基于AFLP标记的中国西藏近缘野生大麦遗传多样性分析

选取7对引物组合, 构建了36份西藏近缘野生大麦和4份栽培大麦的AFLP指纹图谱, 共获得清晰可辨的条带227条, 其中多态性带194条, 占85.46％, 从DNA分子水平显示出所试材料遗传多样性较为丰富。计算得各样品间的遗传距离(欧氏距离)介于2.646~10.488之间, 应用离差平方和法对供试材料的AFLP数据结果进行聚类, 建立了40份大麦材料的AFLP树状图, 聚类结果将40份材料分为5类, 进一步揭示出供试材料间遗传背景的相似性和复杂性。结合Nei’s遗传一致性分析结果, 发现近缘六棱野生大麦较之近缘二棱野生与栽培大麦的亲缘关系更近, 支持栽培大麦是从野生二棱大麦起源, 而野生六棱大麦是进化过程中的过渡类型的大麦系统发生观点。     

野生大麦与栽培大麦醇溶蛋白遗传多样性比较

利用A-PAGE(acid-polyacrylamide gel electrophoresis)法对采自以色列的野生大麦的一个野生自然群体的15个系和来自世界不同国家的14份栽培大麦品种醇溶蛋白的遗传多样性进行了分析.结果表明:在所有的29份供试材料中,共发现52条相对迁移率不同的谱带.52条谱带的出现频率为3.44%～93.1%,多样性指数为0.066～0.368;以中国春醇溶蛋白为标准,ω区大麦醇溶蛋白的谱带数最多,其次是β区;野生大麦Shannon多样性指数依次为β区>ω区>α区>γ区,而栽培大麦Shannon多样性指数依次为ω区β>区>γ区>a区;野生大麦自然群体和栽培大麦品种间的遗传相似系数变幅相当,且聚类分析结果显示,野生大麦自然群体和来自全球不同区域栽培大麦品种间的醇溶蛋白遗传多样性同样丰富.以上结果说明,野生大麦中保存了较栽培大麦更为丰富的基因资源,今后栽培大麦的品质改良应该重视野生大麦资源的合理利用.     

我国大麦醇溶蛋白多肽的多态性研究

对22个省的大麦农家品种487份和3个近缘野生种的10份材料,应用SDS-PAGE、IEF及双向电泳(IEF、SDS-PAGE)进行了醇溶蛋白多肽多态性的研究,主要结果如下:(1)B醇溶蛋白多肽多态性十分显著。经SDS-PAGE可分出16种带型,归为α、β、γ、ω四组,不同的多肽带型中间也有一些多肽是共有的;(2)5种带型的地理分布表明,具有同种带型的品种分布于一定的地理区域;各区域品种所具有带型类型的数目虽然不同,但表现出一定的趋向,这种趋向似与大麦进化和生态区域有关;(3)近缘野生大麦的带型有的与农家品种带型相似,有的则相异;但一些农家品种的带型在供试近缘野生种中还未找到与之相近的带型;(4)初步讨论了醇溶蛋白多肽多态性在品种鉴定上的应用和作为栽培大麦生态区域划分辅助手段的可能性。     

野生垂穗鹅观草醇溶蛋白遗传多样性研究

利用酸性聚丙烯酰胺凝胶电泳(Acid-polyacrylamidegel electrophoresis)法对来源于内蒙古、山西、甘肃、新疆、云南、四川和西藏的25份垂穗鹅观草材料进行醇溶蛋白检测。研究结果表明,供试材料可分离出30条相对迁移率不同的谱带,每份材料可电泳出11～22条谱带,其中只有1条(3.45%)带纹是25份材料共有的,其余29条谱带均具有不同程度的多态性,多态性谱带数目占分离出总条带的96.67%,说明野生垂穗鹅观草具有较丰富的遗传多样性。聚类分析发现,材料的遗传相似系数(GS)变异范围为0.111～0.9286,平均值为0.4821。在GS值为0.4821的水平上供试材料可聚为6个类群,某些具有相同地理来源的材料聚成一类或亚类,即醇溶蛋白图谱类型与材料的生态地理环境具有相关性。     

