High Protein Lines Research Articles

Characterization of high protein soybean using mass spectrometry based proteomic and metabolomic analyses.

We have created several high protein (HPBL) and low protein breeding lines (LPBL) by crossing between NC-Raleigh and PI407228. The NC-Raleigh is a low protein cultivar widely grown in North Carolina with excellent yield potential and a high protein wild soybean (Glycine soja) with low yield. We analyzed the F6 generation of this cross for protein and oil compositions using near-infrared spectroscopy (NIR). Two soybean lines HPBL and LPBL were selected for biochemical analysis using mass spectrometry-based proteomic and metabolomic techniques. We identified 4,538 proteins and 267 metabolites in these lines. The HPBL showed the enrichment of proteins associated with protein synthesis, processing, and storage in addition to higher content of primary and secondary metabolites. The mapping of metabolites on the global metabolic pathways revealed the increased protein content in the HPBL that may have resulted from increased arginine content routed from citrulline-argininosuccinic acid. This information will be useful for breeders and biotechnologists to produce a value-added soybean trait.

High-protein soybean lines with stable seed protein content under heat and drought stresses

A significant factor determining the value of soybean is protein content, in addition to its importance as an oilseed. Increasing the seed protein content in soybean will make it a more valuable food and feedstock and enhance its importance in the international market. Due to this reason, developing high-protein soybean varieties is gaining increased industrial interest. To ensure profitable production, the stability of the high-protein trait needs to be verified under environmental stresses. The objectives of this study were to test whether the high-protein soybean genotypes can maintain their high seed protein content under heat and drought stresses imposed during seed filling and to assess the changes in their oil composition under stress conditions. The changes in seed protein and oil contents and oil composition of the high-protein lines NLM09–77, N14-7017, and N16-9924 under heat and drought stresses were assessed and compared against those of a recently released high-yielding, high-protein variety Benning HP; elite cultivars NC-Dunphy, NC-Raleigh, and NC-Dilday; low-protein breeding lines N11–9228 and N09-2505; heat-tolerant line DS25-1; and heat-susceptible line DT97-4290. We found that the high-protein genotypes NLM09–77, N16–9924, N14-7017, and Benning HP maintained their high seed protein contents under heat and drought stresses (46–55 %). Among them, NLM09–77 and N14-7017 were unique with no apparent negative relationship between the seed protein and oil contents. In terms of oil composition, N14-7017, NLM09-77, and Benning HP were similar to the elite cultivars NC-Dunphy and NC-Dilday. Additionally, the high-protein genotypes N14-7017, NLM09-77, and Benning HP did not exhibit any yield drag (2993–3150 kg ha−1vs 3289–3368 kg ha−1 for the elite cultivars). Taken together, our study demonstrates the usefulness of the advanced breeding lines NLM09–77 and N14-7017 and cultivar Benning HP for developing new varieties with enhanced nutritional value under climate change-associated stresses.

Identification of High-Yielding Soybean Lines with Exceptional Seed Composition Qualities

In current markets, the primary uses for soybean seeds are in products derived from their oil or protein content. However, growers are compensated based on seed yield, so a more valuable crop is one that does not compromise on yield when compared with existing options, with an optimum combination of protein and oil. A negative correlation of seed protein with seed yield and oil makes the simultaneous improvement of these traits difficult but not impossible through conventional breeding. Selections of lines with exceptional yield and seed composition were made from two recombinant inbred line (RIL) soybean mapping populations to identify high protein and/or high oil lines with yields comparable to elite cultivars. The performance of these RILs was evaluated in multiple environments, and several genotypes were identified with yields comparable to those of high-yielding check cultivars with seed protein and/or oil content superior to the checks. These genotypes will provide breeders with additional sources of germplasm for continuing efforts to improve seed composition traits without compromising seed yield and provide growers with more profitable cultivars.

Comprehensive survey of transposon mPing insertion sites and transcriptome analysis for identifying candidate genes controlling high protein content of rice.

