Genetic diversity of four new species related to southwestern Sichuan buckwheats as revealed by karyotype,ISSR and allozyme characterization

The genetic diversity of four new species related to southwestern Sichuan buckwheats was examined using karyotypes, allozymes and intersimple sequence repeats (ISSR). Karyotype analysis showed that Fagopyrum wenchuanense, F. qiangcai and F. pugense are diploid (2n = 2x = 16) while F. crispatofolium is tetraploid (2n = 4x = 32). The karyotype evolutionary trend of buckwheat indicated that F. crispatofolium, F. gracilipes var. odontopterum and F. gracilipes seemed to be more advanced than F. wenchuanense, F. qiangcai, F. pugense, F. esculentum, F. cymosum and F. tataricumare. Three polymorphic enzyme systems were used for the examination of variation, including peroxidase, esterase and superoxide dismutase. Consistent banding patterns were obtained for esterase and superoxide dismutase, while peroxidase produced a large number of sharp bands. A total of 18 ISSR primers were selected for the analysis and showed high variations among the species. Allozymes and ISSR markers were utilized to estimate the genetic distance among accessions and to draw phylogenetic trees. Our data provide evidence of a high degree of genetic diversity among southwestern Sichuan buckwheats. In addition, both cultivars and wild types showed a high degree of divergence suggesting a complex domestication process in this crop. This study provides a better understanding of evolutionary mechanisms and genetic relationships in four new species of buckwheat.     

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.     

中国荞麦属（蓼科）一新种——密毛野荞麦

描述了蓼科一新种——密毛野荞麦（Fagopyrum densovillosum J. L. Liu）,本种与细柄野荞麦（F. gracilipes(Hemsl.) Damm. et Diels）相近似,不同在于植株全体密被白色直立长毛,茎枝较粗壮,节较密集,节间较短, 叶较大,长（0.9～）1.7～6 cm,宽（0.7～）1.2～5.1 cm,阔卵形,心形,阔心形,阔卵状心形,卵形,长卵形,三角状卵形或卵状三角形,在上面具细皱纹,明显小泡状突起,叶柄较长,长 (0.5～) 2.6～7.5 cm,果实较小,长1.8～2.5 mm,直径1.5～2 mm,易于区别。     

Species relationships in Fagopyrum revealed by PCR-based DNA fingerprinting

Random amplified polymorphic DNA (RAPD) markers were used to distinguish between 28 different accessions belonging to 14 species and two sub-species of Fagopyrum. Of the 75 random 10-mer primers tested, only 19 generated robust, easily interpretable amplification products. A total of 364 bands were observed with an average of 19.15 bands per primer, of which 99.45% were polymorphic. Primer OPN-08 produced the maximum number of fragments and UBC-183 produced the minimum number of fragments. The data were utilized to elucidate genetic relationships among 14 species and two sub-species of Fagopyrum. Cluster analysis using the unweighted paired group method of arithmetic means (UPGMA) showed four main clusters, two each of the cymosum and urophyllum groups. The results showed that Fagopyrum tataricum is closer to its wild ancestor F. tataricum ssp. potanini Batalin, closely followed by Fagopyrum giganteum. Cultivated common buckwheat ( Fagopyrum esculentum) showed affinity with its putative wild ancestor F. esculentum ssp. ancestrale and the other closely related diploid species Fagopyrum homotropicum. In the urophyllum group, Fagopyrum macrocarpum and Fagopyrum pleioramosum formed one cluster, whereas Fagopyrum capillatum, Fagopyrum gracilipes and Fagopyrum gilessii clustered separately. Except for a few cases, our results correspond with previously reported studies on Fagopyrum using the isozyme, RFLP and RAPD methods. Species-diagnostic amplification products specific to some species in the cymosum and urophyllum groups were identified. Our results show that RAPDs can be successfully used to analyze species relationships in Fagopyrum and also for constructing linkage maps.     

