Puroindoline genes and proteins in tetraploid and hexaploid species of Triticum

Six tetraploid (5 Triticum turgidum and 1 Triticum timopheevii) and four hexaploid (three Triticum aestivum and one Triticum kiharae) taxa of Triticum were studied in order to identify novel variation in Pin genes and proteins which can be exploited in the improvement of cultivated wheat. Western blotting with a highly specific antibody showed that puroindoline proteins were present in all of the hexaploid lines but were absent from the tetraploids. The immunoreactive bands differed slightly in their relative mobilities and their relative amounts, which could have resulted from variation in the allelic forms of Pin a and Pin b. This was supported by HPLC analyses which showed differences in the retention times and peaks heights of the putative puroindoline components in T. kiharae and T. timopheevii. Sequence analyses of cDNAs also showed variation in the sequences of expressed puroindoline genes. In particular, a sequence encoding a new form of Pin b was present in T. aestivum ssp. macha.     

Effect of the grain protein content locus Gpc-B1 on bread and pasta quality

Grain protein concentration (GPC) affects wheat nutritional value and several critical parameters for bread and pasta quality. A gene designated Gpc-B1, which is not functional in common and durum wheat cultivars, was recently identified in Triticum turgidum ssp. dicoccoides. The functional allele of Gpc-B1 improves nitrogen remobilization from the straw increasing GPC, but also shortens the grain filling period resulting in reduced grain weight in some genetic backgrounds. We developed isogenic lines for the Gpc-B1 introgression in six hexaploid and two tetraploid wheat genotypes to evaluate its effects on bread-making and pasta quality. In common wheat, the functional Gpc-B1 introgression was associated with significantly higher GPC, water absorption, mixing time and loaf volume, whereas in durum wheat, the introgression resulted in significant increases in GPC, wet gluten, mixing time, and spaghetti firmness, as well as a decrease in cooking loss. On the negative side, the functional Gpc-B1 introgression was associated in some varieties with a significant reduction in grain weight, test weight, and flour yield and significant increases in ash concentration. Significant gene × environment and gene × genotype interactions for most traits stress the need for evaluating the effect of this introgression in particular genotypes and environments.     

Discriminant analysis of selected yield components and fatty acid composition of chosen Triticum monococcum, Triticum dicoccum and Triticum spelta accessions

This study analyses the variability of key yield components, the content of protein and crude fat in grain and the fatty acid composition of 50 spring accessions of Triticum monococcum, Triticum dicoccum and Triticum spelta of various origins. The average protein content of the grain of T. monococcum was 20.8%, of T. dicoccum 19.7%, and of spelt 17.0%. The crude fat content of T. monococcum grain (2.7%) was significantly higher compared with T. spelta (2.4%) and T. dicoccum (2.3%). In crude fat, fatty acids C18:2, C18:1 and C16 predominated. T. spelta was characterised by the highest concentrations of C18:2 and C16 (55.89% and 18.77% respectively), while T. monococcum had the highest content of C18:1 (26.35%). The structure of analysed fatty acids proved to be highly desirable in this species. A discriminant analysis performed separately for five variables: protein and fat content and three biometrical characters and separately for fatty acid composition enabled three Triticum species to be distinguished. These species also differed significantly with respect to the C18:1/C16 ratio which was equal to 1.78, 1.06, 1.47 and 0.99 in T. monococcum, T. dicoccum, T. spelta and Triticum aestivum respectively.     