野生垂穗披碱草种质的醇溶蛋白遗传多样性分析

用酸性聚丙烯酰胺凝胶电泳(A-PAGE) 对采集自中国新疆、青海、四川、西藏四省区的33份垂穗披碱草Elymus nutans材料进行醇溶蛋白分析,获得下述结果:（1）供试材料共分离出38条带纹,多态率达92.10%。4个电泳分区（α、β、γ、ω）的平均Shannon指数为0.55,Nei-Li遗传相似系数（GS）变异范围为0.36~0.93,平均值为0.63。这些结果说明供试野生垂穗披碱草材料具有较为丰富的醇溶蛋白遗传多样性。（2）对所有材料的聚类分析和主成分分析发现,在GS值为0.67的水平上供试材料可聚成7个类群,绝大部分来自于相同或相似生态地理环境的材料聚成一类,即醇溶蛋白图谱类型与材料的生态地理环境具有一定的相关性。（3）基于Shannon 多样性指数估算了5个垂穗披碱草地理类群内和类群间的遗传分化,发现类群内遗传变异占总变异的42.94 %,而类群间的遗传变异占总变异的57.06 %。这可能与该草以自花传粉为主的繁育系统有关。（4）对各地理类群基于Nei氏无偏估计的遗传一致度的聚类分析表明,各地理类群间的遗传分化与其所处的地理生态环境具有较高的相关性。 .     

偃麦草属植物醇溶蛋白和谷蛋白多态性及系统学研究

运用酸性聚丙烯酰胺凝胶电泳(A-PAGE)和十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对偃麦草属(Elytrigia)24个物种的醇溶蛋白和谷蛋白进行了研究。以中国春为参照,按醇溶蛋白和谷蛋白电泳图谱条带迁移距离大小和条带多态性对供试材料进行聚类分析。结果显示,24份供试材料均呈现不同的醇溶蛋白和谷蛋白电泳图谱,共分离出醇溶蛋白带纹83条和谷蛋白带纹53条,多态性均达100%,并且在相同染色体组组成的物种中,染色体数目越多,其蛋白带纹越多;偃麦草属24个物种的醇溶蛋白和谷蛋白图谱均有明显差异,蛋白图谱可作为鉴定偃麦草属物种的指纹图谱。聚类分析结果显示,在材料间的醇溶蛋白遗传相似性系数为0.66时,24份材料被划分为6个主要类群,含E和St基因组的物种具有较近的亲缘关系;在谷蛋白遗传相似性系数为0.62时,24份材料被聚为4大类。聚类结果表明,染色体组成未知的物种Et.pachynera可能为异源多倍体物种。     

应用微卫星标记研究西藏野生大麦的遗传多样性

以西藏不同地区的106份野生大麦为材料,其中包括50份野生二棱大麦（HS）,27份野生瓶形大麦（HL）和29份野生六棱大麦（HA）,用Liu等（1996）发表的SSR连锁图的每个连锁群的两个臂的不同位置上选取3～5个共30个SSR标记,研究了西藏3类野生大麦的遗传多样性。结果表明,这3类野生大麦在遗传组成及等位变异频率分布上存在着明显的遗传分化。在总样本中,共检测到229个等位变异,平均每个SSR位点检测到7．6个等位变异,其中70个为这3类野生大麦间共同的等位变异,等位变异数在这3类野生大麦间有明显的差异,亚种问的遗传多样性明显高于亚种内的遗传多样性。其遗传多样性大小顺序为HS〉HL〉HA。聚类分析表明,野生二棱大麦、野生六棱大麦分别聚在不同的两类,而野生瓶形大麦中各有约50％的材料分别聚在这两类。根据本研究及前人研究结果,我们认为中国栽培大麦是从野生二棱大麦经野生瓶形大麦向野生六棱大麦进化的。该结果支持了栽培大麦起源的“野生二棱大麦单系起源论”的观点。     

波斯小麦醇溶蛋白遗传多样性分析

应用APAGE技术对来源于16个国家(地区)96份波斯小麦材料进行醇溶蛋白遗传多样性分析,结果表明,波斯小麦具有丰富的醇溶蛋白等位变异,共分离出55条迁移率不同的带纹。每份材料可电泳出13～26条,平均18.6条,带纹多态性为100%。96份材料共出现了75种电泳图谱类型,其中31份材料共同拥有10种图谱,剩余的65份材料的电泳图谱各不相同。供试材料间GS值变异范围为0.176～1.000,平均值为0.538。聚类分析表明,在GS值0.538水平上供试材料可聚为五大类群,且遗传聚类关系与材料的地理来源有一定的相关性。     

