Purple Grain Research Articles

Understanding the molecular regulation of flavonoid 3’-hydroxylase in anthocyanin synthesis: insights from purple qingke

BackgroundThe Flavonoid 3’-hydroxylase gene(F3’H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear.ResultsIn order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3’H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3’H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein.ConclusionsThe results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke.

Identification and development of functional markers for purple grain genes in durum wheat (Triticum durum Desf.).

Two allelic variants of Pp-A3 and Pp-B1 were identified in purple durum wheat. Molecular markers at both loci were developed and validated on an independent panel, offering a breakthrough for wheat improvement. Purple wheats are a class of cereals with pigmented kernels of particular interest for their antioxidant and anti-inflammatory properties. Although two complementary loci (Pp-B1 and Pp-A3), responsible for purple pericarp have been pinpointed in bread wheat (Triticum aestivum L.), in durum wheat (Triticum durum Desf.) the causative genes along with functional and non-functional alleles are still unknown. Here, using a quantitative trait loci (QTL) mapping approach on a RIL population derived from purple and non-purple durum wheat genotypes, we identified three major regions on chromosomes 2A, 3A, and 7B explaining the highest phenotypic variation (> 50%). Taking advantage of the Svevo genome, a MYB was reannotated on chromosome 7B and reported as a candidate for Pp-B1. An insertion of ~ 1.6kb within the first exon led to a non-functional allele (TdPpm1b), whereas the functional allele (TdPpm1a) was characterized and released for the first time in durum wheat. Pp-A3 was instead identified as a duplicated gene, of which only one was functional. The promoter sequencing of the functional allele (TdPpb1a) revealed six 261-bp tandem repeats in purple durum wheat, whereas one unit (TdPpb1b) was found in the yellow once. Functional molecular markers at both loci were developed to precisely discriminate purple and not purple genotypes, representing a valuable resource for selecting superior purple durum lines at early growth stages. Overall, our results expand the understanding of the function of MYB and bHLH activators in durum wheat, paving new ways to explore cis-regulatory elements at the promoter level.

Unveiling the mysteries of HvANS: a study on anthocyanin biosynthesis in qingke (hordeum vulgare L. var. Nudum hook. f.) seeds

BackgroundBased on our previous research, a full-length cDNA sequence of HvANS gene was isolated from purple and white Qingke. The open reading frame (ORF) in the purple variety Nierumuzha was 1320 base pairs (bp), encoding 439 amino acids, while the ORF in the white variety Kunlun 10 was 1197 bp, encoding 398 amino acids. A nonsynonymous mutation was found at the position of 1195 bp (T/C) in the coding sequence (CDS) of the HvANS gene. We carried out a series of studies to further clarify the relationship between the HvANS gene and anthocyanin synthesis in Qingke.ResultsThe conservative structural domain prediction results showed that the encoded protein belonged to the PLN03178 superfamily. Multiple comparisons showed that this protein had the highest homology with Hordeum vulgare, at 88.61%. The approximately 2000 bp promoter sequence of the HvANS gene was identical in both varieties. The real-time fluorescence PCR (qRT-PCR) results revealed that HvANS expression was either absent or very low in the roots, stems, leaves, and awns of Nierumuzha. In contrast, the HvANS expression was high in the seed coats and seeds of Nierumuzha. Likewise, in Kunlun 10, HvANS expression was either absent or very low, indicating a tissue-specific and variety-specific pattern for HvANS expression. The subcellular localization results indicated that HvANS was in the cell membrane. Metabolomic results indicated that the HvANS gene is closely related to the synthesis of three anthocyanin substances (Idaein chloride, Kinetin 9-riboside, and Cyanidin O-syringic acid). Yeast single hybridization experiments showed that the HvANS promoter interacted with HvANT1, which is the key anthocyanin regulatory protein. In a yeast two-hybrid experiment, we obtained two significantly different proteins (ZWY2020 and POMGNT2-like) and verified the results by qRT-PCR.ConclusionsThese results provide a basis for further studies on the regulatory mechanism of HvANS in the synthesis of anthocyanins in Qingke purple grains.

Metabolic profiling of (poly)phenolic compounds in mouse urine following consumption of hull-less and purple-grain barley.

