Effect of the three waxy null alleles on enzymes associated to wheat starch granules using proteomic approach

In bread wheat (Triticum aestivum L.), waxy genes are present at three loci (Wx-A1, Wx-B1 and Wx-D1) and are responsible for amylose synthesis in the grain. Near isogenic lines (NILs) of the agronomic cultivar Tremie with either one, two or three waxy null alleles were used to study the genome response to these null alleles through proteomic analysis of starch granule associated proteins of mature grain. Among the 352 spots that were revealed, 86 varied significantly between NILs. The main differences were observed for the granule bound starch synthase (GBSS), product of the waxy genes. A strong relation (R² = 0.94) between the amount of GBSS per gram of starch granule and the amylose quantity in the grain was revealed through quantification of the GBSS in each NIL. The analysis of GBSS quantity for each NIL invalidated the additive hypothesis for the expression of this enzyme in (Wx-A1a Wx-D1a Wx-B1b) (Wx-D1a Wx-B1a Wx-A1b) and the normal form of Tremie. In addition, four proteins identified as inactive GBSS were increased (+144.9%) in the triple null isogenic form compared to the normal form of Tremie. The quantitative variations of the proteins present in starch granules support the idea of regulatory mechanisms between genomes.     

Molecular characterization of two novel null waxy alleles in Mexican bread wheat landraces

Starch is composed of two glucose polymers: amylose, formed by long linear chains, and amylopectin, which is larger and highly branched. The ratio between the two polymers (usually 22–35% amylose, 68–75% amylopectin) affects the starch properties, thereby determining different aspects of wheat quality. Waxy protein, which is the sole enzyme responsible for amylose synthesis, showed polymorphism in a collection of one hundred and three Mexican landraces, including null alleles at Wx-A1 and Wx-B1 loci, respectively. Molecular characterization of these alleles showed that the null Wx-A1 allele presented in only one accession was novel (Wx-A1o) and involved a deletion spanning the three and a half last exons of the gene. Some of the accessions lacking the Wx-B1 protein contained the common allele Wx-B1b (12.6%) (deletion of approximately 67 kb), while four others (3.8%) possessed the novel Wx-B1l, characterized by the deletion of one cytosine in the second exon of the gene and leading to a change in the ORF. This novel allele is particularly interesting because the absence of the Wx-B1 protein in this case was not associated with the lack of the other genes included in the approximately 67 kb region lost with the common null allele Wx-B1b.     

面条品质性状相关基因在四川小麦中的分布

为给面条小麦亲本材料的选择提供依据,利用多重PCR技术分析了Wx-B1和2个主效PPO位点的等位基因在104份四川主栽小麦和骨干亲本中的分布,并检测了59份新育成品系的淀粉糊化特性.结果表明,控制低PPO活性的等位基因PPO-2Ab和PPO-2Da的频率分别为42.3%和83.6%,Wx-B1蛋白缺失基因Wx-B1b的频率为26.9%,四川小麦淀粉特性整体表现较优良.Wx-B1缺失小麦在峰值粘度、稀懈值、最终粘度和反弹值方面与正常类型间差异显著.绵阳26 、川麦107、川麦43等是面条小麦育种的优良亲本.准确、实用、高效多重PCR技术适合于多位点控制的品质性状的标记辅助选择.     

一些小麦品种的淀粉特性分析

为了筛选出淀粉品质优良的面条小麦品种,分析了200份国内小麦品种(系)和6份澳大利亚小麦品种的淀粉特性及Waxy蛋白亚基组成.与国外优质面条小麦相比,国内小麦的淀粉品质总体上有一定差距,但主要淀粉特性的变异范围比较广,如直链淀粉含量变幅为17.79%～27.14%,膨胀势为3.77～10.45g/g,峰值粘度为491.50～4 497.00 cP,低谷粘度为74.00～3 042.00 cP,崩解值为584.50～1 986.50 cP.国内小麦品种(系)中有15份缺失Wx-B1蛋白亚基,其直链淀粉含量、峰值粘度和崩解值的平均值优于正常类型品种(系),如花培18-5直链淀粉含量低于19%,峰值粘度高于4000 cP,崩解值高于1 500 cP.河南省小麦品种(系)的淀粉特性优于其他省市.     