利用RAPD标记分析大麦种质资源的遗传多样性

利用RAPD标记对19份西藏近缘野生大麦材料、33份我国不同省市的地方品种以及8份国外引进大麦品种共60份大麦种质资源的遗传多样性进行检测.结果表明材料间遗传差异明显.32个RAPD引物中,有25个引物(占78.13%)可扩增出清晰且具多态性的条带,另外7个引物能扩增出1～3条清晰但无多态性的条带.每个引物可扩增出1～8条多态性带,平均为3.72条.32个引物共产生119条DNA片段,其中87条具有多态性,多态性比率(PPB)为73.11%,平均多态信息量(PIC)为0.434;每个位点平均有效等位基因数(Ne)为2.304;材料间遗传相似系数GS变化范围为0.757～0.981,平均值为0.871.19份来源于西藏的近缘野生大麦材料间GS值变幅为0.818～0.969,平均为0.892;33份我国栽培大麦地方品种间的GS值变化范围为0.783～0.981,平均为0.879;8份分别来自8个国家的栽培大麦品种间的GS值变幅为0.820～0.956,平均为0.882.根据RAPD标记分析的结果,对60份大麦种质资源进行聚类分析,在平均GS值0.871水平上60份大麦材料可聚为5类,聚类结果能在一定程度上反应材料的地理分布关系,但某些相同地理来源的材料也较分散地分布在整个聚类树中.本研究从分子水平上进一步证明了我国栽培大麦丰富的遗传多样性,是世界栽培大麦的遗传多样性中心之一.     

赖草属植物醇溶蛋白的遗传多态性

用酸性聚丙烯酰胺凝胶电泳 (A PAGE)对小麦族赖草属 2 0种和 1亚种的 45份材料进行醇溶蛋白遗传分析 ,结果表明 :(1 ) 4 5份材料共出现 43种不同的醇溶蛋白图谱 ,从中分离出的 38条带纹多态性高达 1 0 0 % ;(2 )赖草属植物具有丰富的醇溶蛋白遗传多态性 ,其种间和种内不同居群间均存在明显的醇溶蛋白遗传变异 ,其种间变异大于种内变异 ,醇溶蛋白图谱可以作为鉴定赖草属植物的指纹图谱 ;(3)醇溶蛋白图谱的聚类结果与形态学结果基本一致 ,表明醇溶蛋白资料可以运用于赖草属植物种间、种内遗传差异及亲缘关系研究     

Hordein variation in the genus Hordeum as recognized by monoclonal antibodies.

The composition of the major storage protein, hordein, in wild barley species has been studied by using gel electrophoresis, Coomassie staining, and immunoblot assays. We have shown earlier that it is possible to obtain cross-reaction outside the cultivated barley, with monoclonal antibodies raised against hordeins from the barley cultivar Bomi. These antibodies have now been used to investigate the hordein composition in all species of the Hordeum genus. The results showed that polypeptides similar to the two major hordein groups of cultivated barley, the B- and C-hordeins, are produced in all wild Hordeum species, and that there are both similarities and differences between the two hordein groups. The similarities indicate a common evolutionary origin, while the distinction between B- and C-hordeins in the entire genus clearly shows that the divergence of their coding genes preceded the divergence of the Hordeum species. The presence of the same antigenic site in two different species indicates that they are evolutionarily related. Among the wild species, two rarely occurring sites were exclusively found in H. vulgare ssp. spontaneum and H. bulbosum, which confirms that they are the cultivated barley's closest relatives. Some of the antibodies also gave an extensive reaction pattern with H. murinum, which suggests a fairly close relationship to H. vulgare, though not as close as between H. vulgare and H. bulbosum.     

Analysis of genetic diversity of hordein in wild close relatives of barley from Tibet

We analyzed genetic diversity in the storage protein hordein encoded at Hor-1, Hor-2 and Hor-3 loci in seeds from 211 accessions of wild close relatives of barley, Hordeum vulgare ssp. agriocrithon and H. vulgare ssp. spontaneum. Altogether 32, 27 and 13 different phenotypes were found for Hor-1, Hor-2 and Hor-3, respectively. A comparison of our results with those of previous studies indicates that Tibetan samples reflect the highest diverse level of hordein phenotypes when compared to samples from Israel and Jordan. This high degree of polymorphism supports the hypothesis that Tibet is one of the original centers of H. vulgare L.Communicated by H.F. Linskens     

Elucidation of the origin of ‘agriocrithon’ based on domestication genes questions the hypothesis that Tibet is one of the centers of barley domestication

