Multiple QTL-effects of wheat Gpc-B1 locus on grain protein and micronutrient concentrations

Micronutrient malnutrition afflicts over three billion people worldwide and the numbers are continuously increasing. Developing genetically micronutrient-enriched cereals, which are the predominant source of human dietary, is essential to alleviate malnutrition worldwide. Wheat chromosome 6B derived from wild emmer wheat [ Triticum turgidum ssp. dicoccoides (Körn.) Thell] was previously reported to be associated with high Zn concentration in the grain. In the present study, recombinant chromosome substitution lines (RSLs), previously constructed for genetic and physical maps of Gpc-B1 (a 250-kb locus affecting grain protein concentration), were used to identify the effects of the Gpc-B1 locus on grain micronutrient concentrations. RSLs carrying the Gpc-B1 allele of T. dicoccoides accumulated on average 12% higher concentration of Zn, 18% higher concentration of Fe, 29% higher concentration of Mn and 38% higher concentration of protein in the grain as compared with RSLs carrying the allele from cultivated wheat ( Triticum durum ). Furthermore, the high grain Zn, Fe and Mn concentrations were consistently expressed in five different environments with an absence of genotype by environment interaction. The results obtained in the present study also confirmed the previously reported effect of the wild-type allele of Gpc-B1 on earlier senescence of flag leaves. We suggest that the Gpc-B1 locus is involved in more efficient remobilization of protein, zinc, iron and manganese from leaves to the grains, in addition to its effect on earlier senescence of the green tissues.     

Physical map of the wheat high-grain protein content gene Gpc-B1 and development of a high-throughput molecular marker

Grain protein content (GPC) is important for human nutrition and has a strong influence on pasta and bread quality. A quantitative trait locus, derived from a Triticum turgidum ssp. dicoccoides accession (DIC), with an average increase in GPC of 14 g kg(-1) was mapped on chromosome 6BS. Using the wheat-rice colinearity, a high-density map of the wheat region was developed and the quantitative trait locus was mapped as a simple Mendelian locus designated Gpc-B1. A physical map of approx. 250 kb of the Gpc-B1 region was developed using a tetraploid wheat bacterial artificial chromosome library. The constructed physical map included the two Gpc-B1 flanking markers and one potential candidate gene from the colinear rice region completely linked to Gpc-B1. The relationship between physical and genetic distances and the feasibility of isolating genes by positional cloning in wheat are discussed. A high-throughput codominant marker, Xuhw89, was developed. A 4-bp deletion present in the DIC allele was absent in a collection of 117 cultivated tetraploid and hexaploid wheat germplasm, suggesting that this marker will be useful to incorporate the high GPC allele from the DIC accession studied here into commercial wheat varieties.     

Precise mapping of a locus affecting grain protein content in durum wheat

Grain protein content (GPC) is an important factor in pasta and breadmaking quality, and in human nutrition. It is also an important trait for wheat growers because premium prices are frequently paid for wheat with high GPC. A promising source for alleles to increase GPC was detected on chromosome 6B of Triticum turgidum var. dicoccoides accession FA-15-3 (DIC). Two previous quantitative trait locus (QTL) studies found that the positive effect of DIC-6B was associated to a single locus located between the centromere and the Nor-B2 locus on the short arm of chromosome 6B. Microsatellite markers Xgwm508 and Xgwm193 flanking the QTL region were used in this study to develop 20 new homozygous recombinant substitution lines (RSLs) with crossovers between these markers. These 20 RSLs, plus nine RSLs developed in previous studies were characterized with four new RFLP markers located within this chromosome segment. Grain protein content was determined in three field experiments organized as randomized complete block designs with ten replications each. The QTL peaks for protein content were located in the central region of a 2.7-cM interval between RFLP markers Xcdo365 and Xucw67 in the three experiments. Statistical analyses showed that almost all lines could be classified unequivocally within low- and high- protein groups, facilitating the mapping of this trait as a single Mendelian locus designated Gpc-6B1. The Gpc-6B1 locus was mapped 1.5-cM proximal to Xcdo365 and 1.2-cM distal to Xucw67. These new markers can be used to reduce the size of the DIC chromosome segment selected in marker-assisted selection programs. Markers Nor-B2 and Xucw66 flanking the previous two markers can be used to select against the DIC segment and reduce the linkage drag during the transfer of Gpc-6B1 into commercial bread and pasta wheat varieties. The precise mapping of the high GPC gene, the high frequency of recombinants recovered in the targeted region, and the recent development of a tetraploid BAC library including the Gpc-6B1 DIC allele are the first steps towards the map-based cloning of this gene.Communicated by J. Dvorak     

