苦荞地方种质资源的遗传多样性分析

利用SSR分子标记技术对中国苦荞主产区陕西、云南、四川、西藏等地的82份苦荞地方种质的遗传多样性进行了分析,以揭示中国特有的作物种质--苦荞地方种质资源的遗传多样性,促进苦荞优良品种的选育.结果显示:(1)所用25个SSR引物中有13个引物在苦荞地方品种中具有多态性,且扩增条带的稳定性较好,共扩增出208条条带,其中多态性条带200条,占总数的96.2%;(2)聚类分析结果显示,82份苦荞材料的遗传相似系数(GS)分布于0.52～0.85之间,平均值为0.69,在GS值为0.722的水平上,82份材料被聚为10大类群.研究表明82份苦荞品种间遗传多样性明显,具有丰富的遗传基础.     

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。     

荞麦属种间13S球蛋白基因序列比较研究

荞麦13S球蛋白是荞麦种子中的一类主要贮藏蛋白。本研究选用荞麦属植物甜荞栽培种及其野生类型、苦荞栽培种及其野生类型、毛野荞、左贡野荞、细柄野荞和硬枝万年荞6个种共44份材料,进行PCR特异性扩增、测序得到荞麦13S球蛋白基因的保守片段序列。对序列进行差异分析,结果发现44份供试材料13S球蛋白基因片段的285个排列位点中不变位点为24个,多态性位点S为261个(含简约信息位点数198个和单型可变位点63个),序列总突变位点Eta为503个。野生甜荞种内13S球蛋白基因序列差异明显高于栽培甜荞,但野生苦荞种内13S球蛋白基因序列差异仅稍高于栽培苦荞。推测其一方面可能与繁殖方式有关,另一方面可能与荞麦驯化过程中通常只有少数野生型群体被驯化有关。系统聚类分析发现栽培甜荞与野甜荞亲缘关系近,与左贡野荞亲缘关系次之;栽培苦荞与野苦荞亲缘关系近,与毛野荞亲缘关系次之;细柄野荞和硬枝万年荞的13S蛋白基因片段序列差异较小,说明其亲缘关系较近。上述结果为荞麦属种间遗传多样性与进化关系研究提供了理论依据。     

荞麦及其野生种遗传多样性分析

荞麦起源于我国西南地区,该地区分布有大量荞麦地方品种和野生近缘种。本研究利用SSR分子标记,对从我国西南地区收集的81份荞麦及其野生资源进行遗传多样性分析。结果显示,利用19对SSR引物共检测出84个等位基因,每对SSR引物平均扩增出4.421个等位基因,19对SSR引物的平均Shannon's信息指数为0.985,平均PIC为0.478。81份荞麦及其野生资源的相似系数范围为0.500~1.000,分析发现大粒组荞麦种(甜荞及其近缘种、苦荞及小米荞、金荞)与小粒组硬枝万年荞亲缘关系比较近。通过聚类分析在遗传相似系数为0.732时,可将81份荞麦种质分为4个类群,第Ⅰ类由小粒组齿翅野荞、细柄野荞、小野荞麦、疏穗小野荞麦和硬枝万年荞组成;第Ⅱ类由甜荞和甜荞近缘种组成;第Ⅲ类都是由金荞组成;第Ⅳ类是由苦荞和小米荞组成。在遗传系数为0.920时,小粒组荞麦种可以明显区分出疏穗小野荞麦、硬枝万年荞、齿翅野荞及其细柄野荞。研究表明,19对SSR引物多态性较高,能较好反应荞麦及其野生种质的遗传多样性,本研究的结果为荞麦属种之间的亲缘关系分析和荞麦起源进化研究提供科学依据。     

甜荞品种内与品种间的遗传多样性研究

以9个甜荞品种为材料,1个苦荞品种为对照,每个品种随机取10粒种子发芽,提取基因组DNA,采用长随机引物和内含子切接点引物的PCR分子标记技术,对甜荞品种内和品种间的遗传多样性进行了分析.结果表明:(1)所使用的引物均能在 9份甜荞品种和1份苦荞品种中稳定扩增出条带,共获得了45条条带,其中多态性条带42条,占总数的93.3%;(2)甜荞同一品种内存在一定的遗传差异,但遗传距离较小,大部分不同甜荞品种间的遗传差异大于其品种内的遗传差异.因而,在甜荞品种鉴定和遗传多样性研究中,通过品种内适当混合取样可以较好地代表和反映甜荞品种特性.     

