Strong presence of the high grain protein content allele of NAM-B1 in Fennoscandian wheat

Grain protein content in wheat has been shown to be affected by the NAM-B1 gene where the wildtype allele confers high levels of protein and micronutrients but can reduce yield. Two known non-functional alleles instead increase yield but lead to lower levels of protein and micronutrients. The wildtype allele in hexaploid bread wheat is so far mainly known from historical specimens and a few lines with an emmer wheat introgression. Here we report a screening for the wildtype allele in wheats of different origin. First, a worldwide core collection of 367 bread wheats with worldwide origin was screened and five accessions carrying the wildtype NAM-B1 allele were found. Several of these could be traced to a Fennoscandian origin and the wildtype allele was more frequent in spring wheat. These findings, together with the late maturation of spring wheat, suggested that the faster maturation caused by the wildtype allele might have preserved it in areas with a short growing season. Thus a second set consisting of 138 spring wheats of a northern origin was screened and as many as 33?% of the accessions had the wildtype allele, all of a Fennoscandian origin. The presence of the wildtype allele in landraces and cultivars is in agreement with the use of landraces in Fennoscandian wheat breeding. Last, 22 spelt wheats, a wheat type previously suggested to carry the wildtype allele, were screened and five wildtype accessions found. The wildtype NAM-B1 accessions found could be a suitable material for plant breeding efforts directed towards increasing the nutrient content of bread wheat.     

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.     

Phytochelatin and cadmium accumulation in wheat

Cadmium (Cd) is a nonessential heavy metal that can be harmful at low concentrations in organisms. Therefore, it is necessary to decrease Cd accumulation in the grains of wheats aimed for human consumption. In response to Cd, higher plants synthesize sulphur-rich peptides, phytochelatins (PCs). PC–heavy metal complexes have been reported to accumulate in the vacuole. Retention of Cd in the root cell vacuoles might influence the symplastic radial Cd transport to the xylem and further transport to the shoot, resulting in genotypic differences in grain Cd accumulation. We have studied PC accumulation in 12-day-old seedlings of two cultivars of spring bread wheat (Triticum aestivum), and two spring durum wheat cultivars (Triticum turgidum var. durum) with different degrees of Cd accumulation in the grains. Shoots and roots were analysed for dry weight, Cd and PC accumulation. There were no significant differences between the species or the varieties in the growth response to Cd, nor in the distributions of PC chain lengths or PC isoforms. At 1 μM external Cd, durum wheat had a higher total Cd uptake than bread wheat, however, the shoot-to-root Cd concentration ratio was higher in bread wheat. When comparing varieties within a species, the high grain Cd accumulators exhibited lower rates of root Cd accumulation, shoot Cd accumulation, and root PC accumulation, but higher shoot-to-root Cd concentration ratios. Intraspecific variation in grain Cd accumulation is apparently not only explained by differential Cd accumulation as such, but rather by a differential plant-internal Cd allocation pattern. However, the higher average grain Cd accumulation in the durum wheats, as compared to the bread wheats, is associated with a higher total Cd accumulation in the plant, rather than with differential plant-internal Cd allocation. The root-internal PC chain length distributions and PC–thiol-to-Cd molar ratios did not significantly differ between species or varieties, suggesting that differential grain Cd accumulation is not due to differential PC-based Cd sequestration in the roots.     

A new multiplex PCR test for the determination of Vrn-B1 alleles in bread wheat (Triticum aestivum L.)

Winter wheat requires vernalization, a long exposure to low but non-freezing temperatures, to promote reproductive development. The vernalization requirement in bread wheat (Triticum aestivum L.) is mainly controlled by the Vrn-1 genes that are located on chromosomes 5A, 5B and 5D. Dominant alleles confer spring habit and are epistatic to the recessive winter alleles which means that spring varieties carry at least one dominant allele. To date, two dominant and one recessive Vrn-B1 alleles have been described. Vrn-B1a (formerly designated as Vrn-B1) differs from the winter vrn-B1 allele by a large deletion in intron 1. Vrn-B1b has an additional small deletion and is probably derived from Vrn-B1a. The novel allele described here and designated as Vrn-B1c also has a large deletion within intron 1 but with different breakpoints from Vrn-B1a or b, and sequence duplication, showing that this is an independently derived spring allele. By combining an exon 1 primer with previously published PCR primers it was possible to develop a multiplex PCR that distinguished all four alleles simultaneously. The multiplex PCR was validated by testing 320 winter wheat and 137 spring wheat varieties. This demonstrated that the novel Vrn-B1c allele was present in 25 spring varieties of diverse origin, showing this allele to be widely distributed.     