Content and relative composition of some phytochemicals in diploid,tetraploid and hexaploid Triticum species with potential nutraceutical properties

Seven cultivated Triticum species or subspecies (Triticum monococcum, Triticum turgidum ssp dicoccum, T. turgidum ssp. durum, T. turgidum ssp turanicum, Triticum timopheevii, Triticum aestivum and Triticum zhukovskyi) were compared for their contents of bioactive compounds, mainly 5-n-alkylresorcinols (ARs). Multivariate analysis of variance and principal component analysis were used to evaluate the differences in the phytochemical profiles and to establish the relationships among variables. Significant differences were observed for both total phenol (TP) and AR content. The highest AR level (377 μg/g) was observed in Triticum dicoccum, which also exhibited the highest variability for these compounds (298–436 μg/g). By contrast, the lowest AR content (286 μg/g) was found in Triticum durum. C21:0 was the main homologue chain in all the samples, its value ranging between 54.5% in T. durum and 41.2% in T. aestivum. The T. dicoccum and T. monococcum wheats had relatively low amounts of TP, whereas Triticum turanicum (215 mg/kg), T. timopheevii (250 mg/kg) and T. zhukovskyi (286 mg/kg) had approximately 3-fold higher TP levels. These results suggest that there are opportunities for breeding wheat varieties with superior health benefits and for promoting the use of ancient Triticum crops as novel sources of healthy food.     

Molecular and Biochemical Characterisation of HMW Glutenin Subunits fromT. tauschiiand the D Genome of Hexaploid Wheat

Electrophoretic (urea SDS–PAGE) and chromatographic (RP–HPLC) analysis was performed on 8 allelic variants of HMW glutenin subunits derived fromTriticum tauschiiand from the D genome of a hexaploid wheat species (Triticum macha) and hexaploid landraces. These subunits had been previously identified using SDS–PAGE. The characterisation revealed that subunits Dy10^tand Dy12^tfromT. tauschiicould be differentiated from their bread wheat counterparts using both urea SDS–PAGE and RP–HPLC. In the latter case, theT. tauschiiy-type subunits were clearly more hydrophobic than the Dy type subunits of bread wheat. The characterisation also suggested that subunit Dx5^t, derived from two separateT. tauschiiaccessions, did not contain the extra cysteine residue characteristic of Dx5 from bread wheat. RFLP analysis of the genes encoding the HMW glutenin subunits of interest suggested that the absence of Dx-type HMW glutenins in two hexaploid landraces was due to lack of expression of their encoding genes. The relationship betweenHindIII DNA fragment size and protein subunit size, as measured by electrophoretic mobility, is examined and discussed. Finally, the solubility properties of a HMW protein designated T1 (derived fromT. tauschiiaccession AUS 18913) suggested that it was not a HMW glutenin subunit as was previously thought. Further studies are needed to clarify the identity of this subunit.     

Development and characterization of a Triticum aestivum-H. villosa T5VS•5DL translocation line with soft grain texture

The Hardness locus on the short arm of chromosome 5D is the main determinant of grain texture in bread wheat. The Pina and Pinb genes are tightly linked at this locus, and the soft kernel texture phenotype results when both genes are present and encode the wild-type puroindoline proteins PINA and PINB. In this study a compensating T5VS•5DL Triticum aestivum-Haynaldia villosa translocation line, NAU415, was characterized by chromosome C-banding, genomic in situ hybridization and molecular markers. Single Kernel Characterization System (SKCS) analysis and scanning electron microscopy indicated that NAU415 had soft endosperm although it lacked the wheat Pina-D1a and Pinb-D1a genes, suggesting the presence of functional Pin gene orthologs on chromosome 5VS. Using a PCR approach, Pina-related (designated Dina) and Pinb-related (Dinb) genes in H. villosa and NAU415 were identified and sequenced. The nucleotide and predicted amino acid sequences showed close similarities to the wild-type puroindolines of T. aestivum cv. Chinese Spring. The tryptophan-rich regions of both Dina and Dinb showed a sequence change from lysine-42 to arginine, a feature that may have an effect on grain texture. The potential of T5VS•5DL translocation line as a source of genes that may be used for modulation of endosperm texture and other valuable traits in wheat breeding is discussed.     