Wild barley forms a two‐rowed spike with a brittle rachis whereas domesticated barley has two‐ or six‐rowed spikes with a tough rachis. Like domesticated barley, ‘agriocrithon’ forms a six‐rowed spike; however, the spike is brittle as in wild barley, which makes the origin of agriocrithon obscure. Haplotype analysis of the Six‐rowed spike 1 (vrs1) and Non‐brittle rachis 1 (btr1) and 2 (btr2) genes was conducted to infer the origin of agriocrithon barley. Some agriocrithon barley accessions (eu‐agriocrithon) carried Btr1 and Btr2 haplotypes that are not found in any cultivars, implying that they are directly derived from wild barley through a mutation at the vrs1 locus. Other agriocrithon barley accessions (pseudo‐agriocrithon) carried Btr1 or Btr2 from cultivated barley, thus implying that they originated from hybridization between six‐rowed landraces carrying btr1Btr2 and Btr1btr2 genotypes followed by recombination to produce Btr1Btr2. All materials we collected from Tibet belong to pseudo‐agriocrithon and thus do not support the Tibetan Plateau as being a center of barley domestication. Tracing the evolutionary history of these allelic variants revealed that eu‐agriocrithon represents six‐rowed barley lineages that were selected by early farmers, once in south‐eastern Turkmenistan (vrs1.a1) and again in the eastern part of Uzbekistan (vrs1.a4).     

Genetic change in the waxy trait of barley under the influence of wild type DNA. Analysis of the composition of starch and the electrophoretic spectrum of caryopsis hordein from altered plants and the retention of these changes to the fourth generation

Injection of DNA isolated from the wild type of barley into grains of recipient mutant plants (waxy mutants) at the milk stage of maturity leads to a change in starch synthesis; type of spikes and hordein composition. In the first generation of injected plants the wild type starch synthesis was observed in some separate plants (these observations were made at a haploid level in pollen cells). In the second generation of transformed plants along with the change in starch and hordein synthesis a modification of the type of spikes was also revealed. Recipient plants had six-rowded (hexastichous) spikes, and donor plants--two rowded (distichous) spikes. Disc-electrophoresis of hordeins of the wild type barley (Yuzhny var.), hordeins of the waxy mutant (defected in synthesis of normal starch) and barley plants transformed under the action of wild type exogenous DNA reveals differences in the protein spectrum between donor, recipient and transformants. In the second generation in many of the transformed plants starch synthesis reverted to the recipient mutant type. Simultaneously a reversion of hordein composition to the initial mutant type was observed, and the distichous pikes became hexastichous. Analysis of the components of starch revealed that donor plant that have amilose and amilopectin in starch, and the recipient plants that lack amilose, can be distinguished by the spectra of light absorption of starch. For characterizing these differences the plot of absoprtions at 490 versus that at 590 nm was used. The tangens of angles of these curves for the waxy mutant were equal to 1.05 +/- 0.07 and 1.81 +/- 0.04 for the wild type barley. All transformants have a 1.78 ratio and for revertants this value was 1.02.     

The polymorphism and structural homology of storage polypeptides (hordein) coded by the Hor-2 locus in barley (Hordeum vulgare L.)

Two-dimensional mapping (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the polypeptide components of “B” hordein fractions from eight barley varieties of widely different ancestry has been carried out. The relative positions of 47 different polypeptides were mapped, there being between 8 and 16 present in any one variety. The individual polypeptides differed in their distribution patterns; some were present in a number of varieties, while others were restricted to one or two. They also differed in their relative contributions to the total hordein fraction, both within and between varieties. The structural homology of the major polypeptides was compared by cleavage at methionine residues with cyanogen bromide and separation of the peptides on gradient gels. The polypeptides were classified into three groups which gave cleavage patterns with either two (class I), four (class II), or five (class III) low molecular weight bands. Class III polypeptides were found in all eight varieties, but in seven of the varieties class I or class II polypeptides were also present. With one exception, polypeptides migrating in the same position in different varieties gave identical or almost identical patterns. The three classes of polypeptides showed different distributions on the two-dimensional gels. Classes II and III polypeptides had a similar range of isoelectric points (pH 6.5–8.0), but all of the class II polypeptides were of slightly lower molecular weight. Class I polypeptides had a wider range of pI and molecular weight; the most alkaline and the lowest molecular weight polypeptides were in this group. The hordein fractions from a number of other barley varieties were compared with that of Julia. All had major polypeptides which migrated with ones present in Julia, but they differed in the relative amounts of these and in the absence of some polypeptides and the presence of others. B hordein is coded for by a single locus which has been suggested to be a complex multigenic family derived by duplication and divergence of a single gene. The data reported here provide support for this hypothesis and suggest that both mutations in the duplicated genes and recombination within the locus may have contributed to the polymorphism of the polypeptides.