Comparative Genomics Of Gynostemma Pentaphyllum And Gynostemma Pentagynum

Gynostemma pentaphyllum(Thunb.)Makino and Gynostemma pentagynum Z.P.Wang are perennial herbaceous vines belonging to the Cucurbitaceae family of the order Cucurbitales.The two species are the representative groups of Subgen.Gynostemma with berry fruit type and Subgen.Trirostellum with capsule fruit type respectively.G.pentaphyllum,as the most widely distributed medicinal plant in the genus Gynostemma,contains dammar saponins,flavonoids and a variety of biologically active substances,and has extremely high medicinal value such as lowering blood pressure,anti-inflammatory and detoxifying,and inhibiting tumors.However,the evolutionary mechanisms of genes related to the development of different fruit types and saponin synthesis pathways in Gynostemma species have been controversial.Therefore,based on high-throughput sequencing technology,this study assembled and annotated the genome sequences of G.pentaphyllum and G.pentagynum,combined with transcriptomic analysis to study the saponin synthesis pathway in different tissues of the two species and the evolutionary patterns of gene families related to fruit development.The main results are as follows:1.High quality chromosome level genomes of G.pentaphyllum and G.pentagynum were obtained.In this study,the genomes of G.pentaphyllum and G.pentagynum were assembled into 11 chromosomes using Illumina II sequencing,Pacbio III sequencing and Hi C assisted assembly.The G.pentaphyllum genome size is 608.88 Mb and contig N50 is 5,045,119 bp.The Scaffold N50 is 56,377,829 bp.The genome assembly size of G.pentagynum is493.37 Mb and the contig N50 length is 43,123,386 bp.The Scaffold N50 length is52,291,176 bp.The evaluation results of BUSCO of G.pentaphyllum and G.pentagynum were 95.1% and 97.5% respectively,indicating that the assembly results were good.27,418 structural genes were predicted in the G.pentaphyllum genome,of which 26,588 were functionally annotated.Of the 26,050 structural genes annotated by G.pentagynum,25,615 were functionally annotated.The proportion of G.pentaphyllum repeat sequences was 60.23%and G.pentagynum was 59.65%.2.Gene family analysis revealed that there were 14,392 unique gene families and 20,880 extended gene families in G.pentaphyllum,most of which and some of the contracted gene families in G.pentaphyllum were significantly enriched in sesquiterpenoid and triterpenoid biosynthesis and monoterpenoid biosynthesis pathway.Phylogenetic analysis based on single-copy lineal homologous genes showed that G.pentaphyllum and G.pentaphyllum were clustered into a monophyletic branch.The analysis of differentiation time showed that Gynostemma and other species of Cucurbitaceae were differentiated at about 64 Mya(Paleocene),G.pentaphyllum and G.pentagynum were differentiated at about 63 Mya.The results of genome doubling analysis showed that G.pentaphyllum and G.pentagynum had a common genome duplication event before their differentiation with pumpkin,and the time was similar to the first whole genome duplication event of pumpkin.At the same time,species differentiation occurred in G.pentaphyllum and G.pentagynum at this time,suggesting that whole gene replication maybe drives species differentiation.3.Combined with genome annotation and transcriptome analysis,the genes related to the fruit cracking and saponin synthesis pathway of G.pentaphyllum and G.pentagynum with different fruit types were screened out.The results showed that the genes related to the b HLH protein family,which are closely related to fruit dehiscence,had significant expression differences in the fruit transcriptome samples of G.pentaphyllum and G.pentagynum,indicating that the fruit dehiscence of Gynostemma was significantly related to the gene expression level.At the same time,the differential expression of genes related to saponin synthesis pathway in different tissues(stem,leaf,flower,fruit,tendril)was explored.The results showed that some saponin synthesis pathway genes of G.pentaphyllum(e.g.Gype02G0716.1、Gype09G2675.1、Gype10G0618.1、Gype07G1535.1、Gype11G1710.1、Gype02G1454.1)had higher expression level in leaves,while in G.pentagynum saponin pathway related genes,evm.model.Scaffold2.1251 、 evm.model.Scaffold3.948 、evm.model.Scaffold9.1164 、 evm.model.Scaffold3.2191 、 evm.model.Scaffold2.1561 、evm.model.Scaffold11.160 and evm.model.Scaffold7.320 showed higher expression levels in leaf samples,suggesting that saponin biosynthesis related genes have differentiated expression levels in different species.In conclusion,the present study obtained high quality genomic data of G.pentaphyllum and G.pentagynum,identified a large number of gynost’ pentaphyllum specific and extended gene families related to saponin synthesis pathway,and identified genome-wide duplication events.At the same time,the b HLH gene family closely related to fruit dehiscence was screened,which plays an important regulatory role in the evolution of fruit types in Gynostemma plants.In addition,several genes related to saponin synthesis pathway were screened in G.pentaphyllum and G.pentagynum,most of which were highly expressed in leaf samples,indicating significant tissue specificity.The results of this study will provide an important basis for further research on the phylogenetic and evolutionary history of Gynostemma genus and the molecular evolutionary mechanism of the development of different fruit types.