The present study attempted for the first time to investigate the metabolic fate of (poly)phenolic compounds provided by a hull-less and purple grain barley genotype biofortified in anthocyanins. Balb/c mice were supplemented either with standard purified diet (SD) or whole-grain barley supplemented diet (WGB) for six weeks. Subsequently, (poly)phenolic metabolites were determined in urine samples by UPLC-MS/MS, and the principal metabolic pathways were elucidated. Thirty-nine (poly)phenolics compounds were identified in WGB which were distributed between the free (58%) and bound (42%) fractions, encompassing anthocyanins, phenolic acids, flavan-3-ols and flavones. Upon WGB intake, forty-two (poly)phenolic metabolites were identified, predominantly comprising phase-II sulphate, glucuronide, and/or methylated conjugates, along with colonic catabolites. Noteworthy metabolites included peonidin-3-O-glucuronide, peonidin-3-O-6''-O-malonylglucoside, and peonidin-3-O-glucoside among anthocyanins; hydroxyphenylpropanoic acid-O-sulphate among phenolic acids; and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone-O-sulphate among flavan-3-ols. Metabolites like phenylpropionic, phenylacetic, hydroxybenzoic, and hippuric acids were found in both WGB and SD groups, with higher levels after barley consumption, indicating both endogenous and polyphenolic metabolism origins. Overall, this study offers valuable insights into the metabolism of (poly)phenols in purple barley, setting the stage for future investigations into the health benefits linked to the consumption of purple grain barley.

Determination of the melanin and anthocyanin content in barley grains by digital image analysis using machine learning methods.

The pigment composition of plant seed coat affects important properties such as resistance to pathogens, pre-harvest sprouting, and mechanical hardness. The dark color of barley (Hordeum vulgare L.) grain can be attributed to the synthesis and accumulation of two groups of pigments. Blue and purple grain color is associated with the biosynthesis of anthocyanins. Gray and black grain color is caused by melanin. These pigments may accumulate in the grain shells both individually and together. Therefore, it is difficult to visually distinguish which pigments are responsible for the dark color of the grain. Chemical methods are used to accurately determine the presence/ absence of pigments; however, they are expensive and labor-intensive. Therefore, the development of a new method for quickly assessing the presence of pigments in the grain would help in investigating the mechanisms of genetic control of the pigment composition of barley grains. In this work, we developed a method for assessing the presence or absence of anthocyanins and melanin in the barley grain shell based on digital image analysis using computer vision and machine learning algorithms. A protocol was developed to obtain digital RGB images of barley grains. Using this protocol, a total of 972 images were acquired for 108 barley accessions. Seed coat from these accessions may contain anthocyanins, melanins, or pigments of both types. Chemical methods were used to accurately determine the pigment content of the grains. Four models based on computer vision techniques and convolutional neural networks of different architectures were developed to predict grain pigment composition from images. The U-Net network model based on the EfficientNetB0 topology showed the best performance in the holdout set (the value of the "accuracy" parameter was 0.821).

Study of the breeding forms of emmer wheat (<i>Triticum dicoccum</i> (Schrank) Schuebl.)

The results of the study of economically useful features of the plants of the semi-naked emmer wheat breeding lines (Triticum dicoccum (Schrank) Schuebl.) with purple pericarp coloring of grain are presented. Seven breeding lines F9, created by two-step hybridization of the purple-grain durum wheat with naked and awnless emmer, and five breeding samples of seminaked emmer wheat were studied. In the field experiments, plants were selected for selection valuable traits. Phenological observations, structural analysis, and evaluation of the quantitative traits of the plants of emmer wheat breeding forms with purple grain coloration were carried out from 2020 to 2022. Two-way analysis of variance showed significant factors - the breeding lines and the years of study. In the course of the study, it was found that for further breeding work, emmer wheat lines carrying a combination of economically important traits are of value. Short-stalked purple-grain form 27-1 is resistant to lodging, the yield of the grain - 80 %, the weight of 1000 grains – 34–37 g. Line 27–12 has grains with purple color, high grain-unit value of about 800 g/l and good grain productivity for emmer wheat reaching 400 g/m2. Purple emmer line 31–16 has a good bushiness (3–4 pieces), short stalk (71–74 cm), high lodging resistance (4–5 points) and grain threshing capacity (80 %). From 2021 to 2022, the same studies were conducted with emmer wheat from the collection of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR). The selection line of emmer wheat 41/14 was distinguished by the high indicator of threshed grains (85 %). The main disadvantages of the established forms of emmer wheat with purple-colored grains still remain relatively low grain yield, insufficiently strong ear shank, not complete grinding of the grain from the flowering glumes and husks. However, further breeding work can improve these traits, as well as increase the grain yield, the weight of 1000 grains and a more complete release of the grain from the chaff during threshing.

