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Amplified fragment length polymorphism linkage analysis of common buckwheat (Fagopyrum esculentum) and its wild self-pollinated relative Fagopyrum homotropicum.

Common buckwheat (Fagopyrum esculentum) (2n = 2x = 16) and Fagopyrum homotropicum (2n = 2x = 16) were mated in an interspecific cross and amplified fragment length polymorphism (AFLP) linkage maps were constructed by analyzing segregation in the F2 population. Six hundred and sixty-nine bands were identified using 20 AFLP primer combinations, of which 462 (69%) segregated in the F2 population. The map of F. esculentum has eight linkage groups with 223 markers covering a total of 508.3 cM. The map of F. homotropicum has eight linkage groups with 211 markers covering 548.9 cM. There was one to one correspondence of the esculentum and homotropicum linkage groups. Three morphological markers, distylous self-incompatibility, shattering habit, and winged seed, were located on the AFLP map. Distylous self-incompatibility and shattering habit are tightly linked to each other (1.3 cM) and are located near the center of linkage group 1. Winged seed is located on linkage group 4.

Chromosome Mapping↗

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.

Journal Article↗

Two new Fagopyrum (Polygonaceae) species, F. gracilipedoides and F. jinshaense from Yunnan, China.

Two new Fagopyrum (Polygonaceae) species, F. gracilipedoides and F. jinshaense, are described. Fagopyrum gracilipedoides was only found at Baoshan village, Lijiang district, Yunnan province, China. This species resembles the weedy species, F. gracilipes in morphology but they differ in both mating system and ploidy level; F. gracilipedoides is a heterostylous self-incompatible diploid species (2n = 16), whereas F. gracilipes is a self-compatible tetraploid species (2n = 32). Fagopyrum jinshaense was found along the Jinsha River valley near the border of Sichuan, Yunnan, and Tibet in China. Fagopyrum jinshaense is morphologically similar to F. gilesii, but they differ in the morphology of their inflorescences. Fagopyrum jinshaense has long spike-like inflorescences, whereas F. gilesii has a compact head-like one. Both F. jinshaense and F. gilesii are self-incompatible with heterostylous flowers and are diploid. Interspecific crosses between the two new species and already known species in the urophyllum group of Fagopyrum resulted in the production of self- and cross-sterile hybrids in the species combinations attempted. These results indicated that both new species are reproductively isolated from other Fagopyrum species. Molecular phylogenetic analysis based on the nucleotide sequence of the trnK gene intron in chloroplast DNA revealed the following points: 1) F. gracilipedoides, F. rubifolium and F. gracilipes / F. capillatum showed a trifurcating relationship and 2) F. jinshaense formed a clade with F. pleioramosum, F. callanthum, and F. macrocarpum, and was only distantly related to F. gilesii. In the clade of F. capillatum, F. gracilipes, F. gracilipedoides and F. rubifolium, the breakdown of self-incompatibility accompanying genome duplication seems to have occurred twice independently.

China↗

Comparative study of rbcL gene sequences in Fagopyrum and related taxa.

Four species of Fagopyrum, two species of Persicaria and one species of Rumex were analyzed for the DNA sequences of their rbcL genes. In addition to these seven species, we analyzed the published or available sequence data of ten related taxa in Polygonales, Plumbaginales and Caryophyllales. By estimating the base substitution rate between each pair of species, and by constructing phylogenetic trees we concluded that: 1) In Fagopyrum, F. cymosum and F. tataricum are much more closely related than expected previously; this confirms the conclusion of Kishima et al. (1995). 2) The new classification of Fagopyrum species proposed by Ohnishi and Matsuoka (1996) based on morphology, isozyme and cpDNA variability is consistent with the rbcL sequences. 3) The Polygonales and the Plumbaginales should be separated from the Caryophyllales as concluded by Giannasi et al. (1992). 4) The separation of Fagopyrum from other genera in the Polygoneae has a solid molecular basis.

Base Sequence↗

Phylogenetic relationships among Fagopyrum species revealed by the nucleotide sequences of the ITS region of the nuclear rRNA gene.

