum and G.laxiflorum that it could not be an appropriate molecular marker in Gynostemma.