The Use of the Genetic Resources of Tetraploid Wheat <i>Triticum aethiopicum</i> on the Developing of Purple-Grain Common Wheat with a High Content of Anthocyanins

The article considers the results on the developing of purple-grain common wheat and the study of its genetic diversity. Triticum aethiopicum Jakubz. 2n = 28 is the only source of genes for the purple grains color among the wheat species. The trait of purple grain is accompanied by an increase in the content of anthocyanins and micronutrients in the grain. To develop common wheat lines with purple grain, T. aethiopicum k-19068 was first hybridized with the Iranian landrace T. aestivum k-14333. The resulting hybrids were then crossed with the highly productive common wheat cultivar Liza. The developing lines F6 (2n = 42) characterize low diversity on multiple alleles of gliading-coding loci (H = 0.410). They were dominated by alleles inherited from the parent variety Liza. No gliadin alleles that could belong to T. aethiopicum have been identified. Comparison of groups of lines with purple and white grains made it possible to show that they do not significantly differ from each other in terms of yield and elements of its structure. As a result, we have not found a negative correlation between the high content of anthocyanins in grain and grain productivity. This allows us to hope for the possibility of developing high-yielding lines of common wheat with purple grain enriched with anthocyanins.

Marker-assisted breeding of hybrid lines of <i>Triticum dicoccon</i> (Schrank) Schuebl. × <i>Triticum aethiopicum</i> Jakubz. with purple grain

Background. Cereals whose grain contains antioxidants salutary for human health are promising for functional nutrition. Anthocyanins inducing purple grain color are antioxidants, and it is crucial to make efforts towards increasing their content in grain. The objective of this work was to assess the content of anthocyanins in emmer grain and the productivity of breeding lines with purple grain. Materials and methods. The study included the F9 lines developed by two-step hybridization between purple-colored Ethiopian wheat (Triticum aethiopicum Jakubz. var. arraseita) and emmer (Triticum dicoccon (Schrank) Schuebl.), and those isolated in F2 using marker-based selection for homozygous dominant alleles of two genes. The parent forms of the hybrids were a naked-grain emmer cultivar ‘Gremme’, an awnless emmer accession (k-25516) from the VIR collection, and an accession from the collection of IPK Gatersleben (Ethiopian wheat line TRI 15744), the donor of the purple color. The content of anthocyanins in whole-grain flour was measured in 12 lines, a structural analysis of plants was carried out, and their quantitative characteristics were assessed. Results. The highest anthocyanin content (82.5 µg per 1 g) was observed in line No. 10 of the 27-3 hybrid. The largest share of threshed naked grains (86.9 ± 7.3 %) was recorded for line No. 6 of the 31-19 hybrid. Line No. 11 of the 27-12 hybrid had the highest test weight (802 ± 13 g/L). Line No. 9 of the 27-1 hybrid showed the best index of grain productivity (389 ± 25 g/m2). Conclusion. Marker-assisted selection made it possible to identify homozygous dominant alleles of the two complementary interacting genes, Pp3 and Pp-B1, which cause the purple color of the grain pericarp. Breeding lines with purple-colored grain demonstrated different levels of the total anthocyanin content, test weight, and grain yield. The lines with high indices of these traits are valuable for breeding practice.

Extension of the forming process in the selection of winter common wheat for productivity and quality by using the gene pool of related wheat species within the framework of food security

The formation process in the selection of winter common wheat for productivity and quality has been expanded by using the gene pool of related species. The purpose of this study was to expand the formative process in the selection of Triticum aestivum L. winter type of development with the further obtaining of genotypes with increased adaptability, plant productivity and grain quality through the use of winter common wheat with purple grain, whole grain wheat and Ethiopian wheat in hybridization. In the study, the methods of conducting the examination of plant varieties are used for carrying out phenological observations, morphological studies, evaluation of breeding material according to economic and valuable characteristics. In the creation of new forms, methods of hybridization and individual selection were used, and the TVEL method was used for pollination. The protein and gluten content were determined by infrared spectrometry. Parental components were studied and selected to expand the forming process to obtain productive grains from the main ear, thousand kernel weight, high grain quality, resistant to lodging, shedding of grain, drought-resistant and resistant to fungal pathogens, hybrid forms, specifically as a result of involvement of the source material Triticum aestivum L. and Triticum sphaerococcum Perc. in the hybridization. According to the results of the hybridization of winter common wheat and Ethiopian wheat, it was possible to obtain and breed in F4 forms with a long ear (>10-11 cm), an increased amount of grain from the main ear (>55 pcs.) and a different colour of grain from dark brown to purple, as an indicator of increased anthocyanin content and high antioxidant capacity. The practical value of this study lies in the fact that the research results expand information on the use of winter common wheat in the selection of productivity and quality of other types of wheat: Triticum sphaerococcum, T. aethiop. var. decoloratum, and the developed proposals will allow considering the use of parental components of wheat in research, help improve methods of hybridization and selection of new forms with desirable traits in the system of interspecies crossings

Genetic loci regulating the concentrations of anthocyanins and proanthocyanidins in the pericarps of purple and red rice.

