【作者】 杨玉霞；

【导师】 吴卫； 郑有良；

【作者基本信息】 四川农业大学 ， 植物资源的保护与利用， 2008， 博士

【摘要】 荞麦(Buckwheat)是蓼科(Polygonaceae)荞麦属(Fagopyrum Mill.)作物,广泛分布于亚洲和欧洲,仅甜荞(Fagopyrum esculentum)和苦荞(Fagopyrum tartaricum)两个栽培种。荞麦营养成分全面,籽粒富含黄酮、类黄酮、固醇和硫胺类结合蛋白,对一些慢性疾病具有潜在的疗效。荞麦中80%的类黄酮物质是芦丁,芦丁能降低血压和血管的通透性,防止动脉硬化和毛细管脆性增加引起的出血症,并具有抗浮肿和抗氧化活性。与其它作物相比,荞麦具有较高的营养价值和独特的药用价值。本文对荞麦种质资源遗传多样性进行了研究,主要结果如下:1.基于叶绿体基因组中的matk、rbcL及trnL-F序列分别对国产荞麦属24份材料进行克隆和测序,并与已经发表的荞麦属植物相关序列进行了分子系统发育关系研究。结果表明,荞麦属中trnL-F的进化速率比matK快,而matK进化速率又比rbcL的进化速率快。荞麦属大粒组和小粒组组间遗传差异较大,小粒组内遗传多样性比大粒组内的高。用最大简约法和贝叶斯分析方法建构的系统树基本一致,由matK和trnL-F序列构建的树比rbcL构建的树有更好的拓扑结构。除个别材料外,荞麦属各材料形成大粒组和小粒组两个单系分支。苦荞、甜荞及细柄野荞(变)种内各居群间亲缘关系较近;金荞和硬枝野荞的遗传多样性较高,亲缘关系较远。苦荞与金荞的亲缘关系较甜荞近。基于matK和rbcL分析表明,花叶野荞和金荞、甜荞及其野生近缘种等大粒组材料亲缘关系较近;基于trnL-F分析表明,花叶野荞和疏穗野荞、硬枝野荞等小粒组材料亲缘关系较近,而和金荞、甜荞及其野生近缘种亲缘关系较远。2.对国产荞麦属24份材料核基因组内转录间隔区(ITS)进行克隆和测序,并与已经发表的荞麦属植物的相应序列进行系统发育关系研究。结果表明,所有材料的整个ITS区长度变化范围为578～660bp。其中5.8SrDNA为163～164bp,ITS1和ITS2长度变幅分别为195～265bp、216～228bp。序列间的遗传分化距离为0～0.128,平均为0.093;ITS1和ITS2的进化速率为5.8 S的3～9倍。荞麦属大粒组和小粒组组间遗传差异较大,小粒组内遗传多样性比大粒组内的高。用最大简约法和贝叶斯分析方法建构的系统树基本一致。除个别材料外,荞麦属各材料形成大粒组和小粒组两个单系分支。苦荞与金荞的亲缘关系较甜荞近。线叶野荞和长柄野荞、小野荞均在ITS1有68bp的缺失,但线叶野荞和硬枝野荞亲缘关系较近,和长柄野荞、小野荞的亲缘关系较远。F.pleioramosum和F.capillatum为细柄野荞的姊妹群。花叶野荞麦和甜荞及其野生近缘种的亲缘关系较近。3.利用酸性聚丙烯酰胺凝胶电泳(A-PAGE)和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术对76份栽培荞麦(苦荞54份,甜荞22份)的贮藏蛋白遗传多样性进行了评价。A-PAGE结果表明,栽培荞麦存在一定程度的醇溶蛋白等位变异,共分离出18条迁移率不同的醇溶蛋白谱带(多态性带占88.89%)。利用SDS-PAGE共检测到15条清蛋白条带,主要由分子量29～97.2 KDa的大分子寡聚蛋白组成。基于A-PAGE和SDS-PAGE得到材料间平均遗传相似系数(GS)分别为0.784(苦荞、甜荞分别为0.892、0.681)、0.732(苦荞、甜荞分别为0.864、0.633)。栽培荞麦种间存在较大遗传差异,甜荞贮藏蛋白遗传多样性比苦荞丰富。聚类分析可将供试材料分为3大类,相同地理来源的材料能够部分地聚成一类或亚类,表明贮藏蛋白所揭示的遗传关系与地理来源有一定相关性。4.对76份栽培荞麦(苦荞54份,甜荞22份)9个主要农艺性状及蛋白质含量进行了统计分析。结果表明,苦荞和甜荞主要农艺性状存在丰富的遗传变异。供试材料具有中、矮秆,主茎节数、花簇数、蛋白质含量适中,一级分枝数偏多、总分枝数偏少,千粒重、单株粒重偏低等特点。多元回归分析表明,主茎节数、一级分枝数、总分枝数、花簇数、千粒重是影响苦荞单株粒重的主要因素,而影响甜荞单株粒重的主要因素为株高、花簇数、生育期。通径分析表明,花簇数对苦荞、甜荞单株籽粒产量的直接效应最大。主成分分析将苦荞、甜荞9个主要农艺性状分别简化为5个和4个综合指标,其累积贡献率分别为87.77%和92.09%。基于性状表现,供试苦荞可分为低产晚熟型、多分枝高蛋白型和早熟粒重型等三种类型,供试甜荞可分为早熟矮秆粒重型和高产优质型等两种类型。聚类结果与其地理来源并不完全一致。据以上分析,筛选出一批综合性状较好或具有一个以上突出优良性状的材料,可供育种利用。5.采用原子吸收、原子荧光光谱法测定了59份栽培荞麦(苦荞38份,甜荞21份)种子中Ca、Fe、Mg、Mn、Zn、Cr、Cu、Pb、Cd、As及Hg等11种无机元素的含量,并根据各元素含量对供试材料进行聚类分析。结果表明,荞麦富含Ca、Mg、Cu、Cr等元素,Fe、Mn、Zn等含量也颇高。不同材料同一元素含量均达到(极)显著差异。苦荞中除Fe、Cr含量高于甜荞、Pb含量与之相当外,其他元素含量均低于甜荞。聚类分析将供试材料归为6类,聚类结果与材料地理来源并不完全一致。共筛选出PI 427235、PI 481653和PI 600909等9份Fe、Zn、Cr含量均很高的材料。6.研究荞麦初花期不同器官总黄酮含量的日变化趋势,探讨了药用荞麦最佳取样时间及取样部位。采用正交试验对荞麦总黄酮提取工艺条件进行筛选,得到了最佳提取条件:75%乙醇,物料比1∶25,65℃振荡提取2h。据此条件对4份来自不同地区的甜荞和苦荞不同器官总黄酮含量进行了日变化研究。结果表明,一天中同一品种不同器官黄酮含量均表现为花＞叶＞茎＞根,且花、叶中的含量显著大于茎和根。不同品种各器官中总黄酮含量的日变化差异较大。其中,花和叶中总黄酮含量以13:00和16:00时为最高。药用甜荞和苦荞宜在下午13:00时到19:00时采收花和叶为佳。更多还原

【Abstract】 Buckwheat belongs to Fagopyrum Mill.(Polygonaceae) and it distributs in Asia and Europe extensively.In general,only two species of buckwheat,namely common buckwheat(Fagopyrum esculentum) and tartary buckwheat(Fagopyrum eseulentum) are cultivated species.Many kinds of nutritional ingredients are rich in buckwheat.Flavones, flavonoids,sterols,fagopyrin,and thiamin-binding proteins in buckwheat seeds possess potential effects in treating some chronic diseases.In buckwheat,the main component of flavonoids(80%) is