Spectroscopic analysis of diversity in the spatial distribution of arabinoxylan structures in endosperm cell walls of cereal species in the HEALTHGRAIN diversity collection

Two varieties each of spelt (Triticum aestivum var. spelta), durum wheat (Triticum turgidum var. durum), rye (Secale cereale), barley (Hordeum vulgare), oats (Avena sativa), einkorn (Triticum monococcum var. monococcum) and emmer (Triticum turgidum var. dicoccum) (all members of the Pooideae sub-family of grasses) were selected according to variation in their contents of soluble and/or total arabinoxylan (AX) determined during the HEALTHGRAIN diversity screen, together with one genotype of the related “model” grass species Brachypodium distachyon. The spatial distribution of low substituted (LS-AX) and highly substituted arabinoxylan (HS-AX) was determined using FT-IR spectroscopic mapping of transverse thin cross-sections consisting of cell walls only. Variation in cell wall AX composition was observed between the cereals, and compared with that observed for wheat (Triticum aestivum var. aestivum). One line of each cereal type was analysed in more detail using ¹H NMR spectroscopy. The results of the two analyses were consistent, showing variation in the composition and structure of the endosperm cell wall AX that is consistent with the genetic relationships of the cereals studied.     

节节麦大穗大粒相关农艺性状的遗传分析

节节麦(Aegilops tauschii,DD)是六倍体普通小麦D基因组的祖先,其自然类群中含有丰富的抗逆、高产基因,利用其与四倍体硬粒小麦合成的六倍体小麦在现代小麦育种中得到了愈来愈多的应用。本课题在野生节节麦类群中发现了大穗、大粒材料AT462,利用其作母本与节节麦材料AT18(强分蘖)杂交;构建了F2、F3群体,通过调查亲本和群体单株的穗长、小穗数、粒长、粒宽和粒重等表型,对这些穗部性状进行了相关性分析和遗传分析。结果表明:(1)在F2和F3群体中,粒重、粒长与穗长之间不存在显著相关性,而且穗长与粒宽之间在两个群体中的平均相关系数绝对值小于0.1,粒重与小穗数之间的相关系数绝对值小于0.2,表明节节麦大粒相关性状不受穗长的影响,受小穗数影响也较小;(2)采用F2单世代分离分析的方法对节节麦AT462×AT18的F2群体大穗、大粒相关性状进行遗传分析,其中穗长受2对具有加性效应的主效基因控制;粒重和小穗数均同时受2对基因的加性效应、显性效应以及互作效应控制,其中加性效应占主导地位;粒长、粒宽均受2对基因的加性效应、显性效应以及互作效应控制,且三种效应较为均衡。这说明控制节节麦粒重、穗长、小穗数等产量性状相关基因的加性效应在遗传中占主导地位,在育种中较易利用,且其主效基因的遗传力达0.9。     

Allelic variation of polyphenol oxidase genes impacts on Chinese raw noodle color

Polyphenol oxidase (PPO) activity is a factor in time-dependent discoloration of Asian noodles. At least two major genes Ppo-A1 and Ppo-D1 control kernel PPO activity in wheat. The goal was to determine the relative importance of allelic variation for Ppo-A1 and Ppo-D1 on Chinese raw noodle color profile and kernel and flour characteristics using a winter wheat recombinant inbred population segregating for Ppo-A1 and Ppo-D1. Ppo-A1 allelic variation explained 80% and Ppo-D1 allelic variation explained 2% of the variation in kernel PPO activity. The low PPO allele at each locus gave noodles that were brighter (greater L*), more yellow (greater b*) and more red (greater a*) at 24 h, and noodles with less change in L* but more change in a* and b* with time (0-24 h). The two loci combined explained 27, 35, and 17% of variation in L*, a*, and b* at 24 h, and 51, 55, and 58% of the variation in change in L*, a*, and b* with time, respectively. Allelic variation at Ppo-D1 explained a larger percentage of variation than did Ppo-A1 in each case except b* at 24 h where the two loci explained equal amounts.     

