The Molecular Phylogenetic Analysis Of Mulberry Based On The Nucleus ITS And Chloroplast DNA Sequences

As an important woody plant,mulberry has a long cultivation history.Among them,Morus alba is the most widely distributed mulberry species.China is an important original center of mulberry species,with the abundant germplasm resources.The artificial cultivation and natural hybridization of mulberry has caused the emergency of many varieties or subspecies.However,the same mulberry germplasms have different names.Therefore,we tried to find molecular markers to illuminate the classification and phylogenetic relationship of mulberry that exists tremendously controversial in nomenclature.The nuclear internal transcribed spacer(ITS)has been widely applied in species identification and phylogenetic relationships.ITS is a tandem repeat sequence with high copy number in genome,easily to be amplificated and hanving higher resolution.The chloroplast noncoding regions have a relatively high mutation rate,and had been selected as an important tool to study intraspecific phylogenetic analysis.Therefore,the noncoding region of chloroplast DNA sequences and the nuclear ITS sequences were used to reveal the classification and relationship of mulberry in the present study.The contents of this study are as follows:1.The analysis of the nuclear ITS sequence for 40 mulberry resourcesThe ITS sequences of 40 mulberry species had been analysed.Among them,16 mulberries contained polymorphisms in ITS,suggesting the existing imcomplete concerted evolution in 16 mulberries.Subsequently,we analyzed three factors in two types of ITS including GC content,minimum free energy of secondary structure,and the three conservative motif in 5.8S r DNA.The results indicated that ITS-? in 13 mulberries were ITS pseudogenes.MS was near concerted evolved and MY6 and MJPZ were just being concerted evolution.In order to investigate the meaning of ITS-? in mulberry evolution,ITS-? were used to reconducted the phylogenetic tree of Morus.We found that ITS-? and B tribe of Morus were paratactic,and ITS-?1 of MY6 MB and ITS-? of MY6 and MJPZ were clustered together.MY6 MB maybe a new mulberry species,and named it as E tribe.According to the statistics of ITS-? copy and its location in phylogenetic tree,concerted evolution maybe existed in 16 mulberries,and the direction of concerted evolution was M.alba because of its strongerecological potential.In conclusion,we proposed the possibility of concerted evolution in mulberry was the follow: the filial generation of E tribe and B tribe of Morus contains two types of ITS from two parents,due to the strong ecological potential of M.alba.The ITS type of E tribe became vestigial.The manifestation of ITS degeneration showed ITS copy reducing of one parent in genome,truncated ITS,and finally evolved to M.alba.2.The phylogenetic analysis of Morus based on cp DNAAccording to the colinear analysis of six mulberry chloroplast genome,we selected 4 chloroplast DNA intergenic regions to analyze the phylogenetic evolution of Morus,including trn L-trn F,trn T-trn L,trn D-trn T,and psb E-pet G.The results indicated that trn D-trn T and psb E-pet G sequences were not suitable for phylogenetic analysis.trn L-F and trn T-L spacers are high mutation region of mulberry chloroplast genome,which can fully reflect the genetic relationship between mulberry varieties.We reconstructed phylogenetic tree based on trn L-F and trn T-L.Firstly,the species Morus notabilis and Morus yunanensis than other 29 mulberries were grouped together.Secondly,the 29 mulberry resources were clustered into two groups with 100% of the posterior probability.However,the internal relationship of mulberry were still not solved because of lacking of sequence defference.The phylogenetic relationships of Morus based on necular ITS and cp DNA were different.For example,B tribe mulberry contained two types of ITS in ITS phylogenetic tree,but they had no obvious differentiation in cp DNA phylogenetic tree.The frequent hybridization in mulberry presumably caused this inconsistent.In conclution,based on the analysis of ITS sequences,we observed the existence of concerted evolution in mulberry.Mulberry species MY6 MB belonged to a new tribe called E.trn L-Fand trn T-L were selected as molecular marker for the phylogenetic analysis in M.alba.According to the combination analysis of ITS and cpDNA sequence,M.alba were evoluted from two tribes.The results provided by this study was informatic for the study of mulberry origin and evolution,and would be foundation for resourse selection and crossbreeding of mulberry.