The pigmented flavonoids, anthocyanins and proanthocyanidins, have health promoting properties. Previous work determined that the genes Pb and Rc turn on and off the biosynthesis of anthocyanins (purple) and proanthocyanidins (red), respectively. Not yet known is how the concentrations of these pigmented flavonoids are regulated in grain pericarps. Quantitative trait locus (QTL) analysis in a population of rice (Oryza sativa L.) F5 recombinant inbred lines from white pericarp "IR36ae" x red+purple pericarp "242" revealed three QTLs associated with grain concentrations of anthocyanins (TAC) or proanthocyanidins (PA). Both TAC and PA independently mapped to a 1.5 Mb QTL region on chromosome 3 between RM3400 (at 15.8 Mb) and RM15123 (17.3 Mb), named qPR3. Across 2 years, qPR3 explained 36.3% of variance in TAC and 35.8% in PA variance not attributable to Pb or Rc. The qPR3 region encompasses Kala3, a MYB transcription factor previously known to regulate purple grain characteristics. Study of PbPbRcrc progeny showed that TAC of RcRc near isogenic lines (NILs) was 2.1-4.5x that of rcrc. Similarly, study of PbPbRcRc NILs, which had 70% higher PA than pbpbRcRc NILs, revealed a mutual enhancement, not a trade-off between these compounds that share precursors. This suggests that Pb and Rc upregulate genes in a shared pathway as they activate TAC and PA synthesis, respectively. This study provides molecular markers for facilitating marker-assisted selection of qPR3, qPR5, and qPR7 to enhance grain concentrations of pigmented flavonoids and documented that stacking Rc and Pb genes further increases both flavonoid compounds.

Genetic Mapping of Flavonoid Grain Pigments in Durum Wheat.

Pigmented cereal grains with high levels of flavonoid compounds have attracted the attention of nutritional science backing the development of functional foods with claimed health benefits. In this study, we report results on the genetic factors controlling grain pigmentation in durum wheat using a segregant population of recombinant inbred lines (RILs) derived from a cross between an Ethiopian purple grain accession and an Italian amber grain cultivar. The RIL population was genotyped by the wheat 25K SNP array and phenotyped for total anthocyanin content (TAC), grain color, and the L*, a*, and b* color index of wholemeal flour, based on four field trials. The mapping population showed a wide variation for the five traits in the different environments, a significant genotype x environment interaction, and high heritability. A total of 5942 SNP markers were used for constructing the genetic linkage map, with an SNP density ranging from 1.4 to 2.9 markers/cM. Two quantitative trait loci (QTL) were identified for TAC mapping on chromosome arms 2AL and 7BS in the same genomic regions of two detected QTL for purple grain. The interaction between the two QTL was indicative of an inheritance pattern of two loci having complementary effects. Moreover, two QTL for red grain color were detected on chromosome arms 3AL and 3BL. The projection of the four QTL genomic regions on the durum wheat Svevo reference genome disclosed the occurrence of the candidate genes Pp-A3, Pp-B1, R-A1, and R-B1 involved in flavonoid biosynthetic pathways and encoding of transcription factors bHLH (Myc-1) and MYB (Mpc1, Myb10), previously reported in common wheat. The present study provides a set of molecular markers associated with grain pigments useful for the selection of essential alleles for flavonoid synthesis in durum wheat breeding programs and enhancement of the health-promoting quality of derived foods.

Processing affects (decreases or increases) metabolites, flavonoids, black rice pigment, and total antioxidant capacity of purple glutinous rice grains.