Control of barley (Hordeum vulgare L.) development and senescence by the interaction between a chromosome six grain protein content locus, day length, and vernalization

Regulatory processes controlling traits such as anthesis timing and whole-plant senescence are of primary importance for reproductive success and for crop quality and yield. It has previously been demonstrated that the presence of alleles associated with high grain protein content (GPC) at a locus on barley chromosome six leads to accelerated leaf senescence, and to strong (>10-fold) up-regulation of several genes which may be involved in senescence control. One of these genes (coding for a glycine-rich RNA-binding protein termed HvGR-RBP1) exhibits a high degree of similarity to Arabidopsis glycine-rich RNA-binding protein 7 (AtGRP7), which has been demonstrated to accelerate flowering under both long-day (LD) and short-day (SD) conditions, but not after vernalization. Development of near-isogenic barley lines, differing in the allelic state of the GPC locus, was compared from the seedling stage to maturity under both SD and LD and after vernalization under LD. Intriguingly, pre-anthesis plant development [measured by leaf emergence timing and pre-anthesis (sequential) leaf senescence] was enhanced in high-GPC germplasm. Differences were more pronounced under SD than under LD, but were eliminated by vernalization, associating observed effects with floral induction pathways. By contrast, differences in post-anthesis flag leaf and whole-plant senescence between low- and high-GPC germplasm persisted under all tested conditions, indicating that the GPC locus, possibly through HvGR-RBP1, impacts on both developmental stages. Detailed molecular characterization of this experimental system may allow the dissection of cross-talk between signalling pathways controlling early plant and floral development on one side, and leaf/whole-plant senescence on the other side.     

Characterization of grain protein content gene (<Emphasis Type="Italic">GPC</Emphasis>-<Emphasis Type="Italic">B1</Emphasis>) introgression lines and its potential use in breeding for enhanced grain zinc and iron concentration in spring wheat

At least two billion people around the world suffer from micronutrient deficiency, or hidden hunger, which is characterized by iron-deficiency anemia, vitamin A and zinc deficiency. As a key staple food crop, wheat provides 20% of the world’s dietary energy and protein, therefore wheat is an ideal vehicle for biofortification. Developing biofortified wheat varieties with genetically enhanced levels of grain zinc (Zn) and iron (Fe) concentrations, and protein content provides a cost-effective and sustainable solution to the resource-poor wheat consumers. Large genetic variation for Fe and Zn were found in the primitive and wild relatives of wheat, the potential high Zn and Fe containing genetic resources were used as progenitors to breed high-yielding biofortified wheat varieties with 30–40% higher Zn content. Grain protein content (GPC) determines processing and end-use quality of wheat for making diverse food products. The GPC-B1 allele from Triticum turgidum L. var. dicoccoides have been well characterized for the increase in GPC and the associated pleiotropic effect on grain Zn and Fe concentrations in wheat. In this study effect of GPC-B1 allele on grain Zn and Fe concentrations in wheat were measured in different genetic backgrounds and two different agronomic management practices (with- and without foliar Zn fertilization). Six pairs of near-isogenic lines differing for GPC-B1 gene evaluated at CIMMYT, Mexico showed that GPC-B1 influenced marginal increase for grain Zn, Fe concentrations, grain protein content and slight reduction in kernel weight and grain yield. However, the magnitude of GPC and grain Zn and Fe reductions varied depending on the genetic background. Introgression of GPC-B1 functional allele in combination with normal or delayed maturity alleles in the CIMMYT elite wheat germplasm has the potential to improve GPC and grain Zn and Fe concentrations without the negative effect on grain yield due to early senescence and accelerated maturity.     

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Content and Degradation in Diploid,Tetraploid, and Hexaploid Wheat Species during Monocarpic Senescence

Wheat provides a unique genetic system in which variable sink size is available across the ploidies. We characterized monocarpic senescence in diploid, tetraploid, and hexaploid wheat species in flag leaf from anthesis up to full grain maturity at regular intervals. Triticum tauschii Acc. cv. EC-331751 showed the fastest rate of senescence among the species studied and the rate of loss per day was highest in terms of photosynthesis rate, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) content, and flag leaf N content coupled with a higher rate of gain in grain N content. Cultivars Kundan and HD 4530 maintained high flag leaf N content throughout grain filling as compared to the diploids and showed a slower rate of senescence. RuBPCO content was higher in the diploids as compared to Kundan and HD 4530 at anthesis. However, the rate of decline in RuBPCO content per day was also higher in the diploids. This degradation in RuBPCO was mediated by high endoproteolytic activities in the diploids which in turn supported its higher rate of N mobilization as compared to the tetraploid and hexaploid wheat. Acidic endopeptidases were responsible for the mobilization of flag leaf nitrogen in wheat across ploidy levels (r=–0.582, p<0.01).     