Original birthplace of cultivated common buckwheat inferred from genetic relationships among cultivated populations and natural populations of wild common buckwheat revealed by AFLP analysis

The genetic relationships among seven cultivated populations and eight natural populations of wild common buckwheat were analyzed using amplified fragment length polymorphism (AFLP). The genetic distance was estimated for each pair of the 15 populations based on the AFLP data, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on the genetic distance. All the cultivated populations were grouped in a cluster. The natural populations were grouped into two clusters composed of (1) the Sanjiang group (three populations from eastern Tibet and one population from Adong village of Yunnan province) and (2) two populations from Yunnan province and two populations from Sichuan province. The Sanjiang group is more closely related to cultivated populations. These results indicate that the direct ancestor of common buckwheat was natural populations of wild common buckwheat from the Sanjiang area.     

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。     

Buckwheat (Fagopyrum esculentum Moench) has high capacity to take up phosphorus (P) from a calcium (Ca)-bound Source

Two experiments were carried out in a growth chamber to investigate the phosphorus (P)-uptake efficiency of Fagopyrum esculentum Moench (buckwheat) and Triticum aestivum (spring wheat) from a Ca-bound form. The first experiment was based on a sand-culture system with either rock phosphate (RP) or CaHPO₄ (CaHP) as the P source and nitrate or ammonium nitrate as nitrogen source. A highly calcareous soil was used in the second experiment. Buckwheat was shown to be highly efficient in taking up Ca-bound P compared to spring wheat. When plants were supplied with nitrate, the total P uptake by buckwheat from RP was nearly 10-fold higher than that of spring wheat (20.1 compared with 2.1 mg P pot^–1). Changing nitrogen source from nitrate only to ammonium nitrate increased P uptake by spring wheat substantially, but not buckwheat. High P-uptake efficiency of buckwheat was also demonstrated using the field soil, but to a lesser extent, which may be related to the difference in Zn supply between sand culture and field soil. It is suggested that buckwheat may be included in intercropping or crop rotation systems to activate P sources in calcareous soils. The principal mechanism of P uptake efficiency of buckwheat may be its ability to acidify the rhizosphere; however, further study is needed to unravel the regulation of root excretion of H⁺ and its molecular basis in order to exploit buckwheat's genetic capability to utilise sparingly soluble P from soil.     

S-LOCUS EARLY FLOWERING 3 is exclusively present in the genomes of short-styled buckwheat plants that exhibit heteromorphic self-incompatibility

The different forms of flowers in a species have attracted the attention of many evolutionary biologists, including Charles Darwin. In Fagopyrum esculentum (common buckwheat), the occurrence of dimorphic flowers, namely short-styled and long-styled flowers, is associated with a type of self-incompatibility (SI) called heteromorphic SI. The floral morphology and intra-morph incompatibility are both determined by a single genetic locus named the S-locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at the S-locus. Despite recent progress in our understanding of the molecular basis of flower development and plant SI systems, the molecular mechanisms underlying heteromorphic SI remain unresolved. By examining differentially expressed genes from the styles of the two floral morphs, we identified a gene that is expressed only in short-styled plants. The novel gene identified was completely linked to the S-locus in a linkage analysis of 1,373 plants and had homology to EARLY FLOWERING 3. We named this gene S-LOCUS EARLY FLOWERING 3 (S-ELF3). In an ion-beam-induced mutant that harbored a deletion in the genomic region spanning S-ELF3, a phenotype shift from short-styled flowers to long-styled flowers was observed. Furthermore, S-ELF3 was present in the genome of short-styled plants and absent from that of long-styled plants both in world-wide landraces of buckwheat and in two distantly related Fagopyrum species that exhibit heteromorphic SI. Moreover, independent disruptions of S-ELF3 were detected in a recently emerged self-compatible Fagopyrum species and a self-compatible line of buckwheat. The nonessential role of S-ELF3 in the survival of individuals and the prolonged evolutionary presence only in the genomes of short-styled plants exhibiting heteromorphic SI suggests that S-ELF3 is a suitable candidate gene for the control of the short-styled phenotype of buckwheat plants.     

Search for the wild ancestor of buckwheat III. The wild ancestor of cultivated common buckwheat, and of tatary buckwheat

By surveying wild Fagopyrum species and their distribution in southern China and the Himalayan hills, I arrived at the conclusion that the newly discovered subspecies F. esculentum ssp. ancestralis Ohnishi is the wild ancestor of cultivated common buckwheat, while previously known wild tatary buckwheat,F. tataricum ssp. potanini Batalin is the wild ancestor of tatary buckwheat. Their original birthplace is revealed to be northwestern corner of Yunnan province for common buckwheat judging from the distribution of wild ancestor, and to be the northwest part of Sichuan province for tatary buckwheat judging from allozyme variability in wild tatary buckwheat. F. cymosum is not the ancestor of cultivated buckwheat; it is only distantly related to cultivated buckwheat, in morphology, isozymes and cpDNA. Several genetic, ecological and taxonomic categories which should be taken into consideration in examining the origin of buckwheat were discussed. Key Words: Fagopyrum esculentum ssp. ancestralis; Fagopyrum tataricum ssp. potanini; southern China theory of origin of buckwheat. Contribution from Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University. No. 78.     

