Development And Application Of SSR Molecular Markers Of Polygonatum Cyrtonema Hua Sibiricum Based On Full-length Transcriptome

Polygonatum multiflorum is a plant belonging to the genus Polygonatum in the Liliaceae family.It is also a medicinal plant with both ornamental and medicinal value.Due to its rich nutritional value and high economic and medicinal value in tubers,the market demand is increasing day by day.In order to meet market demand,in addition to wild Polygonatum multiflorum,artificially cultivated Polygonatum multiflorum has also emerged.Due to the wide variety of Polygonatum plants currently available,the source information of germplasm during introduction and cultivation in various regions is chaotic,making classification very difficult.This experiment collected 305 samples of Polygonatum multiflorum from Fujian Province,and observed and classified the phenotypic characteristics of the collected materials based on morphological markers;At the same time,the materials of Polygonatum multiflorum provided by Fujian were sequenced,and the full-length transcriptome data of Polygonatum multiflorum obtained by sequencing were developed with SSR primers,sequenced and analyzed with core sites of full-length transcriptome,and verified with ISSR molecular markers.The classification results of SSR,ISSR,and morphological markers were compared and analyzed to study the genetic diversity.The identification of varieties of Polygonatum multiflorum and other studies provide a reference basis.The main experimental research results are as follows:1 Genetic diversity analysis of polygonatum multiflorum based on morphological markersThis experiment collected 305 germplasms of Polygonatum in Fujian Province,including 80 from Shaowu City,75 from Sanming City,35 from Quanzhou City,77 from Longyan City,and 38 from Ningde City.9 quality and 10 quantitative traits of Polygonatum resources in Fujian Province were analyzed to explore the genetic diversity of Polygonatum germplasm in Fujian.The research results show that the coefficient of variation of 10 quantitative traits ranges from 27.17 % to 89.05 %,with an average value of 52.772 %.The order of coefficient of variation is leaf width,total stem length,perianth length,number of leaves,stem length,rhizome diameter,stem diameter,plant height,fruit diameter,and leaf length.The variation amplitude of leaf width is the largest,while the variation amplitude of leaf length is the smallest.The average genetic diversity of nine quality traits is0.795,and the order of genetic diversity index is: leaf shape > rhizome shape > leaf and stem color > floral color > fruit color > leaf order >rhizome color > inflorescence > fruit type.Cluster analysis divided 305 Fujian Polygonatum germplasm into five categories: the first category is mainly composed of Polygonatum multiflorum with rich leaf traits,and the color of leaves and stems is mainly green throughout the plant;The second type mainly includes long stemmed Polygonatum,with an average stem length of 11.2cm;The third type is Poly gonatum arisanense,which has whorled inflorescences;The fourth type is Polygonatum cristatum,which is green throughout the plant;The fifth type is Polygonatum kingianum Collett & Hemsl,which has a large number of leaves and a smaller leaf width.From the classification results combined with the diversity of morphological traits,it can be seen that the four characteristics of leaf shape,rhizome shape,leaf width,leaf and stem color,and total flower stem length have good differentiation effects in the classification of Polygonatum multiflorum.2 Abundant SSR Site Content of Polygonatum multiflorum in Fujian ProvinceThe full-length transcriptome data of Polygonatum multiflorum was obtained by sequencing the sample A21014918 a of Polygonatum multiflorum provided by Sanming in Fujian Province.Through data analysis,63263 SSR loci were found on 34748 sequences.A total of 4516 SSRs loci were identified in 5681 isoforms through structural analysis.The frequency of SSR occurrence in Polygonatum multiflorum is relatively high,with 15986 sequences containing more than one SSR site and 13563 sequences containing complex SSR sites.There are five types of SSR,including two base repeats(47673),three base repeats(10577),four base repeats(1993),five base repeats(1086),and six base repeats(1934).Among them,dinucleotides account for 75.37%,with AG/CT having the highest frequency,accounting for 48.6%.The research results indicate that the SSR loci of Polygonatum multiflorum have rich types and high polymorphism,which can provide a theoretical basis for the development and utilization of SSR molecular markers,genetic diversity analysis of germplasm resources,and molecular marker assisted selection breeding of Polygonatum multiflorum.3 Development of SSR primers based on Full-length transcriptome sequencing data of Polygonatum multiflorumBased on the full-length transcriptome data of Polygonatum