A sucrose-binding protein and ß-conglycinins regulate soybean seed protein content and control multiple seed traits.

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max [L.] Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins (HPs). We isolated 2 high-seed protein soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes ß-conglycinin (GmCG-1) and HP2 encodes sucrose-binding protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits.

CRISPR-based editing of the ω- and γ-gliadin gene clusters reduces wheat immunoreactivity without affecting grain protein quality.

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on ω- and γ-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole-genome sequencing of an edited line showed mutation or deletion of nearly all ω-gliadin and half of the γ-gliadin gene copies and confirmed the lack of editing in the α/ß-gliadin genes. The estimated 75% and 64% reduction in ω- and γ-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to a non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the ω- and γ-gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

TaNAM-6A is essential for nitrogen remobilisation and regulates grain protein content in wheat (Triticum aestivum L.).

Grain protein content (GPC) is a crucial quality trait in bread wheat, which is influenced by the key transcription factor TaNAM. However, the regulatory mechanisms of TaNAM have remained largely elusive. In this study, a new role of TaNAM was unveiled in regulating nitrogen remobilisation which impacts GPC. The TaNAM knockout mutants generated by clustered regularly interspaced short palindromic repeats/Cas9 exhibited significantly delayed senescence and lower GPC, while overexpression of TaNAM-6A resulted in premature senility and much higher GPC. Further analysis revealed that TaNAM directly activates the genes TaNRT1.1 and TaNPF5.5s, which are involved in nitrogen remobilisation. This activity aids in the transfer of nitrogen from leaves to grains for protein synthesis. In addition, an elite allele of TaNAM-6A, associated with high GPC, was identified as a candidate gene for breeding high-quality wheat. Overall, our work not only elucidates the potential mechanism of TaNAM-6A affecting bread wheat GPC, but also highlights the significance of nitrogen remobilisation from senescent leaves to grains for protein accumulation. Moreover, our research provides a new target and approach for improving the quality traits of wheat, particularly the GPC.

Two dwarfing genes Rht-B1b and Rht-D1b show pleiotropic effects on grain protein content in bread wheat (Triticum aestivum L.).

KEY MESSAGE: Five QTL for wheat grain protein content were identified, and the effects of two dwarfing genes Rht-B1b and Rht-D1b on grain protein content were validated in multiple populations. Grain protein content (GPC) plays an important role in wheat quality. Here, a recombinant inbred line (RIL) population derived from a cross between Yangmai 12 (YM12) and Yanzhan 1 (YZ1) was used to identify quantitative trait loci (QTL) for GPC. Two hundred and five RILs and their parents were grown in three years in randomized complete blocks each with two replications, and genotyped using the wheat 55 K SNP array. Five QTL were identified for GPC on chromosomes 1A, 1B, 2D, 4B, and 4D. Notably, QGpc.yaas-4B (co-located with Rht-B1) and QGpc.yaas-4D (co-located with Rht-D1) were consistently detected across all experiments and best linear unbiased estimating, accounting for 6.61-8.39% and 6.05-10.21% of the phenotypic variances, respectively. The effects of these two dwarfing alleles Rht-B1b and Rht-D1b on reducing GPC and plant height were validated in two additional RIL populations and one natural population. This study lays a foundation for further investigating the effects of dwarfing genes Rht-B1b and Rht-D1b on wheat GPC.

Image-based phenomic prediction can provide valuable decision support in wheat breeding.

KEY MESSAGE: Genotype-by-environment interactions of secondary traits based on high-throughput field phenotyping are less complex than those of target traits, allowing for a phenomic selection in unreplicated early generation trials. Traditionally, breeders' selection decisions in early generations are largely based on visual observations in the field. With the advent of affordable genome sequencing and high-throughput phenotyping technologies, enhancing breeders' ratings with such information became attractive. In this research, it is hypothesized that G[Formula: see text]E interactions of secondary traits (i.e., growth dynamics' traits) are less complex than those of related target traits (e.g., yield). Thus, phenomic selection (PS) may allow selecting for genotypes with beneficial response-pattern in a defined population of environments. A set of 45 winter wheat varieties was grown at 5 year-sites and analyzed with linear and factor-analytic (FA) mixed models to estimate G[Formula: see text]E interactions of secondary and target traits. The dynamic development of drone-derived plant height, leaf area and tiller density estimations was used to estimate the timing of key stages, quantities at defined time points and temperature dose-response curve parameters. Most of these secondary traits and grain protein content showed little G[Formula: see text]E interactions. In contrast, the modeling of G[Formula: see text]E for yield required a FA model with two factors. A trained PS model predicted overall yield performance, yield stability and grain protein content with correlations of 0.43, 0.30 and 0.34. While these accuracies are modest and do not outperform well-trained GS models, PS additionally provided insights into the physiological basis of target traits. An ideotype was identified that potentially avoids the negative pleiotropic effects between yield and protein content.

