二倍体和四倍体栽培苦荞的细胞学比较研究

观察和统计了二倍体和四倍体苦荞 (Fagopyrum tataricum)花粉母细胞减数分裂过程 TI、T 时期的异常程度及花粉粒败育性等多个指标 ,并比较分析了二倍体和四倍体苦荞在这些指标上的差异显著性。结果显示 :四倍体苦荞的异常 TI细胞频率、异常 T 细胞频率、TI细胞平均微核数、T 细胞平均微核数及花粉败育率均显著高于二倍体苦荞 ,这表明四倍体苦荞的遗传稳定性和花粉可育性都比二倍体苦荞差。     

Accumulation of Phenolic Compounds at the Initial Steps of Ontogenesis of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Plants That Differ in Their Ploidy Levels

Plants of buckwheat Fagopyrum esculentum possessing diploid and tetraploid genotypes were studied at the initial ontogenetic stages. They were compared in their morphophysiological characteristics, accumulation of phenolic compounds (including their main classes—phenylpropanoids and flavonoids), and activity of L-phenylalanine ammonia-lyase. An apparent resemblance in morphophysiological characteristics of seedlings was found between the two specimens, but diploid plants tended to faster linear growth than tetraploid ones. Differences in the accumulation of phenolic compounds in the hypocotyl and cotyledonous leaves were revealed. In most cases, in the course of seedling growth, the changes in phenylalanine ammonialyase activity did not correlate with the changes in the levels of these secondary metabolites. The effects of gene dosage were established towards accumulation of phenylpropanoids and anthocyanins in hypocotyls of seedlings and flavonoid accumulation in cotyledonous leaves. It is concluded that buckwheat seedlings with a tetraploid genotype have higher capacity than diploid seedlings for biosynthesis of phenolics.     

苦荞和甜荞查尔酮合成酶基因的克隆及序列比较

以苦荞品种‘西农9920’和甜荞品种‘西农9976’为材料,根据其它植物查尔酮合成酶(chalcone synthase,CHS)基因DNA序列的保守区域设计的一对简并引物,进行PCR扩增,从2种荞麦基因组中克隆出了长度均为860 bp的CHS基因片段,对其进行回收、克隆,挑选阳性克隆测序;序列分析表明这2个片段含有CHS基因的N端和C端的结构域,分别为苦荞和甜荞的CHS基因片段,命名为FtCHS和FeCHS。对获得的2种荞麦CHS基因的DNA序列进行比较分析,发现两者间存在多达43处单碱基多态性,这些单碱基多态性可能是苦荞和甜荞种子中类黄酮含量差异的重要原因之一。苦荞和甜荞CHS与其它植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的掌叶大黄和石竹科的满天星的同源性较近。     

苦荞全基因组11S种子储藏蛋白基因的鉴定及表达分析

以苦荞（Fagopyrum tataricum（L.）Gaertn）全基因组数据为平台,采用生物信息学方法,挖掘出9个11S种子储藏蛋白基因,并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明,苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa,等电点位于5.18~9.82之间,分子量为21.27~103.33 kD;定位分析结果显示,这些成员位于苦荞基因组的6条连锁群上（Megascaffold2/5以及scaffold77/344/395/861）;序列比对分析发现,除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外,其余8个都含有2个cupin结构域,并且在cupin保守结构域中,苦荞和拟南芥（Arabidopsis thaliana（L.）Heynh）共有14个保守的氨基酸残基;蛋白结构预测表明,苦荞11S种子储藏蛋白的结构具有2种类型;苦荞与其它6个物种[拟南芥、花生（Arachis hypogaea Linn.）、大豆（Glycine max（Linn.）Merr.）、杏仁（Armeniaca vulgaris Lam.）、胡桃（Juglans regia L.）和芝麻（Sesamum indicum Linn.）]11S种子储藏蛋白以及苦荞过敏蛋白（TBb和TBt）系统发育分析结果表明,这些蛋白可以分为3类,共具有4对旁系同源蛋白和3对直系同源蛋白;与已报道的苦荞过敏性储藏蛋白以及其它5个物种（花生、大豆、杏仁、胡桃和芝麻）的11S过敏蛋白比较发现,5个11S种子储藏蛋白（sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA）与苦荞2个过敏蛋白的同源性较高,同时它们与胡桃11S过敏蛋白的同源性最高,但尚需进一步实验来确定这5个成员是否为食物过敏原;RNA-Seq转录组数据显示,4个基因（sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA）在2种荞麦属植物的灌浆期种子中表达水平较高,且在‘大苦1号’中的表达水平要高于‘大甜1号’。     