A high-grain protein content locus on barley (Hordeum vulgare) chromosome 6 is associated with increased flag leaf proteolysis and nitrogen remobilization

Leaf senescence and nitrogen remobilization from senescing tissues are two important factors determining grain protein content (GPC) in cereals. We compared near-isogenic barley ( Hordeum vulgare L.) germplasm varying in the allelic state of a major GPC quantitative trait locus on chromosome 6, delineated by molecular markers HVM74 and ABG458 and explaining approximately 46% of the variability in this trait. High GPC was consistently associated with earlier whole-plant senescence. SDS–PAGE and immunoblot analysis of flag leaf proteins indicated earlier leaf protein [including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)] degradation in high-GPC germplasm. This was accompanied by enhanced availability of ammonium and glutamine in developing kernels, suggesting increased phloem retranslocation of nitrogen. Based on previous microarray analysis, we performed a detailed expression study of six leaf genes, tentatively involved in plastidial proteolysis, vacuolar proteolysis, intermediary N metabolism and N transport. All of these were upregulated in high-GPC barley, mostly around 21 to 28 days past anthesis, prior to or around the time demonstrating maximal differences in leaf protein (including Rubisco) levels. Therefore, these genes represent potential targets to manipulate grain protein accumulation. It appears likely that their functional analysis will enhance our understanding of whole-plant N recycling. Additionally, earlier leaf (photosynthetic) protein degradation may lead to reduced N carbon assimilation in high-GPC germplasm, explaining past studies demonstrating a negative correlation between GPC and yield.     

Anthesis date mainly explained correlations between post-anthesis leaf senescence, grain yield, and grain protein concentration in a winter wheat population segregating for flowering time QTLs

The genetic variability of the duration of leaf senescence during grain filling has been shown to affect both carbon and nitrogen acquisition. In particular, maintaining green leaves during grain filling possibly leads to increased grain yield, but its associated effect on grain protein concentration has not been studied. The aim of this study was to dissect the genetic factors contributing to correlations observed at the phenotypic level between leaf senescence during grain filling, grain protein concentration, and grain yield in winter wheat. With this aim in view, an analysis of quantitative trait locus (QTL) co-locations for these traits was carried out on a doubled haploid mapping population grown in a large multienvironment trial network. Pleiotropic QTLs affecting leaf senescence and grain yield and/or grain protein concentration were identified on chromosomes 2D, 2A, and 7D. These were associated with QTLs for anthesis date, showing that the phenotypic correlations with leaf senescence were mainly explained by flowering time in this wheat population. Study of the allelic effects of these pleiotropic QTLs showed that delaying leaf senescence was associated with increased grain yield or grain protein concentration depending on the environments considered. It is proposed that this differential effect of delaying leaf senescence on grain yield and grain protein concentration might be related to the nitrogen availability during the post-anthesis period. It is concluded that the benefit of using leaf senescence as a selection criterion to improve grain protein concentration in wheat cultivars may be limited and would largely depend on the targeted environments, particularly on their nitrogen availability during the post-anthesis period.     

The nature of the effects of the aphids Sitobion avenae and Metopolophium dirhodum on the growth of wheat

Field-caged, post-anthesis populations of the aphids Sitobion avenae and Metopolophium dirhodum reduced grain weight of wheat by 14 and 7% respectively and induced changes in the senescence of the flag leaf. Spikelet number and grain number were unaffected as they are normally determined by pre-anthesis factors. Percentage grain protein was significantly reduced by both aphid species. The pattern of grain weight reduction within the ear was consistent with known limitations on the distribution of flag leaf assimilates among the grains. The relative effects of the two aphid species apparently resulted from the degree of nutrient drain imposed at particular feeding sites and the reduction in the leaf area duration of the flag leaf.     