Simultaneous improvement of grain yield and grain protein concentration in durum wheat by using association tests and weighted GBLUP.

KEY MESSAGE: Simultaneous improvement for GY and GPC by using GWAS and GBLUP suggested a significant application in durum wheat breeding. Despite the importance of grain protein concentration (GPC) in determining wheat quality, its negative correlation with grain yield (GY) is still one of the major challenges for breeders. Here, a durum wheat panel of 200 genotypes was evaluated for GY, GPC, and their derived indices (GPD and GYD), under eight different agronomic conditions. The plant material was genotyped with the Illumina 25 k iSelect array, and a genome-wide association study was performed. Two statistical models revealed dozens of marker-trait associations (MTAs), each explaining up to 30%. phenotypic variance. Two markers on chromosomes 2A and 6B were consistently identified by both models and were found to be significantly associated with GY and GPC. MTAs identified for phenological traits co-mapped to well-known genes (i.e., Ppd-1, Vrn-1). The significance values (p-values) that measure the strength of the association of each single nucleotide polymorphism marker with the target traits were used to perform genomic prediction by using a weighted genomic best linear unbiased prediction model. The trained models were ultimately used to predict the agronomic performances of an independent durum wheat panel, confirming the utility of genomic prediction, although environmental conditions and genetic backgrounds may still be a challenge to overcome. The results generated through our study confirmed the utility of GPD and GYD to mitigate the inverse GY and GPC relationship in wheat, provided novel markers for marker-assisted selection and opened new ways to develop cultivars through genomic prediction approaches.

Protein use efficiency and stability of baking quality in winter wheat based on the relation of loaf volume and grain protein content.

KEY MESSAGE: A novel approach based on the loaf volume-grain protein content relation is suggested to consider the static protein use efficiency and stability as efficient quality-related descriptors for wheat varieties. The most important trait for baking quality of winter wheat is loaf volume (V). It is mostly determined by grain protein content (GPC) and quality. New varieties with a high potential of grain protein use efficiency (ProtUE) are very important for reducing the surplus use of nitrogen fertilizer in areas where nitrogen leaching is large. This is also an important goal of agricultural policies in the European Union. Additionally, ProtUE needs to be very stable across environments in the face of progressing climate change with more volatile growing conditions. We evaluated a new approach to assess ProtUE and stability based on the V-GPC relationship instead of using only single traits. The study comprised 11,775 baking tests from 355 varieties grown 1988-2019 in 668 different environments in Germany. V was predicted by quadratic and linear regression functions for quality groups, indicating a reduction of ProtUE from 1988 to 2019. We introduced a dynamic and a static approach to assess ProtUE and stability as potential criteria in variety registration. We found a considerably lower heritability of the dynamic ProtUE (h2 = 43%) compared to the static ProtUE (h2 = 92%) and a lower dynamic stability (h2 = 32%) than for the static stability (h2 = 51%). None of these measures is in conflict with the selection for high V. In particular, V and static ProtUE are strongly genetically associated (r = 0.81), indicating an advantage of the static over the dynamic approach.

Increasing the Grain Yield and Grain Protein Content of Common Wheat (Triticum aestivum) by Introducing Missense Mutations in the Q Gene.