Identification of a new IgE-binding epitope of peanut oleosin that cross-reacts with buckwheat

Peanut and buckwheat induce a severe allergic reaction, anaphylaxis, which is considered to be mediated by immunoglobulin E (IgE). We identified in this study a new IgE-binding epitope of the peanut allergen that cross-reacted with buckwheat. The phosphate-buffered saline-soluble fraction of buckwheat inhibited the binding between IgE and the peanut allergen. A cross-reactive peptide was isolated from the α-chymotrypsin hydrolysate of peanut. Based on the amino acid sequence and mass spectrometric analysis data, the peptide was identified as Ser-Asp-Gln-Thr-Arg-Thr-Gly-Tyr (SDQTRTGY); this sequence is identical to amino acids 2-9 in the N-terminal hydrophilic domain of oleosin 3 which is located on the surface of the lipid storage body. Synthetic SDQTRTGY was found to bind with IgE in the sera of all eight peanut-allergic patients tested. Since many foods of plant origin contain oleosin, the possibility of an anaphylactic cross-reaction in allergic patients should always be considered.     

Identification of a New IgE-Binding Epitope of Peanut Oleosin That Cross-Reacts with Buckwheat

Peanut and buckwheat induce a severe allergic reaction, anaphylaxis, which is considered to be mediated by immunoglobulin E (IgE). We identified in this study a new IgE-binding epitope of the peanut allergen that cross-reacted with buckwheat. The phosphate-buffered saline-soluble fraction of buckwheat inhibited the binding between IgE and the peanut allergen. A cross-reactive peptide was isolated from the α-chymotrypsin hydrolysate of peanut. Based on the amino acid sequence and mass spectrometric analysis data, the peptide was identified as Ser-Asp-Gln-Thr-Arg-Thr-Gly-Tyr (SDQTRTGY); this sequence is identical to amino acids 2–9 in the N-terminal hydrophilic domain of oleosin 3 which is located on the surface of the lipid storage body. Synthetic SDQTRTGY was found to bind with IgE in the sera of all eight peanut-allergic patients tested. Since many foods of plant origin contain oleosin, the possibility of an anaphylactic cross-reaction in allergic patients should always be considered.     

基于ITS和ndhF-rpl32序列的荞麦种间亲缘关系分析

荞麦起源于我国, 演化形成了丰富的物种和遗传多样性。为了有效研究和利用荞麦及其野生种资源, 以从四川、甘肃、贵州等地采集的荞麦属(Fagopyrum)10个种(含变种、亚种和复合体种)共71份材料为对象, 通过ITS和叶绿体ndhF-rpl32序列分析, 利用MEGA5.0构建系统进化树, 探讨了荞麦种内及种间亲缘关系。结果表明, 在ITS序列矩阵中, 序列长度为725 bp, 信息位点为150个, 占序列总长度的20.7%; 在ndhF-rpl32序列矩阵中, 序列长度为940 bp, 信息位点为158个, 占序列总长度的16.8%。由ITS序列和ndhF-rpl32序列构建的两个进化树都可以将71份材料分为大粒荞麦种组和小粒荞麦种组; 其中, 大粒荞麦种组包括栽培苦荞和米苦荞(F. tataricum)、金荞复合体(F. cymosum complex)、栽培甜荞(F. esculentum)和野生甜荞(F. esculentum ssp. ancestralis); 小粒荞麦种组包括齿翅野荞(F. gracilipes var. odontopterum)、疏穗小野荞(F. leptopodum var. grossii)、小野荞(F. leptopodum)、密毛野荞(F. densovillosum)、细柄野荞(F. gracilipes)和硬枝万年荞(F. urophyllum)。而ndhF-rpl32序列构建的系统发育树还能区分栽培甜荞和野生甜荞, 具有更好的聚类效果。另外, 与栽培甜荞相比, 金荞复合体与栽培苦荞的亲缘关系更近。该研究为荞麦属种的分类和条形码研究提供了一定的科学依据。     

Expression and epitope analysis of the major allergenic protein Fag e 1 from buckwheat