Identification of Genomic Regions and the Isoamylase Gene for Reduced Grain Chalkiness in Rice

Grain chalkiness is an important grain quality related to starch granules in the endosperm. A high percentage of grain chalkiness is a major problem because it diminishes grain quality in rice. Here, we report quantitative trait loci identification for grain chalkiness using high-throughput single nucleotide polymorphism genotyping of a chromosomal segment substitution line population in which each line carried one or a few introduced japonica cultivar Nipponbare segments in the genetic background of the indica cultivar ZS97. Ten quantitative trait loci regions were commonly identified for the percentage of grain chalkiness and the degree of endosperm chalkiness. The allelic effects at nine of these quantitative trait loci reduced grain chalkiness. Furthermore, a quantitative trait locus (qPGC8-2) on chromosome 8 was validated in a chromosomal segment substitution line–derived segregation population, and had a stable effect on chalkiness in a multiple-environment evaluation of the near-isogenic lines. Residing on the qPGC8-2 region, the isoamylase gene (ISA1) was preferentially expressed in the endosperm and revealed some nucleotide polymorphisms between two varieties, Nipponbare and ZS97. Transgenic lines with suppression of ISA1 by RNA interference produced grains with 20% more chalkiness than the control. The results support that the gene may underlie qPGC8-2 for grain chalkiness. The multiple-environment trials of the near-isogenic lines also show that combination of the favorable alleles such as the ISA1 gene for low chalkiness and the GS3 gene for long grains considerably improved grain quality of ZS97, which proves useful for grain quality improvement in rice breeding programs.     

Trends and annual fluctuations in selenium concentrations in wheat grain

Selenium concentrations were determined in grain from four varieties of winter and spring wheat grown in the same area during 1918–1979 and 1930–1980, respectively. Concentrations in the four longest series tended to decrease over time. The annual variation, expressed as the coefficient of variation, ranged between 51%–62% for the four investigated series. According to regression calculations, an additional 100 mm of precipitation during the growing season (May-August) would lead to a 7.2 ng Se g^–1 increase in the Se concentration of spring wheat grain. For winter wheat grain the calculated increase was not significant. No correlation was found between grain yield and grain Se concentration. Although there was a significant difference in grain Se concentrations between spring wheat varieties. there was no indication that older varieties differed from newer ones in this regard.     

Leaf Regulation of the Nitrogen Concentration in the Grain of Wheat Plants

The regulation of the final grain N concentration in wheat (Triticumaestivum) plants was studied through the alteration of the source/sinkratio. Plants were grown in a greenhouse in pots with soil,and fertilized with a supraoptimal N supply. The plants were divided into six groups. In one treatment, plantsremained untouched as a control (Treatment 1). In another group,all the ears except that of the main tiller were removed atflowering (Treatment 2). All other plants were de-tillered afterthe emergence of the third leaf, leaving only one tiller perplant. At flowering, one plant set was left untouched (Treatment3). In a second group, all the leaves were excised (Treatment4). In another group half the spikelets of the ear were excised(Treatment 5) and in the last group three-quarters of the spikeletswere excised (Treatment 6). Ear excision produced an increase in individual grain weightand the grain N concentration above the normal N concentrationobserved in this cultivar. The final N concentration was correlatedwith the concentration of free amino acids in the flag leaf34 d after flowering. It is concluded that in intact plants grain protein synthesisis substrate-limited by the amino acid export pool in the leaves,and grain excision increases the availability of amino acidsto be transported to the remaining grains. Key words: Amino acids, grain N concentration, nitrogen, remobilization, wheat     

Wheat Grain Filling Is Limited by Grain Filling Capacity rather than the Duration of Flag Leaf Photosynthesis: A Case Study Using NAM RNAi Plants

It has been proposed that delayed leaf senescence can extend grain filling duration and thus increase yields in cereal crops. We found that wheat (Triticum aestivum) NAM RNAi plants with delayed senescence carried out 40% more flag leaf photosynthesis after anthesis than control plants, but had the same rate and duration of starch accumulation during grain filling and the same final grain weight. The additional photosynthate available in NAM RNAi plants was in part stored as fructans in the stems, whereas stem fructans were remobilised during grain filling in control plants. In both genotypes, activity of starch synthase was limiting for starch synthesis in the later stages of grain filling. We suggest that in order to realise the potential yield gains offered by delayed leaf senescence, this trait should be combined with increased grain filling capacity.     

