Antitumor Effects of an Anthocyanin-Rich Grain Diet in a Mouse Model of Lewis Lung Carcinoma.

Functional foods enriched with plant polyphenol anthocyanins attract particular attention due to their health-promoting properties, including antitumor activity. We evaluated the effects of a grain diet rich in anthocyanins in a mouse model of Lewis lung carcinoma. Mice of the C57BL/6 strain were fed with wheat of near-isogenic lines differing in the anthocyanin content for four months prior to tumor transplantation. Although a significant decrease in the size of the tumor and the number of metastases in the lungs was revealed in the groups with both types of grain diet, the highest percentage of animals without metastases and with attenuated cell proliferation in the primary tumor were observed in the mice with the anthocyanin-rich diet. Both grain diets reduced the body weight gain and spleen weight index. The antitumor effects of the grain diets were associated with the activation of different mechanisms: immune response of the allergic type with augmented interleukin(IL)-9 and eotaxin serum levels in mice fed with control grain vs. inhibition of the IL-6/LIF system accompanied by a decrease in the tumor-associated M2 macrophage marker arginase 1 gene mRNA levels and enhanced autophagy in the tumor evaluated by the mRNA levels of Beclin 1 gene. Thus, anthocyanin-rich wheat is suggested as a promising source of functional nutrition with confirmed in vivo antitumor activity.

Analysis of Functional Single-Nucleotide Polymorphisms (SNPs) and Leaf Quality in Tea Collection under Nitrogen-Deficient Conditions.

This study discusses the genetic mutations that have a significant association with economically important traits that would benefit tea breeders. The purpose of this study was to analyze the leaf quality and SNPs in quality-related genes in the tea plant collection of 20 mutant genotypes growing without nitrogen fertilizers. Leaf N-content, catechins, L-theanine, and caffeine contents were analyzed in dry leaves via HPLC. Additionally, the photochemical yield, electron transport efficiency, and non-photochemical quenching were analyzed using PAM-fluorimetry. The next generation pooled amplicon-sequencing approach was used for SNPs-calling in 30 key genes related to N metabolism and leaf quality. The leaf N content varied significantly among genotypes (p ≤ 0.05) from 2.3 to 3.7% of dry mass. The caffeine content varied from 0.7 to 11.7 mg g-1, and the L-theanine content varied from 0.2 to 5.8 mg g-1 dry leaf mass. Significant positive correlations were detected between the nitrogen content and biochemical parameters such as theanine, caffeine, and most of the catechins. However, significant negative correlations were observed between the photosynthetic parameters (Y, ETR, Fv/Fm) and several biochemical compounds, including rutin, Quercetin-3-O-glucoside, Kaempferol-3-O-rutinoside, Kaempferol-3-O-glucoside, Theaflavin-3'-gallate, gallic acid. From our SNP-analysis, three SNPs in WRKY57 were detected in all genotypes with a low N content. Moreover, 29 SNPs with a high or moderate effect were specific for #316 (high N-content, high quality) or #507 (low N-content, low quality). The use of a linear regression model revealed 16 significant associations; theaflavin, L-theanine, and ECG were associated with several SNPs of the following genes: ANSa, DFRa, GDH2, 4CL, AlaAT1, MYB4, LHT1, F3'5'Hb, UFGTa. Among them, seven SNPs of moderate effect led to changes in the amino acid contents in the final proteins of the following genes: ANSa, GDH2, 4Cl, F3'5'Hb, UFGTa. These results will be useful for further evaluations of the important SNPs and will help to provide a better understanding of the mechanisms of nitrogen uptake efficiency in tree crops.

WAX INDUCER 1 Regulates ß-Diketone Biosynthesis by Mediating Expression of the Cer-cqu Gene Cluster in Barley.

Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with ß-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the ß-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

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.

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene.

BACKGROUND: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. RESULTS: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. CONCLUSIONS: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high ß-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

The mechanism of potato resistance to Globodera rostochiensis: comparison of root transcriptomes of resistant and susceptible Solanum phureja genotypes.