Rice is the most important crop species in the world, being staple food of more than 80% of people in Asia. About 80% of rice grain is composed of carbohydrates (starch), with its protein content as low as 7–8%. Therefore, increasing the protein content of rice offers way to create a stable protein source that contributes to improving malnutrition and health problems worldwide. We detected two rice lines harboring a significantly higher protein content (namely, HP5-7 and HP7-5) in the EG4 population. The EG4 strain of rice is a unique material in that the transposon mPing has high transpositional activity and high copy numbers under natural conditions. Other research indicated that mPing is abundant in the gene-rich euchromatic regions, suggesting that mPing amplification should create new allelic variants, novel regulatory networks, and phenotypic changes in the EG4 population. Here, we aimed to identify the candidate genes and/or mPing insertion sites causing high protein content by comprehensively identifying the mPing insertion sites and carrying out an RNA-seq-based transcriptome analysis. By utilizing the next-generation sequencing (NGS)-based methods, ca. 570 mPing insertion sites were identified per line in the EG4 population. Our results also indicated that mPing apparently has a preference for inserting itself in the region near a gene, with 38 genes in total found to contain the mPing insertion in the HP lines, of which 21 and 17 genes were specific to HP5-7 and HP7-5, respectively. Transcriptome analysis revealed that most of the genes related to protein synthesis (encoding glutelin, prolamin, and globulin) were up-regulated in HP lines relative to the control line. Interestingly, the differentially expressed gene (DEG) analysis revealed that the expression levels of many genes related to photosynthesis decreased in both HP lines; this suggests the amount of starch may have decreased, indirectly contributing to the increased protein content. The high-protein lines studied here are expected to contribute to the development of high protein-content rice by introducing valuable phenotypic traits such as high and stable yield, disease resistance, and abundant nutrients.

Identification of Quantitative Trait Loci Associated with Seed Protein Concentration in a Pea Recombinant Inbred Line Population.

This research aimed to identify quantitative trait loci (QTLs) associated with seed protein concentration in a recombinant inbred line (RIL) population of pea and aimed to validate the identified QTLs using chromosome segment-introgressed lines developed by recurrent backcrossing. PR-25, an RIL population consisting of 108 F7 bulked lines derived from a cross between CDC Amarillo (yellow cotyledon) and CDC Limerick (green cotyledon), was used in this research. The RIL population was genotyped using an Axiom 90K SNP array. A total of 10,553 polymorphic markers were used for linkage map construction, after filtering for segregation distortion and missing values. The linkage map represents 901 unique loci on 11 linkage groups which covered a map distance of 855.3 Centimorgans. Protein concentration was assessed using near-infrared (NIR) spectroscopy of seeds harvested from field trials in seven station-years in Saskatchewan, Canada, during the 2019–2021 field seasons. Three QTLs located on chromosomes 2, 3 and 5 were identified to be associated with seed protein concentration. These QTLs explained 22%, 11% and 17% of the variation for protein concentration, respectively. The identified QTLs were validated by introgression lines, developed by marker-assisted selection of backcross lines for introgression of corresponding chromosome segments (~1/4 chromosome) harboring the QTL regions. Introgression line PR-28-7, not carrying any protein-related QTLs identified in this study, was 4.7% lower in protein concentration than CDC Amarillo, the lower protein parent of PR-25 which carried one identified protein-related QTL. The SNP markers located at the peak of the three identified QTLs will be converted into breeder-friendly KASP assays, which will be used for the selection of high-protein lines from segregating populations.

Assessment of soybean test weight among genotypes, environments, agronomic and seed compositional traits

AbstractThe value of soybean [Glycine max (L.) Merr.] depends on quantity (yield) and quality, such as seed composition and test weight (TW). Test weight is a bulk density measurement for grain quality evaluation, and higher TW grains are preferred for storage, transport, and export. Thus, soybean breeding should focus on improvement of TW. The objectives of this study were to determine genotypic and environmental effects on TW of soybean and explore relationships of TW with yield, maturity, 100 seed weight (SDWT), seed quality (SQ), seed oil concentration (SOC), and seed protein concentration (SPC). Three sets of breeding populations (BPs), two mapping populations (MPs), and five different high vs. low seed protein near‐isogenic lines populations (NILPs) were used. The BPs and MPs had an average range of 3.5 kg hectoliter (hL)–1 in TW among genotypes. The average ranges of TW in the NILPs were equal for both low protein (LP) and high protein (HP) lines (2.7 kg hL–1). Broad‐sense heritability estimates for TW ranged from .62 to .95. The genotypic and G × E effects on TW were significant in all trials. The SDWT, SQ, SOC, and SPC had strong significant relationships with TW in multiple trials, but were inconsistent overall. Test weight of NILPs differing by presence of ‘Danbaekkong’ high protein allele on chromosome 20 had mostly significant positive and negative relationships with SPC and SOC, respectively.