自交可育甜荞、金苦荞、米苦荞不同品系总黄酮、粗蛋白及其蛋白组分含量研究北大核心CSCD

为探究荞麦新品种的营养保健价值,该文对自交可育甜荞、金苦荞、米苦荞共56个不同品系荞麦种子的粗蛋白、总黄酮、蛋白组分含量及其果实性状的变异进行了研究。结果表明:(1)甜荞、金苦荞、米苦荞种子中粗蛋白含量平均值分别为13.19%、15.44%、11.75%,总黄酮含量平均值分别为0.14%、2.50%、2.09%,清蛋白含量的平均值分别为5.22%、6.13%、4.56%,球蛋白含量的平均值分别为1.29%、1.15%、0.91%,醇溶蛋白含量的平均值分别为0.42%、0.58%、0.55%,谷蛋白含量的平均值分别为2.66%、3.36%、2.80%,三种荞麦的蛋白组分均符合清蛋白>谷蛋白>球蛋白>醇溶蛋白。(2)果实性状中,甜荞果实千粒重、果实面积、果实直径的变异系数最大,米苦荞果实周长、果实长宽比、果实长、果实宽和50 mL容重的变异系数最大。(3)相关分析表明甜荞种子粗蛋白含量与果实长宽比、果实长,金苦荞种子粗蛋白含量与果实周长、果实长,米苦荞种子粗蛋白含量与果实宽、总黄酮含量与果实面积、果实宽、果实直径、50 mL容重的相关性均达到了显著或极显著水平。(4)该研究筛选出甜荞(1808-166贵甜2号优系)、金苦荞(多苦74、多苦78)、米苦荞(1906-136黑米荞麦、43-2)等高蛋白含量或高黄酮含量的荞麦品系。该研究结果对荞麦优良品种的选育和荞麦新产品的开发具有一定指导意义。     

低海拔地区黑苦荞酚类物质含量、组成及抗氧化活性研究

为探究黑苦荞的市场利用价值,该研究选择种植于湖北江汉平原低海拔地区的川荞1号和九江苦荞作为材料,分析苦荞籽粒中游离酚、结合酚、总酚、游离黄酮、结合黄酮和总黄酮的含量,利用DPPH自由基法、ABTS自由基法和铁离子还原抗氧化法(FRAP)三种抗氧化测试模型综合评价其体外抗氧化活性,并运用高效液相色谱(HPLC)技术对其酚类物质的组成进行鉴定。结果表明:(1)川荞1号籽粒的总酚和总黄酮含量显著高于九江苦荞,分别为27.38 mg GAE·g^-1DW、31.46 mg RE·g^-1DW和12.71 mg GAE·g^-1DW、14.68 mg RE·g^-1DW;其中游离酚与游离黄酮含量显著高于结合酚与结合黄酮含量,均占总酚和总黄酮含量的79%以上,且九江苦荞中结合酚和结合黄酮的含量高于川荞1号。(2)苦荞籽粒中酚类物质主要由芦丁、槲皮素、表儿茶素、山奈酚、山奈酚-3-芸香糖苷和槲皮素-3-O-芸香糖苷-3’-O-吡喃葡萄糖苷等黄酮类化合物组成,其中游离酚以芦丁和槲皮素为主,结合酚以表儿茶素和芦丁为主。(3)...     

The Influence of Seed Size Variation on Seed Germination and Seedling Vigour in Diploid and Tetraploid Dactylis glomerata L.

In this study the effects of seed size variation on germinationand seedling vigour have been investigated within and betweenploidy levels of diploid and related autotetraploid Dactylixglomerata. Rates of seed germination and seedling growth werecompared in two contrasting environments using diploid and tetraploidseeds of equal and also different biomass. Within each ploidylevel, seed biomass had no effect on either the overall percentagenor the rate of germination. In contrast, the comparison ofseeds of equal biomass but differing in ploidy level showedthat seeds from tetraploid plants germinated faster and to ahigher percentage than those from diploid plants. With respectto seedling growth, heavier seeds from the tetraploid genotypesgave seedlings of significantly higher biomass than those fromlighter tetraploid and diploid seeds throughout the 2 monthsof study. Interestingly, seeds of equal biomass but from differentploidy levels produced seedlings more similar than those fromthe extreme seed weight categories. These differences were maintainedin two different environmental conditions. These results suggestthat there is a complex interdependance of seed size and ploidyon seed germination and seedling growth but is not a simpleconsequence of differences in seed size between diploids andtheir related tetraploids.Copyright 1995, 1999 Academic Press Dactylis glomerata, polyploidy, seed size, germination, seedling     

