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Winter field survival (WFS) in autumn-seeded winter cereals is a complex trait associated with low temperature tolerance (LTT), prostrate growth habit (PGH), and final leaf number (FLN). WFS and the three sub-traits were analyzed by a genome-wide association study of 96 rye (Secale cereal L.) genotypes of different origins and winter-hardiness levels. A total of 10,244 single nucleotide polymorphism (SNP) markers were identified by genotyping by sequencing and 259 marker-trait-associations (MTAs; p < 0.01) were revealed by association mapping. The ten most significant SNPs (p < 1.49e-04) associated with WFS corresponded to nine strong candidate genes: Inducer of CBF Expression 1 (ICE1), Cold-regulated 413-Plasma Membrane Protein 1 (COR413-PM1), Ice Recrystallization Inhibition Protein 1 (IRIP1), Jasmonate-resistant 1 (JAR1), BIPP2C1-like protein phosphatase, Chloroplast Unusual Positioning Protein-1 (CHUP1), FRIGIDA-like 4 (FRL4-like) protein, Chalcone Synthase 2 (CHS2), and Phenylalanine Ammonia-lyase 8 (PAL8). Seven of the candidate genes were also significant for one or several of the sub-traits supporting the hypothesis that WFS, LTT, FLN, and PGH are genetically interlinked. The winter-hardy rye genotypes generally carried additional allele variants for the strong candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions., (© 2022. The Author(s).)

The nutritional quality of common wheat-based foods can be improved by adding flours from whole pulses or their carbohydrate and protein constituents. Faba bean ( Vicia faba L.) is a pulse with high protein concentration. In this study, prepared faba bean (FB) flours were added to wheat based baked crackers. Wheat cracker recipes were modified by substituting forty percent wheat flour with flours from whole faba bean, starch enriched flour (starch 60%), protein concentrate (protein 60%) or protein isolate (protein 90%). Baked crackers were ground into meal and analyzed for their macronutrient composition, starch characteristics and in vitro starch hydrolysis. Faba bean supplemented crackers had lower ( p ≤ 0.001) total starch concentrations, but proportionally higher protein (16.8-43%), dietary fiber (6.7-12.1%), fat (4.8-7.1%) and resistant starch (3.2-6%) ( p ≤ 0.001) than wheat crackers (protein: 16.2%, dietary fiber: 6.3%, fat: 4.2, resistant starch: 1.2%). The increased amylose, amylopectin B1- chain and fat concentration from faba bean flour and starch flour supplementation in cracker recipe contributed to increased resistant starch. Flours from whole faba bean, starch or protein fractions improved the nutritional properties and functional value of the wheat-based crackers. The analytical analysis describing protein, starch composition and structure and in vitro enzymatic hydrolysis advance understanding of factors that account for the in vivo benefits of faba bean flours added to crackers in human physiological functions as also previously shown for pasta. The findings can be used to guide development of improve nutritional quality of similar wheat-based food products.

Overwintering cereals accumulate low temperature tolerance (LTT) during cold acclimation in the autumn. Simultaneously, the plants adjust to the colder season by making developmental changes at the shoot apical meristem. These processes lead to higher winter hardiness in winter rye varieties ( Secale cereale L.) adapted to Northern latitudes as compared to other cereal crops. To dissect the winter-hardiness trait in rye, a panel of 96 genotypes of different origins and growth habits was assessed for winter field survival (WFS), LTT, and six developmental traits. Best Linear Unbiased Estimates for WFS determined from five field trials correlated strongly with LTT (r = 0.90, p < 0.001); thus, cold acclimation efficiency was the major contributor to WFS. WFS also correlated strongly ( p < 0.001) with final leaf number (r = 0.80), prostrate growth habit (r = 0.61), plant height (r = 0.34), but showed weaker associations with top internode length (r = 0.30, p < 0.01) and days to anthesis (r = 0.25, p < 0.05). The heritability estimates ( h 2 ) for WFS-associated traits ranged from 0.45 (prostrate growth habit) to 0.81 (final leaf number) and were overall higher than for WFS ( h 2 = 0.48). All developmental traits associated with WFS and LTT are postulated to be regulated by phytohormone levels at shoot apical meristem.

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 F 2 and BC 1 F 2 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., Competing Interests: Conflicts of Interest No potential conflict of interest relevant to this article was reported., (© The Korean Society of Plant Pathology.)