rutin.Rutin can reduce high blood pressure,decrease the permeability of the blood vessels,reduce the risk of arteriosclerosis,antagonize the increase of capillary fragility associated with haemorrhagic disease,and have an antioedema effect and antioxidant activity.Genetic diversity of buckwheat germplasms were studied based on biochemistry and morphological markers The main results were described as following:1.Three segments of chloroplast DNA(cpDNA),namely matK,rbcL and trnL-F spacer were determined in 24 taxon of Fagopyrum were cloned and sequenced,some related published sequences of Fagopyrum were analyzed in this paper.Molecular phylogenetic relationships of the related taxon of Fagopyrum were studied.The result indicated that evolutionary rate of trnL-F spacer was faster than that of matK,and evolutionary rate of matK was faster than that of rbcL in Fagopyrum,the genetic diversity between the cymosurn group and the urophyllum group was larger,and within the urophyllum group was higher than that of within the cymosum group.The phylogenetic relationships were inferred using the maximum-parsimony and Bayesian analyses,and the two methods produced trees with largerly congruent topology.Better phylogenetic resolution was obtained from the matK and trnL-F dataset compared to the rbcL dataset. Except for very few materials,the cymosum group and the urophyllum group formed two monophyletic branches,respectively,matK,rbcL and trnL-F spacer sequences within the species of F.tataricum and F.tataricum ssp.potanini,F.esculentum and F.esculentum ssp. ancestralis,F.gracilipes and F.gracilipes var.odantopterum were homologous and there had close relationships within these species and their subspecies.The genetic diversity of F. cymosum and F.urophyllum was higher,and the relationships within two species were farther,relatively.F.eymosum and F.tataricum were much more closely related than that of F.cymosum and F.eseulentum.Based on matK and rbcL analysis showed that F. polychromofolium had close relationships with the cymosum group,such as F.cymosum,F. esculentum and their wild relatives,based on trnL-F analysis showed that F. polychromofolium had closed relationships with F.caudatum and F.urophyllum which belong to the urophyllum group,while had distant relationships with F.cymosum,F. esculentum and their wild relatives.2.The internal transcribed spacer(ITS) regions of the rDNA were determined in 24 taxones of Fagopyrum were cloned and sequenced;some related published sequences of genus Fagopyrum were analyzed in this paper.The result showed that the sequence of ITS ranged from 578 to 660 bp in length.The 5.8S subunit of Fagopyrum was found to consist of 163 or 164 bp nucleotides,while the length of ITS1 and ITS2 varied from 199 to 265bp and from 216 to 228 bp,respectively.The sequences of ITS(ITS1 and ITS2) were about 3～8 times more variable than those of the 5.8S subunit.The genetic distances varied from 0 to 0.128,with an average of 0.093.The genetic diversity between the cymosum group and the urophyllum group was larger,and within the urophyllum group was higher than that of within the cymosum