云南苦荞种质资源主要性状的遗传多样性分析

为了从云南苦荞种质资源中挖掘优异种质资源,拓宽苦荞遗传基础,以48份苦荞种质资源为材料研究了6个主要农艺性状和5个品质性状的遗传多样性。结果表明,云南的苦荞资源存在着丰富的遗传多样性,6个农艺性状中株粒重的变异系数为34.4%最大,品质性状中总黄酮含量的变异系数为51.72%最大。聚类结果表明,将48份材料聚为3大类,可区分为低产型、矮秆高产型和中秆高产型。6个主要农艺性状和5个品质性状的主成分分析结果表明,前3个累计贡献率分别达84.105%和80.332%,各主成分性状载荷值反映了主要数量性状的育种选择潜力。综合分析种质资源的主要农艺性状,可为云南苦荞种质资源的利用提供有效的科学依据。     

苦荞重组自交系群体籽粒黄酮含量与产量性状分析

为揭示影响苦荞籽粒黄酮含量的主要因素,获得高黄酮含量的苦荞种质,该文以"小米荞×晋荞2号"重组自交系(RILs)群体为材料,采用酶标仪检测籽粒黄酮含量,调查了百粒重、籽粒长宽比、株高、分枝数及籽粒产量等性状的变异,并探究籽粒黄酮含量与产量性状之间的相关关系,比较了不同粒色、不同粒型苦荞株系的籽粒黄酮含量的差异,基于聚类...     

染料结合法测定荞麦种子蛋白质含量的研究

以考马斯亮蓝G250为染料,结合经典的凯氏定氮法测定结果,对影响染料结合法测定蛋白质含量的振荡时间、温度、考马斯亮蓝溶液浓度等因素进行了研究,并分析了由考马斯亮蓝染料和蛋白质结合后染料结合量(OD值差)与凯氏法测得的蛋白质百分含量之间的相关性。结果表明:测定的适宜条件是:温度15℃,处理时间50min,考马斯亮蓝溶液的浓度是0.06mg/mL。此条件形成的络合物较稳定,重复性较好,并且所测的染料结合量与凯氏定氮法测得的蛋白质含量间呈极显著的一元线性回归和相关关系。栽培甜荞和栽培苦荞的回归方程分别为:y=15.364x+3.865和y=10.769x+6.287,这两个回归方程差异显著,不能合并,分别适合于快速估计甜荞和苦荞种子的蛋白质含量。     

Ecological factors influencing tetraploid speciation in snow buttercups (Ranunculus Adoneus): niche differentiation and tetraploid establishment

Chromosome doubling plays an important role in generating new species of flowering plants. However, reproductive incompatibilities between newly formed tetraploid plants and their diploid progenitors are expected to create a significant barrier to the persistence and establishment of neopolyploid populations. Ecological differentiation can reduce this barrier via prezygotic isolation arising from spatial separation. Alternatively, superior viability or fecundity of neotetraploid plants might compensate for the reproductive cost of incompatible pollen from diploid neighbors. The performance of plants of both cytotypes can be assessed in their respective habitats through reciprocal transplants, although such experiments have not been used previously in the study of tetraploid speciation. We used a series of seed and seedling transplant experiments to assess ecological differentiation and competitive ability during early establishment phases for tetraploid and diploid forms of the snow buttercup (Ranunculus adoneus). At two sites, seeds from diploids and tetraploids had similar germination probabilities. Tetraploid snow buttercup seedlings had a significant growth advantage in a controlled environment chamber experiment. However, in the field diploid and tetraploid buttercup seedlings did not differ consistently in survival or growth, nor did the two cytotypes show reciprocal advantages in performance, as expected if ecological differentiation has occurred. At the seed and seedling stages, neither niche differentiation nor tetraploid competitive superiority appears sufficient to explain neotetraploid success in the presence of their diploid progenitors.     

播种季节和种植方式对多年生苦荞主要农艺性状的影响

该研究选取六个多年生苦荞新品系,对春季、秋季直播与秋季再生其主要农艺性状进行调查。结果表明:(1)不同播种季节对多年生苦荞新品系主花序的花粉可育率、总结实率、有效结实率、植株株高、主茎粗、主茎分枝数、主茎节数、籽粒百粒重、单株粒数、单株产量的影响均达到显著或极显著水平;秋播主花序花粉可育率、总结实率、有效结实率、植株主茎分枝数、籽粒百粒重、单株粒数、单株产量均极显著高于春播;植株株高、主茎粗、主茎节数均极显著低于春播;主花序花朵大小、籽粒种子长宽比无显著差异。(2)不同种植方式对主花序花粉可育率、有效结实率、植株主茎节数及籽粒百粒重的影响达到显著或极显著水平;秋季再生主花序花粉可育率、籽粒单株粒数显著高于秋季直播;主花序有效结实率、植株主茎粗、主茎节数、籽粒百粒重显著低于秋季直播;主花序花朵大小、总结实率、植株株高、主茎分枝数、籽粒种子长宽比、单株产量无显著差异;相关分析表明,各生长季节下主花序有效结实率及单株粒数与单株产量的相关系数均最高。(3)所有参试品系中,1612-241秋季直播的单株产量显著高于其他品系; 1612-16、1612-33秋季再生单株产量较正季优势显著。该研究结果有助于筛选出适宜一季播种两季收获的优良品系,为今后多年生苦荞的选择育种提供线索基础。     