Variation in seed protein subunits among species of the genus Fagopyrum Mill

Seed protein subunits of 75 accessions belonging to ten species of buckwheat (seven species of the big-achene group and three of the small-achene group) were studied by means of SDS-PAGE. The subunits varied greatly both within a species and among different species. The seven buckwheat species of the big-achene group have 42 different subunits whereas those of the small-achene group have only 16. Each buckwheat species has at least a few unique subunits, which could be used for species identification in the genus Fagopyrum. In the small-achene group, F. gracillipes and F. pleioramosum are closely related. Based on the number, distribution, and cluster analysis of the seed protein subunits, common buckwheat, wild common buckwheat, F. esculentum var. homotropicum, F. zuogongense, and F. megaspartanium are close to one another and tartary buckwheat, wild tartary buckwheat, F. plius, F. cymosum, and F. giganteum are also close to each other, supporting the hypothesis that F. megaspartanium and F. pilus are ancestral species of common buckwheat and tartary buckwheat, respectively.     

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。     

野生荞麦细柄野荞麦的繁殖生物学特性研究

该研究通过野外观察和人工控制实验相结合的方法,从开花动态、花部基本特征、繁育系统、传粉生物学及种子性状等方面对荞麦属(Fagopyrum Mill.)植物细柄野荞麦(Fagopyrum gracilipes)的繁殖生物学特性进行了探究,并分析了各性状对其繁殖的贡献。结果表明:在贵州威宁,细柄野荞麦的花果期常为每年的6—10月,单花序和单花的花期分别为13～21 d和1～3 d。花较小,直径为(3.99±0.12) mm,花柱和花药高分别为1.30和1.65 mm,花直径与花被片长和花被片宽呈显著正相关,花柱高与花药高呈极显著正相关。细柄野荞麦花粉胚珠比为371±16.40,杂交指数为2,套袋实验显示其自交、异交亲和,表明其繁育系统为兼性自交,部分异交亲和。细柄野荞麦的访花昆虫较少,主要为膜翅目(Hymenoptera)、双翅目(Diptera)和鞘翅目(Coleoptera) 7个科的9种昆虫,食蚜蝇科(Syrphidae)昆虫是其主要传粉昆虫。细柄野荞麦果实存在有翅和无翅两种类型,有利于其适应不同的传播方式,种子较小,千粒重为(1.05±0.04) g,萌发率较低,播种后30 d的累积萌发率为(19.60±2.14)%,但萌发整齐,主要集中在前5 d。综上所述,细柄野荞麦灵活的繁育系统为其产生大量种子提供了保障,多样的果实传播方式和整齐的种子萌发特性为其占据更广阔的生境成为群落优势种创造了基础。     

Progress and prospects for interspecific hybridization in buckwheat and the genus Fagopyrum

Cultivated buckwheat, such as common (Fagopyrum esculentum Moench.) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat, is one of the most versatile crops for forage and food and has several benefits for human health. Interspecific hybridization between Fagopyrum species is of great importance to improvement of buckwheat. Hybridization would allow the transfer of agronomical beneficial characteristics from wild Fagopyrum species, including self-pollination and increased fertility, frost tolerance, and higher content of beneficial compounds. However, conventional breeding methods are only partially applicable because of the self-incompatibility and incompatibility barriers between different species. Present review summarizes the morphology of self-incompatibility, the genetic and cellular basis of incompatibility between different Fagopyrum species. In many interspecific crosses hybrid embryos are aborted after successful pollination due to post-zygotic incompatibility. The use of in vitro embryo rescue after interspecific hybridization has been successful in circumventing breeding barriers between Fagopyrum species. Methods applied successfully for the construction of interspecific hybrids are discussed in detail.     