Analyses of albumins, globulins and amphiphilic proteins by proteomic approach give new insights on waxy wheat starch metabolism

Starch is composed of two types of glucose polymers: amylose and amylopectin. The Waxy (Wx) locus controls amylose synthesis in the wheat kernel. Hexaploid wheat has three Wx loci located on chromosomes 7A (Wx-A1), 4A (Wx-B1), and 7D (Wx-D1). Eight near isogenic lines (NILs) of Triticum aestivum cv. Tremie with one, two or three Wx null alleles were used. The albumin–globulin fraction, and amphiphilic proteins were separated using 2-dimensional electrophoresis (2DE) allowing the changes in the waxy kernel to be identified. Albumin–globulin fraction showed overexpression of sucrose synthases in the waxy NILs compared to the normal form of Tremie and a decrease in many proteins related to stress and defence metabolism such as serpins. A subunit of ADP-glucose pyrophosphorylase (AGPase), which is known to play a major role in starch synthesis, was also shown to be down regulated in the waxy NILs. Amphiphilic proteins confirmed the observations made on the albumin–globulin fraction with a decrease in a stress-related protein. These different regulations linked to observations made on wheat kernel (thousand kernel weight (TKW), protein amount per grain, size and distribution of the starch granules) led to formulation of the hypothesis that waxy endosperm does not reach maturity of the wild-type endosperm.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

Functionality of chemically modified wild-type,partial waxy and waxy starches from tetraploid wheats

Partial waxy (reduced-amylose) and fully waxy (amylose-free) tetraploid durum wheats (Triticum turgidum L. var durum) were developed by introgression of null alleles at the Wx-A1 and Wx-B1 loci from common hexaploid wheat (Triticum aestivum L.). Purified starches were obtained from each genotype, and chemically modified by: 1) cross-linking with phosphorus oxychloride, 2) substitution with propylene oxide, and 3) sequential cross-linking with phosphorus oxychloride followed by substitution with propylene oxide. Functional properties were compared to blends of waxy and wild-type durum starches of known amylose contents. Significant differences in functionality were observed amongst the genotypes and blends after each modification. Waxy (0% amylose) and wild-type (30% amylose) typically were at the extremes of the observed ranges of functional properties. In general, the functional properties of the chemically modified starches were dependent upon amylose content. Starches from Wx-B1 null lines (24% amylose), were an exception. After substitution, such starches had the significantly highest value for RVA final viscosity, and generally performed in a manner similar to starch blends of 12–18% amylose.     

Y-type gene specific markers for enhanced discrimination of high-molecular weight glutenin alleles at the Glu-B1 locus in hexaploid wheat

A number of primers were designed which target DNA sequence variation of the coding and /or promoter regions of wheat HMW glutenin y-type genes located at the Glu-B1 locus. This allowed the development of a set of PCR-based markers for specific HMW glutenin genes encoding By-subunits for which no markers were previously available. Markers were validated using test cultivars containing specific Glu-B1 alleles confirmed by SDS-PAGE and RP-HPLC analysis. Among the specific markers developed, primer pair ZSBy8F5/R5 was specific for the By8 gene, which exists in Glu-B1b (Bx7+By8) and Glu-B1u (Bx7*+By8) alleles. This marker allows discrimination of alleles containing By8 and By8* that are usually difficult to distinguish using SDS-PAGE. Since the over-expressed Glu-B1 allele (Glu-Bl al.) contains the By8* subunit, it is possible to use this marker in breeding programs for selecting for the over-expression of subunit Bx7 in crosses that segregate between normal Bx7 and over-expressed Bx7 subunits. This marker also represents an alternative for distinguishing two common Glu-B1 alleles: Glu-B1i (Bx17+By18) and Glu-B1b (Bx7+By8). Two primer pairs ZSBy9aF1/R3 and ZSBy9F7/R6 both gave characteristic banding patterns for Glu-B1c (Bx7+By9) and can therefore be used to discriminate By9 - containing alleles from non - By9 alleles. Primer pair ZSBy9F2/R2 produced amplicons with a diagnostic banding pattern for allele Glu-B1f (Bx13+By16) and also permitted the discrimination of Glu-B1h (Bx14+By15) and Glu-B1e (Bx20) that have opposing genetic effects on wheat quality and are difficult to discriminate by SDS-PAGE.     