group.The phylogenetic relationships were constructed using the maximum-parsimony and Bayesian analyses,and the two methods produced trees with largely congruent topology.Except for very few materials,the cymosum group and the urophyllum group formed two monophyletic branches,respectively.F.cymosum and F. tataricum were much more closely related than that of F.cymosum and F.esculentum. Although a 68 bp gap in ITS1 found in F.lineare,F.statice and F.leptopodum,yet a close relationship between F.lineare and F.urophyllum and a more farther relationship among F. lineare with F.statice and F.leptopodum.F.pleioramosum and F.capillatum were the sister species of F.gracilipes.Based on ITS analysis,it was showed that F.polychromofolium had close relationships with F.esculentum and F.esculentum ssp.ancestralis.3.The genetic diversity of the storage proteins of 76 cultivated buckwheat accessions (54 accessions of tartary buckwheat and 22 accessions of common buckwheat) was characterized by A-PAGE and SDS-PAGE,respectively.A total of 18 prolamin bands were detected by A-PAGE,among which 88.89%bands were polymorphic,indicating that some prolamin genetic variation was observed in cultivated buckwheat.15 albumn bands based on SDS-PAGE were observed in accessions.Most intense bands were in the range of molecular weights from 29 to 97.2 kDa.The average of genetic similarity coefficient based on prolamin bands was 0.784(in F.tataricum and F.esculentum were 0.892 and 0.681, respectively),while on prolamin and albumn bands was 0.742(in F.tataricum and F. esculentum were 0.864 and 0.633,respectively).Accessions of F.tataricum and F. esculentum showed significant interspecific variation in the A-PAGE and SDS-PAGE profile of the storage proteins.The cluster analysis indicated that all the accessions could be divided into 3 groups.The genetic variations among cultivated buckwheat accessions were associated with their geographic origins in some degree.4.Nine mainly agronomic traits and grain protein content of 76 cultivated buckwheat accessions(54 accessions of tartary buckwheat and 22 accessions of common buckwheat) collected from seven countries,were investigated based on analysis of variance,correlation, principal component and cluster.Higher variations of the nine agronomic traits were observed.It has shown that these materials had mid-low stalk,moderate number of main stem nodes,number of inflorescences and grain protein content,more number of primary branches,shorter total number of branches,lower 1000-seed yield and seed yield per plant. Multiple regression analysis showed that node number of branch,number of primary branches,total number of branches,number of inflorescences and 1000-seed yield were the main traits affected seed yield per plant in tartary buckwheat,while in common buckwheat were the plant height,number of inflorescences and period of duration.The direct effect of number of inflorescences was strongest both in tartary buckwheat and in common buckwheat.9 mainly agronomic characters were changed into 5 and 4 indexes through principal components analysis and the principal factors were