荞麦属2个野生荞麦种的染色体核型研究

对荞麦属皱叶野荞麦(Fagopyrum crispatifolium J.L.Liu)及其近缘种细柄野荞麦[F.gracilipes (Hemsl.) Dam.et Diels]的染色体核型进行了分析.结果表明,2个野生荞麦的染色体数目均为2n=4x=32,但二者在随体数目、染色体长度等方面差异明显,染色体核型不同,皱叶野荞麦的核型公式为:30m(4SAT)+2sm,细柄野荞麦的核型公式为:32m,属首次报道.     

荞麦属植物淀粉酶和甲酸脱氢酶同功酶研究(英文)

以聚丙烯酰胺凝胶电泳方法研究了荞麦属植物8个种42个收集系干种子和发芽种子的淀粉酶和甲酸脱氢酶同功酶。结果表明,荞麦淀粉酶在于种子中缺乏活性,但是在发芽种子中活性很强。在供试材料的发芽种子中共发现23个淀粉酶谱带,其中甜荞和苦荞分别有10条和8条。不同荞麦种间淀粉酶谱带差异很大,但是同种内不同收集系间差异较小。谱带聚类分析表明大野荞和毛野荞分别与甜荞和苦荞较近缘,支持它们分别为甜荞和苦荞祖先种的假说。在干种子和发芽种子中,发现所有荞麦种类均只有1条位置一致的甲酸脱氢酶谱带,暗示该酶在进化中具有高度稳定性。     

The lignin synthesis related genes and lodging resistance of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

Lignin is closely related to the lodging resistance of common buckwheat (Fagopyrum esculentum Moench.). However, the characteristics of lignin synthesis related genes have not yet been reported. We investigated the lignin biosynthesis gene expression, activities of related enzymes, and accumulation of lignin monomers during branching stage, bloom stage, and milky ripe stage by real-time quantitative PCR, UVspectrophotometry, and gas chromatography-mass spectrometry in the 2^nd internode of three common buckwheat cultivars with different lodging resistance. The results showed that lignin content and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were closely related to the lodging resistance of common buckwheat. Further, we studied gene expression of cinnamate 4-hydroxylase (C4H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), and caffeic acid O-methyltransferase (COMT). The lignin biosynthesis genes were divided into three classes according to their expression pattern: 1) expression firstly increasing and then descending (PAL, 4CL, CAD, C4H, CCoAOMT, F5H, and CCR), 2) expression remaining constant during maturation (C3H), and 3) expression decreasing with maturation (COMT). The present study provides preliminary insights into the expression of lignin biosynthesis genes in common buckwheat, laying a foundation for further understanding the lignin biosynthesis.     

Morphological and Physiological Aspects of Evolution in Common Buckwheat

Comparative morphological and physiological studies of the ancestral forms (Fagopyrum homotropicumO. and F. esculentumssp. ancestraleO.) and the various morphogenotypes of common buckwheat (F. esculentumMoench.) were carried out in glasshouse experiments in a soil culture. A considerable reduction of plant morphogenesis, a restriction of the growth of the vegetative organs, and the ecological and agricultural specialization (accompanied by the change in the primary adaptive life strategy from competitive (c) to ruderal (r) or to combined c/rtype) occurred in the evolution of common buckwheat. The reduction of morphogenesis was accompanied by changes in some of the morphological and anatomical structures and their functions, primarily, the source–sink relationships among plant organs. Compensatory mechanisms were developed, and the nutritional compounds became reutilized as an additional source for fruit formation and ripening.     

中国毛茛科翠雀属的分类学研究(六):拟川西翠雀花二新异名

通过标本室和野外观察, 发现根据四川丹巴标本描述的毛茛科光果拟螺距翠雀花(Delphinium bulleyanum Forrest ex Dielsvar. leiogynum W. T. Wang)和根据四川汶川标本描述的汶川翠雀花(D. wenchuanense W. T. Wang)与此前发现分布于四川宝兴、都江堰、汶川一带的拟川西翠雀花(D. pseudotongolense W. T. Wang)没有区别, 故将二者均处理为拟川西翠雀花的异名。     