小麦Wx-A1和Wx-B1亚基的生化与分子标记检测

为了解小麦品种(系)Wx亚基的缺失类型,为品质育种提供依据,利用SDS-PAGE和STS分子标记技术对99份国内外小麦品种(系)Wx-A1和Wx-B1亚基的缺失型进行检测.结果表明,供试小麦品种(系)中Wx-B1亚基缺失的有19份,未检测出Wx-A1亚基缺失材料.STS标记结果中,Wx-7A位点的显性标记引物在野生型中扩增出一条1 172 bp的特异带,即Wx-A1a基因出现;Wx-7A位点的共显性标记引物在野生型中扩增出一条593 bp的特异带,在缺失类型中扩增出一条574 bp的特异带.Wx-4A位点的显性标记引物在野生型中扩增出一条425 bp的特异带,即Wx-B1a亚基出现,而在缺失该亚基的突变材料中没有扩增出该特异带.     

A novel hardness-related and starch granule-associated protein marker in wheat: LMW-GS-‘S’

Starch granule (SG)-associated proteins are involved in starch synthesis and the interaction between SGs and the endosperm protein matrix. In this study, SG proteins were sequentially extracted with the chaotropic reagent, urea from 1 M to 4 M, and then profiled using an integrated proteomic approach including one- and two-dimensional electrophoresis, mass spectrometry and antibody-based enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the SG-associated proteins were dominated by granule-bound starch synthase (GBSS), gliadin, low molecular weight glutenin subunits (LMW-GS), serine protease inhibitors, α-amylase inhibitors and puroindolines. A protein with an apparent molecular mass of 50 kDa, expressed in cultivar hard wheat Kukri but not in soft wheat Triller was identified as a novel member of the ‘S’ group of LMW-GS, designated as LMW-GS-‘S’. Further characterization using a broad wheat population revealed that LMW-GS-‘S’ was selectively expressed in hard wheat cultivars while deleted in all soft wheats tested. Its relationship with hardness was confirmed by its expression in tetraploid durum wheats, which are among the hardest wheats around the world. Monoclonal antibody (MAb) F8-14E6 against LMW-GS-‘S’ was developed and used in an ELISA to screen 90 Glu allele-defined doubled haploid Janz/Kukri wheat lines. The allele that encodes LMW-GS-‘S’ was mapped to GluB3h (p < 0.001).     

PCR-based markers for identification of HMW-GS at Glu-B1x loci in common wheat

The bread wheat elasticity, which is very important for bread-making quality, is largely determined by the composition of high-molecular-weight glutenin subunits (HMW-GS). The HMW-GS encoded by Glu-B1 loci are highly polymorphic and the combinations 17+18 and 14+15 are good for bread making. Thus it is very important to identify the alleles at Glu-B1 loci for wheat quality improvement. In this study, the five common HMW-GS types encoded by Glu-B1x locus carried by 18 Chinese bread wheat cultivars (or lines) were analyzed by SDS-PAGE. Two pairs of PCR primers which could distinguish the Glu-Blx alleles of the five common HMW-GS types were designed based on the Glu-B1x gene sequences (Reddy and Appels, 1993; Genbank accession: X13927; Genbank accession:AY367771). 22 recombinant inbred lines (RILs) derived from Jing711 (contains 17 subunit on Glu-B1x) and Pm97034 (contains 14 subunit on Glu-B1x) were used to validate the accuracy of the primers, which showed that the two specific markers could be used together to distinguish alleles at Glu-B1x locus and accelerate wheat quality breeding by marker assisted selection.     

Both genetic and environmental conditions affect wheat grain texture: Consequences for grain fractionation and flour properties

This review summarizes the results of studies on near-isogenic common wheat lines differing in the Pinb-D1 allele encoding puroindoline B or durum wheat into which both wild-type puroindoline genes were introduced. The material was grown in different environments to evaluate the respective effect of puroindoline genes or of the environmental factors on grain characteristics and milling behavior.Environmental conditions were found to impact grain porosity (=1/vitreousness) and the presence of both wild-type puroindoline genes was found to reduce the vitreousness threshold under 60%. Hardness measurements with single kernel characterization system were found to differ from near-infrared reflectance spectroscopy analysis and were linearly related to vitreousness but differently depending on the puroindoline allele carried.Puroindoline genes were found to play a major role in the grain porosity, breaking energy, size of generated particles and in the concentration of phytic acid and damaged starch into flour whereas vitreousness introduced variations in the ability to break and in the level of damaged starch.Finally, the highest flour yield is obtained from either vitreous common wheat grains carrying the wild-type puroindoline alleles or carrying mutated alleles and displaying low vitreousness. This result was confirmed using common French wheat cultivars whose puroindoline genes were identified.     