BACKGROUND: Globodera rostochiensis belongs to major potato pathogens with a sophisticated mechanism of interaction with roots of the host plants. Resistance of commercial varieties is commonly based on specific R genes introgressed from natural populations of related wild species and from native potato varieties grown in the Andean highlands. Investigation of molecular resistance mechanisms and screening the natural populations for novel R genes are important for both fundamental knowledge on plant pathogen interactions and breeding for durable resistance. Here we exploited the Solanum phureja accessions collected in South America with contrasting resistance to G. rostochiensis. RESULTS: The infestation of S. phureja with G. rostochiensis juveniles resulted in wounding stress followed by activation of cell division and tissue regeneration processes. Unlike the susceptible S. phureja genotype, the resistant accession reacted by rapid induction of variety of stress response related genes. This chain of molecular events accompanies the hypersensitive response at the juveniles' invasion sites and provides high-level resistance. Transcriptomic analysis also revealed considerable differences between the analyzed S. phureja genotypes and the reference genome. CONCLUSION: The molecular processes in plant roots associated with changes in gene expression patterns in response to G. rostochiensis infestation and establishment of either resistant or susceptible phenotypes are discussed. De novo transcriptome assembling is considered as an important tool for discovery of novel resistance traits in S. phureja accessions.

Effects of Grape Polyphenols on the Life Span and Neuroinflammatory Alterations Related to Neurodegenerative Parkinson Disease-Like Disturbances in Mice.

Functional nutrition is a valuable supplementation to dietary therapy. Functional foods are enriched with biologically active substances. Plant polyphenols attract particular attention due to multiple beneficial properties attributed to their high antioxidant and other biological activities. We assessed the effect of grape polyphenols on the life span of C57BL/6 mice and on behavioral and neuroinflammatory alterations in a transgenic mouse model of Parkinson disease (PD) with overexpression of the A53T-mutant human α-synuclein. C57BL/6 mice were given a dietary supplement containing grape polyphenol concentrate (GPC-1.5 mL/kg/day) with drinking water from the age of 6-8 weeks for life. Transgenic PD mice received GPC beginning at the age of 10 weeks for four months. GPC significantly influenced the cumulative proportion of surviving and substantially augmented the average life span in mice. In the transgenic PD model, the grape polyphenol (GP) diet enhanced memory reconsolidation and diminished memory extinction in a passive avoidance test. Behavioral effects of GP treatment were accompanied by a decrease in α-synuclein accumulation in the frontal cortex and a reduction in the expression of neuroinflammatory markers (IBA1 and CD54) in the frontal cortex and hippocampus. Thus, a GP-rich diet is recommended as promising functional nutrition for aging people and patients with neurodegenerative disorders.

Structural and functional divergence of the Mpc1 genes in wheat and barley.

BACKGROUND: The members of the Triticeae tribe are characterised by the presence of orthologous and homoeologous gene copies regulating flavonoid biosynthesis. Among transcription factors constituting a regulatory MBW complex, the greatest contribution to the regulation of flavonoid biosynthetic pathway is invested by R2R3-Myb-type TFs. Differently expressed R2R3-Myb copies activate the synthesis of various classes of flavonoid compounds in different plant tissues. The aim of this research was the identification, comparison and analysis of full-length sequences of the duplicated R2R3-Myb Mpc1 (Myb protein c1) gene copies in barley and wheat genomes. RESULTS: The Mpc1 genes were identified in homoeologous group 4 and 7 chromosomes: a total of 3 copies in barley (Hordeum vulgare L.) and 8 copies in bread wheat (Triticum aestivum L.) genomes. All Mpc1 genes have a similar two-exon structure, and almost all of them are transcriptionally active. The calculation of the divergence time revealed that first duplication between 4 and 7 chromosomes of the common ancestor of the Triticeae tribe occurred about 35-46 million years ago (MYA); the last duplication arised about 16-19 MYA before the divergence Triticum and Hordeum genera The connection between gene expression and the appearance of anthocyanin pigmentation was found for three genes from homoeologous group 4 chromosomes: TaMpc1-A2 (5AL) in wheat coleoptile, HvMpc1-H2 (4HL) in barley lemma and aleurone layer, and HvMpc1-H3 (4HL) in barley aleurone layer. TaMpc1-D4 (4DL) from the wheat genome showed a strong level of expression regardless of the colour of coleoptile or pericarp. It is assumed, that this gene regulates the biosynthesis of uncoloured flavonoids in analysed tissues. CONCLUSIONS: The regulatory R2R3-Myb genes involved in anthocyanin synthesis were identified and characterised in Triticeae tribe species. Genes designated HvMpc1-H2 and HvMpc1-H3 appeared to be the main factors underlying intraspecific variation of H. vulgare by lemma and aleurone colour. TaMpc1-A2 is the co-regulator of the Mpc1-1 genes in bread wheat genome controlling anthocyanin synthesis in coleoptile.