Phenolic acids composition,total polyphenols content and antioxidant activity of Triticum monococcum, Triticum turgidum and Triticum aestivum: A two-years evaluation

Wheat is a good source of polyphenols, plant metabolytes with beneficial effects on human health. However, little information is available on phenolic acid composition and concentration in different Triticum species, as well as on possible environmental effects. To shed some light on this issue, thirty-nine wheat accessions cropped for two years and belonging to different Triticum species (Triticum monococcum, Triticum turgidum ssp. dicoccum, turanicum and durum, Triticum aestivum ssp. spelta and aestivum) were assessed for phenolic acids (ferulic, p-coumaric, vanillic, syringic, p-hydroxybenzoic, caffeic acids and syringaldehyde), total polyphenols and antioxidant activity in soluble conjugated and insoluble bound extracts. Ferulic acid was the most abundant compound in both extracts. Insoluble bound phenolic acids represented >90% of total phenolic acids. Einkorn showed the maximum concentration of conjugated phenolic acids (50.5 mg/kg DM), while durum and bread wheats presented the highest content of bound phenolic acids (651.8 and 629.2 mg/kg DM, respectively). Cropping year influenced the concentration of conjugated but not of bound phenolic acids. A survey of phenolic acid distribution in the kernel showed that they are rare in endosperm, but abundant in germ and bran. Total polyphenols and antioxidant activity were highly correlated to phenolic acid content.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Variation in kernel characteristics and protein molecular weight distribution of Langdon durum-wild emmer wheat chromosome substitution lines

Triticum turgidum L. var. dicoccoides (DIC) provides a useful source of genes to improve agronomic and quality characteristics of durum wheat. Research was performed to identify DIC chromosomes that carry useful genes for quality improvement. Langdon-T. dicoccoides substitution lines consisting of 13 lines based on DIC accession PI 481521, 10 lines on PI 478742, and 2 lines on Israel A were evaluated for kernel characteristics and protein molecular weight distribution. DIC chromosomes 3A from PI 481521, and 1A and 7A from PI 478742 increased kernel hardness. Chromosome 2A from PI 481521 increased kernel weight, which resulted in increased semolina yield. Some substituted DIC chromosomes also affected grain protein concentration and protein molecular weight distribution. For example, chromosomes 2A, 5B, and 7B from PI 481521, and 6B from Israel A increased total protein concentration, which was primarily attributed to an increase of SDS soluble gliadins. Chromosome 6B from PI 478742 was unique in that it led to an increase in SDS insoluble high molecular weight polymeric proteins, which contributed to increased dough mixing strength. Results from this research indicated that chromosome 6B from PI 478742 is a potential gene source to improve dough characteristics of durum wheat by increasing insoluble high molecular weight polymeric protein concentration.     

Quality Characteristics of Durum Wheat Lines Deriving High Protein From aTriticum dicoccoides(6b) Substitution

One of the objectives in the Canadian durum wheat (Triticum turgidumL.) breeding programs has been the selection of lines having higher protein content. The Langdon-dicoccoides(Triticum dicoccoides6B) substitution, a source of high protein, has been introgressed into two high yielding, but lower protein Canadian lines of durum wheat. The resulting lines with protein content similar to registered cultivars were evaluated for protein quality. The introgression had no detrimental effects upon pasta cooking quality, and thus the dicoccoides 6B chromosome substitution will be a valuable route to increasing protein level for the durum wheat breeding program.     

Molecular characterization of dimeric alpha-amylase inhibitor genes in wheat and development of genome allele-specific primers for the genes located on chromosome 3BS and 3DS

Alpha-amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. For weevil control, alpha-amylase inhibitors and their genes could be used to genetically engineer weevil resistant seeds. Thirty genes encoding dimeric alpha-amylase inhibitors were isolated from Triticum aestivum L. ‘Chinese Spring’ and characterized by nucleotide and amino acid sequence analysis. Eleven representative alpha-amylase inhibitor genes were identified, and the deduced amino acid sequences of these genes were of high coherence (95.1%). These inhibitors and others obtained from the wheat EST database were clustered into three groups, the genes from ‘Chinese Spring’ were present in each group. Specific primer sets were designed for each group, based on the SNPs of these genes, and the chromosome locations of each group of inhibitor genes investigated by amplification of the ‘Chinese Spring’ ditelosomic lines. There were two and one groups of inhibitor genes on chromosomes 3BS and 3DS, respectively, whereas no group of inhibitor genes was found on chromosome 3AS. Thus, the primer set for each group of inhibitor genes was genome allele-specific. The two known inhibitors, 0.53 and 0.19, were located on chromosomes 3BS and 3DS, respectively. The validity of the three genome allele-specific primer sets was confirmed by amplifications in 15 accessions of Triticum urartu, Triticum monococcum, Aegilops tauschii and Triticum dicoccoides. These results gave further support at the molecular level, that the 24 kDa dimeric alpha-amylase inhibitors in cultivated wheat are encoded by a multigene family.     