Detection of stable QTLs for grain protein content in rice (Oryza sativa L.) employing high throughput phenotyping and genotyping platforms

Lack of appropriate donors, non-utilization of high throughput phenotyping and genotyping platforms with high genotype × environment interaction restrained identification of robust QTLs for grain protein content (GPC) in rice. In the present investigation a BC3F4 mapping population was developed using grain protein donor, ARC10075 and high-yielding cultivar Naveen and 190 lines were genotyped using 40 K Affimetrix custom SNP array with the objective to identify stable QTLs for protein content. Three of the identified QTLs, one for GPC (qGPC1.1) and the other two for single grain protein content (qSGPC2.1, qSGPC7.1) were stable over the environments explaining 13%, 14% and 7.8% of the phenotypic variances, respectively. Stability and repeatability of these additive QTLs were supported by the synergistic additive effects of multi-environmental-QTLs. One epistatic-QTL, independent of the main effect QTL was detected over the environment for SGPC. A few functional genes governing seed storage protein were hypothesised inside these identified QTLs. The qGPC1.1 was validated by NIR Spectroscopy-based high throughput phenotyping in BC3F5 population. Higher glutelin content was estimated in high-protein lines with the introgression of qGPC1.1 in telomeric region of short arm of chromosome 1. This was supported by the postulation of probable candidate gene inside this QTL region encoding glutelin family proteins.

Biofortification strategies to increase wheat nutrition and sustaining yield simultaneously

Genetic biofortification is a strategy that uses plant breeding techniques to produce staple food crops with higher micronutrient levels and can be a feasible and costeffective means of delivering micronutrients to population that may have limited access to diverse diets. The present study reports the development of wheat with high grain protein, yellow pigment and high zinc content in addition to introgression of rust resistance genes to ensure biofortification as well as yield sustainability. A convergent cross for combining gene(s) for high protein, high yellow pigment, high zinc and rust resistance was performed. This included crossing BC1F2 introgression lines of cross PBW698/BF22//PBW698 carrying grain zinc QTL introgressed from Triticum monococcum with high protein line BWL3560 having Gpc-B1. A set of 192 F3 lines were evaluated for agronomic and quality traits. Molecular marker analysis of F3 progenies for Gpc-B1 gene (Xucw108) revealed 129 F3 lines to be homozygous positive. The progenies showed the range of 8.96-13.92% for grain protein content, 25.1-36.3mg/Kg for grain iron concentration, 34.0-54.0 mg/Kg for grain zinc concentration and 2.01-4.57ppm for grain yellow pigment content compared to 9.65%, 26.7 mg/Kg, 42.9 mg/Kg and 3.74ppm respectively in recipient line PBW698. Therefore, this population constituted a useful material for identifying the nutritionally enhanced lines.

Genetic diversity and variability for protein and micro nutrients in advance breeding lines and chickpea varieties grown in Andhra Pradesh

An investigation was taken up to study the nutritional value, extent of genetic variability and genetic diversity in advance breeding lines and chickpea varieties grown in Andhra Pradesh, India. Protein and micronutrient content (iron, zinc, copper and manganese) varied significantly among 54 genotypes. Protein content ranged from 9.5% to 24.9% while micro nutrients varied from 2.6 mg/100 g to 14.6 mg/100 g for iron, 3.5 mg/100 g to 7.7 mg/100 g for zinc, 0.5 mg/100 g to 3.2 mg/100 g for copper and 1.6 mg/100 g to 3.4 mg/100 g for manganese. Moderate to high genotypic variability for protein and micro nutrient content with high heritability and genetic advance indicated the scope for enhancement of traits through selection. Genetic diversity studies revealed five different clusters and that high protein lines are grouped in cluster I and lines with higher concentration of micro nutrients are grouped in clusters IV and V. Systematic hybridization between promising lines for protein and micronutrients chosen from these clusters is suggested to study their combining ability and subsequent use in breeding programmes intended to breed for superior chickpea cultivars.