SNPs associated with barley resistance to isolates of Pyrenophora teres f. teres.

BACKGROUND: Net blotch caused by Pyrenophra teres f. teres is a major foliar disease of barley. Infection can result in significant yield losses of susceptible cultivars of up to 40%. Of the two forms of net blotch (P. teres f. teres and P. teres f. maculata), P. teres f. teres (net form of net blotch) is the dominant one in Russia. The goal of the current study was to identify genomic regions associated with seedling resistance to several pathotypes of the net form of net blotch in Siberian spring barley genotypes. For this, a genome-wide association study of a Siberian barley collection, genotyped with 50 K Illumina SNP-chip, was carried out. RESULTS: Seedling resistance of 94 spring barley cultivars and lines to four Pyrenophora teres f. teres isolates (S10.2, K5.1, P3.4.0, and A2.6.0) was investigated. According to the Tekauz rating scale, 25, 21, 14, and 14% of genotypes were highly resistant, and 19, 8, 9, and 16% of genotypes were moderate-resistant to the isolates S10.2, K5.1, P3.4.0, and A2.6.0, respectively. Eleven genotypes (Alag-Erdene, Alan-Bulag, L-259/528, Kedr, Krymchak 55, Omsky golozyorny 2, Omsky 13709, Narymchanin, Pallidum 394, Severny and Viner) were resistant to all studied isolates. Nine additional cultivars (Aley, Barkhatny, Belogorsky, Bezenchuksky 2, Emelya, G-19980, Merit 57, Mestny Primorsky, Slavaynsky) were resistant to 3 of the 4 isolates. The phenotyping and genotyping data were analysed using several statistical models: GLM + Q, GLM + PCA, GLM + PCA + Q, and the MLM + kinship matrix. In total, 40 SNPs in seven genomic regions associated with net blotch resistance were revealed: the region on chromosome 1H between 57.3 and 62.8 cM associated with resistance to 2 isolates (to P3.4.0 at the significant and K5.1 at the suggestive levels), the region on chromosome 6H between 52.6 and 55.4 cM associated with resistance to 3 isolates (to P3.4.0 at the significant and K5.1 and S10.2 at the suggestive levels), three isolate-specific significant regions (P3.4.0-specific regions on chromosome 2H between 71.0 and 74.1 cM and on chromosome 3H between 12.1 and 17.4 cM, and the A2.6.0-specific region on chromosome 3H between 50.9 and 54.8 cM), as well as two additional regions on chromosomes 2H (between 23.2 and 23.8 cM, resistant to S10.2) and 3 (between 135.6 and 137.5 cM resistant to K5.1) with suggestive SNPs, coinciding, however, with known net blotch resistance quantitative trait loci (QTLs) at the same regions. CONCLUSIONS: Seven genomic regions on chromosomes 1H, 2H, 3H, and 6H associated with the resistance to four Pyrenophora teres f. teres isolates were identified in a genome-wide association study of a Siberian spring barley panel. One novel isolate-specific locus on chromosome 3 between 12.1 and 17.4 cM was revealed. Other regions identified in the current study coincided with previously known loci conferring resistance to net blotch. The significant SNPs revealed in the current study can be converted to convenient PCR markers for accelerated breeding of resistant barley cultivars.

Myc-like transcriptional factors in wheat: structural and functional organization of the subfamily I members.

