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.

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.

Storage protein activator controls grain protein accumulation in bread wheat in a nitrogen dependent manner.

The expression of cereal grain storage protein (GSP) genes is controlled by a complex network of transcription factors (TFs). Storage protein activator (SPA) is a major TF acting in this network but its specific function in wheat (Triticum aestivum L.) remains to be determined. Here we generated an RNAi line in which expression of the three SPA homoeologs was reduced. In this line and its null segregant we analyzed GSP accumulation and expression of GSP and regulatory TF genes under two regimes of nitrogen availability. We show that down regulation of SPA decreases grain protein concentration at maturity under low but not high nitrogen supply. Under low nitrogen supply, the decrease in SPA expression also caused a reduction in the total quantity of GSP per grain and in the ratio of GSP to albumin-globulins, without significantly affecting GSP composition. The slight reduction in GSP gene expression measured in the SPA RNAi line under low nitrogen supply did not entirely account for the more significant decrease in GSP accumulation, suggesting that SPA regulates additional levels of GSP synthesis. Our results demonstrate a clear role of SPA in the regulation of grain nitrogen metabolism when nitrogen is a limiting resource.

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.

Wheat Grains as a Sustainable Source of Protein for Health.

Protein deficiency is recognized among the major global health issues with an underestimation of its importance. Genetic biofortification is a cost-effective and sustainable strategy to overcome global protein malnutrition. This study was designed to focus on protein-dense grains of wheat (Triticum aestivum L.) and identify the genes governing grain protein content (GPC) that improve end-use quality and in turn human health. Genome-wide association was applied using the 90k iSELECT Infinium and 35k Affymetrix arrays with GPC quantified by using a proteomic-based technique in 369 wheat genotypes over three field-year trials. The results showed significant natural variation among bread wheat genotypes that led to detecting 54 significant quantitative trait nucleotides (QTNs) surpassing the false discovery rate (FDR) threshold. These QTNs showed contrasting effects on GPC ranging from -0.50 to +0.54% that can be used for protein content improvement. Further bioinformatics analyses reported that these QTNs are genomically linked with 35 candidate genes showing high expression during grain development. The putative candidate genes have functions in the binding, remobilization, or transport of protein. For instance, the promising QTN AX-94727470 on chromosome 6B increases GPC by +0.47% and is physically located inside the gene TraesCS6B02G384500 annotated as Trehalose 6-phosphate phosphatase (T6P), which can be employed to improve grain protein quality. Our findings are valuable for the enhancement of protein content and end-use quality in one of the major daily food resources that ultimately improve human nutrition.

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.

Within-leaf chloroplasts and nitrogen allocation to thylakoids in relation to photosynthesis during grain filling in maize.

Nitrogen (N) is an important contributor to photosynthetic rate (Pn). However, during grain-filling stage in maize, some leaf N is remobilized to meet demands for grain protein accumulation rather than photosynthetic demands. Therefore, plants that can maintain a relatively high Pn during the N remobilization process would have the key to achieving both high grain yields (HGY) and high grain protein concentrations (HGPC). In this study, we investigated two high-yielding maize hybrids in photosynthetic apparatus and N allocation in a two-year field experiment. During grain filling, XY335 had a higher Pn and photosynthetic N-use efficiency than ZD958 had in the upper leaf, but not in the middle or lower leaves. In the upper leaf, the diameter and area of the bundle sheath (BS) were larger and the distance between bundle sheaths was greater in XY335 than in ZD958. XY335 had more bundle sheath cells (BSCs) and a larger BSC area, as well as a larger chloroplast area in the BSC, resulting in a higher total number and total area of chloroplasts in the BS. XY335 also had higher stomatal conductance (gs), intercellular CO2 concentration, and N allocation to the thylakoids. No genotypic differences were found in mesophyll cell ultrastructure, N content and starch content in the three types of leaves. Therefore, a trifecta of higher gs, greater N allocation to thylakoids for photo-phosphorylation and electron transport, and more and larger chloroplasts promoting CO2 assimilation in the BS confers a high Pn to simultaneously achieve HGY and high HGPC in maize.

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.

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.

The interacting effects of irrigation, sowing date and nitrogen on water status, protein and yield in pea (Pisum sativum L.).

