Sequential expression of raffinose synthase and stachyose synthase corresponds to successive accumulation of raffinose, stachyose and verbascose in developing seeds of Lens culinaris Medik.

Raffinose, stachyose and verbascose form the three major members of the raffinose family oligosaccharides (RFO) accumulated during seed development. Raffinose synthase (RS; EC 2.4.1.82) and stachyose synthase (STS; EC 2.4.1.67) have been associated with raffinose and stachyose synthesis, but the precise mechanism for verbascose synthesis is not well understood. In this study, full-length RS (2.7 kb) and STS (2.6 kb) clones were isolated by screening a cDNA library prepared from developing lentil seeds (18, 20, 22 and 24 days after flowering [DAF]) to understand the roles of RS and STS in RFO accumulation in developing lentil seeds. The nucleotide sequences of RS and STS genes were similar to those reported for Pisum sativum. Patterns of transcript accumulation, enzyme activities and RFO concentrations were also comparable to P. sativum. However, during lentil seed development raffinose, stachyose and verbascose accumulation corresponded to transcript accumulation for RS and STS, with peak transcript abundance occurring at about 22-24 DAF, generally followed by a sequential increase in raffinose, stachyose and verbascose concentrations followed by a steady level thereafter. Enzyme activities for RS, STS and verbascose synthase (VS) also indicated a sudden increase at around 24-26 DAF, but with an abrupt decline again coinciding with the subsequent steady state increase in the RFO. Galactan:galactan galactosyl transferase (GGT), the galactinol-independent pathway enzyme, however, exhibited steady increase in activity from 24 DAF onwards before abruptly decreasing at 34 DAF. Although GGT activity was detected, isolation of a GGT sequence from the cDNA library was not successful.

Faba bean meal, starch or protein fortification of durum wheat pasta differentially influence noodle composition, starch structure and in vitro digestibility.

Faba bean (Vicia faba L.) flour, starch concentrate (60% starch), protein concentrate (~60% protein) and protein isolate (~85% protein) were added to replace one-quarter of durum wheat semolina to enrich the nutritional quality and physiological functions of durum wheat (Triticum turgidum L.) pasta. The raw pasta samples prepared with protein concentrate or isolate had higher (p ≤ 0.001) protein and lower (p ≤ 0.001) total starch concentrations, along with increased total dietary fiber and slowly digestible starch (p ≤ 0.001) than durum wheat semolina control or those with added whole faba-bean flour or isolated starch. The faba bean fortified pasta had altered starch with increased proportion of medium B-type glucan chains and long C-type glucan chains, reduced starch digestibility and were associated with glycaemia related effects in the human diet. The faba bean fortified pasta had increased protein and dietary fiber that influenced food intake and satiety. The results suggest differential contributions of food ingredients in human health outcomes.

In Vitro Wheat Immature Spike Culture Screening Identified Fusarium Head Blight Resistance in Wheat Spike Cultured Derived Variants and in the Progeny of Their Crosses with an Elite Cultivar.

Fusarium head blight (FHB) is a devastating fungal disease of wheat (Triticum aestivum L.). The lack of genetic resources with stable FHB resistance combined with a reliable and rapid screening method to evaluate FHB resistance is a major limitation to the development of FHB resistant wheat germplasm. The present study utilized an immature wheat spike culture method to screen wheat spike culture derived variants (SCDV) for FHB resistance. Mycotoxin concentrations determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) correlated significantly (P < 0.01) with FHB severity and disease progression during in vitro spike culture. Selected SCDV lines assessed for FHB resistance in a Fusarium field disease nursery in Carman, Manitoba, Canada in 2016 showed significant (P < 0.01) correlation of disease severity to the in vitro spike culture screening method. Selected resistant SCDV lines were also crossed with an elite cv. CDC Hughes and the progeny of F2 and BC1F2 were screened by high resolution melt curve (HRM) analyses for the wheat UDPglucosyl transferase gene (TaUGT-3B) single nucleotide polymorphism to identify resistant (T-allele) and susceptible (G-allele) markers. The progeny from the crosses were also screened for FHB severity using the immature spike culture method and identified resistant progeny grouped according to the HRM genotyping data. The results demonstrate a reliable approach using the immature spike culture to screen for FHB resistance in progeny of crosses in early stage of breeding programs.