BACKGROUND: Myc-like regulatory factors carrying the basic helix-loop-helix (bHLH) domain belong to a large superfamily of transcriptional factors (TFs) present in all eukaryotic kingdoms. In plants, the representatives of this superfamily regulate diverse biological processes including growth and development as well as response to various stresses. As members of the regulatory MBW complexes, they participate in biosynthesis of flavonoids. In wheat, only one member (TaMyc1) of the Myc-like TFs family has been studied, while structural and functional organization of further members remained uncharacterized. From two Myc-subfamilies described recently in the genomes of Triticeae tribe species, we investigated thoroughly the members of the subfamily I which includes the TaMyc1 gene. RESULTS: Comparison of the promoter regions of the Myc subfamily I members in wheat suggested their division into two groups (likely homoeologous sets): TaMyc-1 (TaMyc-A1/TaMyc1, TaMyc-B1, TaMyc-D1) and TaMyc-2 (TaMyc-A2 and TaMyc-D2). It was demonstrated that the TaMyc-D1 copy has lost its functionality due to the frame shift mutation. The study of functional features of the other four copies suggested some of them to be involved in the biosynthesis of anthocyanins. In particular, TaMyc-B1 is assumed to be a co-regulator of the gene TaC1-A1 (encoding R2R3-Myb factor) in the MBW regulatory complex activating anthocyanin synthesis in wheat coleoptile. The mRNA levels of the TaMyc-A1, TaMyc-B1, TaMyc-A2 and TaMyc-D2 genes increased significantly in wheat seedlings exposed to osmotic stress. Salinity stress induced expression of TaMyc-B1 and TaMyc-A2, while TaMyc-A1 was repressed. CONCLUSIONS: The features of the structural and functional organization of the members of subfamily I of Myc-like TFs in wheat were determined. Myc-like co-regulator (TaMyc-B1) of anthocyanin synthesis in wheat coleoptile was described for the first time. The Myc-encoding genes presumably involved in response to drought and salinity were determined in wheat. The results obtained are important for further manipulations with Myc genes, aimed on increasing wheat adaptability.

Purple-grained barley (Hordeum vulgare L.): marker-assisted development of NILs for investigating peculiarities of the anthocyanin biosynthesis regulatory network.

BACKGROUND: Anthocyanins are plants secondary metabolites important for plant adaptation to severe environments and potentially beneficial to human health. Purple colour of barley grain is caused by the pigments synthesized in pericarp. One or two genes determine the trait. One of them is Ant2 mapped on chromosome 2HL and is known to encode transcription factor (TF) with a bHLH domain. In plants, bHLH regulates anthocyanin biosynthesis together with TF harboring an R2R3-MYB domain. In wheat, the R2R3-MYBs responsible for purple colour of grain pericarp are encoded by the homoallelic series of the Pp-1 genes that were mapped on the short arms of chromosomes 7. In barley, in orthologous positions to wheat's Pp-1, the Ant1 gene determining red colour of leaf sheath has been mapped. In the current study, we tested whether Ant1 has pleiotropic effect not only on leaf sheath colour but also on pericarp pigmentation. RESULTS: А set of near isogenic lines (NILs) carrying different combinations of alleles at the Ant1 and Ant2 loci was created using markers-assisted backcrossing approach. The dominant alleles of both the Ant1 and Ant2 genes are required for anthocyanin accumulation in pericarp. A qRT-PCR analysis of the Ant genes in lemma and pericarp of the NILs revealed that some reciprocal interaction occurs between the genes. Expression of each of the two genes was up-regulated in purple-grained line with dominant alleles at the both loci. The lines carrying dominant allele either in the Ant1 or in the Ant2 locus were characterized by the decreased level of expression of the dominant gene and scant activity of the recessive one. The Ant1 and Ant2 expression was barely detected in uncolored line with recessive alleles at both loci. The anthocyanin biosynthesis structural genes were differently regulated: Chs, Chi, F3h, Dfr were transcribed in all lines independently on allelic state of the Ant1 and Ant2 genes, whereas F3'h and Ans were activated in presence on dominant alleles of the both regulatory genes. CONCLUSIONS: The R2R3-MYB-encoding counterpart (Ant1) of the regulatory Ant2 gene was determined for the first time. The dominant alleles of both of them are required for activation of anthocyanin synthesis in barley lemma and pericarp. The R2R3-MYB + bHLH complex activates the synthesis via affecting expression of the F3'h and Ans structural genes. In addition, positive regulatory loop between Ant1 and Ant2 was detected. Earlier the interaction between the anthocyanin biosynthesis regulatory genes has been revealed in dicot plant species only. Our data demonstrated that the regulatory mechanism is considered to be more common for plant kingdom than it has been reported so far.

Biochemical and genetic polymorphism of Bromopsis inermis populations under chronic radiation exposure.