Brassinosteroids (BR) are plant-based steroids which influence several morphogenetic and developmental processes. A barley (Hordeum vulgare L.) genotype Kinai Kyoshinkai-2 (KK-2) carrying the uzu mutation exhibited altered starch granule size distribution. Hybridizing KK-2 with a barley genotype CDC Kendall with bi-modal starch granules produced progeny lines (116, 144 and 168) with almost uni-modal starch granules. Bioassays correlated uzu mutation with defective BR perception. DNA sequence analysis of the BR receptor-1 (BRI-1) gene detected a single-nucleotide A > G substitution at the position 2612 in the kinase domain which resulted in the change of His (CAC) to Arg (CGC) at residue 857 in subdomain IV of the kinase domain of the respective polypeptide. The study focused on the development of barley grain, accumulation of starch and composition influenced by defective BR perception due to the mutation detected in KK-2 and three other barley-breeding lines (116, 144 and 168). Aberrant BRI-1 delayed grain development, amylose synthesis and starch accumulation in the endosperm. The barley breeding lines 116, 144 and 168 carrying the aberrant BRI-1, exhibited altered granule size distribution with significant shift in the diameter maxima, but insignificant differences in amylose concentration. The BRI-1 mutation also altered amylopectin fine structure in both B- and C- type small starch granules, resulting in an increased fraction of short A-type glucan chains (<10 DP) and decreased fraction of B2 chains (25-36 DP) in genotypes carrying the BRI-1 mutation. The results show the influence of BR on barley grain development, starch accumulation, granule size distribution and amylopectin structure., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 .

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.

In human diet, the products of starch digestion are a major energy source. Starch is stored as water insoluble granules composed of amylose and amylopectin. The susceptibility of starch granule to digestive enzymes is affected by starch granule size, shape, and composition. In this study, starch characteristics and in vitro enzymatic hydrolysis in three rice ( Oryza sativa L.) mutants (RSML 184, RSML 278 and RSML 352) with similar amylose concentration (24.3-25.8%) was compared to parent ADT 43 (21.4%). The three mutants had reduced thousand grain weight and starch concentration but higher protein and dietary fiber concentrations. The mutant RSML 352 had small starch granules and reduced short glucan chains [Degree of polymerization (DP) 6-12] compared to the other two mutants (RSML 184 and RSML 278). The mutant RSML 352 had the highest hydrolytic index (HI) and least concentration of resistant starch (RS) compared to the other two mutants and parent rice ADT 43. The two rice mutants (RSML 184 and RSML 278) had reduced HI and increased RS concentration than the parent ADT 43. The results showed that starch granule size and amylopectin structure influence starch enzymatic digestibility and RS concentration.

Wheat is the primary grain fed to poultry in western Canada, but its nutritional quality, including the nature of its starch digestibility, may be affected by wheat market class. The objectives of this study were to determine the rate and extent of starch digestibility of wheat market classes in broiler chickens, and to determine the relationship between starch digestibility and wheat apparent metabolizable energy (AME). In vitro starch digestion was assessed using gastric and small intestinal phases mimicking the chicken digestive tract, while in vivo evaluation used 468 male broiler chickens randomly assigned to dietary treatments from 0 to 21 d of age. The study evaluated 2 wheat cultivars from each of 6 western Canadian wheat classes: Canadian Prairie Spring (CPS), Canadian Western Amber Durum (CWAD), CW General Purpose (CWGP), CW Hard White Spring (CWHWS), CW Red Spring (CWRS), and CW Soft White Spring (CWSWS). All samples were analyzed for relevant grain characteristics. Data were analyzed as a randomized complete block design and cultivars were nested within market class. Pearson correlation was used to determine relationships between measured characteristics. Significance level was P ≤ 0.05. The starch digestibility range and wheat class rankings were: proximal jejunum - 23.7 to 50.6% (CWHWSc, CPSbc, CWSWSbc, CWRSab, CWGPa, CWADa); distal jejunum - 63.5 to 76.4% (CWHWSc, CPSbc, CWSWSbc, CWRSab, CWGPa, CWADa); proximal ileum - 88.7 to 96.9% (CWSWSc, CPSbc, CWHWSbc, CWRSb, CWGPb, CWADa); distal ileum - 94.4 to 98.5% (CWSWSb, CWHWSb, CPSb, CWRSab, CWGPab, CWADa); excreta - 98.4 to 99.3% (CPSb, CWRSb, CWHWSb, CWSWSab, CWGPab, CWADa). Wheat class affected wheat AMEn with levels ranging from 3,203 to 3,411 kcal/kg at 90% DM (CWRSc, CWSWSc, CPSb, CWGPb, CWADa, CWHWSa). Significant and moderately strong positive correlations were observed between in vitro and in vivo starch digestibility, but no correlations were found between AME and starch digestibility. In conclusion, rate and extent of starch digestibility and AME were affected by western Canadian wheat class, but starch digestibility did not predict AME.

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.