Seeds of common buckwheat (Fagopyrum esculentum) contain valuable nutritive substances but also allergenic proteins that cause hypersensitive reactions. Thus, the development of hypoallergenic buckwheat would make this important pseudo-cereal available to allergic people. A major allergenic protein of buckwheat is Fag e 1. We isolated the respective cDNA, coding for a 22 kDa protein, from a recently developed autogamous strain of common buckwheat and confirmed its immunoglobulin E (IgE)-binding activity using recombinant Fag e 1 and sera of allergic patients. The derived amino acid sequence from Fag e 1 cDNA was used to synthesize an overlapping peptide library on nitrocellulose membranes for the determination of the Fag e 1 epitopes. We identified eight epitopes and the critical amino acids for IgE-binding within the epitopes. This epitope analysis of a major allergenic protein of buckwheat should help therapeutic efforts and aid in the development of hypoallergenic buckwheat.     

基于PAL基因序列的地方苦荞品种遗传多样性分析

苦荞在中国具有广泛的栽培种植地区,长时间演化形成了丰富的遗传多样性。为了研究和利用苦荞资源,以国内北方苦荞产区(内蒙古、青海、陕西、山西、甘肃)、西南苦荞产区(西藏、四川、贵州、云南)、国内其他地方品种(江西、安徽、湖北、湖南、广西)及国外品种(尼泊尔)共计67份苦荞材料为研究对象,PCR扩增其PAL基因并测序。在此基础上分析苦荞的遗传多样性,并采用NJ法(neighbor-joining)对67份苦荞材料构建系统进化树。结果表明,供试的67份苦荞材料的PAL基因序列长度为2011 bp,其中,变异位点为160个,占序列总长度的7.9%,简约信息位点为33个,占序列总长度的1.64%,突变的类型主要是碱基的转换与颠换,高变异位点均集中在外显子2的N端。不同来源的苦荞材料间的遗传距离分布于0.002~0.016之间,来源于中国四川组的苦荞材料种内平均遗传距离最大(0.016),中国内蒙古组的最小(0.002)。中国四川地区的材料与其他地区来源的材料间的遗传距离位于0.010~0.016之间,而其他地区间的遗传距离为0.004~0.013。67份苦荞材料的平均π值和θ值分别为0.0034和0.0143。其中,中国四川材料的π值为0.0148。基于PAL基因序列构建的NJ进化树中,67份苦荞材料分为7个类群,分类与地理来源无关。仅中国西藏来源的5份材料聚集为一类,说明PAL基因序列较为稳定,多数材料间变化差异较小。中国四川地区的苦荞材料具有丰富的遗传多样性,中国西藏地区的某一材料中有较多的SNP位点,推测中国西藏的部分材料可能存在突变的热点区,预示着PAL基因新的突变位点区域。     

Close genetic relationship between cultivated and natural populations of common buckwheat in the Sanjiang area is not due to recent gene flow between them-An analysis using microsatellite markers

Natural populations of wild common buckwheat have been found growing adjacent to cultivated populations of common buckwheat. Gene flow between the cultivated and natural populations would be expected in such cases. To evaluate the amount of gene flow, two sets composed of a cultivated buckwheat population and an adjacent natural population of wild common buckwheat were chosen, one from Yanjing village in the Sanjiang area, which is presumed to be the original birthplace of common buckwheat, and one from Jinhe village, Yanyuan district of Sichuan province in China. The genotypes of 45 individuals from each population were examined at eight microsatellite marker loci to estimate the magnitude of gene flow between the cultivated and wild common buckwheat populations. The Bayesian method with a Markov chain Monte Carlo approach estimated that the magnitude of gene flow between the populations in the Sanjiang area at 0.002-0.008 was not significantly different from that found in Yanyuan district at 0.002-0.008. The gene flow between cultivated populations was higher, usually at 0.002-0.044 (exceptionally high at 0.255 between cultivated populations of Yanjing and Jinhe), than that found between a cultivated population and a natural population (0.002-0.008) or between two natural populations (0.002-0.003). Therefore, the genetic similarity found between the cultivated populations and natural populations observed in the Sanjiang area (Konishi et al., 2005) was not due to recent gene flow between them. This in turn suggests that the close genetic relationship found in the Sanjiang area may be due to the common ancestry of the natural populations and cultivated common buckwheat.     