Spike culture derived wheat (Triticum aestivum L.) variants exhibit improved resistance to multiple chemotypes of Fusarium graminearum.

Fusarium head blight (FHB) in wheat (Triticum aestivum L.), predominantly caused by Fusarium graminearum, has been categorized into three chemotypes depending on the major mycotoxin produced. The three mycotoxins, namely, 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV) also determine their aggressiveness and response to fungicides. Furthermore, prevalence of these chemotypes changes over time and dynamic changes in chemotypes population in the field have been observed. The objective of this study was to identify spike culture derived variants (SCDV) exhibiting resistance to multiple chemotypes of F. graminearum. First, the optimal volume of inoculum for point inoculation of the spikelets was determined using the susceptible AC Nanda wheat genotype. Fifteen µL of 105 macroconidia/mL was deemed optimal based on FHB disease severity assessment with four chemotypes. Following optimal inoculum volume determination, five chemotypes (Carman-NIV, Carman-705-2-3-ADON, M9-07-1-3-ADON, M1-07-2-15-ADON and China-Fg809-15-ADON) were used to point inoculate AC Nanda spikelets to confirm the mycotoxin produced and FHB severity during infection. Upon confirmation of the mycotoxins produced by the chemotypes, 55 SCDV were utilized to evaluate FHB severity and mycotoxin concentrations. Of the 55 SCDV, five (213.4, 244.1, 245.6, 250.2 and 252.3) resistant lines were identified with resistance to multiple chemotypes and are currently being utilized in a breeding program to develop wheat varieties with improved FHB resistance.

Faba bean protein flours added to pasta reduce post-ingestion glycaemia, and increase satiety, protein content and quality.

The hypothesis that adding faba bean (FB) flour and its macronutrient concentrated flours to pasta reduces postprandial glycaemia and increases satiety was tested in 54 young adult males. Each consumed a serving of pasta made from durum wheat semolina (DWS) alone, or DWS flour with 25% of flours from whole FB (FBF), starch concentrate (FBS), protein concentrate (FBPC), or protein isolate (FBPI). Post-consumption measurements included postprandial blood glucose, insulin, C-peptide, GLP-1 and PYY, and subjective appetite, over 120 min. Second meal effects of treatments were assessed after participants consumed either an ad libitum or fixed size meal (12 kcal kg-1) at a pizza meal at 120 min. Additions of FB flours from FBPC and FBPI reduced postprandial glycaemia and appetite, increased protein content and quality of the pastas and PYY and C-peptide responses, but had no effect on plasma insulin or GLP-1. In conclusion, DWS pastas with added faba bean protein flour reduce postprandial BG and appetite and have higher nutritional quality. The clinical trial registry number is NCT02658591 .

Co-localization of genomic regions associated with seed morphology and composition in a desi chickpea (Cicer arietinum L.) population varying in seed protein concentration.

KEY MESSAGE: Major QTL on LG 1 and 3 control seed filling and seed coat development, thereby affecting seed shape, size, color, composition and weight, key determinants of crop yield and quality. A chickpea (Cicer arietinum L.) population consisting of 189 recombinant inbred lines (RILs) derived from a cross between medium-protein ICC 995 and high-protein ICC 5912 genotypes of the desi market class was analyzed for seed properties. Seed from the parental lines and RILs was produced in four different environments for determination of seed shape (SS), 100-seed weight (100-SW), protein (PRO) and starch (STA) concentration. Polymorphic genetic markers for the population were identified by Genotyping by Sequencing and assembled into a 522.5 cM genetic map. Phenotype data from the different growth environments were analyzed by QTL mapping done by single and multi-environment analyses and in addition, single marker association mapping. The analyses identified in total 11 QTL, of which the most significant (P < 0.05) loci were located on LG 1 (q-1.1), LG 2 (q-2.1), LG 3 (q-3.2, q-3.3), LG 4 (q-4.2), and LG 5 (q-5.1). STA was mostly affected by q-1.1, which explained 19.0% of the phenotypic variance for the trait. The largest QTL effects were demonstrated by q-3.2 that explained 52.5% of the phenotypic variances for 100-SW, 44.3% for PRO, and 14.6% for SS. This locus was also highly associated with flower color (COL; 95.2% explained) and showed q-3.2 alleles from the ICC 5912 parent conferred the blue flower color and production of small, round seeds with relatively high protein concentration. Genes affecting seed filling at q-1.1 and seed coat development at q-3.2, respectively, were considered to underlie differences in seed composition and morphology in the RIL population.

