Morphological And Anatomical Studies And SCoT Molecular Markers Analysis Of 8 Species In Polygonatum

Polygonatum is a perennial herb of the Polygonatum genus of the Liliaceae family.It is often used as medicine with dried rhizomes,which has the effects of lowering blood sugar and blood lipids,anti-oxidation,and anti-tumor.With the increase of market demand for Polygonatum,the problem of germplasm confusion has become increasingly prominent.In this study,P.kingianum,P.zanlanscianense,P.kingianum var.grandifolium,P.cyrtonema,P.punctatum,P.hookeri,P.sibiricum and P.filipes were used as test objects.They were collected by Hunan Academy of Agricultural Sciences and planted in the germplasm conservation nursery of Chinese medicinal materials.By comparing the phenological period,morphological traits,anatomical traits and genetic differences of 8 species of Polygonatum,in order to provide a reference for the identification,development and utilization of Polygonatum.The main results are as follows:1.By comparing the phenophase of 8 species of Polygonatum,it was found that the phenophase was 206～252 d,and the phenological period of Polygonatum is susceptible to temperature,and the ambient temperature is low in the early stage of growth and development.The response of different species of Polygonatum to low temperature will be different,which leads to the difference in phenological period between species of this genus.In the process of growth and development,with the increase of ambient temperature,the growth and development of plants were accelerated,and the differences in phenological periods of Polygonatum were reduced.2.The morphological traits of 8 species of Polygonatum were counted and analyzed.It was found that there were abundant variations among the species of Polygonatum.According to the plant height,ground diameter,leaf length,leaf width,leaf number per plant,flower number per plant and fruit number per plant,8 species of Polygonatum can be roughly distinguished.The results of grading evaluation of seven growth traits showed that Polygonatum cyrtonema,P.kingianum var.grandifolium,P.punctatum and P.zanlanscianense could grow well in the climatic environment of Hunan Province.3.The anatomical structures of leaf epidermis,leave,stems,roots and rhizomes of 8 species of Polygonatum were observed by surface preparation method and paraffin sectioning method.It was found that the basic structure of 8 species of Polygonatum were generally similar,but some anatomical structures and parameters were different.(1)The shape of the epidermal cells on the leaves was flat quadrilateral,and the cells were closely arranged without stomatal distribution.The lower epidermis of the leaves can be divided into regular and irregular shapes.The pattern of the anticlinal wall can be divided into three types:straight,straight-curved,deep waveform,among which the anticlinal wall pattern of the lower epidermis of P.punctatum is deep waveform.(2)The leaves were composed of epidermis,mesophyll and veins.The leaves were equilateral leaves,without the differentiation of palisade tissue and spongy tissue.The difference of main vein thickness was the most obvious,the thickest was P.punctatum(1005.12 μm),and the thinnest was P.sibiricum(260.19 μm).(3)The stem was composed of epidermis,cortex,fiber ring,vascular bundles and basic tissues,but the number and distribution of vascular bundles in stem were different,and the number of cell layers in fiber ring was also slightly different.Among them,the number of vascular bundles in the stem of P.kingianum var.grandifolium was the largest and most of them were scattered around the periphery of the stem.There was a large space in the center without vascular bundles,only parenchyma cells were distributed.The vascular bundles in the stems of the remaining seven species of Polygonatum were scattered in the basic tissues,and only a small amount of vascular bundles were distributed on the fiber ring.The number of ’fibrous ring’ cells of P.kingianum var.grandifolium and P.punctatum was more,with 4～5 layers,P.cyrtonema has only 1～2 layers.(4)Roots were composed of epidermis,cortex and vascular cylinder.Among them,the xylem vessels of roots are quite different.According to the difference of vessels,8 species of Polygonatum can be divided into three categories:the first category,the distribution of vessels is concentrated,the number is large and the pore size is large,including P.kingianum var.grandifolium,P.punctatum,P.sibiricum;the second category,the distribution of vessels is more dispersed,the number is less and the aperture is smaller,including P.kingianum,P.zanlanscianense,P.cyrtonema,P.filipes;the third category,the number of vessels is small and the aperture is small,such as P.hookeri.(5)Rhizome is composed of epidermis,basic tissue and vascular bundles,but the vascular bundles of rhizome are quite different.Rhizome vascular bundles can be divided into three types:collateral vascular bundles(P.kingianum,P.zanlanscianense,P.kingianum var.grandifolium,P.cyrtonema,P.sibiricum and P.filipes),amphivasal vascular bundles(P.punctatum),the collateral vascular bundles were the main type,and occasionally amphivasal vascular bundles(P.hookeri).4.The optimal SCoT-PCR reaction system of Polygonatum was established by orthogonal test.1.50 mmol/L Mg2+,0.125 mmol/L dNTPs,1.50 U DNA polymerase,0.40μmol/L primer,20.00 ng template DNA,Supplement with ddH2O to 20 μL.These five factors together restrict the sensitivity of amplification and the yield of amplification products,and their influence from large to small is:Mg2+>DNA template>primer concentration>dNTPs>DNA polymerase.On this basis,the optimal annealing temperature of the selected primers was discussed.5.Twelve SCo T primers were obtained by screening 50 primers.The genomic DNA of Polygonatum samples were amplified by using the optimized reaction system and the optimal annealing temperature of each primer.A total of 649 bands were obtained,with an average of 54.08 bands per primer,there were 629 polymorphic bands,and the percentage of polymorphism was 96.92%.It shows that the polymorphism of SCoT molecular markers in Polygonatum is rich,which is suitable for the genetic analysis of this genus.The results of cluster analysis and principal coordinate analysis were consistent.8 species of Polygonatum were divided into three categories.The first category was P.kingianum var.grandifolium and P.punctatum.The second category is P.zanlanscianense,P.kingianum,P.filipes;the third category is P.cyrtonema,P.sibiricum and P.hookeri.