不同小麦进化材料生育后期的光合特性及其对环境因子响应的比较

为了解不同小麦近缘材料光合特性,以二倍体节节麦、四倍体硬粒小麦、六倍体普通小麦、八倍体小黑麦为材料,在生育后期测定了其光合气体交换参数、叶绿素含量、叶面积指数等光合相关性状,并对净光合速率在不同光照、CO_2浓度、温度、湿度条件下的变化进行了分析。结果表明,八倍体小黑麦的光合气体交换参数和叶绿素含量比其他三个材料高,六倍体小麦在叶面积上具有明显优势;四倍体和六倍体小麦具有较高的光能利用效率,而二倍体小麦和八倍体小黑麦对强光的抗性较强;六倍体小麦和八倍体小黑麦对CO_2的利用率和抗逆性都较强;八倍体小黑麦的光合速率受温度的影响较小,而二倍体和四倍体小麦的光合速率受温度的影响比较大;二倍体小麦的光合速率受湿度的影响较大,四倍体小麦的光合速率受湿度的影响较小,六倍体小麦和八倍体小黑麦光合速率在高湿条件下变化较小。这说明不同小麦近缘种具有不同的光合特性,是小麦进行小麦高光合育种的重要基因资源。     

Detection of mutations in the 7A allele of wheat (Triticum aestivum) granule-bound starch synthase (Wx-7A) with a monoclonal antibody produced by targeted peptide immunisation

A peptide sequence unique for the granule-bound starch synthase (GBSS1) of hexaploid wheat (Triticum aestivum) was identified by sequence alignment of the three isoforms. Multiple antigenic peptides (MAP) of different length (16 and 20 AA) were synthesised against the selected sequence. The assessment of the immune response in mice against differently presented forms (resin-bound, free) and two types of adjuvants indicated that the free MAP with the longer peptide is more immunogenic. A 20AA MAP that elicited a strong immune response was used to produce a monoclonal antibody for the 7A isoform of GBSS 1. Epitope mapping of the selected Mab (F5-1F2) revealed that a 12mer partial sequence of the immunising peptide was specifically detected. Based on this Mab, a simple high throughput ELISA was developed that allows the quick identification of wheat lines carrying the 7A allele of GBSS 1 with minute amounts of sample.     

Novel allelic variants encoded at the Glu-D3 locus in bread wheat

Low-molecular weight glutenin subunits (LWM-GS) are important components of wheat (Triticum aestivum L.) gluten, with important effects on end-use quality. The LMW-GS are encoded at Glu-3 loci (Glu-A3, Glu-B3 and Glu-D3, on the short arms of chromosomes 1A, 1B and 1D), each of which exhibits extensive allelic variation. Each locus encodes numerous LMW-GS, some of which have similar electrophoretic mobilities, making it difficult to distinguish among Glu-3 loci. Alleles of the Glu-D3 locus of bread wheat are considered the most problematic to assign. To date, six Glu-D3 alleles, designated a, b, c, d, e and f, have been reported. We report five previously undescribed alleles (g, h, i, j and k), and describe a method for characterizing them using a combination of SDS-PAGE and multiplexed PCR-based DNA markers. This method could be used for accurate identification of Glu-D3 alleles, permitting the estimation of the effects of these alleles on end-use quality and the selection of desirable alleles and allelic combinations in wheat breeding.     

Proteomic analysis of the mature kernel aleurone layer in common and durum wheat

The aleurone layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties (T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed.