Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)

Buckwheat (Fagopyrum esculentum) is a heterostylous self-incompatible crop that requires outcrossing for seed formation. Pollen parents influence the traits of seeds in many plants, but their influence in buckwheat is unknown. Here, we crossed self-incompatible (SI) and self-compatible (SC) lines with different rutin contents. The average rutin contents of SI leading cultivars were all 0.15–0.21 mg/g, and a SI high rutin content line that we had developed was 0.6 mg/g, although it has wide range SDs (0.12–0.41). On the other hand, the average rutin contents of SC lines were 0.01–0.06 mg/g, with stable SDs (0.02–0.03). In crosses between high- and low-rutin parents, the average rutin content of F1 seeds was significantly lower than that of the high-rutin parent and higher than that of the low-rutin parent, indicating that the pollen parent influences the rutin content in seeds of buckwheat. RNA-seq analysis confirmed that alleles of several genes encoding enzymes involved in rutin synthesis derived from pollen parents were expressed during seed formation.

九州で栽培されたソバ遺伝資源の子実の化学成分の特徴を明らかにするため,九州の在来種を含む内外の遺伝資源を2年間栽培し,ルチン,粗蛋白および粗脂肪の含量について調査した.ルチン含量の品種間の変異幅は粗蛋白および粗脂肪の含量と比較して大きかった.ルチンおよび粗脂肪の含量は品種間差異よりも年次間差異が大きかった.ネパールの品種にはルチン含量の高い品種が認められたが,ネパール以外では原産地別の品種の特徴が明らかでなかった.一方,ルチンの蓄積には環境要因として登熟期間中の温度が高いと有利であることが示唆された.子実重とルチン含量の間には両年とも有意な負の相関が,粗蛋白含量と粗脂肪含量の間には両年とも有意な正の相関が認められたが,千粒重や製粉歩留等と各成分含量との関係は明らかでなかった.

Buckwheat is a famous edible and medicinal coarse cereal which contain abundant of bioactive flavonoids, such as rutin. In this study, the composition and diversity of endophytic fungi in eight different buckwheat seeds were analyzed by high-throughput sequencing of ITS rDNA. Results showed that, the fungal sequences reads were allocated to 272 OTUs, of them, 49 OTUs were shared in eight buckwheat seeds. These endophytic fungi could be classified into 6 phyla, 19 classes, 41 orders, 79 families, 119 genera, and 191 species. At genus level, Alternaria sp. was the domain fungal endophyte. Besides, fungal endophytes belonged to the genera of Epicocum, Cladosporium, Botrytis, Filbobasidium, Stemphylium, and Vishniacozyma were highly abundant in buckwheat seeds. The total flavonoids and rutin contents in tartary buckwheat cultivars (CQ, XQ, CH, K2) were much higher than those in common buckwheat cultivars (HT, T2, T4, T8). For tartary buckwheat cultivars, the total flavonoids and rutin contents were ranging from 2.6% to 3.3% and 0.9% to 1.3%, respectively. Accordingly, the tartary buckwheat samples displayed stronger antioxidant activity than the common buckwheat. Spearman correlation heat map analysis was successfully found that certain fungal species from the genera of Alternaria, Botryosphaeria, Colletorichum and Diymella exhibited significant positive correlation with flavonoids contents. Results of this study preliminary revealed the fungi-plant interaction relationship at secondary metabolite level, and could provide novel strategy for increasing the flavonoids accumulation of buckwheat seeds, as well as improving their quality.

The aim of this study was to determine the variation of phenolic compounds in common and tartary buckwheats collected from China, Japan and Korea. Two buckwheat varieties of each country were cultivated using nutrient solution and vermiculite in the greenhouse from February to May, 2012 and harvested at two-week intervals. Phenolics such as chlorogenic acid, C-glycosylflavones (orientin, isoorientin, vitexin, and isovitexin), rutin, and quercetin were quantified using high performance liquid chroma-tography (HPLC). At 17, 29, 44, 58, and 72 days after sowing (DAS), before harvesting each organs, rutin content of cultivars from China and Korea at 17 DAS was the highest (mean 43.63 and 39.95 mg?g–1 DW, respectively) than that of 29 and 44 DAS. However, two varieties from Japan at 44 DAS were documented maximum; whereas, the amount of rutin in “Hokkai T10” harvested at 44 DAS (58.36 mg?g–1 DW) was the highest of all cultivars. Rutin level in the leaves and stems at 72 DAS were comparatively higher than at 58 DAS. The highest of rutin content was found at 72 DAS (73.33 mg?g–1 DW) in “Hokkai T10” leaves followed by “Daegwan No.3-3” (61.13 mg?g–1 DW), “rice tartary” (53.89 mg?g–1 DW). Rutin content of flowers was presented as the highest amount in “Hokkai T10” at 72 DAS (88.3 mg?g–1 DW) was approximately 14-fold higher than that of 58 DAS (6.44 mg?g–1 DW). However, rutin content of flower in “Xiqiao No.2” was 3-fold higher at 72 DAS than at 58 DAS. Rutin and total phenolic compounds content in flowers, leaves, and stems were higher in the order. The content of four C-glycosylflavones in common buckwheat was higher than those in tartary buckwheat, but rutin content which accounted >90% of the total phenolic compounds was higher in tartary buckwheat. The highest amounts of chlorogenic acid and quercetin were measured in the flower of “Xiqiao No.2” (6.85 and 11.69 mg?g–1 DW, respectively). Based on these results, the presence of different phenolic compounds in all the varieties of buckwheat confirmed that it can be regarded as a potent source of functional foods.

To improve the quality of buckwheat flour, we investigated the effect of the cropping season on the rutin content in various cultivars of common buckwheat (Fagopyrum esculentum Moench.). Thirty cultivars of buckwheat were grown under both long day conditions (the summer cropping) and short day conditions (the late summer cropping). The inter-varietal variations and the effect of the cropping season on the rutin content were examined. The rutin content was determined by high performance liquid chromatography (HPLC). Results show that the average rutin content in the late summer cropping was about half that of the summer cropping. There were wide inter-varietal variations of rutin content in the summer cropping. The differences in rutin content in the late summer cropping were less pronounced. Rutin content was closely correlated with the flowering period in summer (r=0.735***). The later flowering cultivars produced a higher content of rutin than the earlier flowering cultivars in the summer cropping. The differences in the cumulative solar radiation received in the two seasons seems to be a reasonable explanation for this increase in the rutin content of the buckwheat flour. The rutin content of early flowering cultivars, which are suitable for long day conditions, is positively correlated with yield in the summer cropping. However, the rutin content in the intermediate and late summer ecotype cultivars, which are suitable for short day conditions, shows no correlation with yield in the late-summer cropping. In conclusion, there are wide inter-varietal variations of rutin content in buckwheat flour and the rutin content increases under long day conditions.

Rutin and flavonoid contents in three buckwheat species Fagopyrum esculentum, F. tataricum, and F. homotropicum and their protective effects against lipid peroxidation

The concentration of flavonoids rutin and quercetin in flours of common and Tartary buckwheat was investigated. In Tartary buckwheat, concentration of rutin is much higher compared to common buckwheat. In Tartary buckwheat it was measured 1.17 to 1.75% rutin in dry matter, while in common buckwheat it was only 0.003%. After direct contact of buckwheat flour with water, different biochemical activities in Tartary buckwheat developed with rutin. After the time (5 minutes or two hours), the concentration of rutin is in the flour-water mixtures much lowered, and quercetin appeared. However, after quick initial changes, some rutin remained in flour-water mixtures even after 24 hours. In any way, after 24 hours of direct contact of flour particles with water, the concentration of quercetin is higher than that of rutin. It is established that the concentration of rutin in flour-water mixtures is the result of two processes. One is the release of rutin from grain structures and its dissolving in water, and the second is the release of rutin degrading enzymes from grain structures and their activity in solution.

Chapter Eighteen - Cultivation of Fagopyrum tataricum and Fagopyrum esculentum in Order to Obtain Raw Material with High Rutin Content in the Far East of Russia

Common buckwheat (Fagopyrum esculentum) is a heterostylous self-incompatible (SI) species with two different flower morphologies, pin and thrum. The SI trait is controlled by a single gene complex locus, S. Self-compatible (SC) lines were developed by crossing F. esculentum and F. homotropicum; these lines have an SC gene, Sh, which is dominant over the s allele and recessive to the S allele. S-ELF3 has been identified as a candidate gene in the S locus and is present in the S and Sh but not s alleles. A single-nucleotide deletion in the S-ELF3 gene of the Sh allele results in a frame shift. To develop co-dominant markers to distinguish between ShSh and Shs plants, we performed a next-generation sequencing analysis in combination with bulked-segregant analysis. We developed four co-dominant markers linked to the S locus. We investigated the polymorphism frequency between a self-compatible line and leading Japanese buckwheat cultivars. Linkage between a developed sequence-tagged-site marker and flower morphology was confirmed using more than 1000 segregating plants and showed no recombination. The developed markers would be useful for buckwheat breeding and also to produce lines for genetic analysis such as recombinant inbred lines.