Glu-D1d与Wx-B1b基因聚合在强筋小麦育种中的利用

Glu-D1d与Wx-B1b基因遗传效应是面包面条兼用型强筋小麦的主要遗传基础，二者聚合可实现蛋白质(面筋)质量和淀粉特性的同步改良，并拓宽和提升强筋小麦的加工用途和商品价值。本文总结了Glu-D1d与Wx-B1b基因及其聚合对强筋小麦品质的影响，并对两个基因聚合育种策略和注意事项进行探讨，以期为面包面条兼用型强筋小麦育种提供理论依据和实践经验。     

Waxy endosperm accompanies increased fat and saccharide contents in bread wheat (Triticum aestivum L.) grain

The contents of fat, starch, pentosan, fructan, β-glucan and several mono- and oligosaccharides in grain were evaluated to find out the possible effects of the Wx-D1 gene of bread wheat using two sets of near-isogenic waxy and non-waxy lines and two low-amylose mutant lines with a common genetic background of Kanto 107. These materials have two non-functional Wx-A1b and Wx-B1b alleles in common. Waxy near-isogenic lines with a non-functional Wx-D1d allele showed consistently increased contents of fat, total fructan, β-glucan, glucose, fructose, sucrose, 1-kestose, 6-kestose, neokestose, nystose and bifurcose compared with non-waxy lines with a functional Wx-D1a allele throughout three growing/harvest seasons. Starch and total pentosan contents were inconsistently influenced by the allelic status of the Wx-D1 locus, while water-soluble pentosan and raffinose contents were not affected. The compositional changes of a low-amylose mutant line with an almost non-functional Wx-D1f allele were closely similar to those of waxy near-isogenic lines, while significantly different changes were barely observed in another low-amylose mutant line with a partly functional Wx-D1g allele in two seasons. These results showed that the Wx-D1 gene has pleiotropic effects on the fat and saccharide contents of bread wheat grain.     

Modeling plant carbon flow and grain starch accumulation in wheat

The process of starch accumulation in grain directly influences the yield and quality formation in wheat. Since few studies have been aimed at modeling the grain starch accumulation, this study was undertaken to develop a simplified process model for predicting the rate of starch accumulation in wheat grain by focusing on the variation of plant carbon dynamics post-anthesis. Five different experiments involving genotypes, nitrogen rates and water regimes were conducted to support model development and model evaluation. The model proposed that the starch accumulation rate (STR) in individual grain was determined by the availability of carbon source in plant (GCA_i) and the ability of starch synthesis in grain (f(A_i)), as influenced by the factors of temperature, water and nitrogen conditions within plants. The f(A_i) could be described in a two-section curve with post-anthesis growing degree days (GDD), first exponential increase and then linear decrease. The GCA_i was determined by post-anthesis carbon assimilation and carbon remobilization from vegetative organs to grains. A genotypic parameter was incorporated into the model algorithm, i.e. the maximum rate of individual grain starch accumulation, to differentiate the ability of starch accumulation among cultivars. The overall performance of the model was validated with different data sets from three field experiments spanning 3 years and comprising various genotypes, nitrogen and water levels. The RMSE values for all treatments were averaged as 12.51%, indicating a good fit between the simulated and observed data. It appears that the model can give a reliable prediction for grain starch accumulation of different wheat cultivars under various growing conditions.     

Characterization of a wheat mutant missing low-molecular-weight glutenin subunits encoded by the B-genome

DH20, a new wheat mutant missing low-molecular weight glutenin subunits encoded by the Glu-B3 locus, was discovered among double haploid lines obtained from a cross between the Korean wheat cultivars Keumkang and Olgeuru. Absence of the Glu-B3 LMW-GS proteins was determined by one-dimensional gel electrophoresis (SDS-PAGE) and confirmed by two-dimensional gel electrophoresis (2-DGE) and tandem mass spectrometry (MS/MS). The deletion of Glu-B3 genes was also demonstrated using allele specific DNA markers. Basic agronomic traits, protein content, dough mixing properties and bread loaf volume of DH20 and parental wheat cultivars were evaluated in field-grown wheat over a two year period. This mutant is a valuable resource for understanding the function of individual LMW-GS alleles or genes at the Glu-B3 locus using either breeding or biotechnology approaches.     