Starch granule size, shape and structure of amylopectin are species specific and influence starch properties and end-use of starch. Amylopectin glucan chain structure was used to predict the starch botanical sources. Mathematical probability for accumulation of small glucan chains DP 6-10 reveal exponential fit curve with maximum R 2 in smallest granule size starches (Chlamydomonas, quinoa, buckwheat). Cereal and cassava showed R 2 of 0.81-0.96 while in pulses and tubers it was less than 0.7. The amylopectin small glucan chains form a unique 'finger print region' that identified starch botanical source. Differential amylopectin chain length distribution (APCLD) graphs between DP 6-80 of all species from Chlamydomonas starch distinguished five structural groups that clustered the 31 analyzed starches into four major patterns. APCLD analyses of amylopectin combined with characteristic pattern of small linear DP (6-9) glucan chains predicted the starch botanical source., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The structure of β-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 β-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 β-glucan in two environments. Results showed that concentrations of β-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-β-glucan lines. The concentrations of β-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&#95;4105 was associated with β-glucan concentration, whereas Bmac504 and Bmac211 were associated with DP3:DP4 ratio. Bmac273e was associated with both β-glucan concentration and DP3:DP4 ratio., Conclusion: The grain β-glucan concentration and DP3:DP4 ratio are strongly affected by genotype and environment. Single-marker analyses suggested that the genetic control of β-glucan concentration and DP3:DP4 ratio was linked to separate chromosomal regions on barley genome. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Background: Glycemic response, a trait that is tedious to be assayed in cereal staples, has been identified as a factor correlated with alarmingly increasing prevalence of Type II diabetes. Reverse genetics based discovery of allelic variants associated with this nutritional trait gains significance as they can provide scope for genetic improvement of this factor which is otherwise difficult to target through routine screening methods., Results: Through EcoTILLING by sequencing in 512 rice accessions, we report the discovery of six deleterious variants in the genes with potential to increase Resistant Starch (RS) and reduce Hydrolysis Index (HI) of starch. By deconvolution of the variant harbouring EcoTILLING DNA pools, we discovered accessions with a minimum of one to a maximum of three deleterious allelic variants in the candidate genes., Conclusions: Through biochemical assays, we confirmed the potential role of the discovered alleles alone or in combinations in increasing RS the key factor for reduction in glycemic response.

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., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Wheat grain development is a complex process and is characterized by changes in physicochemical and structural properties of starch. The present study deals with endosperm starch physicochemical properties and structure during development in different granule-bound starch synthase I (GBSSI) null also known as waxy (Wx) genotypes. The study was conducted with pure starch isolated from wheat grains at 3-30 days post anthesis (DPA), at 3-day intervals. Amylose concentration increased throughout grain development in non-waxy (7.2-30.5%) and partial waxy genotypes (6.0-26.8%). Completely waxy genotype showed 7.0% amylose at 3 and 6 DPA, which declined during development and reached non-detectable quantities by 30 DPA. Amylopectin structure had a higher content of short chains at 3 DPA, which decreased continuously until 12 DPA, after which there were only minor changes in amylopectin chain length distribution. Similarly, the average degree of polymerization (DP) increased from 3 DPA (12.3) to 12 DPA (15.0), and then did not differ significantly up to 30 DPA (15.0). This suggests the formation of basic amylopectin architecture in wheat by 12 DPA. Wx-B and Wx-D affected amylopectin short chains mostly of DP 6-8 at 3 and 6 DPA. Wx-A affected the same fraction of chains at 9 and 12 DPA, and Wx-D affected DP 18-25 chains from 18 to 30 DPA, suggesting differential effect of waxy isoproteins on amylopectin structure formation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Microscope analysis of starches prepared from 139 barley genotypes identified a Japanese genotype, Kinai Kyoshinkai-2 (KK-2), with altered starch granule size distribution. Compared to normal barley starch, KK-2 produced consistently higher volumes of starch granules with 5-15 μm diameter and reduced volumes of starch granules with >15 μm diameter when grown in different environments. A cross between KK-2 and normal starch cultivar CDC Kendall was made and led to the production of 154 F5 lines with alterations to the normal 7:3:1 distribution for A-:B-:C-type starch granule volumes. Three F5 lines showed unimodal starch granule size distribution due to apparent lack of very small (<5.0 μm diameter) C-type starch granules, but the phenotype was accompanied by reduced grain weight and total starch concentration. Five F5 lines produced a significantly larger population of large (>15 μm diameter) A-type starch granules as compared to normal starch and showed on average a 10:4:1 distribution for A-:B-:C-type starch granule volumes. The unusual starch phenotypes displayed by the F5 lines confirm starch granule size distribution in barley can be genetically altered.