Development of DNA barcodes for selected Acacia species by using rbcL and matK DNA markers

Acacia species are very important tree species in tropical and subtropical countries of the World for their economic and medicinal benefits. Precise identification of Acacia is very important to distinguish the invasive species from rare species however, it is difficult to differentiate Acacia species based on morphological charcters. In addition, precise identification is also important for wood charcterization in the forest industry as these species are declining due to illegal logging and deforestation. To overcome thsese limitations of morphological identification, DNA barcoding is being used as an efficient and quick approach for precise identification of tree species. In this study, we selected two chloroplast and plastid base DNA markers (rbcL and matK) for the identification of five selected tree species of Acacia (A. albida, A. ampliceps, A. catechu, A. coriacea and A. tortilis). The genomic DNA of the selected Acacia species was extracted, amplified through PCR using specific primers and subsequently sequenced through Sanger sequencing. In matK DNA marker the average AT nucleotide contents were higher (59.46%) and GC contents were lower (40.44%) as compared to the AT (55.40%) and GC content (44.54%) in rbcL marker. The means genetic distance K2P between the Acacia species was higher in matK (0.704%) as compared to rbcL (0.230%). All Acacia species could be identified based on unique SNPs profile. Based on SNP data profiles, DNA sequence based scannable QR codes were developed for accurate identification of Acacia species. The phylogenetic analysis based on both markers (rbcL and matK) showed that both A. coriacea and A. tortilis were closely related with each other and clustered in the same group while other two species A. albida and A. catechu were grouped together. The specie A. ampliceps remained ungrouped distantly, compared with other four species. These finding highlights the potential of DNA barcoding for efficient and reproducible identification of Acacia species.     

Limited honeybee hive placement balances the trade-off between biodiversity conservation and crop yield of buckwheat cultivation

An increasing number of farmland initiatives aim to aid biodiversity conservation through alternative farming practices such as nature-inclusive farming. However, these approaches frequently lead to trade-offs between biodiversity conservation and crop yield. For example, buckwheat (Fagopyrum esculentum) is a melliferous crop that flowers for a long period in the summer when nectar in agricultural areas is generally scarce, and buckwheat cultivation could therefore contribute to wild pollinator conservation. However, honeybees (Apis mellifera) are placed to ensure sufficient crop pollination, which potentially increases resource competition with wild pollinators in and around the crop. Here, we have studied this trade-off by surveying pollinators in and around 16 small-scale (∼1 ha) flowering buckwheat fields and we determined the contribution of pollinator density to crop yield in a nature-inclusive farming project. We found that the buckwheat pollinator community was diverse, albeit dominated by honeybees. We found no clear indications of resource competition between honeybees and wild pollinators within the buckwheat fields. Honeybee density in the surroundings was generally low, and increased minimally during honeybee-hive placement. While densities of honeybees decreased non-linearly over the day in buckwheat fields, they did not (temporarily) move into the surroundings of the field, suggesting limited competition for resources with wild pollinators. Crop yield was largely dependent on crop pollinator density, notably of honeybees, and to a lesser extent crop biomass (as a proxy for agricultural management). Our results show that buckwheat cultivation fits well within nature-inclusive farming if some simple precautionary measures are being taken, such as limiting the honeybee-hive densities and placing hives only during the main flowering period. The introduction of buckwheat cultivation into crop rotation could then contribute to fill an important nectar gap in the summer, which potentially boosts wild pollinator populations in the long term.     