MAIN CONCLUSION: For the subsequent assessment of the genetic mechanisms responsible for the resistance of plants to chronic irradiation, the analysis of RAPD-cDNA with the subsequent isolation, cloning, and sequencing of expressed polymorphic sequences is a promising technique. A study was conducted on Bromopsis inermis populations that have been growing for a long time in the EURT area. Using RAPD primers, we studied the genetic spectra of plants. In analysing the UPGMA algorithm, we identified two well-distinguishable clusters with a high level of bootstrap support (> 85%): background samples hit the first, and impact samples hit the second. Our data indicate a decrease in diversity in the most polluted population, as well as the appearance of new alleles in chronically irradiated samples of the B. inermis. Smooth brome seedlings were characterised by the content of anthocyanins, comparable with other types of cereals. In the gradient of chronic irradiation, the relative content of anthocyanins was not significantly changed. For the first time, the partial nucleotide sequences of the key genes of anthocyanin biosynthesis (Chi and F3h) in the brome were determined, these sequences were found to be 191 and 356 bp in length, respectively, and were cloned and sequenced. Three copies of the Chi gene were identified in the B. inermis genome. One copy (BiChi-1) clustered with the sequences of the Aegilops tauschii gene (D genome), and the other two copies (BiChi-2 and BiChi-3) formed a separate cluster in the Pooideae subfamily adjacent to Hordeum vulgare. In the copy of BiChi-1, a complete deletion of intron 1 was detected. For the F3h gene, one copy of the B. inermis gene was obtained, which forms a separate branch in the subfamily Pooideae.

Genetic control of anthocyanin pigmentation of potato tissues.

BACKGROUND: The cultivated potato Solanum tuberosum L. is the fourth most important crop worldwide. Anthocyanins synthesis and accumulation in potato tissues are considered as one of important traits related to stress resistance and nutritional value. It is considered that the major regulatory gene for anthocyanin biosynthesis is R2R3 MYB-encoding gene StAN1. However, the genetic control of pigmentation of different potato tissues is substantially under investigated. The development of genetic markers for breeding of potato with specific pigmentation pattern remains an actual task. RESULTS: We investigated 36 potato varieties and hybrids with different pigmentation of tubers and leaves. Sequence organization of regulatory R2R3 MYB (StAN1, StMYBA1, StMYB113), bHLH (StbHLH1, StJAF13) and WD40 (StWD40) genes potentially controlling anthocyanin biosynthesis has been evaluated. The results demonstrated a high variability in the StAN1 third exon and promoter region with the exception for 35 bp, containing elements for the transcription start and activation of gene expression in roots. The analysis of transcriptional activity of genes coding R2R3 MYBs, bHLHs and WD40 transcriptional factors in leaves of eight potato genotypes with different anthocyanin pigmentation was performed. The results showed a relation between the gene expression level and plant pigmentation only for the StAN1 and StWD40 genes, while other studied genes had either strong expression in all varieties and hybrids (StMYBA1, StbHLH1 and StJAF13) or they were not expressed at all (StMYB113). CONCLUSIONS: It was found that StAN1 is the major regulatory gene controlling potato anthocyanin synthesis. However, diagnostic markers developed for the functional StAN1 alleles (StAN1777 and StAN1816) can not be used efficiently for prediction of potato pigmentation patterns. It is likely that the sequence organization of StAN1 promoter is important for anthocyanin synthesis control and the development of additional diagnostic markers is necessary.

Organization and evolution of the chalcone synthase gene family in bread wheat and relative species.

BACKGROUND: Flavonoid compounds are secondary plant metabolites, having a functional importance in plant development, protection from pathogens and unfavorable environmental factors. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; it is involved in biosynthesis of all classes of flavonoid compounds. Nevertheless, the Chs gene family in bread wheat (Triticum aestivum L.) has been not characterized yet. The aim of the current study was to investigate structural and functional organization of the Chs genes and evolution of this gene family in bread wheat and relative species. RESULTS: The nucleotide sequences of the eight Chs copies in T. aestivum were identified. Among them, two homoeologous sets of the Chs genes were located on the short (Chs-A1, -B1, -D1) and the long (Chs-A4, -B4, -D4) arms of homoeologous group 2 chromosomes. Two paralogous gene copies in the B-genome (Chs-B2, -B3) were located in the distal regions of 2BS chromosome. To clarify the origin of Chs duplications in the B-genome the phylogenetic analysis with the Chs sequences of Triticum and Aegilops species carrying ancestral genomes was conducted. It was estimated that the first duplication event occurred in the genome of the common ancestor of Triticum and Aegilops genera about 10-12 million years ago (MYA), then another copy was formed in the ancestor of the B-genome about 6-7 MYA. A homology modeling revealed high sequence similarity of bread wheat CHS enzymes. A number of short deletions in coding regions of some Chs sequences are not expected to have any significant functional effects. Estimation of transcriptional activity of the Chs copies along with a comparative analysis of their promoters structure suggested their functional specialization, which likely contributed to the maintaining of the duplicated Chs genes in wheat genome. CONCLUSIONS: From possible ten Chs copies in bread wheat genome, eight members of this family retained their intact structure and activity, while two copies appear to be lost at the level of diploid and tetraploid ancestors. Transcriptional assay along with a comparative analysis of the cis-regulatory elements revealed their functional diversification. The multiple functions supported by the Chs family are assumed to be a driving force for duplications of the Chs gene and their retention in plant genome.