Chapter thirty three - Genetic Analyses of the Heteromorphic Self-Incompatibility (S) Locus in Buckwheat

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.

Purification and characterization of flavonol 3-glucosidase, and its activity during ripening in tartary buckwheat seeds

Summary Buckwheat ( Fagopyrum sp.) is a well-known source of many health beneficial components such as starch, dietary fibres, proteins, and polyphenols such as flavonoid glycosides, especially rutin, and phenolic acids. Use of buckwheat for medicinal and health purposes demands that the content of these components are as high as possible. In this study, we have investigated the influence of species, variety, nitrogen (N) fertilization, and harvest time on the content and yield of polyphenols in buckwheat aerial parts and seeds. Experiments were performed as a joint project in both Denmark and Germany; however, all treatments were not repeated in both locations. Three varieties of normal buckwheat ( F. esculentum ) and one of tartary buckwheat ( F. tataricum ) were included in the experiments on the two locations. Experiments with high and low N fertilization were performed on both locations and in Germany combined with three harvest times of aerial parts. Seeds at maturity were only harvested and analysed in the Danish experiments. For aerial parts of both normal and tartary buckwheat from both locations there was no significant effect on the concentration of flavonoids with either increasing at high and low N-application. N-fertilization did not show a conclusive affect on the concentration of phenolic acids in the aerial parts as well. Concentration of rutin in aerial parts harvested at flowering stage in the Danish experiments varied between 1.7 and 3.9% dry matter content (DM) in tartary buckwheat and between 2.2 and 2.4% DM in normal buckwheat. Harvest time or development stage had a highly significant influence on the concentration of polyphenols in the aerial parts, as concentrations at harvest before flowering were up to four times higher than for harvest after flowering. In aerial parts the species/varieties did not differ significantly in content of polyphenols, except for kaempferol-3- O -rutinoside, which was higher in the tartary buckwheat 'Lifago' than in the other normal buckwheat varieties. Seeds of tartary buckwheat had a significantly higher concentration of flavonoids than normal buckwheat. In tartary buckwheat seeds, the kaempferol-3- O -rutinoside content decreased with increasing N-fertilization, but no significant effect on flavonoids in normal buckwheat seeds was found. Concentration of rutin in normal and tartary buckwheat seeds differed significantly and was between 0.03% and 0.11% DM and 1.4% and 1.5% DM, respectively.

Two sporophytic self-incompatibility (SI) alleles were introgressed from Brassica napus rapid cycling material into five self-compatible (SC) lines of oilseed summer rape. The field performance of segregating BC2F2 lines (SC:SI 1:1) was compared with the corresponding SC lines using a split-plot field design. Plants of the SC-SI mixed stands produced, in comparison to plants of the SC plots, more siliques on the main raceme, but the siliques contained fewer seed suggesting that SI plants might not have been fully fertilised. Key words: Rape (summer), sporophytic self-incompatibility

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by a single S-locus with multi allelic variety. The male S determinant, SP11/SCR (S-locus protein 11/S-locus cysteine-rich protein), is a small cysteine-rich protein, and the female S determinant, SRK (S-locus receptor kinase), functions as a receptor for SP11 at the surface of stigma papilla cells. Although a few of the following downstream factors in the SP11-SRK signaling cascade have been identified, a comprehensive understanding of the SI mechanism still remains unexplained in Brassicaceae. Analysis of self-compatible (SC) mutants is significant for understanding the molecular mechanism in SI reactions, thus we screened SC lines from a variety of Japanese bulk-populations of B. rapa vegetables. Two lines, TSC4 and TSC28, seem to have disruptions in the SI signaling cascade, while the other line, TSC2, seems to have a deficiency in a female S determinant, SRK. In TSC4 and TSC28, known SI-related factors, i.e. SRK, SP11, MLPK (M-locus protein kinase), THL (thioredoxin-h-like), and ARC1 (arm repeat containing 1), were expressed normally, and their expression levels were comparable with those in SI lines. On a B. rapa genetic linkage map, potential SC genes in TSC4 and TSC28 were mapped on linkage groups A3 and A1, respectively, whereas MLPK, ARC1, and THL were mapped on A3, A4, and A6, respectively. Although potential SC genes of TSC4 and MLPK were on the same linkage group, their positions were apparently independent. These results indicate that the SC genes of TSC4 and TSC28 are independent from the S-locus or known SI-related genes. Thus, the SC lines selected here have mutations in novel factors of the SI signaling cascade, and they will contribute to fill pieces in a signal transduction pathway of the SI system in Brassicaceae.