Grain yield (GY) and grain protein content (GPC) are important traits for wheat breeding and production; however, they are usually negatively correlated. The Q gene is the most important domestication gene in cultivated wheat because it influences many traits, including GY and GPC. Allelic variations in the Q gene may positively affect both GY and GPC. Accordingly, we characterized two new Q alleles (Qs1 and Qc1-N8) obtained through ethyl methanesulfonate-induced mutagenesis. Compared with the wild-type Q allele, Qs1 contains a missense mutation in the sequence encoding the first AP2 domain, whereas Qc1-N8 has two missense mutations: one in the sequence encoding the second AP2 domain and the other in the microRNA172-binding site. The Qs1 allele did not significantly affect GPC or other processing quality parameters, but it adversely affected GY by decreasing the thousand kernel weight and grain number per spike. In contrast, Qc1-N8 positively affected GPC and GY by increasing the thousand kernel weight and grain number per spike. Thus, we generated novel germplasm relevant for wheat breeding. A specific molecular marker was developed to facilitate the use of the Qc1-N8 allele in breeding. Furthermore, our findings provide useful new information for enhancing cereal crops via non-transgenic approaches.

Genome-Wide Survey and Functional Verification of the NAC Transcription Factor Family in Wild Emmer Wheat.

The NAC transcription factor (TF) family is one of the largest TF families in plants, which has been widely reported in rice, maize and common wheat. However, the significance of the NAC TF family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of NAC genes was conducted in the wild emmer genome and 249 NAC family members (TdNACs) were identified. The results showed that all of these genes contained NAM/NAC-conserved domains and most of them were predicted to be located on the nucleus. Phylogenetic analysis showed that these 249 TdNACs can be classified into seven clades, which are likely to be involved in the regulation of grain protein content, starch synthesis and response to biotic and abiotic stresses. Expression pattern analysis revealed that TdNACs were highly expressed in different wheat tissues such as grain, root, leaves and shoots. We found that TdNAC8470 was phylogenetically close to NAC genes that regulate either grain protein or starch accumulation. Overexpression of TdNAC8470 in rice showed increased grain starch concentration but decreased grain Fe, Zn and Mn contents compared with wild-type plants. Protein interaction analysis indicated that TdNAC8470 might interact with granule-bound starch synthase 1 (TdGBSS1) to regulate grain starch accumulation. Our work provides a comprehensive understanding of the NAC TFs family in wild emmer wheat and establishes the way for future functional analysis and genetic improvement of increasing grain starch content in wheat.

Ensuring Global Food Security by Improving Protein Content in Major Grain Legumes Using Breeding and 'Omics' Tools.

Grain legumes are a rich source of dietary protein for millions of people globally and thus a key driver for securing global food security. Legume plant-based 'dietary protein' biofortification is an economic strategy for alleviating the menace of rising malnutrition-related problems and hidden hunger. Malnutrition from protein deficiency is predominant in human populations with an insufficient daily intake of animal protein/dietary protein due to economic limitations, especially in developing countries. Therefore, enhancing grain legume protein content will help eradicate protein-related malnutrition problems in low-income and underprivileged countries. Here, we review the exploitable genetic variability for grain protein content in various major grain legumes for improving the protein content of high-yielding, low-protein genotypes. We highlight classical genetics-based inheritance of protein content in various legumes and discuss advances in molecular marker technology that have enabled us to underpin various quantitative trait loci controlling seed protein content (SPC) in biparental-based mapping populations and genome-wide association studies. We also review the progress of functional genomics in deciphering the underlying candidate gene(s) controlling SPC in various grain legumes and the role of proteomics and metabolomics in shedding light on the accumulation of various novel proteins and metabolites in high-protein legume genotypes. Lastly, we detail the scope of genomic selection, high-throughput phenotyping, emerging genome editing tools, and speed breeding protocols for enhancing SPC in grain legumes to achieve legume-based dietary protein security and thus reduce the global hunger risk.

Rapid improvement of rice eating and cooking quality through gene editing toward glutelin as target.

Rice eating and cooking quality (ECQ) is a major concern of breeders and consumers, determining market competitiveness worldwide. Rice grain protein content (GPC) is negatively related to ECQ, making it possible to improve ECQ by manipulating GPC. However, GPC is genetically complex and sensitive to environmental conditions; therefore, little progress has been made in traditional breeding for ECQ. Here, we report that CRISPR/Cas9-mediated knockout of genes encoding the grain storage protein glutelin rapidly produced lines with downregulated GPC and improved ECQ. Our finding provides a new strategy for improving rice ECQ.