The DNA sequences of the internal transcribed spacer (ITS) region of the rRNA gene were determined in 20 taxa of 12 species of Fagopyrum. By comparing the sequence data and constructing phylogenetic trees, the phylogenetic relationships among the Fagopyrum species were established. The sequences of ITS (ITS1 and ITS2) were about 3-7 times more variable than those of the 5.8S subunit of the rRNA gene. The classification of Fagopyrum species based on the DNA sequences of the rRNA gene almost coincides with the classification based on morphology, allozyme variability, and nucleotide sequences of the rbcL-accD region of the chloroplast DNA. A close relationship of F. lineare with the F. statice-F. leptopodum clade was suggested and this was strongly supported by a 68-bp gap in ITS1 found in these three species. A tetraploid F. cymosum from Kathi, India, has two distinct rRNA genes probably on homoeologous chromosomes, and was distinguished from the clade of other diploid/tetraploid F. cymosum with a high bootstrap value. This strengthened the hypothesis of the polyphyletic origin of the tetraploid F. cymosum.

Base Sequence↗

Expression, cloning, and immunological analysis of buckwheat (Fagopyrum esculentum Moench) seed storage proteins.

cDNA of buckwheat (Fagopyrum esculentum Moench) was isolated from immature seeds harvested 14 days after pollination. Two genes, designated FA02 and FA18, were found to encode legumin-like proteins and were expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which was believed to be a major buckwheat allergen (Urisu, A.; Kondo, Y.; Morita, Y.; Yagi, E.; Tsuruta, M.; Yasaki, T.; Yamada, K.; Kuzuya, H.; Suzuki, M.; Titani, K.; Kurosawa, K. Isolation and characterization of a major allergen in buckwheat seeds. In Current Advances in Buckwheat Research; Shinshu University Press: Matsumoto, Japan, 1995; pp 965--974). It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. Antiserum was raised against the deduced FA02 beta-subunit, and immunoblotting of total protein from buckwheat seeds (F. esculentum M. and Fagopyrum tartaricum Gaertn.) revealed that several groups of proteins reacted with the antiserum. Polypeptides in the 23--25 kDa range displayed the greatest reactivity.

Allergens↗

Two distinct groups of natural populations of Fagopyrum urophyllum (Bur. et Franch.) Gross revealed by the nucleotide sequence of a noncoding region in chloroplast DNA.

Fagopyrum urophyllum is a cross-pollinating perennial woody shrub species belonging to the urophyllum group of Fagopyrum. Natural populations of F. urophyllum were morphologically classified into two distinct groups, the Dali group and the Kunming group without exception. This grouping was verified by molecular phylogeny based on the nucleotide sequence of chloroplast DNA. The reproductive isolation found between the two groups was almost perfect as the distribution of two groups did not overlap each other. The net nucleotide substitution rate (Da) between the two groups was at the same level as between two distinct species. These data suggests that the two groups should be classified into distinct species if future studies on F. urophyllum confirm complete reproductive isolation and no ambiguously classified populations in the border area of the central Yunnan province of China.

DNA, Chloroplast↗

[The study of Fagopyrum tataricum complex prescription on type II diabetes rats].

OBJECTIVE: To observe the effect of Fagopyrum tatarium complex prescription on insulin resistance and indexes of blood stasis such as TG, TCh in type II diabetes rats. METHOD: To obtain type II diabetes rat model, rats were injected 25 mg/kg STZ and fed on high energy feed. RESULTS: Fagopyrum tataricum complex prescription (FTCP) was close or super to glyburide on relieving symptoms, preventing obesity, decreasing blood glucose level, improving insulin-sensitivity and blood stasis symptoms. FTCP could also prevent chronic complication of diabetes. No toxicity or side effect of FTCP was found.

Animals↗

[The ultracytochemical localization of ATPase activity in pollen tube and stigma of Fagopyrum esculentum after compatible and incompatible pollination].