Single Nucleotide Polymorphisms in B-Genome Specific UDP-Glucosyl Transferases Associated with Fusarium Head Blight Resistance and Reduced Deoxynivalenol Accumulation in Wheat Grain.

An in vitro spike culture method was optimized to evaluate Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) and used to screen a population of ethyl methane sulfonate treated spike culture-derived variants (SCDV). Of the 134 SCDV evaluated, the disease severity score of 47 of the variants was ≤30%. Single nucleotide polymorphisms (SNP) in the UDP-glucosyltransferase (UGT) genes, TaUGT-2B, TaUGT-3B, and TaUGT-EST, differed between AC Nanda (an FHB-susceptible wheat variety) and Sumai-3 (an FHB-resistant wheat cultivar). SNP at 450 and 1,558 bp from the translation initiation site in TaUGT-2B and TaUGT-3B, respectively were negatively correlated with FHB severity in the SCDV population, whereas the SNP in TaUGT-EST was not associated with FHB severity. Fusarium graminearum strain M7-07-1 induced early expression of TaUGT-2B and TaUGT-3B in FHB-resistant SCDV lines, which were associated with deoxynivalenol accumulation and reduced FHB disease progression. At 8 days after inoculation, deoxynivalenol concentration varied from 767 ppm in FHB-resistant variants to 2,576 ppm in FHB-susceptible variants. The FHB-resistant SCDV identified can be used as new sources of FHB resistance in wheat improvement programs.

Genotype, environment and G × E interaction influence (1,3;1,4)-ß-d-glucan fine structure in barley (Hordeum vulgare L.).

BACKGROUND: The structure of ß-glucan influences its use in cereal-based foods and feed. The objective of this study was to determine the effect of environment (E) and genotype (G) on ß-glucan fine structure and its genetic control in two-row spring barley with normal starch characteristics. RESULTS: A population of 89 recombinant inbred lines, derived from the cross of two-row spring barley genotypes Merit × H93174006 (H92076F1 × TR238), was characterized for concentration and structure of grain ß-glucan in two environments. Results showed that concentrations of ß-glucan, DP3, DP4 and DP3 + DP4 were positively correlated with each other, suggesting no preference for DP3 or DP4 subunit production in high- or low-ß-glucan lines. The concentrations of ß-glucan, DP3, DP4 and DP3:DP4 ratios were significantly influenced by genotype and environment. However, only DP3:DP4 ratio showed a significant effect of G × E interaction. Association mapping of candidate markers in 119 barley genotypes showed that marker CSLF6_4105 was associated with ß-glucan concentration, whereas Bmac504 and Bmac211 were associated with DP3:DP4 ratio. Bmac273e was associated with both ß-glucan concentration and DP3:DP4 ratio. CONCLUSION: The grain ß-glucan concentration and DP3:DP4 ratio are strongly affected by genotype and environment. Single-marker analyses suggested that the genetic control of ß-glucan concentration and DP3:DP4 ratio was linked to separate chromosomal regions on barley genome. © 2016 Society of Chemical Industry.

Differential expression of two galactinol synthase isoforms LcGolS1 and LcGolS2 in developing lentil (Lens culinaris Medik. cv CDC Redberry) seeds.