Starch phosphorylation associated SNPs found by genome-wide association studies in the potato (Solanum tuberosum L.).

BACKGROUND: The natural variation of starch phosphate content in potatoes has been previously reported. It is known that, in contrast to raw starch, commercially phosphorylated starch is more stable at high temperatures and shear rates and has higher water capacity. The genetic improvement of phosphate content in potato starch by selection or engineering would allow the production of phosphorylated starch in a natural, environmentally friendly way without chemicals. The aim of the current research is to identify genomic SNPs associated with starch phosphorylation by carrying out a genome-wide association study in potatoes. RESULTS: A total of 90 S. tuberosum L. varieties were used for phenotyping and genotyping. The phosphorus content of starch in 90 potato cultivars was measured and then statistically analysed. Principal component analysis (PCA) revealed that the third and eighth principal components appeared to be sensitive to variation in phosphorus content (p = 0.0005 and p = 0.002, respectively). PC3 showed the correlation of starch phosphorus content with allelic variations responsible for higher phosphorylation levels, found in four varieties. Similarly, PC8 indicated that hybrid 785/8-5 carried an allele associated with high phosphorus content, while the Impala and Red Scarlet varieties carried alleles for low phosphorus content. Genotyping was carried out using an Illumina 22 K SNP potato array. A total of 15,214 scorable SNPs (71.7% success rate) was revealed. GWAS mapping plots were obtained using TASSEL based on several statistical models, including general linear models (GLMs), with and without accounting for population structure, as well as MLM. A total of 17 significant SNPs was identified for phosphorus content in potato starch, 14 of which are assigned to 8 genomic regions on chromosomes 1, 4, 5, 7, 8, 10, and 11. Most of the SNPs identified belong to protein coding regions; however, their allelic variation was not associated with changes in protein structure or function. CONCLUSIONS: A total of 8 novel genomic regions possibly associated with starch phosphorylation on potato chromosomes 1, 4, 5, 7, 8, 10, and 11 was revealed. Further validation of the SNPs identified and the analysis of the surrounding genomic regions for candidate genes will allow better understanding of starch phosphorylation biochemistry. The most indicative SNPs may be useful for developing diagnostic markers to accelerate the breeding of potatoes with predetermined levels of starch phosphorylation.

The factors affecting the evolution of the anthocyanin biosynthesis pathway genes in monocot and dicot plant species.

BACKGROUND: The available data demonstrate that even in universal metabolic pathways, some species-specific regulatory features of structural genes are present. For instance, in the anthocyanin biosynthesis pathway (ABP), genes may be regulated by ABP-specific regulatory factors, and their expression levels may be strongly associated with anthocyanin pigmentation, or they may be expressed independently of pigmentation. A dataset of orthologous ABP genes (Chs, Chi, F3h, F3'h, Dfr, Ans) from monocot and dicot plant species that have distinct gene regulation patterns and different types of pollination was constructed to test whether these factors affect the evolution of the genes. RESULTS: Using a maximum likelihood approach, we demonstrated that although the whole set of the ABP genes is under purifying selection, with greater selection acting on the upstream genes than on the downstream genes, genes from distinct groups of plant species experienced different strengths of selective pressure. The selective pressure on the genes was higher in dicots than in monocots (F3h and further downstream genes) and in pollinator-dependent plants than in pollinator-independent species (Chi and further downstream genes), suggesting an important role of pollination type in the evolution of the anthocyanin biosynthesis gene network. Contrasting effects of the regulation patterns on evolution were detected for the F3h and Dfr genes, with greater selective pressure on the F3h gene in plant species where the gene expression was not strongly associated with pigmentation and greater selective pressure on Dfr in plant species where the gene expression was associated with pigmentation. CONCLUSIONS: We demonstrated the effects of pollination type and patterns of regulation on the evolution of the ABP genes, but the evolution of some of the genes could not be explained in the framework of these factors, such as the weaker selective pressure acting on Chs in species that attract pollinators or the stronger selective pressure on F3h in plant species where the gene expression was not associated with pigmentation. The observations suggest that additional factors could affect the evolution of these genes. One such factor could be an effect of gene duplication with further division of functions among gene copies and relaxed selective pressure acting on them. Additional tests with an appropriate dataset combining data on duplicated gene sequences and their functions in the flavonoid biosynthesis pathway are required to test this hypothesis.