Evaluation of genetic relationships in the genus Houttuynia Thunb. in China based on RAPD and ISSR markers

The genetic relationships among 70 accessions of Houttuynia Thunb. from Sichuan, Chongqing, Guizhou and Jiangsu provinces in China were tested using RAPD and ISSR markers. The results showed that the polymorphism of Houttuynia germplasm was high at the DNA level. ISSR markers are more efficient than RAPD markers at uncovering the polymorphism of the genus Houttuynia. The genetic variation between the cultivated and the wild Houttuynia cordata accessions was insignificant according to RAPD and ISSR markers. The results of cluster analysis by using UPGMA method showed that the groups based on ISSR GS was correlated with chromosome numbers and many accessions with the same chromosome numbers could be classified together. Analysis based on RAPD GS was more related to geographic distribution. Furthermore, the cluster analysis based on RAPD and ISSR markers also showed that the genetic diversity in mountainous and margin areas of Sichuan Basin was more plentiful than that at the bottom of the Basin and its surrounding highlands or hills. Houttuynia emeiensis accession could not be separated completely from H. cordata accessions, it was closely related to H. cordata cytotype A with the chromosome number of 36. Within H. cordata, the genetic similarities between each pair of cytotypes C, D, E, F, G, H, I, J, K and L were higher, but the genetic similarities between each of them to the cytotype A were relatively lower. The phylogeny of the germplasm resources of the genus Houttuynia was also discussed.     

A study of resources of Fagopyrum (Polygonaceae) native to China

Morphology, taxonomy, reproductive biology, esterase isozyme patterns and chromosome number were studied in 16 accessions of cultivated and wild buckwheat native to Tibet, Sichuan, Guizhou and Yunnan. Among these are four established species: Fagopyrum esculentum, F tataricum, F. pleioramosum and F gracilipes. The first three species are diploid (2 n = 16) and the last one is tetraploid (2 n = 32). In addition to the above, three new species are described: F. zuogongense, F. megaspartanium and E pilus. There are clear differences in esterase isozyme patterns between these species. The zymographs of esterase isozyme of diploid F. megaspartanium (native to Sichuan) and F. pilus (native to Tibet) are similar to those of F. esculentum and F tataricum respectively. F. gracilipes , in the small achene group of the genus, has a zymograph of esterase isozyme that is very different from the species in the large-achene group. The diploid accessions of F. pilus and F. megaspartanium may be ancestors of cultivated buckwheat.     

川西北荞麦种间亲缘关系初步研究

采用RAPD和同工酶技术对川西北具有代表性的10份荞麦材料进行分析.结果表明,筛选出的15个RAPD引物扩增出388条带.其中352条具有多态性,多态性比率为90.72%;过氧化物同工酶分析获得15条酶带,酯酶同工酶获得10条酶带.3种方法聚类结果基本一致,10份材料可初步聚为4个类群.其中,与金荞亲缘关系相比,甜荞较近而苦荞较远;普格县野荞和齿翅野荞、细柄野荞亲缘关系较近,划为同类;阿坝野荞单独划为一类,对其在分类中的地位有待进一步研究.     

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.     

Molecular characterization of endangered endemic plant Aloe pseudorubroviolacea using chloroplast matK and plastid rbcL gene

An endangered and rare species Aloe pseudorubroviolacea from the plant family Asphodelaceae which is presently recorded as endangered in Saudi Arabia collected from Al-Baha region of Saudi Arabia its GPS Latitude and Longitude coordinates 19.8345, 41.5481. The chloroplast matK and rbcL gene was considered in this study based on molecular identification the size is about 571 and 664 bp respectively. From the sequence analysis the gene matK and rbcL confirm that this species is very much closely related with A. rubroviolacea and also inter related with the species Astroloba rubriflora, Chrysopogon gryllus, Chortolirion angolense shows about 98.7% sequence homology. The partial matK and rbcL gene sequence discriminate Aloe pseudorubroviolacea from the closely related plant species, A. rubroviolacea. The gene sequence of rbcL discriminates the species from Chrysopogon gryllus and Chortolirion angolense, demonstrates the nucleotide variations in 3 different sites (623C/T; 653C/T; 700C/A). This study showed that matK and rbcL sequence region of chloroplast gene used to authenticate the samples of A. pseudorubroviolacea and which provide to help in correct identification and conservation process of this medicinally valuable endangered plant species.