multiflorum,DNAMAN was used to design SSR primers for the selected SSR sites,56 pairs of primers were randomly selected for synthesis,and46 pairs of SSR marker primers were screened to amplify the target fragment of Polygonatum multiflorum.After rescreening,19 pairs of SSR marker primers with high polymorphism and good repeatability were selected.Using these 19 pairs of primers to amplify 96 germplasms with significant morphological differences in Polygonatum multiflorum,all of them were able to amplify the target band,with good repeatability and high accuracy.Cluster analysis of 60 Fujian Polygonatum germplasm with significant differences in electrophoretic bands can be divided into five categories: the first category is mainly composed of Polygonatum multiflorum,with lanceolate leaves and short flower pedicels;The second type is long stemmed Polygonatum germplasm,with purple plants and long stemmed flower stalks;The third category includes the large leaved Polygonatum multiflorum and Poly gonatum arisanense,with wider leaves and a basic green color between the leaves and stems;The fourth type mainly consists of Polygonatum multiflorum,with plant morphological traits mostly consisting of green leaves and purple stems,and a variety of leaf forms;The fifth category includes Polygonatum kingianum Collett &Hemsl and Polygonatum multiflorum,with lower plant height.The classification results are basically consistent with those of morphological markers.In summary,the SSR markers of Polygonatum multiflorum have good repeatability and rich polymorphism.4 Genetic Diversity Analysis of Fujian Polygonatum multiflorum Based on ISSR Molecular MarkersThis experiment used these two Fujian Polygonatum germplasm with higher DNA quality(TF,YS11)to screen ISSR primers for Polygonatum multiflorum.Based on UBC universal primers,nine primers with high polymorphism were screened,and these 9 ISSR primers were used to amplify the DNA of 96 screened Polygonatum multiflorum germplasm.The results showed that a total of 185 polymorphic bands were amplified by 9 primers,with an average polymorphism percentage of 98.95% and an average of 20.55 loci amplified by each primer.The primer with the highest number of amplified loci among the 9 primers was UBC855,with 23 loci;The primer with the least number of amplified loci is UBC843,which has13 loci.The experimental results showed that primer UBC855 can effectively reveal the genetic diversity between different Polygonatum materials,and also indicate that the genome of Polygonatum multiflorum in this region has high genetic variation,and the amplification site has rich genetic diversity.Cluster analysis was conducted on 60 germplasm of Polygonatum multiflorum with significant differences,which can be roughly divided into four categories: the first category is mainly composed of green leaves,purple stems,and lanceolate leaves,identified as Polygonatum multiflorum;The second type has an average total flower stalk exceeding 10 cm,and the color of the leaves and rhizomes is purple.Based on morphological characteristics,it is identified as long stemmed Polygonatum;The third type is the large leaved multi flowered Polygonatum,which is a plant with green leaves and purple stems,with wider leaves;The fourth category includes Polygonatum Ali and Polygonatum multiflorum,most of which are green plants with some green leaves and purple stems.The clustering results are similar to the morphological marker clustering results of Polygonatum multiflorum,indicating that the ISSR marker primer of Polygonatum multiflorum can be applied to the identification of Polygonatum multiflorum germplasm.5 Comparative Analysis of SSR Molecular Markers,ISSR Molecular Markers,and Morphological Markers of Polygonatum multiflorum in FujianFrom the ISSR amplification results of Fujian Polygonatum cyrtonema,a total of 185 polymorphic bands were amplified using 9 ISSR primers,with an average polymorphism percentage of 98.95%.Comparative analysis shows that both the SSR and ISSR molecular markers of Polygonatum multiflorum exhibit high polymorphism,while the polymorphism of SSR primer markers is higher than that of ISSR.SSR markers divide Polygonatum multiflorum germplasm into five major categories,while ISSR markers divide Polygonatum multiflorum germplasm into four major categories.The coincidence rate of the two markers is 70 %,indicating that the developed SSR primers for Polygonatum multiflorum have certain reliability and stability in research fields such as phylogenetic analysis.Based on the analysis of morphological traits,amplification results,and ISSR molecular markers,it can be concluded that the clustering results of SSR markers from Polygonatum multiflorum are more similar to their morphological classification results.The development of SSR molecular markers from Polygonatum multiflorum can be used in research fields such as variety identification,classification,and phylogenetic analysis.