Near-isogenic wheat ( Triticum aestivum L.) lines differing at the Waxy locus were studied for the influence of genome-specific granule-bound starch synthase I (GBSSI/Waxy; Wx-A, Wx-B, Wx-D) on starch composition, structure, and in vitro starch enzymatic hydrolysis. Grain composition, amylose concentration, amylopectin unit-chain length distribution, and starch granule size distribution varied with the loss of functional GBSSI. Amylose concentration was more severely affected in genotypes with GBSSI missing from two genomes (double nulls) than from one genome (single nulls). Unit glucan chains (DP 6-8) of amylopectin were reduced with the complete loss of GBSSI as compared to wheat starch with a full complement of GBSSI. Wx-A and Wx-B had an additive effect toward short-chain phenotype of waxy amylopectin. Loss of Wx-D isoprotein alone significantly (p < 0.05) reduced the C-type starch granules. However, the absence of Wx-D in combination with Wx-A or Wx-B increased the B-type and C-type starch granules but decreased the volume of A-type starch granules. The rate of in vitro starch enzymatic hydrolysis was highest in completely waxy grain meal and purified starch. However, the presence of Wx-D reduced wheat starch hydrolysis as it increased the large A-type starch granule content (volume %) and reduced short chains (DP 6-8) in amylopectin. Factors such as small C-type starch granules, amylose concentration, and long chains of amylopectin (DP 23-45) also influenced wheat starch hydrolysis.

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.

Granule bound starch synthase 1 (GBSS1) accumulation within starch granules and structure of Gbss1 alleles were determined for nine barley ( Hordeum vulgare L.) genotypes producing amylose-free (undetectable), near-waxy (1.6-4.5%), normal (25.8%), and increased (38.0-40.8%) amylose grain starches. Compared to normal starch granules, GBSS1 accumulation was severely reduced in three near-waxy, slightly reduced in two waxy, and slightly elevated in three increased amylose starches. Gbss1 nucleotide sequence analysis for the nine genotypes distinguished them into three Gbss1 groups with several single-nucleotide polymorphisms. A new unique Q312H substitution within GBSS1 was discovered in near-waxy genotype SB94912 with reduced amylose (1.6%) concentration relative to the other two near-waxy lines, CDC Rattan and CDC Candle (4.5%). The two waxy genotype GBSS1 showed a previously described D287V change for CDC Alamo and a new G513W change for CDC Fibar. Both amino acid alterations are conserved residues within starch synthase domains involved in glucan interaction. The increased amylose genotypes showed several unique nucleotide changes within the second and fourth Gbss1 introns, but only SB94893 GBSS1 showed a unique amino acid substitution, A250T in exon 6. The Gbss1 nucleotide differences were used to design genetic markers to monitor Gbss1 alleles in genotypes with various amylose grain starches.

Chickpea (Cicer arietinum L.) is an important pulse crop grown and consumed all over the world, especially in the Afro-Asian countries. It is a good source of carbohydrates and protein, and protein quality is considered to be better than other pulses. Chickpea has significant amounts of all the essential amino acids except sulphur-containing amino acids, which can be complemented by adding cereals to the daily diet. Starch is the major storage carbohydrate followed by dietary fibre, oligosaccharides and simple sugars such as glucose and sucrose. Although lipids are present in low amounts, chickpea is rich in nutritionally important unsaturated fatty acids such as linoleic and oleic acids. β-Sitosterol, campesterol and stigmasterol are important sterols present in chickpea oil. Ca, Mg, P and, especially, K are also present in chickpea seeds. Chickpea is a good source of important vitamins such as riboflavin, niacin, thiamin, folate and the vitamin A precursor β-carotene. As with other pulses, chickpea seeds also contain anti-nutritional factors which can be reduced or eliminated by different cooking techniques. Chickpea has several potential health benefits, and, in combination with other pulses and cereals, it could have beneficial effects on some of the important human diseases such as CVD, type 2 diabetes, digestive diseases and some cancers. Overall, chickpea is an important pulse crop with a diverse array of potential nutritional and health benefits.

Background: An immature wheat spike culture system was used to monitor cadmium (Cd) accumulation in grains, hulls and awns of bread wheat and durum wheat. Immature spikes were cultured prior to anthesis in a medium containing 50 g L(-1) sucrose and 0.4 g L(-1) L-glutamine, supplemented with 0, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20 or 25 mg L(-1) cadmium chloride (CdCl(2)). Grains were collected at maturity and their Cd accumulation was determined using inductively coupled plasma mass spectrometry (ICP-MS)., Results: Cd accumulation at CdCl(2) concentrations of 3 mg L(-1) and above was higher in grains of durum wheat compared with bread wheat. In hulls a similar trend was observed at CdCl(2) concentrations above 15 mg L(-1) . Starch concentration in grains increased slightly at 3 and 4 mg L(-1) CdCl(2). Cd accumulation negatively affected grain protein concentration. Expression patterns of Cd-related genes glutathione reductase (TaGR), metallothionein (MT) and phytochelatin synthase (PCS) in spikes cultured in media containing 0, 5, 10, 15 and 25 mg L(-1) CdCl(2) at 5 days post-anthesis showed that TaGR and PCS expression in bread wheat was up-regulated at 5 mg L(-1) CdCl(2) but down-regulated at other CdCl(2) concentrations. However, in durum wheat, expression of all three genes was down-regulated or remained unchanged., Conclusion: This study demonstrates that immature spike culture can be used to study Cd accumulation in grains and can delineate hyper-accumulating durum wheat from bread wheat at CdCl(2) concentrations of 2 mg L(-1) and above., (Copyright © 2011 Society of Chemical Industry.)