Identification of a Novel Allele of TaCKX6a02 Associated with Grain Size,Filling Rate and Weight of Common Wheat

Cytokinin oxidase (CKX) plays a crucial role in plant growth and development by reversibly inactivating cytokinin (CTK). Twenty-four primer pairs, designed from ESTs of the TaCKX genes family of common wheat, were used to identify their allelic variations associated with grain size, weight, and filling rate in 169 recombinant inbred lines (RIL) derived from Jing 411 × Hongmangchun 21. TaCKX6a02, a member of TaCKX gene family, amplified by primer pair T31–32, showed a close association with grain traits in this RIL population. Statistical analysis indicated that allelic variation of TaCKX6a02 had significant correlation with grain size, weight, and filling rate (GFR; P < 0.001) under varied environments. The TaCKX6a02-D1a allele from Jing411 significantly increased grain size, weight and grain filling rate, compared with TaCKX6a02-D1b from Hongmangchun 21. TaCKX6a02 was located on chromosome 3DS in the interval of Xbarc1119 and Xbarc1162, with a genetic distance of 1.4 cM. The location was further confirmed using Chinese Spring nulli–tetrasomic lines. A major QTL (quantitative trait locus) tightly linked to TaCKX6a02 was detected in the RIL population, explaining 17.1~38.2% of phenotype variations for grain size, weight, GFRmax and GFRmean in different environments. In addition, significant effects of variations of TaCKX6a02 on grain weight and GFR were further validated by association analysis among 102 wheat varieties in two cropping seasons. 12.8~35.1% of phenotypic variations were estimated for these genotypes. A novel 29-bp InDel behind the stop codon was detected by DNA sequence analysis between the two alleles of TaCKX6a02-D1. The gene-specific marker, TKX3D, was designed according to the novel variation, and can be used in marker-assisted selection (MAS) for grain size, weight, and GFR in common wheat.     

Photosynthetic Gas Exchange Characteristics of Wheat Flag Leaf Blades and Sheaths during Grain Filling: The Case of a Spring Crop Grown under Mediterranean Climate Conditions

The rate of net CO₂ assimilation (A), the stomatal (g_s) and residual (g_r) conductances to CO₂, the intercellular CO₂ concentration, the CO₂ compensation points at 21% O₂ (Γ₂₁) and at 2% O₂ (Γ₂), and the amounts of dry matter, nitrogen, and carbohydrates were determined, from anthesis through grain filling, in the flag leaf blade and sheath of spring wheat (Triticum aestivum L. cv Kolibri). The nitrogen content and the rate of net CO₂ assimilation declined slowly until the onset of senescence in both organs, about 3 weeks after anthesis. During senescence the reduction of A in both organs was not primarily caused by a decrease in g_s; the main factor is the decrease in g_r. From values of Γ₂₁ and Γ₂ it is suggested that the rate of respiration in the light contributing to the CO₂ compensation point is higher in sheaths than in blades irrespective of the O₂ level considered. The role of sheaths storing and later transporting assimilates to the developing grains seems to be more important for shoot yield than that of sheaths functioning as photosynthetic organs after the onset of senescence occurs. It is suggested that accumulation of carbohydrates in leaves might somehow trigger senescence in the flag leaf blade and sheath simultaneously.     

Nutrient partitioning and grain yield of TaNAM-RNAi wheat under abiotic stress

Aims

Decreased expression of TaNAM genes by RNAi results in delayed senescence and decreased grain protein, iron, and zinc concentrations. Here, we determined whether NAM expression level alters onset of senescence under stress conditions, whether delayed senescence in the TaNAM-RNAi line resulted in improved tolerance to post-anthesis abiotic stress, and determined the effects of post-anthesis abiotic stress on N and mineral remobilization and partitioning to grain.

Methods

Greenhouse-grown WT and TaNAM-RNAi wheat were characterized in two studies:three levels of N fertility or water limitation during grain fill. Studies were conducted under both optimal and heat stress temperatures. Senescence onset was determined by monitoring flag leaf chlorophyll.

