Studies On The Genetic Relationships Of Cultivars And Rhizosphere Soil Microbial Diversity In Rehmannia Glutinose

Rehmannia glutinosa(named 'DiHuang' in Chinese),one of the most famous medicinal plants,is a perennial herbaceous plant of the Scrophulariacea family.Owing to its nourishing functions to human body, its tuberous root is frequently used for Traditional Chinese Medicines in three different forms,i.e.fresh,dry and boil processed.Many cultivars or varieties were selected and cultivated in China in a long history of R. glutinosa cultivation practices.According to the report,there are more than 50 cultivars of R.glutinosa in China,but only few of them are described in detail in agronomical and morphological characters.Most of them are promiscuous and with unclear genetic relationship.In this work,cultivars,varieties and some sexual raise up seeds of R.glutinosa were collected and planted in the garden of Institute of Medicinal Plant Development.Field investigation was carried out in the morphological diversity including the shape,color, edges of leaves,color of anther,cornal and root,and the yield of medicinal part of root.Two molecular markers,random amplified polymorphism DNA(RAPD ) and amplified fragment length polymorphism (AFLP ),were employed for determine the genetic relationship of R.glutinosa,and the ribosome DNA internal transcribed spacer(ITS ) sequences was also used for analyzing sequence variations and phylogenetic history.The results showed that 48 accessions belong to 21 clusters were identified from 89 collections of R.glutinosa by UPGMA cluster analysis using RAPD in combination with AFLP markers. Among the 48 accessions,the germplasms of 2,20,39,50,53,71,82 and 86 have differential genotype as well as obviously phenotype,and these materials might be pay more attention while mining specific gene or crossbreeding.According to the distances in the dendrogram of R.glutinosa established from 1137 polymorphic bands in RAPD and AFLP markers combination,rDNA ITS sequences in several accessions were amplified and sequenced.The analysis of ITS sequences indicated that ITS1-5.8S-ITS2 of R.glutinosa were informative in its.611-613bp long sequence and have 22 variable sites including mostly occurred C-T transversion.Two phylogenetic trees were generated by Neighbour-jioning and UPGMA methods based on ITS sequences and were compared.In addition,R.glutinosa is an important medicinal plant,but there is a serious problem with its continuous cropping on the same land.We hypothesized some relationships between this problem and the disturbed soil ecosystem.In this work,two community-based microbiological measurements,community level physiological profiling(CLPP ) using Biolog sole C source utilization tests and phospholipid ester-linked fatty acid(PLFA) profiles,were used to evaluate soil microbial community function and composition of different R.glutinosa cropping soils.Field investigation showed that the problems with continuous cropping occurred not only in two-year continuous fields,but also in five-year rotation fields. Soil basal respiration and metabolic quotient were significantly higher in R.glutinosa cropping soils than in the non-cropping controls.On the contrary,the Shannon index and the average well color development (AWCD) from the Biolog data set were lower in R.glutinosa cropping soils,and we also found a seasonal change in AWCD and soil microbial biomass carbon(SMBC ),higher in cropping season but lower in harvest season.CLPP-based principal component analyses(PCA ) explained 87.47%variation of soil microbial communities,PC1 67.42%and PC2 14.05%,and these showed distinct groupings of soil microbial communities in R.glutinosa rhizosphere in PCA scatter plot.Eighteen PLFAs account for 88-90%aboundant in 33 PLFAs used in this work were main component in R.glutinosa rhizosphere soil.PLFA-based PCA also showed distinct composition of soil microbial communities in R.glutinosa rhizosphere,and 11 PLFAs representing different microbes were identified from the principal component scores of PLFAs.Among these, an abundance of PLFA 18:2ω6,9,which is a biomarker of soil fungi,was significantly higher (1.36-2.62times) in R.glutinosa cropping soils than control soils.These results suggest an alteration of soil microbial community following R.glutinosa cropping,and this might be an important reason for the constraints associated with continuous cropping.