虫媒传粉植物荞麦的生物学特性与研究进展

荞麦是禾本科之外的谷物类作物, 具有较高的营养和药用价值。栽培荞麦有甜荞(Fagopyrum esculentum)和苦荞(F. tartaricum), 这两种一年生草本分别为自交不亲和的二型花柱、自交亲和的同型花柱植物; 前者结实依赖昆虫传粉。根据国内外调查研究, 前人对蓼科荞麦属(Fagopyrum)记录了30个物种名, 已有形态学和遗传多样性的调查表明该属的物种多样性中心位于我国西南地区, 特别是长江上游的三江并流区域; 甜荞和苦荞的起源地和祖先物种也认为在该区域。本文在论述前人研究的基础上, 指出对荞麦属的分类修订、野生种质资源的分布、种间关系的调查、优良品种的选育亟待研究。孢粉学和考古学的证据显示在我国长江流域, 人们在4,500年前就开始种植荞麦。荞麦可能曾经是山区人民的主粮, 为孕育长江流域文明提供了食物资源。加强对荞麦基础生物学特性的研究, 运用现代基因组学的方法有望澄清栽培荞麦的起源并探究产量不高的原因, 挖掘和利用其经济和药用价值的性状, 为荞麦成为一类优良的粮食作物提供参考依据。     

Genetic relationships and population structure of the endangered Steamboat buckwheat, Eriogonum ovalifolium var. williamsiae (Polygonaceae)

Eriogonum ovalifolium var. williamsiae (Steamboat buckwheat) is a narrow endemic subshrub, known from a single locality in Washoe County, Nevada. We examined genetic structure of the only known population by analyzing patterns of allozyme variation. Our results suggest that Steamboat buckwheat has high genetic variability, with levels of variation similar to that typical of a widespread species rather than a narrow endemic. Genotype frequencies suggest that mating is random. We detected no genetic subdivision of the population. Several clones spanning up to 67 cm were found, but we do not know if such clones are common. We used allozyme data to assess the genetic similarity of var. williamsiae to five other varieties of E. ovalifolium. All six varieties are very similar allozymically with var. williamsiae being the most similar to the widespread var. ovalifolium. Although var. williamsiae and var. ovalifolium are morphologically distinct, their genetic similarity warrants further study to determine whether or not they should be treated as separate taxa. Evidence of male sterility in var. williamsiae plus other data leads us to hypothesize that this taxon might be either a hybrid or undergoing cytoplasmic introgression. Information gathered from this study, in concert with ongoing work on the breeding system of Steamboat buckwheat, should be helpful in forming management strategies for this plant.     

The role of gene tepal-like bract (TLB) in the separation between the bracts and perianth in Fagopyrum esculentum moench

The morphological and genetic studies of the tlb mutant of common buckwheat Fagopyrum esculentum from the collection of the All-Russia Research Institute of Legumes and Groat Crops have demonstrated that gene TLB controls an important stage of flower development-it determines the lower boundary of simple perianths. The loss of TLB gene function leads to changes in the structure of the bract from scale-like to tepal-like. Gene TLB is assumed to limit, on the basal side, the region of the expression of genes determining the development of flower organs as petals.     

Genetic Diversity of Buckwheat Cultivars (<Emphasis Type="Italic">Fagopyrum tartaricum</Emphasis> Gaertn.) Assessed with SSR Markers Developed from Genome Survey Sequences

Tartary buckwheat is an important edible crop as well as medicinal plant in China. More and more research is being focused on this minor grain crop because of its medicinal functions, but there is a paucity of molecular markers for tartary buckwheat due to the lack of genomics. In this study, a genome survey was carried out in tartary buckwheat, from which SSR markers were developed for analysis of genetic diversity. The survey generated 21.9 Gb raw sequence reads which were assembled into 348.34 Mb genome sequences included 204,340 contigs. The genome size was estimated to be about 497 Mb based on K-mer analysis. In total, 24,505 SSR motifs were identified and characterised from this genomic survey sequence. Most of the SSR motifs were di-nucleotide (67.14 %) and tri-nucleotide (26.05 %) repeats. AT/AT repeat motifs were the most abundant, accounting for 78.60 % of di-nucleotide repeat motifs. SSR fingerprinting of 64 accessions yielded 49.71 effective allele loci from a total of 63 with the 23 polymorphic SSR primer combinations. Analyses of the population genetic structure using SSR data strongly suggested that the 64 accessions of tartary buckwheat clustered into two separate subgroups. One group was mainly distributed in Nepal, Bhutan and the Yunnan-Guizhou Plateau regions of China; the other group was mainly derived from the Loess Plateau regions, Hunan and Hubei of China and USA. The cluster analysis of these accession’s genetic similarity coefficient by UPMGA methods strongly supported the two subgroup interpretation. However accessions from Qinghai of China could be grouped into either of the two subgroups depending on which classification method was used. This region is at the intersection of the two geographical regions associated with the two subgroups. These results and information could be used to identify and utilize germplasm resources for improving tartary buckwheat breeding.