Omics Data Reveal Putative Regulators of Einkorn Grain Protein Composition under Sulfur Deficiency.

Understanding the molecular mechanisms controlling the accumulation of grain storage proteins in response to nitrogen (N) and sulfur (S) nutrition is essential to improve cereal grain nutritional and functional properties. Here, we studied the grain transcriptome and metabolome responses to postanthesis N and S supply for the diploid wheat einkorn (Triticum monococcum). During grain filling, 848 transcripts and 24 metabolites were differentially accumulated in response to N and S availability. The accumulation of total free amino acids per grain and the expression levels of 241 genes showed significant modifications during most of the grain filling period and were upregulated in response to S deficiency. Among them, 24 transcripts strongly responded to S deficiency and were identified in coexpression network analyses as potential coordinators of the grain response to N and S supply. Sulfate transporters and genes involved in sulfate and Met metabolism were upregulated, suggesting regulation of the pool of free amino acids and of the grain N-to-S ratio. Several genes highlighted in this study might limit the impact of S deficiency on the accumulation of grain storage proteins.

Grain protein content and thousand kernel weight QTLs identified in a durum × wild emmer wheat mapping population tested in five environments.

KEY MESSAGE: Genetic dissection of GPC and TKW in tetraploid durum × WEW RIL population, based on high-density SNP genetic map, revealed 12 GPC QTLs and 11 TKW QTLs, with favorable alleles for 11 and 5 QTLs, respectively, derived from WEW. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, WEW) was shown to exhibit high grain protein content (GPC) and therefore possess a great potential for improvement of cultivated wheat nutritional value. Genetic dissection of thousand kernel weight (TKW) and grain protein content (GPC) was performed using a high-density genetic map constructed based on a recombinant inbred line (RIL) population derived from a cross between T. durum var. Svevo and WEW acc. Y12-3. Genotyping of 208 F6 RILs with a 15 K wheat single nucleotide polymorphism (SNP) array yielded 4166 polymorphic SNP markers, of which 1510 were designated as skeleton markers. A total map length of 2169 cM was obtained with an average distance of 1.5 cM between SNPs. A total of 12 GPC QTLs and 11 TKW QTLs were found under five different environments. No significant correlations were found between GPC and TKW across all environments. Four major GPC QTLs with favorable alleles from WEW were found on chromosomes 4BS, 5AS, 6BS and 7BL. The 6BS GPC QTL coincided with the physical position of the NAC transcription factor TtNAM-B1, underlying the cloned QTL, Gpc-B1. Comparisons of the physical intervals of the GPC QTLs described here with the results previously reported in other durum × WEW RIL population led to the discovery of seven novel GPC QTLs. Therefore, our research emphasizes the importance of GPC QTL dissection in diverse WEW accessions as a source of novel alleles for improvement of GPC in cultivated wheat.

Introgression of an expressed HMW 1Ay glutenin subunit allele into bread wheat cv. Lincoln increases grain protein content and breadmaking quality without yield penalty.

KEY MESSAGE: An expressed HMW glutenin subunit Glu-Ay showed positive impacts on a range of wheat processing quality and yield traits. The grain protein compositions are significantly optimised for baking, resulting in a better breadmaking quality. The unique breadmaking properties of wheat flour are related to the quality and quantity of high-molecular weight glutenin subunits (HMW-GSs) present in the grain. In the current study, the silent 1Ay HMW-GS allele, present in most bread wheat cultivars, was replaced by the expressed 1Ay21* allele, which was introgressed into Australian bread wheat cultivar Lincoln by a backcrossing and selfing scheme. Stability of gene expression and the effect of the introgressed 1Ay21* subunit on protein composition, agronomic traits, flour functionality, and breadmaking quality were studied using BC4F5 grain grown in glasshouse and field. Field phenotyping and grain quality testing showed that the 1Ay21* gene conferred significant improvements to a range of traits, including an increase in grain protein content by up to 9%, UPP% by up to 24%, bread volume by up to 28%. The glasshouse experiment and one of the field trials showed positive 1Ay21* effects on yield, while one field trial showed one significant effects. This indicates that expression of the 1Ay21* gene has the potential of simultaneously increasing protein content and grain yield under certain environment. The qualitative improvements of the grain also led to a reduction of the energy required during the baking process in addition to the significant positive effects on bread quality.

