Studies On Genetic Diversity Of Buckwheat Gelasms With Norganic Elements, Storage Prorage Protein And SSR Marker

The buckwheat belongs to the Fagopyrum of Polygonaceae. It is known as a kind of green food material and medicinal plant for its unique nutrition and medicinal value. China is the origin center of buckwheat, where have extremely abundant germplasm resources. It is reported that there are about 23 species of the Fagopyrum Mill. in the world. The reserch method of genetic diversity of plant germplasm resources is diversiform, such as morphology, isozyme and all kinds of molecular markers, etc. In this papers, we study on genetic diversity of buckwheat gelasms with norganic elements, storage protein and SSR marker.1. Nine metal elements (Fe, Mn, Zn, Cu, Ca, Mg, Mo, Cd and Se) were determined by flame and graphite furnace atomic absorption spectrometry, the principal component analysis and the hierarchical cluster analysis were first used to study the data of metal element in buckwheat. Four principal components which accounted for 73.64% of total variance were extracted from the original data. The first factor and the second factor accounted for 44.41% of the total variance, which means that copper, magnesium, molybdenum and cadmium were the characteristic elements in buckwheat. The results of cluster analysis showed that twenty-eight buckwheat samples could cluster ultimately into two groups, the eight Fagopyrum esculentum samples from different places were fully clustered together; the Fagopyrum tataricum samples, two Fagopyrum dibotrys samples and all wild small-acheme group samples were clustered as the second group. There were five Fagopyrum tataricum samples, however, were clustered together with the Fagopyrum esculentum sample, which could identify the genuineness of the Fagopyrum Mill to some extent. In general, the results of the principal component analysis and the cluster analysis based on the contents of metal elements in buckwheat might provide a certain reference to the genuineness and the classification of buckwheat species.2. The albumin and salt dissolve globulin sites of 19 buckwheat samples, which contain three species and from different habitats of China, were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). According to the electrophoresis of the storage Protein, the data of albumin, globulin, globulin and albumin were observed with clustering analysis, respectively. The results show that:(1) according to its migration rate, the albumin and globulin spectrum can be divided into three areas, the rapid electrophoresis area C (14.4-28KDa), the medium-speed electrophoresis area B (28-50KDa) and the thickly and dyeing deeply slow electrophoresis A area (50-94KDa). The Albumin was mainly consist of A area, and the globulin was mainly consist of A area and C area. (2) neither analysis with the data of albumin or globulin, the results all revealed that the origin of buckwheat is closely related to the genetic diversity and the interspecific genetic variation was richer than intraspecific. (3) the 19 materials of buckwheat can be divided into seven groups basing on the clustering analysis of the albumin data; Similarly, five groups can be gained with the globulin data, and four groups can be gained based on albumin and globulin data. Over all, the results, which comprehensive analysis with several protein components, were more intuitive and reliable than use the single component, the storage protein SDS-PAGE belt type can be used as one tool of genetic diversity research in buckwheat.3. Orthogonal design was used to optimize cpSSR-PCR amplification system of the genus Fagpyrum Mill in five factors at four levels, respectively. Experiment was also conducted with four universal cpSSR primers. The optimization of main influential factors of cpSSR reaction system in buckwheat was studied. The results showed that the optimum concentrations of Mg2+, Taq DNA polymerase, dNTPs, template DNA, and primers in 25μL reaction system were 1.5 mmol·L-1,0.3 U,1.5 mmol·L-1,2.0μl(about 20ng) and 1.5μmol·L-1, respectively. Moreover, it was successful in studying genetic relationship of Fagpyrum Mill.4. Chloroplast simple sequence repeat(cpSSR) and nuclear genome simple sequence repeat(nSSR) markers were used to reveal the buckwheat genera phylogenetic relationship and to clarify the genetic background of some unique buckwheat germplasms collected from Sichuan. Four pairs of cpSSR universal primers generated 8 bands with an average of 2 alleles perlocus; Fifteen pairs of nSSR primers produced 55 bands with the mean of 3.6 alleles per locus. According to cpSSR and nSSR analysis showed that:(1) wild small-acheme group samples and big-acheme group had a different cpDNA, respectively, the genetic relationship between the Fagopyrum dibotrys and small-acheme group and big-acheme group was very complex, the Fagopyrum dibotrys more like a hybrid and excessive specie from them. (2)the genetic variation among small-acheme group buckwheat varietiesis was low in cpDNA; and the intraspecific genetic variation of Fagopyrum dibotrys was also lower. However, the results by nDNA indicated that no matter intraspecific and interspecific, or small-acheme and big-acheme, the differences were significant. (3) Fagopyrum crispatofolium and Fagopyrum densovilloum no matter from the cpSSR, or nSSR data, showed that they had a relatively closer affinity; The genetic background of two subspecies of Fagopyrum gracilipes couldn't distinguish by cpSSR, but their genetic variation were clear by nSSR. Therefore, for some genetic background unclear materials, combining cpSSR and nSSR marker analysis was a effective kind of identification method.