Studies On Germplasm Resources Of Rhizoma Dioscorea

Rhizoma Dioscorea is the tubers of Dioscorea opposita Thunb. and its close relatives.China’s Ministry of Public Health publicized it as Chinese medicine used for both food and medicine. It has been cultivated for more than2,500years in many areas of China, where people often exchange their variety with each other. Rhizoma Dioscorea tend to be influenced by the surroundings. Some varieties are close in external features and overlapped in the range of variation. The bulbils, flowers, fruits, seeds are not formed in the same cultivated area. As a result, it is difficult to identify whether any two species are the same kind or the same class group just in terms of shape. The present study applied a combination of morphological markers, ISSR and SRAP molecular markers to construction of fingerprint and analysis of genetic diversity of90Rhizoma Dioscorea germplasm resources in13provinces (regions) in China, including Shangdong, Anhui, Jiangxi, Zhejiang, Shanxi, Henan, Guangxi, Shanxi, Guangdong, Hainan, etc. Then the main chemical compositions were extracted and analyzed. Finally core collection was constructed. Major findings are as follows.1. Analysis of morphological characteristics:With the average value of the19morphological characters obtained in a3-year investigation as the index, we adopted the botanical means of classification to make a phylogenetic classification of Rhizoma Dioscorea germplasm resources. The90resources were then classified into four main types, i.e. D. opposita, D. persimilis, D. alata and D. fordii. Based on this, the present study analyzed and evaluated their main characteristics.2. Construction of ISSR and SRAP fingerprint:The ISSR and SRAP markers were used to construct90germplasm resources by using two independent methods respectively (namely, a particular marker for a single primer and a combination of polymorphic band types for multiple primers) to effectively identify the germplasm resources. The result shows that among90germplasm resources,11resources were identified by using ISSR key band,8resources were identified by using SRAP special sites. The principle is to use the minimum number of primers to identify as more resources as possible. Then10ISSR and10pairs of SRAP primers were selected to construct Rhizoma Dioscorea resource fingerprint respectively and the result shows that75resources can be identified by ISSR-DNA fingerprint and15cannot;82resources can be identified by SRAP-DNA fingerprint and8cannot.3. ISSR and SRAP analysis of genetic diversity:The ISSR molecular markers were used to analyze the genetic diversity of the90germplasm resources.22polymorphic primers were screened from200ISSR primers to amplify the bands of the90resources for test. And226polymorphic sites were obtained, which were used to calculate genetic distances. A phylogenetic tree of the90germplasma resources was constructed using the UPGMA clustering method with DPS Software. At the genetic distance of0.43.90germplasm resources were clustered into two large groups, one with48resources and the other42; at the genetic distance of0.35, the first large group was subdivided into two subgroups, one with28, the other20; at the genetic distance of0.27, the first subgroup of28resources was furt(?)er divided into two minor groups, one with22, the other with5. Therefore, the90resources were divided into4groups by ISSR markers.30primer pairs that can amplify stable and clear bands were screened from49SRAP primer pairs, which amplified616polymorphic bands. Meanwhile an analysis of genetic diversity was made to the germplasm resources by using DPS Software. The resul(?)shows:90germplasma resources can be divided into5types:Type Ⅰ includes20resources of D. opposita, Type Ⅱ includes42resources of D. persimilis, Type Ⅲ includes6resources of D.fordii and Type Ⅳ and Type V include3and19resources of D. alata respectively.4. Comparison of ISSR and SRAP analysis of genetic diversity:To make better use of ISSR and SRAP markers, they are compared in the analysis of germplasm resources. The result shows that the polymorphism rate of the two types of markers reach79.2%and80.47%respectively, which indicates that the genetic diversity between two germplasm resources is high. The total number of amplified bands and polymorphic sites and the average number of amplified bands in SRAP markers are much higher than those in ISSR markers. The results of both markers show that about52%of total genetic diversity of germplasm resources occurs in different species groups and48%occurs in the same species group. The range of genetic distance of SRAP markers is wider than that of ISSR markers. ISSR and SRAP markers divide90germplasm resources into4and5types respectively and SRAP reveals its unusual genetic structure. In terms of fingerprint, SRAP-DNA fingerprint has higher capability of identification.4. Analysis of principal chemical compositions:The HPLC method, UV colorimetric method, and Phenol-sulphate acid method were used to measure the content of allantoin, flavone,polysaccharides and adenosine of the90resources respectively, and also made a comparative analysis of these four components in the six groups, i.e. the national resource, Fujian resource, D. opposita, D. persimilis, D. alata and D. fordii resources. The result shows: The content of various compositions differs greatly among different resources. No.38(Baoding, Hebei) has the highest level of allantoin while No.24(Ninghua, Fujian) has the lowest level (0.1030%), with the former20.56times higher than the latter; No.27(Anyuan, Fujian) has the highest level of flavone (0.9102%) while No.42(Yanzhou, Shandong) has the lowest level (0.0508%), with the former17.92times higher than the latter; No.48(Wenzhou, Zhejiang) has the highest level of polysaccharides (9.1522%) while No.56(Yunxiao, Fujian) has the lowest level (0.7388%), with the former12.39higher than the latter; No.69(Shaowu, Fujian) has the highest level of adenosine (0.2163%) while No.33(Jiaozuo, Henan) has the lowest (0.0185%). In terms of the average level of the four components in all groups, the average level of allantoin in decreasing order is0.6277%in D. opposita resource,0.4969%in the national resource,0.4794%in D. persimilis,0.4590%in Fujian resource,0.4415%in D. alata and0.2777%in D. fordii. The average level of flavone in decreasing order is0.1789%in D. persimilis,0.1696%in D. alata,0.1666%in the national resource,0.1646%in D. fordii,0.1605%in Fujian resource, and0.1356%in D. opposita. The average level of polysaccharides in decreasing order is3.1913%in D. alata,2.3493%in Fujian resource,2.3279%in the national resource,2.2136%in D. persimilis,1.8075%in D. opposita and1.7387%in D. fordii. The average level of adenosine in decreasing order is0.1250%in D. persimilis,0.1096%in Fujian resource,0.1083%in the national resource,0.0949%in D. fordii,0.0946%in D. opposita and0.0850%in D. alata.6. Construction of core collection:Firstly, data of morphological traits, ISSR markers and SRAP markers were collected to create core sample banks of Rhizoma Dioscorea germplasm resources separately, and then the three categories of data were accumulated; The resources that can be identified by the above three categories of data are core collections. The resources that cannot be identified by any two categories of data and any individual category are regarded as backup resource pools and the resources that cannot be identified by all the three categories of data are regarded as redundant resources. Based on the above principle, we constructed core collections and backup resource pools, among which there are69core collections divided into4sample banks;20backup resource pools; and1redundant resource.