Yield reduction historically associated with the Aegilops ventricosa 7DV introgression is genetically and physically distinct from the eyespot resistance gene Pch1.

KEY MESSAGE: Yield penalty and increased grain protein content traits associated with Aegilops ventricosa 7D introgression have been mapped for the first time, and they are physically distinct from the eyespot resistance locus Pch1. Wheat wild relatives represent an important source of genetic variation, but introgression of agronomically relevant genes, such as for disease resistance, may lead to the simultaneous introduction of genetically linked deleterious traits. Pch1 is a dominant gene, conferring resistance to eyespot and was introgressed to wheat from Aegilops ventricosa as part of a large segment of the 7DV chromosome. This introgression has been associated with a significant yield reduction and a concomitant increase in grain protein content. In this study, we evaluated both traits and their relationship to the location of the Pch1 gene. We found that both QTLs were clearly distinct from the Pch1 gene, being located on a different linkage group to Pch1. In addition, we found that the QTL for increased grain protein content was strong and consistent across field trials, whereas the yield penalty QTL was unstable and environmentally dependent. The yield and grain protein content QTLs were genetically linked and located in the same linkage group. This finding is due in part to the small size of the population, and to the restricted recombination between wheat 7D and Ae. ventricosa 7Dv chromosomes. Although recombination in this interval is rare, it does occur. A recombinant line containing Pch1 and 7D_KASP6, the marker associated with increase in grain protein content, but not Xwmc221, the marker associated with the yield penalty effect, was identified.

Functional Validation of Glutamine synthetase and Glutamate synthase Genes in Durum Wheat near Isogenic Lines with QTL for High GPC.

Durum wheat (Triticum turgidum L. ssp. durum) is a minor crop grown on about 17 million hectares of land worldwide. Several grain characteristics determine semolina's high end-use quality, such as grain protein content (GPC) which is directly related to the final products' nutritional and technological values. GPC improvement could be pursued by considering a candidate gene approach. The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a bottleneck in the first step of nitrogen assimilation. QTL for GPC have been located on all chromosomes, and several major ones have been reported on 2A and 2B chromosomes, where GS2 and Fd-GOGAT genes have been mapped. A useful and efficient method to validate a putative QTL is the constitution of near-isogenic lines (NILs) by using the marker found to be associated to that QTL. Here, we present the development of two distinct sets of heterogeneous inbred family (HIF)- based NILs segregating for GS2 and Fd-GOGAT genes obtained from heterozygous lines at those loci, as well as their genotypic and phenotypic characterizations. The results allow the validation of the previously identified GPC QTL on 2A and 2B chromosomes, along with the role of these key genes in GPC control.

Genome-Wide Identification of QTLs for Grain Protein Content Based on Genotyping-by-Resequencing and Verification of qGPC1-1 in Rice.

To clarify the genetic mechanism underlying grain protein content (GPC) and to improve rice grain qualities, the mapping and cloning of quantitative trait loci (QTLs) controlling the natural variation of GPC are very important. Based on genotyping-by-resequencing, a total of 14 QTLs were detected with the Huanghuazhan/Jizi1560 (HHZ/JZ1560) recombinant inbred line (RIL) population in 2016 and 2017. Seven of the fourteen QTLs were repeatedly identified across two years. Using three residual heterozygote-derived populations, a stably inherited QTL named as qGPC1-1 was validated and delimited to a ~862 kb marker interval JD1006-JD1075 on the short arm of chromosome 1. Comparing the GPC values of the RIL population determined by near infrared reflectance spectroscopy (NIRS) and Kjeldahl nitrogen determination (KND) methods, high correlation coefficients (0.966 and 0.983) were observed in 2016 and 2017. Furthermore, 12 of the 14 QTLs were identically identified with the GPC measured by the two methods. These results indicated that instead of the traditional KND method, the rapid and easy-to-operate NIRS was suitable for analyzing a massive number of samples in mapping and cloning QTLs for GPC. Using the gel-based low-density map consisted of 208 simple sequence repeat (SSR) and insert/deletion (InDel) markers, the same number of QTLs (fourteen) were identified in the same HHZ/JZ1560 RIL population, and three QTLs were repeatedly detected across two years. More stably expressed QTLs were identified based on the genome resequencing, which might be attributed to the high-density map, increasing the detection power of minor QTLs. Our results are helpful in dissecting the genetic basis of GPC and improving rice grain qualities through molecular assisted selection.