Results

Under optimal tempertures, TaNAM-RNAi plants had a yield advantage at lower N. TaNAM-RNAi plants had delayed senescence relative to the WT and lower grain protein and mineral concentrations, N remobilization efficiency, and partitioning of N and most minerals to grain.

Conclusions

Nutritional quality of TaNAM-RNAi grain was consistently lower than WT. Delayed senescence of TaNAM-RNAi plants provided a yield advantage under optimal temperatures but not under water or heat stress. Discovery of specific NAM protein targets may allow separation of the delayed senescence and nutrient partitioning traits, which could be used for improvement of wheat.     

Natural Variations in SLG7 Regulate Grain Shape in Rice

Rice (Oryza sativa) grain shape, which is controlled by quantitative trait loci (QTL), has a strong effect on yield production and quality. However, the molecular basis for grain development remains largely unknown. In this study, we identified a novel QTL, Slender grain on chromosome 7 (SLG7), that is responsible for grain shape, using backcross introgression lines derived from 9311 and Azucena. The SLG7 allele from Azucena produces longer and thinner grains, although it has no influence on grain weight and yield production. SLG7 encodes a protein homologous to LONGIFOLIA 1 and LONGIFOLIA 2, both of which increase organ length in Arabidopsis. SLG7 is constitutively expressed in various tissues in rice, and the SLG7 protein is located in plasma membrane. Morphological and cellular analyses suggested that SLG7 produces slender grains by longitudinally increasing cell length, while transversely decreasing cell width, which is independent from cell division. Our findings show that the functions of SLG7 family members are conserved across monocots and dicots and that the SLG7 allele could be applied in breeding to modify rice grain appearance.     

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.     

Quantitative trait loci for resistance to fusarium head blight in a Chinese wheat landrace Haiyanzhong

Fusarium head blight (FHB) of wheat causes not only significant reduction in grain yield and end-use quality, but also the contamination of the grain with mycotoxins that are detrimental to human and animal health after consumption of infected grain. Growing resistant varieties is an effective approach to minimize the FHB damage. The Chinese wheat landrace Haiyanzhong (HYZ) shows a high level of resistance to FHB. To identify quantitative trait loci (QTL) that contribute to FHB resistance in HYZ, 136 recombinant inbred lines (RIL) were developed from a cross of HYZ and Wheaton, a hard spring wheat cultivar from the USA. The RIL and their parents were evaluated for percentage of scabbed spikelets (PSS) in both greenhouse and field environments. Five QTL were detected for FHB resistance in HYZ with one major QTL on 7DL. The 7DL QTL peaked at SSR marker Xwmc121, which is flanked by the SSR markers Xcfd46 and Xwmc702. This QTL explained 20.4?C22.6% of the phenotypic variance in individual greenhouse experiments and 15.9% in a field experiment. Four other minor QTL on 6BS (two QTL), 5AS and 1AS each explained less than 10% of the phenotypic variance in individual experiments. HYZ carried the favorable alleles associated with FHB resistance at the QTL on 7DL, 6BS and 5AS, and the unfavorable allele at the QTL on 1AS. The major QTL on 7D can be used to improve the FHB resistance in wheat breeding programs and add diversity to the FHB resistance gene pool.     

Identification and development of a functional marker of TaGW2 associated with grain weight in bread wheat (Triticum aestivum L.)

The OsGW2 gene is involved in rice grain development, influencing grain width and weight. Its ortholog in wheat, TaGW2, was considered as a candidate gene related to grain development. We found that TaGW2 is constitutively expressed, with three orthologs expressing simultaneously. The coding sequence (CDS) of TaGW2 is 1,275?bp encoding a protein with 424 amino acids, and has a functional domain shared with OsGW2. No divergence was detected within the CDS sequences in the same locus in ten varieties. Genome-specific primers were designed based on the sequence divergence of the promoter regions in the three orthologous genes, and TaGW2 was located in homologous group 6 chromosomes through CS nulli-tetrasomic (NT). Two SNPs were detected in the promoter region of TaGW2-6A, forming two haplotypes: Hap-6A-A (?593A and ?739G) and Hap-6A-G (?593G and ?769A). A cleaved amplified polymorphic sequence (CAPS) marker was developed based on the ?593 A-G polymorphism to distinguish the two haplotypes in TaGW2-6A. This gene was fine mapped 0.6?cM from marker cfd80.2 near the centromere in a recombinant inbred line (RIL) population. Two hundred sixty-five Chinese wheat varieties were genotyped and association analysis revealed that Hap-6A-A was significantly associated with wider grains and higher one-thousand grain weight (TGW) in two crop seasons. qRT-PCR revealed a negative relationship between TaGW2 expression level and grain width. The Hap-6A-A frequencies in Chinese varieties released at different periods showed that it had been strongly positively selected in breeding. In landraces, Hap-6A-A is mainly distributed in southern Chinese wheat regions. Association analysis also indicated that Hap-6A-A not only increased TGW by more than 3?g, but also had earlier heading and maturity. In contrast to Chinese varieties, Hap-6A-G was the predominant haplotype in European varieties; Hap-6A-A was mainly present in varieties released in the former Yugoslavia, Italy, Bulgaria, Hungary and Portugal.     