Background: The effects of genotype and environment and their interaction on the concentrations of starch and protein in, and the amylose content and thermal and pasting properties of starch from, pea and fababean are not well known., Results: Differences due to genotype were observed in the concentrations of starch and protein in pea and fababean, in the onset temperature (To) and peak temperature (Tp) of gelatinization of fababean starch, and in the pasting, trough, cooling and final viscosities of pea starch and fababean starch. Significant two-way interactions (location × genotype) were observed for the concentration of starch in fababean and the amylose content, To, endothermic enthalpy of gelatinization (ΔH) and trough viscosity of fababean starch. Significant three-way interactions (location × year × genotype) were observed for the concentration of starch in pea and the pasting, trough, cooling and final viscosities of pea starch., Conclusion: Differences observed in the concentrations of starch and protein in pea and fababean were sufficient to be of practical significance to end-users, but the relatively small differences in amylose content and physicochemical properties of starch from pea and fababean were not., (Copyright © 2011 Society of Chemical Industry.)

The relationship between starch physical properties and enzymatic hydrolysis was determined using ten different hulless barley genotypes with variable carbohydrate composition. The ten barley genotypes included one normal starch (CDC McGwire), three increased amylose starches (SH99250, SH99073, and SB94893), and six waxy starches (CDC Alamo, CDC Fibar, CDC Candle, Waxy Betzes, CDC Rattan, and SB94912). Total starch concentration positively influenced thousand grain weight (TGW) (r(2) = 0.70, p < 0.05). Increase in grain protein concentration was not only related to total starch concentration (r(2) = -0.80, p < 0.01) but also affected enzymatic hydrolysis of pure starch (r(2) = -0.67, p < 0.01). However, an increase in amylopectin unit chain length between DP 12-18 (F-II) was detrimental to starch concentration (r(2) = 0.46, p < 0.01). Amylose concentration influenced granule size distribution with increased amylose genotypes showing highly reduced volume percentage of very small C-granules (<5 μm diameter) and significantly increased (r(2) = 0.83, p < 0.01) medium sized B granules (5-15 μm diameter). Amylose affected smaller (F-I) and larger (F-III) amylopectin chains in opposite ways. Increased amylose concentration positively influenced the F-III (DP 19-36) fraction of longer DP amylopectin chains (DP 19-36) which was associated with resistant starch (RS) in meal and pure starch samples. The rate of starch hydrolysis was high in pure starch samples as compared to meal samples. Enzymatic hydrolysis rate both in meal and pure starch samples followed the order waxy > normal > increased amylose. Rapidly digestible starch (RDS) increased with a decrease in amylose concentration. Atomic force microscopy (AFM) analysis revealed a higher polydispersity index of amylose in CDC McGwire and increased amylose genotypes which could contribute to their reduced enzymatic hydrolysis, compared to waxy starch genotypes. Increased β-glucan and dietary fiber concentration also reduced the enzymatic hydrolysis of meal samples. An average linkage cluster analysis dendrogram revealed that variation in amylose concentration significantly (p < 0.01) influenced resistant starch concentration in meal and pure starch samples. RS is also associated with B-type granules (5-15 μm) and the amylopectin F-III (19-36 DP) fraction. In conclusion, the results suggest that barley genotype SH99250 with less decrease in grain weight in comparison to that of other increased amylose genotypes (SH99073 and SH94893) could be a promising genotype to develop cultivars with increased amylose grain starch without compromising grain weight and yield.