一种苦荞麦种子蛋白酶抑制剂的纯化、特性及其抗虫活性

蛋白酶抑制剂广泛存在于生物体内, 是自然界含量最为丰富且具有一定防御作用的蛋白种类之一. 本文采用离子交换层析和凝胶层析等方法,从苦荞麦种子中分离出一种胰蛋白酶抑制剂（TBTI-Ⅱ）. SDS-PAGE分析表明,TBTI Ⅱ的分子量约9.0 kD,由80个氨基酸残基组成,分子中含有较多的 Glu, Asp 和Arg. TBTI-Ⅱ具有较高热稳定性.当在100℃加热处理10 min后,仍保留有67.6%的抑制剂活性. 动力学测定显示,来自苦荞麦中的TBTI-Ⅱ对胰蛋白酶的抑制作用常数(Ki)为1.01×10－4 mol/L. 另外,将含有不同活力单位的苦荞麦蛋白酶抑制剂掺入到棉铃虫的饲料中进行饲养试验显示,TBTI-Ⅱ具有明显的抑制棉铃虫生长的作用. 这些结果表明,来自苦荞麦种子中的小分子蛋白酶抑制剂可能是一种潜在的抗虫因子.     

苦荞地方资源籽实主要性状的遗传变异研究

通过对我国苦荞主产区171份苦荞地方资源籽实的千粒重、百粒米重、百粒皮壳重、皮壳率、籽粒及皮壳黄酮含量测定,运用频数分布、相关性和聚类分析,考察它们的遗传变异情况。结果表明,千粒重、百粒米重、百粒皮壳重、皮壳率、籽粒及皮壳黄酮含量变异范围较大,分别为21.73～42.13 g、0.974～2.606 g、0.468～1.363 g、17.594～38.017 %、1.008～4.368 %和0.002～0.986 %,均值分别为29.43 g、1.988 g、0.729 g、24.702 %、2.019 %和0.144 %。相关分析表明,苦荞千粒重分别与百粒米重（r12 = 0.579**,r12.其它 = 0.315**）、百粒皮壳重（r13 = 0.746**,r13.其它 = 0.989**）、皮壳率（r14 = 0.216**,r14.其它 = -0.980**）之间,百粒皮壳重与皮壳率（r34= 0.807**,r34.其它 = 0.993**）之间,分别呈极显著相关;百粒米重分别与百粒皮壳重（r23 = 0.521**）、皮壳率（r24= 0.247**）之间,皮壳率与皮壳黄酮含量（r46= 0.187*）之间,分别呈显著相关。聚类分析表明,171份苦荞地方资源聚为5类,6个性状聚为3类。筛选出了千粒重、籽粒和皮壳黄酮含量较高的苦荞地方资源。以上结果可作为苦荞遗传研究与育种亲本选配提供参考。     

UV-B radiation affects flavonoids and fungal colonisation in <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> and <Emphasis Type="Italic">F. tataricum</Emphasis>

In the present study, we have evaluated the effects of increased UV-B radiation that simulates 17% ozone depletion, on fungal colonisation and concentrations of rutin, catechin and quercetin in common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum). Induced root growth and reduced shoot:root ratios were seen in both of these buckwheat species after enhanced UV-B radiation. There was specific induction of shoot quercetin concentrations in UV-B-treated common buckwheat, whereas there were no specific responses for flavonoid metabolism in tartary buckwheat. Root colonisation with arbuscular mycorrhizal fungi significantly reduced catechin concentrations in common buckwheat roots, and induced rutin concentrations in tartary buckwheat, but did not affect shoot concentrations of the measured phenolics. Specific UV-B-related reductions in the density of microsclerotia were observed in tartary buckwheat, indicating a mechanism that potentially affects fungus-plant interactions. The data support the hypothesis that responses to enhanced UV-B radiation can be influenced by the plant pre-adaptation properties and related changes in flavonoid metabolism.     

Purification and characterization of a 24 kDa protein from tartary buckwheat seeds

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

Determination of Anomeric Configuration of Furanoside Derivatives by Carbon-13 NMR

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment

Background and Aims

Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.

Methods

Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of ‘Tardivo di Ciaculli’ mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype (‘Carrizo’ citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries.

Key Results

Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area.

Conclusions

Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.