Cloning And Screening Of Low Copy Nuclear Gene Markers In Ranunculus Cantonensis Polyploid Complex And Its Allied Species

Ployploidy is prevalent in plants, which found that more than 70% species in angiosperms have experienced at least once polyploidization event.The Ranunculus cantonensis polyploid complex and its allied species belong to Ranunculaceae Ranunculus. The polyploidy complex mainly distribute in the East Asian, is composed of four diploids, two tetraploidsa pentaploid and a hexaploid. It is a good model to study the polyploid complex evolution, phylogenetic, genetic constitution and polyploid origin.Previous studies about the Ranunculus cantonensis polyploid complex mainly focused on aspects of palynology, isoenzyme, karyotype analysis, FISH, DNA（trnL-F, ITS) sequence analysis and SSR molecular markers. But because of the limitations of these techniques, relationship between the members and genetic constitution of the polyploidy complex is not clear.This study used tetraploid R. cantoniensis as a sample material, to clone multiple low-copy nuclear genes which can provide abundant phylogenetic information. The objective is to screen the suitable nuclear gene markers which could be useful in Ranunculus cantoniensis polyploid complex, in order to make a thorough discussion of the phylogenetic relationship between the complex and its allied species, understanding of the formation and evolution of polyploid taxa.The main results are as follows:(1) This is the first time to achieve 12 fragment sequences of 11 nuclear genes from R. cantoniensis, which can be used for the research of Ranunculus cantonensis polyploid complex.These are the 11 genes:ADH gene, CHS gene, DHAR gene, G3pdh gene, GBSSI gene, ncpGS gene, LEAFY gene, PgiC gene, PHY A gene, PI gene and 4CL gene.(2) We have obtained 152 clonies from 9 species by utilized partion of GBSSI gene intron sequences. This fragment is length of 512 bp, has 70 simple information site, and the evolutionary rate is 13.6% which is moderate. So it is suitable for the phyletic evolution study of Ranunculus.(3) We have reconstructed the reticulate evolution of Ranunculus cantoniensis polyploid complex and its allied species by partion of GBSSI gene intron sequences. The results show that R. repens closely associated with polyploid complex, participating in the origins and evolution of polyploid complex; R. cantoniensis originated from hybridization of R. chinensis and R. silerifolius var. silerfolius, R. sieboldii originated from hybridization of R. chinensis and R. repens, R. repens might originated from hybridization of R. chinensis and R. silerifolius var. dolicathus; R. chinensis was a key species in this complex, which may play a important role as pivotal genome.