Chlorophyll a fluorescence during flag leaf senescence of field-grown winter wheat plants under drought conditions

Improving wheat grain yield plays a significant role in ensuring global food security. Wheat production could be increased by the genetic improvement of wheat genotypes where delayed senescence with enhanced post-anthesis capacity and staygreen traits could have an important role. In this study, chlorophyll a fluorescence (ChlF) rise kinetics from the early until late senescence of flag leaves, grain yield and other agro-morphological characteristics were compared for three winter wheat advanced lines (Osk.4.312/10-18, Osk.4.330/6-18 and Osk.4.354/12-18) under natural drought conditions. The differences between lines were observed when considering the heading date which was 1 and 4 days earlier for the line Osk.4.354/12-18, than lines Osk.4.312/10-18 and Osk.4.330/6-18, respectively. Furthermore, line Osk.4.354/12-18 had the highest test weight (kg hl⁻¹), while line Osk.4.330/6-18 showed a tendency of decreased grain yield, compared to the other two lines. Analysis of ChlF transients and several JIP-test parameters indicated that all three lines had a generally similar course of changes in the photosynthetic performance of flag leaves during senescence under drought conditions. However, at the point when a decrease in photosynthetic performance was initiated, it was slightly less intensive in line Osk.4.354/12-18 accompanied by longer preservation of functionality and connectivity of PSII units, than in the other two lines, which contributed to its better agronomical performance. These results indicated that even delicate variations in the functioning of the photosynthetic apparatus of the flag leaf during grain filling were agronomically important, especially when plants were exposed to drought stress, and could be used to differentiate otherwise similar wheat genotypes. Even small genotype-specific differences in the photosynthetic performance of senescing flag leaves, along with earlier heading dates, could assist in the selection of genotypes with a better ability to cope with unfavourable environmental conditions.     

Senescence and net photosynthesis of the flag leaf and grain growth of wheat in response to fungicide

Green leaf area and net photosynthesis of the flag leaves of Avalon and Maris Huntsman winter wheat crops were studied in relation to grain growth following the application of the fungicide propiconazole at flag leaf emergence. Disease levels were low during grain-fill, and were not significantly affected by the fungicide. Propiconazole significantly maintained green leaf area and photosynthesis per unit area during the period of rapid flag leaf senescence, but it had no effect on stem weight, grain growth or yield.     

Molecular mapping of QTLs for wheat flag leaf senescence under water-stress

A segregating population from the cross between drought sensitive (Variant-2) and drought tolerant (Cham-6) genotypes was made to identify molecular markers linked to wheat (Triticum aestivum L.) flag leaf senescence under water-stress. From 38 random amplified polymorphic DNA (RAPD) primers, 25 inter-simple sequence repeat (ISSR) primers and 46 simple sequence repeat (SRR) primers, tested for polymorphism among parental genotypes and F2 population. Quantitative trait locus (QTL) for flag leaf senescence was associated with 1 RAPD marker (Pr9), 4 ISSR markers (Pr8, AD5, AD2 and AD3), and 1 SSR marker (Xgwm382) and explained 44, 50, 35, 31, 22 and 73 % phenotypic variation, respectively. The genetic distance between flag leaf senescence gene and Pr9 was 10.0 cM (LOD score 22.9). The markers Pr8, AD5, AD2 and AD3 had genetic distances of 10.5, 14.6, 15.6 and 18.1 cM, respectively (LOD scores 22.6, 17.8, 17.5 and 14.6). The genetic distance between Xgwm382 was 3.9 cM (LOD score 33.8). Therefore, the RAPD, ISSR and SSR markers linked to the QTL for the drought-induced flag leaf senescence can be further used in breeding for drought tolerance in wheat.     

Influence of reproductive organs on plant senescence in rice and wheat

The chlorophyll and protein contents of the flag, second and third leaves gradually decreased during the reproductive development of rice (Oryza sativa L. cv. Rasi) and wheat (Triticum aestivum L. cv. Sonalika) plants, whereas proline accumulation increased up to the grain maturation stage and slightly decreased thereafter. In rice plant, the rate of decrease in chlorophyll and protein and increase in proline level were higher in the flag leaf than in the second leaf. It was opposite in wheat plant. The export of [³²P]-phosphate from leaves to grains gradually increased reaching a maximal stage at the grain development stage, and then declined. The export of this radioisotope was greater in rice than in wheat. Removal of panicle at the anthesis and grainfilling stages delayed leaf senescence of rice plant, while in wheat the ponicle removal at any stage did not have a marked effect on delaying leaf senescence. The contents of chlorophyll and protein of glumes were higher in wheat than in rice. The variation of such source-sink relationship might be one of the possible reasons for the above effect on leaf senescence.     