Towards the Properties of Different Biomass-Derived Proteins via Various Extraction Methods.

This study selected three representative protein-rich biomass-brewer's spent grain (BSG), pasture grass (PG), and cyanobacteria (Arthrospira platensis; AP) for protein extraction with different extraction methods (alkaline treatment, aqueous extraction, and subcritical water extraction). The yield, purity, molecular weight, oil-water interfacial tension, and thermal stability of the obtained proteins derived from different biomass and extraction methods were comprehensively characterized and compared. In the view of protein yield and purity, alkaline treatment was found optimal for BSG (21.4 and 60.2 wt.%, respectively) and AP (55.5 and 68.8 wt.%, respectively). With the decreased oil-water interfacial tension, the proteins from all biomass showed the potential to be emulsifier. BSG and AP protein obtained with chemical treatment presented excellent thermal stability. As a novel method, subcritical water extraction is promising in recovering protein from all three biomass with the comparable yield and purity as alkaline treatment. Furthermore, the hydrolyzed protein with lower molecular weight by subcritical water could promote its functions of foaming and emulsifying.

Association mapping reveals multiple QTLs for grain protein content in rice useful for biofortification.

Rice is the staple food for majority of the global population. But, rice grain has low protein content (PC). Mapping of QTLs controlling grain PC is essential for enhancement of the trait through breeding programs. A shortlisted panel population for grain protein content was studied for genetic diversity, population structure and association mapping for grain PC. Phenotyping results showed a wide variation for grain PC. The panel population showed a moderate level of genetic diversity estimated through 98 molecular markers. AMOVA and structure analysis indicated linkage disequilibrium for grain PC and deviation of Hardy-Weinberg's expectation. The analysis showed 15% of the variation among populations and 73% among individuals in the panel population. STRUCTURE analysis categorized the panel population into three subpopulations. The analysis also revealed a common primary ancestor for each subpopulation with few admix individuals. Marker-trait association using 98 molecular markers detected 7 strongly associated QTLs for grain PC by both MLM and GLM analysis. Three novel QTLs qPC3.1, qPC5.1 and qPC9.1 were detected for controlling the grain PC. Four reported QTLs viz., qPC3, QPC8, qPC6.1 and qPC12.1 were validated for use in breeding programs. Reported QTLs, qPC6, qPC6.1 and qPC6.2 may be same QTL controlling PC in rice. A very close marker RM407 near to protein controlling QTL, qProt8 and qPC8, was detected. The study provided clue for simultaneous improvement of PC with high grain yield in rice. The strongly associated markers with grain PC, namely qPC3, qPC3.1, qPC5.1, qPC6.1, qPC8, qPC9.1 and qPC12.1, will be useful for their pyramiding for developing protein rich high yielding rice.

Cisgenic overexpression of cytosolic glutamine synthetase improves nitrogen utilization efficiency in barley and prevents grain protein decline under elevated CO2.

Cytosolic glutamine synthetase (GS1) plays a central role in nitrogen (N) metabolism. The importance of GS1 in N remobilization during reproductive growth has been reported in cereal species but attempts to improve N utilization efficiency (NUE) by overexpressing GS1 have yielded inconsistent results. Here, we demonstrate that transformation of barley (Hordeum vulgare L.) plants using a cisgenic strategy to express an extra copy of native HvGS1-1 lead to increased HvGS1.1 expression and GS1 enzyme activity. GS1 overexpressing lines exhibited higher grain yields and NUE than wild-type plants when grown under three different N supplies and two levels of atmospheric CO2 . In contrast with the wild-type, the grain protein concentration in the GS1 overexpressing lines did not decline when plants were exposed to elevated (800-900 µL/L) atmospheric CO2 . We conclude that an increase in GS1 activity obtained through cisgenic overexpression of HvGS1-1 can improve grain yield and NUE in barley. The extra capacity for N assimilation obtained by GS1 overexpression may also provide a means to prevent declining grain protein levels under elevated atmospheric CO2 .