Molecular Identification Of Two Natural Hybrids Of Hoya

Natural hybridization plays an important role in the formation and evolution of plant species,and it is also possible to use the hybrids produced by natural hybrids to breed a new varieties of plant.The genus Hoya(Hoya R.Br.),a genus of the tribe Marsdensieae(Asclepiadaceae),which is including 200-300 species.There are about 39 species in C hina,which are mainly distributed in Guangdong,Yunnan,Guangxi.At present,the study of the Hoya is focused on tissue culture,pharmacy value and morphology.Although there are many species of Hoya,there is have no stud y been reported on the hybrids.In this study,we used molecular methods to identifysp1,suspected hybrids(ZJ)and suspected new species(XZ).First,we screened a low-copy nuclear gene(N ic)and the chloroplast gene spacer(trnL-trnF)with mutation sites in the sequence.In order to find out the genetic relationship between them,we analyzed their haplotypes.Then we discussed the genetic diversity and genetic relationship of three species by ISSR molecular marker technique.The results are followed:(1)The haplodtype analysis results of the low-copy nuclear gene(N ic)shown: there are 11 variable sites inthree Hoya species and they constitute 6 haplotypes.Haplotype1 and 3 are shared by three species.XH own allHaplotypes but Haplotype5;XZ own Haplotype1 and 3;ZJ own Haplotypes1,3,4,5.Therefore three species own Haplotype1,3 and Haplotype4 is owned by XH and ZJ.Haplotype5 only have ZJ,and Haplotype1,6 belong to XH.Though they are sharing some Haplotypes,There is a large difference in haplotype frequencies between different groups.According to the data of the chloroplast gene spacer(trnL-trnF),we find that there are some individuals' sequences miss 6bp including three species.And it make 2 Haplotypes which frequencies are closer in the three species.(2)We select primers fromISSR randomly and choose 10 primers which with bands after amplification.There are 49 bands in DNA fingerprints.The polymorphic band is 14 and polymorphism is 28.6%.Weused POPGEN E calculated gst value is 0.1175.We analyze the genetic similarity coefficient by NTSYS 2.10 e.The maximum value is 1 and minimum value is 0 between 60 samples.Thenwebuildaclustering tree whichcanhelpusto classify theHoya.When the GS(Genetic Similarity)is 0.34,it can be divided into two categories.One of them consist of a large number of XH and a small amount of ZJ,what is no any XZ.The results are shown us sp1 and suspected new species is different,and it almost the same as the morphological results.In summary,the results show thatsuspected hybridsis hybridization ofsp1 and suspected new species and it provide the molecular evidence of the hybridization.Nuclear geneand the chloroplast genehave shown that there are frequent hybrids mediated between these two species.The result by ISSR is also supportit,in the second largest branch of suspected new species including sp1 that obviously infiltrate from the suspected new species to sp1.