The effect of thidiazuron on the yield of wheat grown with varying nutrient supply

The effect of the synthetic cytokinin thidiazuron on the yield of wheat growing under conditions of varying nutrient supply was investigated. Applications of thidiazuron during the early growth stages of wheat promoted tillering but reduced yield. Applications of thidiazuron during flag leaf senescence had little effect on yield. However, the yield of plants was increased at all levels of nutrient supply by treating plants with paclobutrazol during the early growth stages followed by thidiazuron during flag leaf senescence. Yield increases were greatest in moderately nutrient stressed plants, but were accompanied by a reduction in the N concentration of the grain. Possible reasons for the interaction between thidiazuron and paclobutrazol in increasing the yield of wheat are discussed.     

Monocarpic senescence in wheat: Influence of sterile glumes and ear

The senescence of sterile glumes, flag leaf and the other two leaves below the ear of wheat ( Triticum aestivum L. cv. Sonalika) was studied in relation to grain development and surgical manipulation. The senescence of sterile glumes was faster than that of the leaves in terms of chlorophyll and protein degradation. The flag leaf senesced later than the other two leaves below it. Removal of sterile glumes markedly reduced the harvest index (crop: straw ratio) and average dry weight per grain as compared to removal of the flag leaf. Maximum grain weight was achieved after the glumes had senesced completely. Removal of the ear delayed senescence of all the three leaves. It is concluded that sterile glumes are important suppliers of assimilate for grain filling and that nutrient drainage is the primary cause of the monocarpic senescence in wheat.     

控灌对小麦植株体内激素含量与籽粒灌浆速率的影响

利用防雨棚研究了控灌对小麦植株体内激素含量与籽粒不乐速率的影响。结果表明：随控水加重,小麦旗叶、根系和籽粒中ＡＢＡ含量迅速达到高峰,然后快速降低;控水愈重,ｉ－ＰＡｓ峰值和籽粒灌浆速率峰值出现愈早,尔后下降,从而加速了植株衰老进程,导致产量降低。籽粒灌浆速率变化与旗叶,根主籽粒中激素含量变化呈极显著相关,且上述性状在相对含水量６０％处理下与８０％（对照）相比无显著差异,从而为小麦节水栽培提供了依据     

不同植物生长调节剂对小麦衰老及产量构成的调节效应

研究了叶面喷施GA3、NBT、6－BA、TIBA、PP333对小麦旗叶衰老过程中若干生理指标的影响,分析了它们对成产要素的不同作用。结果表明：不同生长调节剂可以以不同的方式调节小麦旗叶的衰老,影响小麦产量构成因素水平。其中6－BA和PP333能够提高单位面积穗数,明显延缓叶片衰老,增加了穗粒数和千粒重,使成产3因素间比例协调合理,产量较高;TIBA可以明显提高叶绿素含量并显著提高千粒重。GA3和NBT对延缓旗叶衰老有一定作用,但增产效果不显著。研究指出,根据小麦生育状况有选择性地使用生长调节剂,协调成产因素关系,是一个有效的增产途径。     

施氮量和花后土壤含水量对小麦旗叶衰老及粒重的影响

在防雨池栽培条件下,研究了施氮量和花后土壤含水量对小麦旗叶衰老和粒重的影响.结果表明：各氮肥处理下,小麦旗叶SPAD值、可溶性蛋白质含量、超氧化物歧化酶（SOD）活性、过氧化氢酶（CAT）活性和光合速率（P_n）均表现为：花后土壤含水量60%～70%处理>80%～90%处理>40%～50%处理,小麦旗叶丙二醛（MDA）含量表现为：花后土壤含水量40%～50%处理>80%～90%处理>60%～70%处理,表明花后土壤含水量过高或过低均可导致小麦旗叶早衰,影响籽粒灌浆,降低粒重.在花后相同土壤含水量条件下,旗叶SPAD值、可溶性蛋白质含量、SOD活性、CAT活性和P_n均随施氮量的增加而升高,MDA含量随施氮量的增加而降低,表明增施氮肥可以延缓小麦旗叶衰老,但过量施用氮肥则不利于小麦粒重的提高,尤其是在花后土壤缺水的情况下,施用过多氮肥可导致小麦粒重下降.在小麦生产中可以将施用氮肥和控制花后土壤水分含量相结合,延缓小麦植株衰老,提高粒重.