Transport of Recently Assimilated15N Nitrogen to Individual Spikelets in Spring Wheat Grown in Culture Solution

Two cultivars of spring wheat ( Triticum aestivum L.), Sport(high protein) and WL4 (low protein), were grown to maturityin culture solution. Nitrogen in the form of nitrate was addedin daily doses at stepwise-decreasing relative rates to ensurenormal development, and both cultivars received the same totalamount of N during development. At weekly intervals from anthesisto maturity the daily nitrate dose was, for selected groupsof plants, labelled with¹⁵N. After the labelling period theselected plants were harvested and analysed. The cultivar WL4produced more biomass than Sport, as well as more spikeletsand more grains per ear, with a higher mean grain weight, suchthat grain yield of WL4 was 57% greater than Sport. The earsof both cultivars were heterogenous: mean grain weight was highestin middle spikelets, which also contained more grains; the Ncontent followed the pattern of dry weight with more N in themiddle spikelets; but the N concentration was practically thesame in all spikelets (2.15% of d. wt in WL4 and 3.33% in Sport).The distribution of¹⁵N showed that the main stem ear maturedmuch earlier than tiller ears. The results of this nitrogen-labellingexperiment show that, late in development, substantial amountsof recently-absorbed N were immediately assimilated and transportedto the ears. Transport of¹⁵N decreased earlier to the top spikeletsthan to the bottom spikelets. As both cultivars were grown underidentical conditions and both received the same amount of Nit was concluded that the difference in grain N concentrationwas not caused by differences in the capacity of N assimilationand translocation but rather by different rates of accumulationof non-nitrogenous dry matter in the grains. Ear; grain; nitrate; nitrogen transport; Triticum aestivum L.; yield     

Identification of Novel SNP in Promoter Sequence of TaGW2-6A Associated with Grain Weight and Other Agronomic Traits in Wheat (Triticum aestivum L.)

TaGW2 is an orthologue of rice gene OsGW2, which encodes E3 RING ubiquitin ligase and controls the grain size in rice. In wheat, three copies of TaGW2 have been identified and mapped on wheat homoeologous group 6 viz. TaGW2-6A, TaGW2-6B and TaGW2-6D. In the present study, using as many as 207 Indian wheat genotypes, we identified four SNPs including two novel SNPs (SNP-988 and SNP-494) in the promoter sequence of TaGW2-6A. All the four SNPs were G/A or A/G substitutions (transitions). Out of the four SNPs, SNP-494 was causal, since it was found associated with grain weight. The mean TGW (41.1 g) of genotypes with the allele SNP-494_A was significantly higher than mean TGW (38.6 g) of genotypes with the allele SNP-494_G. SNP-494 also regulates the expression of TaGW2-6A so that the wheat genotypes with SNP-494_G have higher expression and lower TGW and the genotypes with SNP-494_A have lower expression but higher TGW. Besides, SNP-494 was also found associated with grain length-width ratio, awn length, spike length, grain protein content, peduncle length and plant height. This suggested that gene TaGW2-6A not only controls grain size, but also controls other agronomic traits. In the promoter region, SNP-494 was present in ‘CGCG’ motif that plays an important role in Ca²⁺/calmodulin mediated regulation of genes. A user-friendly CAPS marker was also developed to identify the desirable allele of causal SNP (SNP-494) for use in marker-assisted selection for improvement of grain weight in wheat. Using four SNPs, five haplotypes were identified; of these, Hap_5 (G_A_G_A) was found to be a desirable haplotype having significantly higher grain weight (41.13g) relative to other four haplotypes (36.33-39.16 g).