Molecular Cytogenetic Studies On Siraitia Grosvenorii And 15 Genera Of Cucurbitaceous Plant

S.grosvenorii,a perennial vine of cucurbitaceous plants,is a traditional Chinese medicine and natural sweetener with a market prospect.In this study,the karyotype,45S rDNA and 5S rDNA distribution characteristics and molecular phylogeny of S.grosvenorii and other 15 genera of the same family were studied by the methods of karyotype analysis,fluorescence in situ hybridization analysis(FISH)and phylogenetic tree analysis included in modern cytology and molecular biology technology.The study of sub-systems provides a certain molecular basis for the genetic breeding,ploidy breeding,and classification of S.grosvenorii.It also provides necessary evidence for the identification,classification and development of the Cucurbitaceae.The main results of this paper are as follows:1.A seed germination system for cucurbitaceous plants was established.2.Based on the modified karyotype analysis method,26 cucurbitaceous plants finished karyotype analysis.The results showed that there were 6 kinds of chromosome bases in cucurbitaceous plants:x=7,x=10,x=11,x=12,x=13 and x=14.Trichosanthes villosa(2n=22),Herpetospermum pedunculosum(2n=20),Lagenaria siceraria(2n=22),Trichosanthes dunniana(2n=22),Trichosanthes ovigera(2n=22),Cucumis bisexualis(2n=24),Momordica dioica(2n=28),Zehneria maysorensis(2n=24),Coccinia grandis(2n=24),Luffa acutangular(2n=26),Sechium edule(2n=28),Cucurbita pepo(2n=40),Trichosanthes kirilowii(Suzhou,Jiangsu)(2n=22)is a new record.There are seven kinds of plants,such as M.cochinchinensis,B.hispida,G.lanatus,G.chinense,L.siceraria,T.kirilowii(Suzhou,Jiangsu)have satellite chromosomes.3.The improved fluorescence in situ hybridization(FISH)method was used for mapping 45S rDNA and 5S rDNA of 20 species of cucurbitaceous such as S.gro.svenorii.The results showed that 13 kinds plants,such as G.chinense,H.pedunculosum,L.siceraria,M.cochinchinensis,M.dioica,C.pepo,T.anguina,T.Maxim,T.dunniana,T.ovigera,Z.maysorensis,C.grandis,S.edule are new records.Except for T.Maxim,19 species of Cucurbitaceae have 45S rDNA signaling sites,which are satellite chromosomes.4.The full-length of LEAFY gene from 6 species of cucurbitaceous were cloned.A transcriptome analysis of the gene expression of S.grosvenorii was carried out to obtain the homologous gene to LEAFY gene,which was used as a template,and Primer RACE was designed to clone the full length of LEAFY gene of cDNA of S.grosvenorii.According to this,specific primers were designed in the non-coding region,and the whole length of the SgLEAFY1 gene was amplified with genomic DNA template.The full-length of LEAFY gene of 6 Cucurbitaceae plants,B.hispida,Lagenaria siceraria var.Hispida,Mukia maderaspatana,T.rubriflos,Melothria sphaerocarpa and Momordica rostrate,were cloned by methods of homologous cloning combined with RACE-polymerase chain reaction(PCR).5.ITS2 and LEAFY intron 2 were used to construct the phylogenetic tree to study S.grosvenorii and Cucurbitaceae plants.According to the LEAGY full-length gene,primers were designed to amplify the LEAFY intron 2 of the Cucurbitaceae plants,and ribosomal DNA internal transcribed spacer 2(ITS2)was amplified by universal primers.A phylogenetic tree was constructed using RAxML method for comparative analysis.Based on the results of ITS2 phylogenetic tree,the development order of the Cucurbitaceae genera were Cucumis,Mukia>Zehneria>Thladiantha,Momordica,Siraitia>Gymnopetalum,Coccinia,Lagenaria,Citrullus>Trichosanthes>Sechium,Cucurbita>Luffa,Herpetospermum.Based on the phylogenetic tree results of LEAFY gene intron 2,the development order of the cucurbitaceae genera were Siraitia>Thladiantha,Momordica>Luffa>Trichosanthes,Gymnopetalum>Cucumis,Mukia,Zehneria>Cucurbita,Lagenaria,Citrullus,Coccinia.6.The analysis of the genetic relationship between the S.grosvenorii and Cucurbitaceae supports the S.grosvenorii as an independent genus,and S.grosvenorii has a higher classification status in the Cucurbitaceae.Compared with the ITS2 phylogenetic tree,the LEAFY intron 2 phylogenetic tree is more clearly defined and the support rate is generally higher.The phylogeny of the Cucurbitaceae by using the LEAFY gene intron 2 loci is better than the ITS2 Location.The results of molecular phylogenetic supported the results of classic morphology classification.The genus of Siraitia is a separate genus and is closely related to phylogeny of the genus Paeoniae and Momordica.