Ultracytochemical localization of adenosine triphosphatase (ATPase) activity in stigmas, pollens and pollen tubes of Fagopyrum esculentum was performed with the cytochemical method of lead phosphate precipitation. The results were as follows: (1) Lower activities of ATPase appeared in stigma cells at 0.5 hour after compatible and incompatible pollination. Stigma surface and pollen grains attached on stigma showed higher ATPase activities after compatible pollination and lower or no activities after incompatible pollination at 0.5 hour. ATPase localized on endoplasmic reticula and sperm cell in pollen grain. (2) Lower ATPase activities appeared both in stigma cells and pollen tubes in style at 1.5 hours after incompatible pollination. Pollen tube stopped growing and the degeneration of its cytoplasma began; on the contrary, at 1.5 hours after compatible pollination, higher ATPase activities were detected both in stigma cells and pollen tube in style. ATPase localized mainly on plasmolemma, in cytoplasmic matrix of stigma cells and in mitochondria, dictyosome, plastid envelop, and on the wall of pollen tube as well. The present study indicated that the stop of pollen tube growth after self-incompatibility of Fagopyrum esculentum resulted not only from the nutrient starvation of pollen tube, but also from its metabolic disorder in incompatible style.

Adenosine Triphosphatases↗

[Phenolic acid derivatives from the rhizome of Fagopyrum cymosum].

OBJECTIVE: To study the chemical constituents of Fagopyrum cymosum. METHOD: The chemical constituents were isolated by various chromatographic methods and their structures were elucidated by the analysis of spectral data and physiochemical properties. RESULT: Four phenolic acid derivatives were isolated from F. cymosum. The chemical structures were elucidated as trans-p-hydroxy cinnamic methyl ester (I), 3, 4-dihydroxy benzamide (II), protocatechuic acid (III), protocatechuic acid methyl ester (IV). CONCLUSION: Compounds I, II, IV were obtained from the genus Fagopyrum for the first time.

Benzamides↗

Inhibition of tumor growth in vitro by the extract of fagopyrum cymosum (fago-c).

Fagopyrum cymosum (Trev.) Meisn has long been used in China to treat various ailments of the lung, including lung tumors. This study investigated whether Fagopyrum cymosum extract (Fago-c) has effects on other organs. Human cancer cells derived from 10 different organs were employed, and their growths as affected by Fago-c were investigated. It was found that the growth of cancer cells from lung, liver, colon, leukocytes and bone is inhibited by Fago-c. However, cancer cells derived from prostate, cervix, ovary and brain are not sensitive to Fago-c, and the extract stimulates the growth of cancer cells from breast (MCF-7). Synergistic inhibition effect of Fago-c and daunomycin was observed in human lung cancer cells (H460). Cellular proteins from H460 cells treated with Fago-c were analyzed by 2D-gel electrophoresis. A protein (M.W./pI = 20K/5.9) was induced. The Fago-c extract was analyzed by High Performance Liquid Chromatography (HPLC). Four major and twenty minor components were identified. These studies indicate that the effect of Fago-c in inhibiting the growth of cell lines derived from certain organs.

Antineoplastic Agents, Phytogenic↗

Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in the urophyllum group of the genus Fagopyrum (Polygonaceae).

We performed phylogenetic analyses of Fagopyrum species in the urophyllum group based on nucleotide sequences of two nuclear genes, FLORICAULA/LEAFY (FLO/LFY) and AGAMOUS (AG), and three segments of chloroplast DNA (cpDNA), rbcL-accD, trnK intron, and trnC-rpoB spacer. The FLO/LFY and AG sequences turned out to be phylogenetically more informative at the intrageneric level than the cpDNA sequences. Congruence among these gene trees, inferred by a maximum-likelihood (ML) method, demonstrated that topologies were partially incongruent between the nuclear and chloroplast DNA phylogenies. The nuclear DNA sequence data supported a monophyletic relation of F. statice, F. gilesii, and F. jinshaense, whereas the former two species formed another monophyletic relation with the F. capillatum-F. gracilipes-F. gracilipedoides-F. rubifolium clade excluding F. jinshaense in the synthetic cpDNA phylogeny. In addition, two divergent sequences of FLO/LFY were found in F. rubifolium (tetraploid). One of these was sister to F. gracilipedoides and another was sister to F. statice, and a monophyletic relation of these two genes was rejected by a bootstrap analysis. These results suggest that hybridization may have occurred during diversification of Fagopyrum species in the urophyllum group, and that F. rubifolium is possibly allotetraploid species.