Identification and characterization of regulatory network components for anthocyanin synthesis in barley aleurone.

BACKGROUND: Among natural populations, there are different colours of barley (Hordeum vulgare L.). The colour of barley grains is directly related to the accumulation of different pigments in the aleurone layer, pericarp and lemma. Blue grain colour is due to the accumulation of anthocyanins in the aleurone layer, which is dependent on the presence of five Blx genes that are not sequenced yet (Blx1, Blx3 and Blx4 genes clustering on chromosome 4HL and Blx2 and Blx5 on 7HL). Due to the health benefits of anthocyanins, blue-grained barley can be considered as a source of dietary food. The goal of the current study was to identify and characterize components of the anthocyanin synthesis regulatory network for the aleurone layer in barley. RESULTS: The candidate genes for components of the regulatory complex MBW (consisting of transcription factors MYB, bHLH/MYC and WD40) for anthocyanin synthesis in barley aleurone were identified. These genes were designated HvMyc2 (4HL), HvMpc2 (4HL), and HvWD40 (6HL). HvMyc2 was expressed in aleurone cells only. A loss-of-function (frame shift) mutation in HvMyc2 of non-coloured compared to blue-grained barley was revealed. Unlike aleurone-specific HvMyc2, the HvMpc2 gene was expressed in different tissues; however, its activity was not detected in non-coloured aleurone in contrast to a coloured aleurone, and allele-specific mutations in its promoter region were found. The single-copy gene HvWD40, which encodes the required component of the regulatory MBW complex, was expressed constantly in coloured and non-coloured tissues and had no allelic differences. HvMyc2 and HvMpc2 were genetically mapped using allele-specific developed CAPS markers developed. HvMyc2 was mapped in position between SSR loci XGBS0875-4H (3.4 cM distal) and XGBM1048-4H (3.4 cM proximal) matching the region chromosome 4HL where the Blx-cluster was found. In this position, one of the anthocyanin biosynthesis structural genes (HvF3'5'H) was also mapped using an allele-specific CAPS-marker developed in the current study. CONCLUSIONS: The genes involved in anthocyanin synthesis in the barley aleurone layer were identified and characterized, including components of the regulatory complex MBW, from which the MYC-encoding gene (HvMyc2) appeared to be the main factor underlying variation of barley by aleurone colour.

Identification of 50 K Illumina-chip SNPs associated with resistance to spot blotch in barley.

BACKGROUND: Spot blotch, caused by Cochliobolus sativus, is one of the most widespread and harmful diseases in barley. Identification of genetic loci associated with resistance to C. sativus is of importance for future marker-assisted selection. The goal of the current study was to identify loci conferring seedling resistance to two different pathotypes of C. sativus in the Siberian spring barley core collection. RESULTS: A total of 96 spring barley cultivars and lines were phenotyped at the seedling stage with two C. sativus isolates (Kr2 and Ch3). According to the Fetch-Steffenson rating scale 16%/17% of genotypes were resistant and 26%/30% were moderate-resistant to the Kr2/Ch3 isolates respectively. A total of 94 genotypes were analyzed with the barley 50 K Illumina Infinium iSELECT assay. From 44,040 SNPs, 40,703 were scorable, from which 39,140 were polymorphic. 27,319 SNPs passed filtering threshold and were used for association mapping. Data analysis by GLM revealed 48 and 41 SNPs for Kr2 and Ch3 isolates, respectively. After application of 5% Bonferroni multiple test correction, only 3 and 27 SNPs were identified, respectively. A total of three genomic regions were associated with the resistance. The region on chromosome 3H associated with Ch3-resistance was expanded between markers SCRI_RS_97417 and JHI-Hv50k-2016-158003 and included 11 SNPs, from which JHI-Hv50k-2016-157070, JHI-Hv50k-2016-156842 had the lowest p-values. These two SNPs were also significant in case of Kr2 isolate. The region on chromosome 2H included 16 loci (7 of them with the lowest p-values were tightly linked to BOPA2_12_11504). Three loci corresponding to this region had suggestive p-values in case of Kr2 tests, so the locus on chromosome 2H may also contribute to resistance to Kr2 isolate. The third region with significant p-value in case of Kr2 tests was identified on chromosome 1H at the locus JHI-Hv50k-2016-33568. CONCLUSIONS: Three genomic regions associated with the resistance to one or both isolates of C. sativus were identified via screening of the Siberian spring barley core collection. Comparison of their location with QTLs revealed previously either with biparental mapping populations studies or with GWAS of distinct germplasm and other isolates, demonstrated that resistance to isolates Kr2 and Ch3 is conferred by known spot blotch resistance loci. Information on SNPs related can be used further for development of DNA-markers convenient for diagnostics of resistance-associated alleles in barley breeding programs.

