| Buckwheat belongs to the Fagopyrum Mill genus of Polygonaceae.China,the largest tartary buckwheat producer,is the center of origin and diversity in the world,containing plenty germplasm resources.Since the 1980 s,nearly 1,000 tartary buckwheat germplasm resources has been collected by the institute of crop science of Chinese Academy of Agricultural Sciences(CAAS) and more than 20 research institutes in China.In this thesis,973 tartary buckwheat materials and 17 Fagopyrum Mill genus plants were used as research materials,provided by the buckwheat gene resources research group of CAAS.In this study,18 main agronomic traits of tartary buckwheat germplasm resources of two populations including wild and cultivated tartary buckwheat were evaluated systematically.And the composition and variation of flavonoids content in tartary buckwheat seeds was observed and studied by HPLC-ESI-Q-TOF/MS.Finally,phylogenetic investigation of seventeen representative buckwheat species was conducted based on ITS sequence and matK gene,psb A-trnH sequence of chloroplast DNA,analysed genetic relationship of Fagopyrum Mill genus plants.The main study results were summarized as follows:1.The evaluation of agronomic traits of Tartary Buckwheat germplasm resources:(1)The sources of tartary buckwheat germplasm resources used in this research are abundant,including 33 abroad accessions and 940 Chinese accessions that are mainly from 270 counties(cities) in 18 provinces(municipality).The accessions are mostly scattered in a broad geographical scope,including E:(79°5528',123°4328'),N:(22°7391',43°2182'),which cover the main producing areas of tartary buckwheat.(2)Great phenotypic variations exist in two tartary buckwheat populations including wild and cultivated tartary buckwheat.Results showed that 973 accessions had abundant phenotypic differences in five main biological characteristics and indicators of seed testing,which provides a solid foundation for establishing core germplasm resources and molecular breeding of tartary buckwheat.2.The analysis of main flavonoids in Tartary Buckwheat germplasm resources:(1)In this study,a rapid and effective method for the extraction and determination of main flavonoids in tartary buckwheat seeds was established by HPLC and UV.The optimum extraction condition was: 70% methanol solution,extracting temperature 50?,ultrasonic time 25 min and ultrasonic frequency 50 k Hz.Meanwhile,methodological validation was carried out for the specificity,precision,stability,repeatability and recovery test.(2)Three main peaks of tartary buckwheat sample solution were detected by gradient elution using aqueous solution and methanol solution(containing 0.1% formic acid) as mobile phase(Fig 3.6a).The mass spectrometric decomposition of rutin(tR 9.1),quercetin(tR 13.5) and compound 3(tR 11.2) were studied by HPLC-ESI-Q-TOF-MS.Structural deduction showed that compound 3 was kaempferol-3-O-rutinoside.(3)Using the established method,the content and variation of main flavonoids in tartary buckwheat germplasm resources were analyzed systematically.The results showed that the average content for rutin of cultivation of tartary buckwheat was 19.96 mg/g,ranging from(14.67,31.00)mg/g;quercetin was 0.65 mg/g,ranging from(0.23,0.92)mg/g;kaempferol-3-O-rutin was 1.87 mg/g,ranging from(0.44,3.95)mg/g.Compared with wild tartary buckwheat,the rutin contents of cultivated had little differences,but quercetin had higher variability.The contents of kaempferol-3-O-rutinoside in tartary buckwheat were always lower than rutin,but higher than quercetin generally.And the contents of this compound differed non-significantly between two populations.3.The genetic relationship analysis of Fagopyrum Mill genus plants:(1)The sequences characters of the ITS,matK and psb A-trnH: There were much more variable sites in ITS and psb A-trnH than matK regions.The ITS sequence has the most Parsim-Info sites,and the matK sequence has the most conserved sites.(2)Based on estimates of evolutionary divergence and topology analysesysis by the ITS,psb A-trnH and matK sequences,we achieved the following results:(1)The special topological trend would be formed when the ITS sequence of nuclear genome and the chloroplast psb A-trnH and matK sequences were analysed respectively.When more gene sequences were analyzed together,the relatively accurate and stable strict consensus tree can be constructed to better analyze the genetic relationship of the Fagopyrum Mill genus plants.(2)The results showed that the pairwise distance of the F.esculentum,F.cymosum and F.tataricum were similar to other species,indicating that the genetic relationships among them were closer.Moreover,the F.cymosum and the F.tataricum were clustered into a small branch first,and then into a branch with the F.esculentum in all the topological graphs.And the pairwise distance between the F.cymosum and the F.tataricum was smaller than that of the F.esculentum in all estimates of evolutionary divergence.The above evidences indicated that although the F.cymosum was a cross-pollinating plant,it was still closer to the F.tataricum.(3)The F.jinshaense and the F.lineare were clustered into a small branch first,and then into a branch with the F.leptopodum in all strict consensus tree based on the chloroplast psb A-trnH and matK sequences.This result indicated that the chloroplast genomes of the three species were closely related,and the F.jinshaense had the closest relationship with the F.lineare.In addition,this study also found that the F.cymosum,F.tataricum and F.esculentum were clustered branch with the F.callianthum and F.gilesii gradually.However,only the results about the close relationship between the F.jinshaense and the F.lineare were reproduced when the ITS sequence of nuclear genome and the chloroplast psb A-trnH and matK sequences were analyzed together,while other conclusions were not shown.(4)All topology analyses supported that the F.pugense and the F.wenchuanense have high degree close and stable genetic relationship.According to the analysis of chloroplast genes,the F.pugense and the F.wenchuanense have the trend of close genetic relationship with the F.rubifolium and the F.macrocarpum,but this conclusion has not been shown when ITS sequence had been analysed together.(5)Finally,all topology analyses about combining data of ITS sequence and chloroplast gene supported that the F.callianthum,F.urophyllum,F.macrocarpum and F.capillatum were clustered into a one branch,with high approval rating.This evidence suggests that there are closed genetic relationship between the four species,compared with other species. |
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