found to contribute 87.77% and 92.09%to yield in tartary buckwheat and in common buckwheat,respectively.By clustering analysis,all tartary buckwheat accessions were divided into four groups,namely low-yield and late-maturing type,multi-branch and high-protein type,early-mature and high weight type,all common buckwheat accessions were clustered into two groups, namely early-mature-low stalk and high weight type and high-yield and high-protein content type.The genetic distances based on agronomic traits were not associated with their geographic distribution.According to these main characters,a batch of accessions with one or some elite agronomic performances were screened out.5.The contents of element such as Ca,Fe,Mg,Mn,Zn,Cr,Cu,Pb,Cd,As and Hg in the seeds of 59 accessions of cultivated buckwheat(38 accessions of tartary buckwheat,21 accessions of common buckwheat) were determined by the method of atomic absorption spectrometry(AAS) and atomic fluorescence spectrometry(AFS).Cluster analysis was made based on the inorganic element’s contents.The results showed that cultivated buckwheat was rich in the contents of Ca,Mg,Cu and Cr,and the contents of Fe,Mn and Zn were quite high.The same element’s average content of different materials had significant or extremely significant deviation.Except that the contents of Fe and Cr were higher than those of in common buckwheat,Pb’s content was approximately consistent to that of in common buckwheat,other element’s contents in tartary buckwheat were all lower than those of in common buckwheat.59 varieties were divided into 6 groups based on the element’s content,and the variation of cluster analysis was not much associated with its geographical distribution.Nine accessions(i.e.PI 427235,PI 481653and PI 600909,etc.) which had higher contents of Fe,Zn and Cr were selected out.6.The diurnal variation trend of the total flavonoids contents in different organs on early flowering of buckwheat were studied,and provided the best collecting time and organs.The orthogonal experimental design was used to screen the extraction process of total flavonoids in buckwheat.The optimal extracting conditions were 75%ethanol as solvent,raw material and solvent ratio 1:25,extracting temperature 65℃,extracting time 2h.The diurnal variation of total flavonoids content in different organs of 4 materials was determined in the optimum extracting conditions.The results showed that the total flavonoids contents in different organs of the same species with various harvesting time were as follows:the contents in flowers were the highest,then the contents in leaves,stems and roots,respectively.And the contents in flowers and leaves were significantly higher than that in stems and roots.The diurnal changes of the total flavonoids contents from different organs in the different varieties were also quite different.It had the highest contents at 13:00 pm and 16:00 pm for total flavonoids of the flowers and leaves of buckwheat.The optimal harvesting time of medicinal common buckwheat and tartary buckwheat were at 13:00 pm to 19:00 pm and the best organs were the flowers and leaves.更多还原