Low-temperature (LT) tolerance in winter wheat (Triticum aestivum L.) is an economically important but complex trait. Four selected wheat genotypes, a winter hardy cultivar, Norstar, a tender spring cultivar, Manitou and two near-isogenic lines with Vrn-A1 (spring Norstar) and vrn-A1 (winter Manitou) alleles of Manitou and Norstar were cold-acclimated at 6°C and crown and leaf tissues were collected at 0, 2, 14, 21, 35, 42, 56 and 70 days of cold acclimation. cDNA-AFLP profiling was used to determine temporal expression profiles of transcripts during cold-acclimation in crown and leaf tissues, separately to determine if LT regulatory circuitries in crown and leaf tissues could be delineated using this approach. Screening 64 primer combinations identified 4,074 and 2,757 differentially expressed transcript-derived fragments (TDFs) out of which 38 and 16% were up-regulated as compared to 3 and 6% that were down-regulated in crown and leaf tissues, respectively. DNA sequencing of TDFs revealed sequences common to both tissues including genes coding for DEAD-box RNA helicase, choline-phosphate cytidylyltransferase and delta-1-pyrroline carboxylate synthetase. TDF specific to crown tissues included genes coding for phospahtidylinositol kinase, auxin response factor protein and brassinosteroid insensitive 1-associated receptor kinase. In leaf, genes such as methylene tetrahydrofolate reductase, NADH-cytochrome b5 reductase and malate dehydrogenase were identified. However, 30 and 14% of the DNA sequences from the crown and leaf tissues, respectively, were hypothetical or unknown proteins. Cluster analysis of up-, down-regulated and unique TDFs, DNA sequence and real-time PCR validation, infer that mechanisms operating in crown and leaf tissue in response to LT are differently regulated and warrant further studies.

An in vitro culture system is useful to study grain development under defined conditions to minimize confounding effects associated with whole plant studies and metabolite movement into the developing grains. The objective of this study was to monitor the expression patterns of carbohydrate metabolism genes during grain development in an in vitro wheat spike culture system. Immature spikes were cultured prior to anthesis, and grains were collected at various days postanthesis (DPA). Grains from cultured spikes showed maximum expression of starch metabolic genes by 10 DPA, with a rapid decline thereafter. The rapid increase and decrease in expression rate in the in vitro system was thought to be due to fructan exohydrolase (1-FEH and 6-FEH) or sucrose transporter 1 (SUT1) and sucrose synthase (SuSy) genes being highly expressed. SUT1 reached peak expression at 8 DPA, two days earlier than the other genes, and may account for the rapid early stage trigger in expression of the other genes. However, expression of 1-FEH and 6-FEH genes in in vitro-cultured spikes peaked at 12 DPA, two days later than the other genes, and could indicate that fructan catabolism was not a factor in the rapid accumulation of starch in the in vitro-cultured spikes. Accumulation of GBSSI polypeptides generally showed similar patterns in both systems, with the maximum amount in the in vitro system observed four days later than in the in planta spikes, reflecting different turnover controls of GBSSI transcripts. The in vitro system offers opportunities for further refinement and detailed grain development studies.

Autumn-seeded winter cereals acquire tolerance to freezing temperatures and become vernalized by exposure to low temperature (LT). The level of accumulated LT tolerance depends on the cold acclimation rate and factors controlling timing of floral transition at the shoot apical meristem. In this study, genomic loci controlling the floral transition time were mapped in a winter wheat (T. aestivum L.) doubled haploid (DH) mapping population segregating for LT tolerance and rate of phenological development. The final leaf number (FLN), days to FLN, and days to anthesis were determined for 142 DH lines grown with and without vernalization in controlled environments. Analysis of trait data by composite interval mapping (CIM) identified 11 genomic regions that carried quantitative trait loci (QTLs) for the developmental traits studied. CIM analysis showed that the time for floral transition in both vernalized and non-vernalized plants was controlled by common QTL regions on chromosomes 1B, 2A, 2B, 6A and 7A. A QTL identified on chromosome 4A influenced floral transition time only in vernalized plants. Alleles of the LT-tolerant parent, Norstar, delayed floral transition at all QTLs except at the 2A locus. Some of the QTL alleles delaying floral transition also increased the length of vegetative growth and delayed flowering time. The genes underlying the QTLs identified in this study encode factors involved in regional adaptation of cold hardy winter wheat.

Unacceptably dark bran color has prevented the white-kernelled variety Argent from meeting grain color marketing standards for hard white wheats (Triticum aestivum L.). The objective of this research was to identify phenolic compounds that negatively affect bran color in white wheat using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and vanillin-HCl and NaOH staining methods. In mature bran, FT-ICR-MS detected derivatives of the flavonol quercetin in varieties Argent and RL4137 (red-kernelled wheat) but not in W98616, a white wheat variety with acceptable grain color. Derivatives of the isoflavone formononetin were more abundant in W98616 relative to RL4137 and Argent. Vanillin-HCl staining indicated that RL4137 sequestered high levels of proanthocyanidin (PA) throughout its entire seed coat, whereas white wheats sequestered PAs as discrete speckles. Argent possessed abundant speckles over its entire seed coat, whereas speckles were almost undetectable in W98616. In mature kernels, flavonoids throughout the seed coat of RL4137 reacted with NaOH, but only the speckles appeared to react in white wheats. W98616 consistently had lighter grain than Argent before and after NaOH treatment. Free and bound phenolic differences in bran samples confirmed that the darker seed coat color of Argent, relative to W98616, was likely due to higher total phenolic acid content. Although isoflavones accumulated in Argent and RL4137, it appears that the majority of the flux through the flavonoid pathway ultimately accumulates quercetin derivatives and PAs. In W98616, PAs accumulate, but it appears that flavonoid biosynthesis ultimately accumulates isoflavones. Argent, compared to W98616, generally accumulated higher levels of total phenolics (flavonols, stilbenes, and PAs) within its darker pigmented bran.