Arabidopsis Proteins↗

[Inhibition of tumor growth in vitro by the extract of Fagopyrum cymosum].

OBJECTIVE: Fagopyrum cymosum (Trev.) Meisn has long been used in China to treat various ailments of the lung, including lung tumors. This study investigated whether Fagopyrum cymosum extract (Fago-c) has effects on other organs. METHODS: Human cancer cells derived from 10 different organs were employed, and their growths as affected by Fago-c were investigated. RESULTS: It was found that the growth of cancer cells from lung, liver, colon, leukocytes and bone is inhibited by Fago-c. However, cancer cells derived from prostate, cervix, ovary and brain are not sensitive to Fago-c, and the extract stimulates the growth of cancer cells from breast (MCF-7). Synergistic inhibition effect of Fago-c and daunomycin was observed in human lung cancer cells (H460). Cellular proteins from H460 cells treated with Fago-c were analyzed by 2D-gel electrophoresis. A protein (M.W./pI=20 K/5.9) was induced. The Fago-c extract was analyzed by High Performance Liquid Chromatography (HPLC). Four major and twenty minor components were identified. CONCLUSION: These studies indicate the effect of Fago-c in inhibiting the growth of cell lines derived from certain organs.

Antibiotics, Antineoplastic↗

Intra- and interspecific phylogeny of wild Fagopyrum (Polygonaceae) species based on nucleotide sequences of noncoding regions in chloroplast DNA.

The intra- and interspecific phylogeny of Fagopyrum (Polygonaceae) species was studied using nucleotide sequence data from two noncoding regions in chloroplast DNA, the trnK (UUU) intron and the trnC (GCA)-rpoB spacer. Thirty-seven accessions of ten species and two unidentified samples in the urophyllum group of Fagopyrum were analyzed. Both of the studied regions showed high variability, including nucleotide substitutions, insertion/deletions, and inversions. Separate parsimony analyses of the two regions generated phylogenies that were largely consistent with each other. A single most parsimonious tree derived from the combined data of the two regions suggested that (1) either F. statice or F. leptopodum was derived from the ancestor more than once, (2) F. gracilipes, a tetraploid species, has recently been derived from diploid ancestor and rapidly spread out to its present distribution areas, and (3) F. pleioramosum, F. macrocarpum, and F. callianthum, three newly discovered species endemic to the upper Min River valley, differentiated from their common ancestral species in the present distribution area.

Journal Article↗

Italian buckwheat (Fagopyrum esculentum) starch: physico-chemical and functional characterization and in vitro digestibility.

A study on the physico-chemical properties and structure together with the evaluation of starch digestibility was carried out on starch isolated from buckwheat (Fagopyrum esculentum) cultivated in different Italian areas. Results showed that buckwheat samples analysed were different among them and from wheat starch used as reference. Buckwheat granules were polygonal in shape and had a smaller diameter than the wheat starch granule. The starch obtained from buckwheat had a higher swelling power than the wheat one, probably as a consequence of the wheaker but more extensive bonding forces in the granule. During cooling, buckwheat samples showed a good paste stability.

Amylases↗

Identification and determination of flavonoids in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) by high-performance liquid chromatography with electrospray ionisation mass spectrometry and photodiode array ultraviolet detection.

An analytical method for flavonoids present in the seed extract of buckwheat (Fagopyrum esculentum Moench, Polygonaceae), using HPLC and a photodiode array detector and interfaced to an electrospray ionisation mass spectrometer, has been developed. Structural information about the flavonols was obtained from the retention time characteristics, the UV-visible spectra and the mass spectra without the need to isolate the individual compounds. The methanol extract of buckwheat contained principally four flavonol glycosides: rutin, quercetin, kaempferol-3-rutinoside and a trace quantity of a flavonol triglycoside.

Chromatography, High Pressure Liquid↗