Management for agronomic practices might improves growth and grain yield in pea. The main objective of this experiment was to assess the interacting effects of different irrigation regimes, sowing date and nitrogen fertilizer treatments on pea traits. We evaluated three irrigation regimes (50, 75, and 100% of the plant irrigation requirement), two sowing dates (February and March), and nitrogen [application of nitroregn (N1) and without nitrogen as control (N0)] in 2019 and 2020 under field conditions. Chlorphyll content, leaf area index, leaf water potential, grain yield and water productivity were higher in the late sowing (March) than in early sowing (February) treatment. Percentage of vegetation cover in late sowing (60%) was significantly higher than in early sowing (52.7%) treatment. Grain yield in 75% water requirement treatment was not significantly different from yield in full irrigation treatment. Application of nitrogen fertilizer significantly reduced grain yield, grain protein and seeds per pod whilst increased chlorophyll content only. The 100% irrigation requirement treatment showed higher evaporation form the soil in N0 than in 50% and 75% irrigation treatments in late sown pea. Leaf evapotranspiration (ET) was lower in 50% water requirement irrigation regime than in the other irrigation treatments. Water use efficiency (WUE) which was higher in the late than early sowing treatment did not differ between 50% and full irrigation treatments in N0. In conclusion, the results of the current study suggested that application of nitrogen fertilizer did not benefit pea growth and that management of irrigation regime in late sowing might improve grain yield in pea and save irrigation water in regions with limited water availability.

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.

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 modify inflorescence branching and increase grain protein content of wheat.

Plant and inflorescence architecture determine the yield potential of crops. Breeders have harnessed natural diversity for inflorescence architecture to improve yields, and induced genetic variation could provide further gains. Wheat is a vital source of protein and calories; however, little is known about the genes that regulate the development of its inflorescence. Here, we report the identification of semidominant alleles for a class III homeodomain-leucine zipper transcription factor, HOMEOBOX DOMAIN-2 (HB-2), on wheat A and D subgenomes, which generate more flower-bearing spikelets and enhance grain protein content. These alleles increase HB-2 expression by disrupting a microRNA 165/166 complementary site with conserved roles in plants; higher HB-2 expression is associated with modified leaf and vascular development and increased amino acid supply to the inflorescence during grain development. These findings enhance our understanding of genes that control wheat inflorescence development and introduce an approach to improve the nutritional quality of grain.

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.

Modulation of Barley (Hordeum vulgare L.) Grain Protein Sink-Source Relations Towards Human Epidermal Growth Factor Instead of B-hordein Storage Protein.

Seeds have evolutionarily developed to store protein without immediately degrading it and constitute ideal tissues for recombinant protein storage. Unfortunately, the production of recombinant protein in seeds is compromised by low yield as compared to other heterologous expression systems. In order to improve the yield of the human epidermal growth factor (EGF) in barley, protein sink-source relations in the developing grain were modulated towards EGF instead of the barley storage protein. The EGF gene, under the control of a B-hordein and a seed-specific oat globulin promoter, was introduced by crossing EGF lines into the Risø 56 mutant deficient in B-hordein storage protein synthesis. Offspring plants were analysed for EGF and Hordein expression and for expression of the unfolded protein response (UPR) genes PDI and CRT to monitor changes in ER stress levels. EGF content was increased significantly in the mature grain of homozygous offspring and PDI and CRT gene expressions were upregulated. We demonstrate, for the first time in barley, that replacement of an abundant seed storage protein with a specific heterologous protein driven by the promoter of the removed gene can accelerate the production of a specific heterologous protein in barley grains.

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.

Prospects of GWAS and predictive breeding for European winter wheat's grain protein content, grain starch content, and grain hardness.

Grain quality traits determine the classification of registered wheat (Triticum aestivum L.) varieties. Although environmental factors and crop management practices exert a considerable influence on wheat quality traits, a significant proportion of the variance is attributed to the genetic factors. To identify the underlying genetic factors of wheat quality parameters viz., grain protein content (GPC), grain starch content (GSC), and grain hardness (GH), we evaluated 372 diverse European wheat varieties in replicated field trials in up to eight environments. We observed that all of the investigated traits hold a wide and significant genetic variation, and a significant negative correlation exists between GPC and GSC plus grain yield. Our association analyses based on 26,694 high-quality single nucleotide polymorphic markers revealed a strong quantitative genetic nature of GPC and GSC with associations on groups 2, 3, and 6 chromosomes. The identification of known Puroindoline-b gene for GH provided a positive analytic proof for our studies. We report that a locus QGpc.ipk-6A controls both GPC and GSC with opposite allelic effects. Based on wheat's reference and pan-genome sequences, the physical characterization of two loci viz., QGpc.ipk-2B and QGpc.ipk-6A facilitated the identification of the candidate genes for GPC. Furthermore, by exploiting additive and epistatic interactions of loci, we evaluated the prospects of predictive breeding for the investigated traits that suggested its efficient use in the breeding programs.

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.

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.

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.