The objective of this study was to understand the effects of processing on metabolites, flavonoids, black rice pigments and total antioxidant capacity of purple grains. The biochemical indicators and metabolites were determined before and after processing of purple grains. The results showed that the total antioxidant capacity, total phenol (TP), flavonoid (PD), oligomeric proanthocyanidin (OPC), ascorbic acid (AsA), cyanidin-3-O-glucoside (C3OG), peonidin 3-glucoside (P3G) contents of purple grains were greatly decreased after brown rice grains were processed into polished rice grains. The TP, PD, OPC, AsA, C3OG, and P3G of Yangzinuo No.1 brown rice (YZN1_B) or polished rice grains (YZN1_H) were higher than those of Yangzinuo No.2 brown rice (YZN2_B) or polished rice grains (YZN2_H). 154 differential metabolites (DMs) were identified between YZN1_B and YZN1_H. 52 DMs were identified between YZN2_B and YZN2_H. Citric acid and isocyanate are key metabolites affected during processing and have good correlations with various biochemical indicators.

Flavonoid Metabolic Profiles and Gene Mapping of Rice (Oryza sativa L.) Purple Gradient Grain Hulls

Rice (Oryza sativa L.) grain hull color is an easily observable trait and regarded as a crucial morphological marker in rice breeding. Here, a purple gradient grain hull mutant (pg) was found from natural mutations of a straw-white grain hull rice variety IARI 6184B (Orzya sativa L. subsp. indica). The color of the mutant grain hulls changed from straw-white to pink, then purple, and finally brownish-yellow. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identified 217 flavonoids, including 18 anthocyanins, among which cyanidin O-syringic acid had the highest concentration in pink (66.2 × 106) and purple (68.0 × 106) grain hulls. The relative contents of hesperetin O-malonyl-hexoside, apigenin derivatives, genistein derivatives, and kaempferol 3-O derivatives were consistently downregulated during pg grain hull development. Conversely, 12 anthocyanins were upregulated in colored hulls, and cyanidin 3-O-malonylhexoside was abundant only in pink and purple grain hulls. Moreover, the candidate gene was mapped into a 1.38 Mb region on chromosome 4 through bulked segregant analysis based on deep sequencing (BSA-seq) and gene mapping approaches. These results increased our understanding of anthocyanin biosynthesis in rice grains, helping rice breeders to select new rice varieties with desirable grain traits.

Metabolite identification in fresh wheat grains of different colors and the influence of heat processing on metabolites via targeted and non-targeted metabolomics

Phenolic antioxidants are phytochemical components in wheat grains that provide a variety of potential health benefits. The metabolites and antioxidant activity of fresh, mature, and heat-treated, wheat grains with black, blue, purple, and white grain coats were identified by targeted and non-targeted metabolomics. The total phenolic (TPC) and flavonoid contents (TFC) and antioxidant activity (AOA) increased with the darkening of grain color, the general trend being black > purple > blue > white. Purple and black wheat are rich in rutin (3916 µg/kg and 3066 µg/kg, respectively) and peonidin-3-O-glucoside chloride (2595 µg/kg and 1740 µg/kg, respectively), while blue wheat is rich in luteolin (2076 µg/kg). In most cases, TPC, TFC, and AOA had the greatest values in fresh grains and the lowest values in mature grains. Using non-targeted metabolomics, a total of 866 metabolites were identified in the tested fresh wheat grains, 106 flavonoids and 39 phenolic acids. In total, the relative abundance of flavonoids in purple and black wheat was higher than in blue wheat, indicating a higher nutritional value of fresh black and purple grains. After heat processing, the content of most metabolites decreased in heat-treated purple grain, whereas heat treatment significantly increased the content of peonidin-3-O-glucoside chloride (2.27-fold) and cynaroside (12.01-fold). This study clarifies that seed coat color and processing treatments impact the metabolite contents and antioxidant activity of wheat grains, providing valuable information for improving the nutritional quality of food during processing.