Metabolic pathways and genes identified by RNA-seq analysis of barley near-isogenic lines differing by allelic state of the Black lemma and pericarp (Blp) gene.

BACKGROUND: Some plant species have 'melanin-like' black seed pigmentation. However, the chemical and genetic nature of this 'melanin-like' black pigment have not yet been fully explored due to its complex structure and ability to withstand almost all solvents. Nevertheless, identification of genetic networks participating in trait formation is key to understanding metabolic processes involved in the expression of 'melanin-like' black seed pigmentation. The aim of the current study was to identify differentially expressed genes (DEGs) in barley near-isogenic lines (NILs) differing by allelic state of the Blp (black lemma and pericarp) locus. RESULTS: RNA-seq analysis of six libraries (three replicates for each line) was performed. A total of 957 genome fragments had statistically significant changes in expression levels between lines BLP and BW, with 632 fragments having increased expression levels in line BLP and 325 genome fragments having decreased expression. Among identified DEGs, 191 genes were recognized as participating in known pathways. Among these were metabolic pathways including 'suberin monomer biosynthesis', 'diterpene phytoalexins precursors biosynthesis', 'cutin biosynthesis', 'cuticular wax biosynthesis', and 'phenylpropanoid biosynthesis, initial reactions'. Differential expression was confirmed by real-time PCR analysis of selected genes. CONCLUSIONS: Metabolic pathways and genes presumably associated with black lemma and pericarp colour as well as Blp-associated resistance to oxidative stress and pathogens, were revealed. We suggest that the black pigmentation of lemmas and pericarps is related to increased level of phenolic compounds and their oxidation. The effect of functional Blp on the synthesis of ferulic acid and other phenolic compounds can explain the increased antioxidant capacity and biotic and abiotic stress tolerance of black-grained cereals. Their drought tolerance and resistance to diseases affecting the spike may also be related to cuticular wax biosynthesis. In addition, upregulated synthesis of phytoalexins, suberin and universal stress protein (USP) in lemmas and pericarps of the Blp carriers may contribute to their increased disease resistance. Further description of the DEGs haplotypes and study of their association with physiological characteristics may be useful for future application in barley pre-breeding.

Differential expression of NBS-LRR-encoding genes in the root transcriptomes of two Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis.

BACKGROUND: The characterization of major resistance genes (R genes) in the potato remains an important task for molecular breeding. However, R genes are rapidly evolving and frequently occur in genomes as clusters with complex structures, and their precise mapping and identification are complicated and time consuming. RESULTS: Comparative analysis of root transcriptomes of Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis revealed a number of differentially expressed genes. However, compiling a list of candidate R genes for further segregation analysis was hampered by their scarce annotation. Nevertheless, combination of transcriptomic analysis with data on predicted potato NBS-LRR-encoding genes considerably improved the quality of the results and provided a reasonable number of candidate genes that provide S. phureja with strong resistance to the potato golden cyst nematode. CONCLUSION: Combination of comparative analyses of tissue-specific transcriptomes in resistant and susceptible genotypes may be used as an approach for the rapid identification of candidate potato R genes for co-segregation analysis and may be used in parallel with more sophisticated studies based on genome resequencing.