宁夏春小麦种质资源Wx基因分子鉴定及其分布

为明确宁夏春小麦种质资源Waxy基因的分布情况,利用 Wx-A1、 Wx-B1和 Wx-D1位点的6个STS标记(PA1、PA2、PB1、PB2、PD1、PD2),对299个宁夏春小麦种质资源进行等位变异检测。结果表明,供试小麦种质的Waxy基因组成以野生型(Wx-A1a/ Wx-B1a/ Wx-D1a)为主,占参试材料总数的88.3%。在 Wx-A1、 Wx-B1和 Wx-D1基因位点上,发现 Wx-A1b、 Wx-B1b和 Wx-D1b突变型材料分别有2、32和2个,分别占材料总数的0.7%、10.7%和0.7%;其中,有1个材料为 Wx-A1b/ Wx-B1b突变型,占材料总数的0.3%。宁夏春小麦种质资源中 Wx-B1基因缺失类型的分布频率相对较高,在新品种和高代品系中该类型逐步增加。该研究有助于了解宁夏优质面粉小麦品种的生产现状,为该区和我国其他相关春麦区优质面条小麦的选育奠定基础。     

Molecular and physico-chemical evaluation of enzymatic browning of whole meal and dough in a collection of tetraploid wheats

Many studies have demonstrated the role of polyphenol oxidase (PPO; E.C. 1.14.18.1) in darkening of wheat products. Even slight browning of pasta is a major hindrance to consumer acceptance. The aim of this research was to evaluate the variability of PPO activity, browning level, and protein content in a collection of more than 100 genotypes of tetraploid wheat, including cultivars and landraces. A molecular approach was followed to evaluate the efficiency of marker PPO18, discovered and used in common wheat, in detecting tetraploid wheat genotypes with low browning level. The data showed a significant genotype influence on the activity of the PPO enzyme, which was correlated with brown index of whole meal and dough, and with protein content. On the whole, the cultivars showed lower PPO activity, brown index, and protein content than landraces, but wide variability was present. Marker PPO18 detected four different alleles for Ppo-A1. The allele Ppo-A1f identified genotypes with high PPO activity and brown index, while the Ppo-A1b and null alleles were associated with the opposite characteristics. Hence, this marker can be used to select new tetraploid wheat cultivars with low browning level.     

Effect of high molecular weight glutenins and rye translocations on soft wheat flour cookie quality

The influence of high molecular weight glutenin subunits (HMW-GS) on wheat breadmaking quality has been extensively studied but the effect of different Glu-1 alleles on cookie quality is still poorly understood. This study was conducted to analyze the effect of HMW-GS composition and wheat-rye translocations on physicochemical flour properties and cookie quality of soft wheat flours. Alleles encoded at Glu-A1, Glu-B1 and Glu-D1 locus had a significant effect over physicochemical flour properties and solvent retention capacity (SRC) profile. The null allele for Glu-A1 locus presented the highest cookie factor observed (CF = 7.10), whereas 1BL/1RS and 1AL/1RS rye translocations had a negative influence on CF. The three cultivars that showed the highest CF (19, 44 and 47) had the following combination: Glu-A1 = null, Glu-B1 = 7 + 8, Glu-D1 = 2 + 12 and no secalins. Two prediction equations were developed to estimate soft wheat CF: one using the HMW-GS composition and the other using physicochemical flour parameters, where SRCsuc, SRC carb, water-soluble pentosans, damaged starch and protein turned out to be better CF predictors. This data suggests that grain protein allelic composition and physicochemical flour properties can be useful tools in breeding programs to select soft wheat of good cookie making quality.     

Genome-wide association mapping of starch granule size distribution in common wheat

Starch is a crucial component in wheat endosperm and plays an important role in processing quality. Endosperm of matured wheat grains contains two distinct starch granules (SG), referred to as larger A- and smaller B-granules. In the present study, 166 Chinese bread wheat cultivars planted in four environments were characterized for variation in SG size. A genome-wide association study (GWAS) using the 90 K SNP assay identified 23 loci for percentage volumes of A- and B-granules, and 25 loci for the ratio of A-/B-granules volumes, distributing on 15 chromosomes. Fifteen MTAs were associated with both the percentage volumes of A-, B-granules and the ratio of A-/B-granules volumes. MTAs IWB34623 and IWA3693 on chromosome 7A and IWB22624 and IWA4574 on chromosome 7B associated with the percentage volumes of A- and B-granules consistently identified in multiple environments were considered to be stable. Linear regression analysis showed a significantly negative correlation of the number of favorable alleles with the percentage volumes of A-granules and a significantly positive correlation between the number of favorable alleles and the percentage volumes of B-granules, respectively. The loci identified in this study and associated markers could provide basis for manipulating SG size to obtain superior noodle quality in wheat.