ОЦЕНКА КАЧЕСТВА ЗЕРНА СОРТОВ ЯРОВОЙ ПШЕНИЦЫ С ФИОЛЕТОВОЙ ОКРАСКОЙ ЗЕРНА В УСЛОВИЯХ ЦРНЗ

The article presents the results of the study of the baking qualities of grain of three varieties of soft spring wheat characterized by by purple grain colouring, and the red grain standard of Zlata in the conditions of the Central region of the Non-Chernozem zone. The years of the study were characterized by contrasting meteorological conditions – excessively humid year 2020 and arid year 2021. Protein content was determined using a spectrophotometer. Gluten was washed manually. Bread baking was carried out according to the modified methodology of the State Commission for Testing and Protection of Breeding Achievements. The allelic composition of high molecular weight glutenins was carried out by electrophoresis according to Singh et al., (1991) in the modification of Branlard et al. (2001). It has been found that all varieties of spring wheat with purple grain colouring are not inferior in quality to the standard, differing from it by two alleles of high-molecular glutenins, affecting the stability of manifestation of baking qualities by year. The quality of the grain is highly dependent on the meteorological conditions of the growing season. All the studied varieties of spring wheat with a purple grain colouring are able to form a grain suitable for baking in the conditions of the Non-Chernozem zone. The quantitative protein and crude gluten content in grain varies by year, but is not inferior to the standard. The quality of gluten varied within groups I and II. At the same time, the gluten was stronger under drought conditions. All varieties showed a high potential of bread volume yield – at or above the standard. The low score of the general bakery evaluation is explained by inclusions of purple bran particles, which caused a darker colouring of the crumb. The positive correlation between the volume yield of bread and the protein content was stronger in the year with reduced protein content in grain.

High maize barrier prevents maize pollen transfer in mixed crops

BACKGROUND: There is no scientifically-based assessment for the safety during co-cultivation of genetically modified and ordinary maize plants in Russia. The influence of the barrier from high maize plants for pollen-mediated gene flow in the 2020 field experiment (South-East of the European part of Russia, Saratov region) was evaluated.
 MATERIALS AND METHODS: We used the high (2,152,90 m) maize hybrids (Kaz LK 178 and ES Regain) as a barrier for pollen-madiated gene flow from pollen donor (Purple Saratovskaya) with purple grain to recipient (Bursting Corn) maize line with yellow grain.
 RESULTS: The analysis of the ears of the recipient maize line showed that not a single purple grain was found on them. It was found that in the presence of a barrier zone with a width of 315 m (depending on the direction), crossing in recipient maize line in all directions from the donor is completely excluded.
 CONCLUSIONS: It was established, for the first time, that the barrier from high maize hybrid plants completely excludes over-pollination between donor and Bursting Corn, recipient maize plants with different flowering time. In the study of barrier plants as pollen recipients, it was found that the percentage of crosses on the cob of barrier plants ranged from 0.1 to 7.1%. The number of crosses exceeding 0.9% is observed mainly in the ES Regain variety at close (15 m) distances from the donor, regardless of the prevailing wind direction. Based on the results of model experiments, it can be recommended to use a barrier for corn pollen from tall maize plants and maize varieties with different flowering periods to exclude uncontrolled over-pollination of maize varieties in the South-East of the European part of Russia conditions.

Metabolomics and Transcriptomics Provide Insights into Anthocyanin Biosynthesis in the Developing Grains of Purple Wheat (Triticum aestivum L.).

Purple wheat is thought to have beneficial effects on humans owing to its high anthocyanin content. However, a systematic understanding of the anthocyanin biosynthesis process in developing wheat grain is lacking. Here, the dynamic changes in anthocyanin components and transcripts in the grain of purple wheat ZNM168 at five developmental stages (10, 15, 20, 25, and 30 DAF) were characterized. Compared with other anthocyanins, four components, cyanidin 3-O-rutinoside, cyanidin 3-O-glucoside, cyanidin 3,5-O-diglucoside, and malvidin 3-O-glucoside, were significantly accumulated with grain development. In particular, the considerable accumulation of cyanidin 3-O-rutinoside indicated that it was the pivotal pigment for the purple grain. Transcriptome analysis revealed that the nine differentially expressed genes related to anthocyanin biosynthesis belonged to the BZ1 group, the homologous enzyme encoded by the maize Bronze-1 locus, which may primarily serve to glucosylate anthocyanidins. By constructing a gene coexpression network based on weighted gene coexpression network analysis, the TaBZ1 UniGene (TraesCS1D02G019200) was predicted as a core gene in anthocyanin biosynthesis. In addition, correlation analysis between the metabolites and transcripts suggested that TraesCS2A01G527700 (TaCHS) and TraesCS6B01G006200 (TaANS) were considered critical structural genes in the anthocyanin biosynthesis pathway. This study provides insights to exploit genes pinpointed as genetic engineering targets, thereby breeding anthocyanin-enriched wheat.

Phytochemical Analysis of Phenolics, Sterols, and Terpenes in Colored Wheat Grains by Liquid Chromatography with Tandem Mass Spectrometry.