A number of COR genes (COld-Regulated genes) have been implicated in the acquisition of low temperature (LT) tolerance in wheat (Triticum aestivum L.). This study compared the relative expression patterns of selected COR genes in leaf and crown tissues of wheat near-isogenic lines to increase understanding of the molecular mechanisms underlying LT acclimation. Reciprocal near-isogenic lines were generated such that the dominant Vrn-A1 and recessive vrn-A1 loci were interchanged in a spring cv. Manitou and a winter cv. Norstar. Phenological development, acquisition of LT tolerance, and WCS120 polypeptide accumulation in these genotypes proceeded at rates similar to those previously reported for 6 degrees C acclimation from 0 to 98 d. However, a differential accumulation of WCS120 polypeptide and expression of the COR genes Wcs120, Wcor410, and Wcor14 was observed in the leaf and crown tissues. COR gene transcript levels peaked at 2 d of the acclimation period in both tissues and differences among genotypes were most evident at this time. COR gene expression was highest for the LT-tolerant and lowest for the tender genotypes. However, expression rates were divergent enough in genotypes with intermediate hardiness that comparisons among tissues and/or times during acclimation often resulted in variable interpretations of the relative expression of the COR genes in the determination of LT tolerance. These observations emphasize the need to pay close attention to experimental conditions, sampling times, and genotype and tissue selection in experiments designed to identify the critical genetic components that interact to determine LT acclimation.

Low-temperature (LT) tolerance is an important economic trait in winter wheat (Triticum aestivum L.) that determines the plants' ability to cope with below freezing temperatures. Essential elements of the LT tolerance mechanism are associated with the winter growth habit controlled by the vernalization loci (Vrn-1) on the group 5 chromosomes. To identify genomic regions, which in addition to vrn-1 determine the level of LT tolerance in hexaploid wheat, two doubled haploid (DH) mapping populations were produced using parents with winter growth habit (vrn-A1, vrn-B1, and vrn-D1) but showing different LT tolerance levels. A total of 107 DH lines were analyzed by genetic mapping to produce a consensus map of 2,873 cM. The LT tolerance levels for the Norstar (LT(50)=-20.7 degrees C) x Winter Manitou (LT(50)=-14.3 degrees C) mapping population ranged from -12.0 to -22.0 degrees C. Single marker analysis and interval mapping of phenotyped lines revealed a major quantitative trait locus (QTL) on chromosome 5A and a weaker QTL on chromosome 1D. The 5A QTL located 46 cM proximal to the vrn-A1 locus explained 40% of the LT tolerance variance. Two C-repeat Binding Factor (CBF) genes expressed during cold acclimation in Norstar were located at the peak of the 5A QTL.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.

We characterized three near-full-length putative homoeologous cDNA (Ss2a-1, Ss2a-2, and Ss2a-3) in wheat endosperm most similar to the maize zSSIIa. Polypeptide sequences deduced from three Ss2a cDNA clones share a 95% overall sequence similarity, and may thus have similar biochemical properties and may make identical contributions to starch biosynthesis in wheat endosperm. The accumulation of RNA transcripts corresponding to three Ss2a genes in developing endosperm varies among three cultivars studied, but usually peaks in young endosperm at about 10 days post anthesis (DPA). The polyclonal antibody for the SSIIa-1 recombinant protein strongly reacted to three previously identified granule-bound starch synthases of 100 to 115 kDa. The polyclonal antibody for the granule-bound starch synthases strongly reacted to the SSIIa-1 recombinant protein. Sequences of the N-terminal and an internal peptide of these three granule-bound starch synthases match well with those of three predicted mature SSIIa polypeptides. These granule-bound starch synthases may therefore be SSIIa polypeptides. The antibodies also recognized a group of three polypeptides with the same gel mobility as the three granule-bound starch synthases, a polypeptide of 90 kDa, and a group of three polypeptides of about 80 to 82 kDa. Thus, the wheat SSIIa may exist in several functional forms in the stroma of amyloplasts.

Two starch granule-bound proteins (SGP), SGP-140 and SGP-145, were preferentially associated with A-type starch granules (>10 microm) in developing and mature wheat (Triticum aestivum) kernels. Immunoblotting and N-terminal sequencing suggested that the two proteins were different variants of SBEIc, a 152-kD isoform of wheat starch-branching enzyme. Both SGP-140 and SGP-145 were localized to the endosperm starch granules but were not found in the endosperm soluble fraction or pericarp starch granules younger than 15 d post anthesis (DPA). Small-size starch granules (<10 microm) initiated before 15 DPA incorporated SGP-140 and SGP-145 throughout endosperm development and grew into full-size A-type starch granules (>10 microm). In contrast, small-size starch granules harvested after 15 DPA contained only low amounts of SGP-140 and SGP-145 and developed mainly into B-type starch granules (<10 microm). Polypeptides of similar mass and immunologically related to SGP-140 and/or SGP-145 were also preferentially incorporated into A-type starch granules of barley (Hordeum vulgare), rye (Secale cereale), and triticale (x Triticosecale Wittmack) endosperm, which like wheat endosperm have a bimodal starch granule size distribution.

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.

The genetic relationships among the five groups of hexaploid wheat: common, spelta, macha, vavilovii, and semi-wild wheat (SWW) are not clear. Random amplified polymorphic DNA (RAPD) analysis was used to assess phylogenetic relationships among these five morphological groups of hexaploid wheat. RAPD data were analyzed using the NTSYS-PC computer program to generate Jaccard genetic similarity coefficients. A dendrogram based on RAPD analysis grouped 15 accessions into five distinct clusters. These results are in agreement with those based on morphological classification, suggesting that common wheat is most closely related to SWW, followed by spelta, vavilovii, and macha.

A wheat gene, denoted Sbe1, encoding a type I starch-branching enzyme (SBEI) was isolated from a genomic library and shown to comprise 14 exons distributed over a 5.7 kb DNA region. Analyses of kernel RNA by 5' rapid amplification of cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a considerable sequence variation at the 5' ends of SBEI gene transcripts. DNA sequence alignments between the 5'-RACE products and the Sbe1 genomic DNA indicated that the first two exons and first intron were differentially processed to generate three classes of the mature transcript. One form of the SBEI gene transcript in 12-day old kernels contained the exon I+II+III combination at the 5' end, whereas other forms differed by inclusion of intron 1 or exclusion of exon II sequences. RT-PCR analysis of Sbe1-uidA::nptII chimeric mRNA produced in transgenic wheat cultured cells confirmed that the isolated Sbe1 was able to produce all three forms of SBEI gene transcripts by alternative splicing of the primary mRNA. The variants of processed Sbe1 mRNA were potentially translated into N-terminal variants of the SBEI precursor with different transit peptide sequences.

A PCR-based screening approach was used to isolate genomic clones from wheat encoding peroxidase isozymes. Three complete genes (pox1, pox2 and pox4) and one truncated gene (pox3) were characterized. The nucleotide sequences predicted mature proteins of 31 kDa, in which all the highly conserved motifs of secreted plant peroxidases were preserved. The coding regions showed 73-83% DNA sequence identity, with the highest level of similarity noted for the tandemly oriented pox2 and pox3. Expression of respective pox genes in various tissues of wheat was assessed by the RT-PCR technique, which showed that all four genes are active. The primary pox1 mRNA was spliced to remove three introns, whereas processing of the other pox transcripts involved only two intervening sequences. Splicing occurred at consensus GU/AG splice sites except for the first introns of pox1, pox2 and pox4 transcripts, where processing took place at unusual GC donor sites. The RNA analysis suggested that the pox1, pox2 and pox4 genes are predominantly expressed in roots. Lower levels of expression were found for pox4 and pox3 in leaves. Infection of wheat by the powdery mildew fungus selectively induced expression of pox2 in leaves.

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli β-glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.

A monoclonal antibody against wheat germ sucrose synthetase is developed and characterized. Its use in studying the effect of cold acclimation on the expression of sucrose synthetase in winter and spring wheat plants is described. The antibody shows cross-reactivity with sucrose synthetase from maize and pea plants, as well as carrot cells. A gradual accumulation of the enzyme as a function of time spent at 2 degrees C is observed in both wheat varieties. In contrast, an initial sharp rise in the mRNA level is observed, which agrees with the previously reported response of maize plants subjected to anaerobic stress.

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 μg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 μg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 μg/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 μg/ml kanamycin, but exhibited reduced rooting ability.

Cationic peroxidase immobilized on sepharose has been used to separate specific antibodies from the antiperoxidase whole rabbit serum immunoglobulins (IgG) raised against it. The IgGs so separated completely pellet the peroxidase activity of the cationic peroxidase at a ratio of 6 : 1 (w/w). This shows the specificity of purified IgGs which is also shown by the single reaction arc obtained by immunoelectrophoresis of the isolated IgGs followed by challenging with crude peroxidase. These specific IgGs when linked to cyanogen bromide activated sepharose have been used to purify the cationic peroxidase from the spent medium proteins and from the cell extract of the peanut cells grown in suspension culture., (Copyright © 1984 Gustav Fischer Verlag, Stuttgart. Published by Elsevier GmbH.. All rights reserved.)