Galactinol synthase (GS, EC 2.4.1.123) catalyzes the transfer of a galactosyl residue from UDP-galactose to myo-inositol to synthesize galactinol, a precursor for raffinose family oligosaccharides (RFO) biosynthesis. Screening, a cDNA library constructed with RNA isolated from developing lentil seeds, with partial GS genes resulted in identification of cDNA clones for two isoforms of GS, LcGolS1 (1336 bp, ORF-1002 bp, 334 amino acids) and LcGolS2 (1324bp, ORF-975bp, 325 amino acids) with predicted molecular weights of 38.7 kDa and 37.6 kDa, respectively. During lentil seed development, LcGolS1 transcripts showed higher accumulation during 26-32 days after flowering (DAF) corresponding to seed desiccation, while LcGolS2 showed maximum accumulation at 24 DAF, prior to increase in LcGolS1 transcripts. GS enzyme activity was maximum at 26 and 28 DAF and corresponded to galactinol accumulation, which also increased rapidly at 22 DAF with maximum accumulation at 26 DAF. Substrates for GS activity, myo-inositol and glucose/galactose were present in high concentrations during early stages of seed development but gradually decreased from 20 DAF to 32 DAF when galactinol concentration increased coinciding with increased GS enzyme activity.

Galactinol synthase enzyme activity influences raffinose family oligosaccharides (RFO) accumulation in developing chickpea (Cicer arietinum L.) seeds.

To understand raffinose family oligosaccharides (RFO) metabolism in chickpea (Cicer arietinum L.) seeds, RFO accumulation and corresponding biosynthetic enzymes activities were determined during seed development of chickpea genotypes with contrasting RFO concentrations. RFO concentration in mature seeds was found as a facilitator rather than a regulating step of seed germination. In mature seeds, raffinose concentrations ranged from 0.38 to 0.68 and 0.75 to 0.99 g/100 g, whereas stachyose concentrations varied from 0.79 to 1.26 and 1.70 to 1.87 g/100 g indicating significant differences between low and high RFO genotypes, respectively. Chickpea genotypes with high RFO concentration accumulated higher concentrations of myo-inositol and sucrose during early seed developmental stages suggesting that initial substrate concentrations may influence RFO concentration in mature seeds. High RFO genotypes showed about two to three-fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentrations. RFO biosynthetic enzymes activities correspond with accumulation of individual RFO during seed development.

A reliable and rapid method for soluble sugars and RFO analysis in chickpea using HPAEC-PAD and its comparison with HPLC-RI.

A high performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) was optimised to separate with precision, accuracy and high reproducibility soluble sugars including oligosaccharides present in pulse meal samples. The optimised method within 20min separated myo-inositol, galactinol, glucose, fructose, sucrose, raffinose, stachyose and verbascose in chickpea seed meal extracts. Gradient method of eluting solvent (sodium hydroxide) resulted in higher sensitivity and rapid detection compared to similar analytical methods. Peaks asymmetry equivalent to one and resolution value ⩾1.5 support column's precision and accuracy for quantitative determinations of soluble sugars in complex mixtures. Intermediate precision determined as relative standard deviation (1.8-3.5%) for different soluble sugars confirms reproducibility of the optimised method. The developed method has superior sensitivity to detect even scarcely present verbascose in chickpea. It also quantifies myo-inositol and galactinol making it suitable both for RFO related genotype screening and biosynthetic studies.

Genotype and growing environment interaction shows a positive correlation between substrates of raffinose family oligosaccharides (RFO) biosynthesis and their accumulation in chickpea ( Cicer arietinum L.) seeds.

To develop genetic improvement strategies to modulate raffinose family oligosaccharides (RFO) concentration in chickpea ( Cicer arietinum L.) seeds, RFO and their precursor concentrations were analyzed in 171 chickpea genotypes from diverse geographical origins. The genotypes were grown in replicated trials over two years in the field (Patancheru, India) and in the greenhouse (Saskatoon, Canada). Analysis of variance revealed a significant impact of genotype, environment, and their interaction on RFO concentration in chickpea seeds. Total RFO concentration ranged from 1.58 to 5.31 mmol/100 g and from 2.11 to 5.83 mmol/100 g in desi and kabuli genotypes, respectively. Sucrose (0.60-3.59 g/100 g) and stachyose (0.18-2.38 g/100 g) were distinguished as the major soluble sugar and RFO, respectively. Correlation analysis revealed a significant positive correlation between substrate and product concentration in RFO biosynthesis. In chickpea seeds, raffinose, stachyose, and verbascose showed a moderate broad sense heritability (0.25-0.56), suggesting the use of a multilocation trials based approach in chickpea seed quality improvement programs.