The colored grain of wheat (Triticum aestivum L.) contains a large number of polyphenolic compounds that are biologically active ingredients. The purpose of this work was a comparative metabolomic study of extracts from anthocyaninless (control), blue, and deep purple (referred to here as black) grains of seven genetically related wheat lines developed for the grain anthocyanin pigmentation trait. To identify target analytes in ethanol extracts, high-performance liquid chromatography was used in combination with Bruker Daltonics ion trap mass spectrometry. The results showed the presence of 125 biologically active compounds of a phenolic (85) and nonphenolic (40) nature in the grains of T. aestivum (seven lines). Among them, a number of phenolic compounds affiliated with anthocyanins, coumarins, dihydrochalcones, flavan-3-ols, flavanone, flavones, flavonols, hydroxybenzoic acids, hydroxycinnamic acids, isoflavone, lignans, other phenolic acids, stilbenes, and nonphenolic compounds affiliated with alkaloids, carboxylic acids, carotenoids, diterpenoids, essential amino acids, triterpenoids, sterols, nonessential amino acids, phytohormones, purines, and thromboxane receptor antagonists were found in T. aestivum grains for the first time. A comparative analysis of the diversity of the compounds revealed that the lines do not differ from each other in the proportion of phenolic (53.3% to 70.3% of the total number of identified compounds) and nonphenolic compounds (46.7% to 29.7%), but diversity of the compounds was significantly lower in grains of the control line. Even though the lines are genetically closely related and possess similar chemical profiles, some line-specific individual compounds were identified that constitute unique chemical fingerprints and allow to distinguish each line from the six others. Finally, the influence of the genotype on the chemical profiles of the wheat grains is discussed.

ОЦЕНКА РИСКОВ ПЕРЕОПЫЛЕНИЯ КУКУРУЗЫ ПРИ СОВМЕСТНОМ ВЫРАЩИВАНИИ НЕСКОЛЬКИХ ЛИНИЙ В УСЛОВИЯХ ЮГО-ВОСТОКА ЕВРОПЕЙСКОЙ ЧАСТИРОССИИ

The new Russian Federal law (No. 358 of 03.07.2016) prohibits the commercial use of GM plants in agriculture, but allows, since 2018, for the first time in Russia their cultivation and testing for research purposes. Consequently, there is a need to assess and develop criteria for safe co-cultivation of non-GM and GM varieties, which are currently absent in Russia. In this paper, it was established for the first time that the 10-15 m distance is sufficient to prevent cross-pollination between maize lines with an acceptable presence of 0.9 % of the donor’s genetic material, regardless of the recipient line, donor and recipient time flowering, and the wind direction in Saratov condition (South-West region of European part of Russia). The work aimed to assess the influence of the distance between pollen donor and recipient, wind direction, donor and recipient time flowering, and a buffer zone presence between them on the crossing frequency in mixed maize crops. The maize lines Korichnevyi marker (KM), GPL-1, Zarodyshevyi marker Saratovskii Purpurnyi (ZMS-P), Purpurnaya Saratovskaya (PS), as well as hybrids Purpurnyi (GP), Raduga and Tester 3 were grown (the experimental field of the Rossorgo, Saratov, South-West region of European Russia, 2018-2019). We planted the GP and ZMS-P lines as pollen donors in 2018 on a 3×80 m2 area with planting density of 7-10 plants per 1 m2. Between the pollen donor area, maize KM and GPL-1 lines were planted, and around them there were a 1290 m2 area of yellow-colored grain recipients (Raduga and Tester 3 hybrids). In September, 5-12 ears from each pollen recipient were harvested. The cross-pollination frequency was calculated as the ratio of purple grains (GP pollination result) or yellow grains with a purple spot (ZMS-P pollination result) to the total grain number in recipient lines. In 2018 it was established that the maximum percentage (from 0.1 to 13.2 %) derived from cross-pollination with two pollen donors depends on different factors. At closer distances (1-4 m), the cross-pollination increased 4-fold for the earlier flowering recipient. The percentage of crosses for recipient Raduga decreased 3 times with a 10 m increase in the distance and 11 times at a 40 m distance from the donor plants. Experiments in 2018 indicate that the 10 m distance from the pollen donor guarantees the percentage of crosses not exceeding the 0.9 % GM threshold in food products accepted in the European Union and Russia. In 2019, we used PS inbred line as a pollen donor. The PS was planted on a 3×5 m plot with Sudanese grass (Sorghum × drummondii) Allegory cultivar as a buffer zone 3 m wide to the East and West and 15 m long to the South-West and North-East. Yellow-grain hybrid Raduga was planted around the buffer zone. The frequency of crosses was calculated as the ratio of the number of purple grains to the total number of Raduga grains per ear. In 2018, the frequency of crosses was also estimated depending on the synchronism of flowering between pollen donors and recipients. The GPL-1 recipient with a 9-day difference from PG (pollen donor) in the beginning of flowering showed a 4 % lower pollination rate compared to a KM line with a flowering period closer to the PG pollen donor (1-day difference). Tall plants of PG donor of pollen prevented spreading pollen from a short ZMS-P donor to the Tester 3 and Raduga recipients in the direction of the wind rose. In 2019, no more than 0.9 % of purple grains were observed for the recipient Raduga when using a buffer zone of 15 m and more from the pollen donor in the wind rose direction. Based on the results of field experiments, the isolation distance from 15 m or more can be recommended to exclude cross-pollination of maize within the threshold of 0.9 % in the conditions of the South-East of the European part of Russia.

ASSESSMENT OF THE RISKS OF CROSS-POLLINATION DURING CO-CULTIVATION OF MAIZE LINES IN THE SOUTH-EAST EUROPEAN RUSSIA

The new Russian Federal law (No. 358 of 03.07.2016) prohibits the commercial use of GM plants in agriculture, but allows, since 2018, for the first time in Russia their cultivation and testing for research purposes. Consequently, there is a need to assess and develop criteria for safe co-cultivation of non-GM and GM varieties, which are currently absent in Russia. In this paper, it was established for the first time that the 10-15 m distance is sufficient to prevent cross-pollination between maize lines with an acceptable presence of 0.9 % of the donor’s genetic material, regardless of the recipient line, donor and recipient time flowering, and the wind direction in Saratov condition (South-West region of European part of Russia). The work aimed to assess the influence of the distance between pollen donor and recipient, wind direction, donor and recipient time flowering, and a buffer zone presence between them on the crossing frequency in mixed maize crops. The maize lines Korichnevyi marker (KM), GPL-1, Zarodyshevyi marker Saratovskii Purpurnyi (ZMS-P), Purpurnaya Saratovskaya (PS), as well as hybrids Purpurnyi (GP), Raduga and Tester 3 were grown (the experimental field of the Rossorgo, Saratov, South-West region of European Russia, 2018-2019). We planted the GP and ZMS-P lines as pollen donors in 2018 on a 3×80 m2 area with planting density of 7-10 plants per 1 m2. Between the pollen donor area, maize KM and GPL-1 lines were planted, and around them there were a 1290 m2 area of yellow-colored grain recipients (Raduga and Tester 3 hybrids). In September, 5-12 ears from each pollen recipient were harvested. The cross-pollination frequency was calculated as the ratio of purple grains (GP pollination result) or yellow grains with a purple spot (ZMS-P pollination result) to the total grain number in recipient lines. In 2018 it was established that the maximum percentage (from 0.1 to 13.2 %) derived from cross-pollination with two pollen donors depends on different factors. At closer distances (1-4 m), the cross-pollination increased 4-fold for the earlier flowering recipient. The percentage of crosses for recipient Raduga decreased 3 times with a 10 m increase in the distance and 11 times at a 40 m distance from the donor plants. Experiments in 2018 indicate that the 10 m distance from the pollen donor guarantees the percentage of crosses not exceeding the 0.9 % GM threshold in food products accepted in the European Union and Russia. In 2019, we used PS inbred line as a pollen donor. The PS was planted on a 3×5 m plot with Sudanese grass (Sorghum × drummondii) Allegory cultivar as a buffer zone 3 m wide to the East and West and 15 m long to the South-West and North-East. Yellow-grain hybrid Raduga was planted around the buffer zone. The frequency of crosses was calculated as the ratio of the number of purple grains to the total number of Raduga grains per ear. In 2018, the frequency of crosses was also estimated depending on the synchronism of flowering between pollen donors and recipients. The GPL-1 recipient with a 9-day difference from PG (pollen donor) in the beginning of flowering showed a 4 % lower pollination rate compared to a KM line with a flowering period closer to the PG pollen donor (1-day difference). Tall plants of PG donor of pollen prevented spreading pollen from a short ZMS-P donor to the Tester 3 and Raduga recipients in the direction of the wind rose. In 2019, no more than 0.9 % of purple grains were observed for the recipient Raduga when using a buffer zone of 15 m and more from the pollen donor in the wind rose direction. Based on the results of field experiments, the isolation distance from 15 m or more can be recommended to exclude cross-pollination of maize within the threshold of 0.9 % in the conditions of the South-East of the European part of Russia.