Field evaluation and break-even analysis of specialty soybeans for biodiesel and meal protein production

ABSTRACTThe economic value of a soybean [Glycine max (L.) Merr.] crop depends on yield and quality. The objectives of this study were to evaluate soybean lines for protein and oil content and to examine the break-even (BE) analysis of these traits combined with yield. Forty lines of maturity group (MG) IV or V were grown at four Arkansas locations for two years. Yield, protein, and oil content of each line were determined and compared to the check average for a BE comparison of the total economic value of the line. Results showed six profitable lines ($8.40 to $54.96 per metric ton based on yield) in 2008 and six profitable lines ($0.49 to $61.19 per metric ton based on yield) in 2009. Overall, in both MG, high-protein lines needed to yield more to be profitable, whereas high-oil lines had competitive yield, protein and oil content; R00-764 (MG IV), R05-71 (MG V), and R02-6185F (MG V) were competitive without added premiums.

Identification and characterization of high protein oat lines from a mutagenized oat population

Oat is an excellent source of high-quality proteins. The protein content in cultivated oat is about 12%–15%. Oats with even higher protein contents would have great potential as a vegetative protein source. From a mutagenized oat population, 230 lines with seed protein levels of 15% or higher were identified from green house grown plants. Fifteen lines with protein levels ranging between 17 and 24% were chosen for further studies. The high protein character was confirmed by two consecutive propagations in the field. Crossings showed that the trait was stable and segregated in a Mendelian fashion. SDS-PAGE analysis revealed that the major increase was in globulin like proteins. In addition, total dietary fibre, β-glucan and lipid levels were quantified. No correlation between any of these characters and the high protein trait could be found. Thousand Kernel Weight was also monitored and shown to be stable in the high protein lines. Thus, starting from the lines presented here a further development of commercial oat varieties with protein levels of 20% or higher is feasible. Such a high protein oat would be very important to meet the consumers need for high-quality high-protein oat for the food and feed industry.

Soybean peptide fractions inhibit human blood, breast and prostate cancer cell proliferation.

In this study, we examined in vitro the bio-activity of peptide fractions obtained from soybeans against blood (CCRF-CEM and Kasumi-3), breast (MCF-7), and prostate (PC-3) cancer cell proliferation. Gastro-intestinal treated peptide fractions (<5, 5-10 and 10-50kDa) prepared from seed proteins of two high oleic acid soybean lines-N98-4445A, S03-543CR and one high protein line-R95-1705, were tested for anticancer activity against human breast, blood and prostate cancer cell lines. Anti-proliferative cell titer assay was conducted to assess the inhibitory effects of the peptide fractions, while trypan blue dye exclusion assay was used to determine the dose response of most effective fractions. Results showed that the peptide fractions inhibited the cancer cell lines up to 68.0% and the minimum concentration to get 50% inhibitory activity (IC50) ranged between 608 and 678µg/mL. This multiple site in vitro cancer inhibition by GI friendly peptides could have the potential use as food ingredients or nutritional supplements in an alternative cancer therapy.

Protein-rich beverage developed using non-GM soybean (R08-4004) and evaluated for sensory acceptance and shelf-life.

Protein beverages have been in demand due to an increasing consumers' interest in healthy eating habit. However, there is an increased concern on the use of genetic modified (GM) ingredient in the food product. This study aimed to develop protein hydrolysate beverages using a non-GM soybean (R08-4004/high protein line) grown in Arkansas. Protein isolate was prepared from the soybean using alkaline method (pH 9.5). Due to its poor solubility in acidic condition, alcalase 2.4L (food grade protease) hydrolyzed soy protein was used to develop a beverage containing 20g protein per serving (500mL). Three flavored beverages: Chai tea (C), tangerine (T), and mixed berries (MB) were prepared using bitter blocker, masking agent, and citric acid to minimize an unpleasant bitter taste developed in the soy hydrolysates. Protein solubility, pH, microbial growth, instrumental color parameters, and turbidity were measured to evaluate the shelf-life stability of the beverages at refrigerated storage (5°C) for 42days. Beverages T and MB received overall highest scores from the sensory panel. Citric acid alone or in combination with bitter blocker or masking agent lowered the bitterness. Pasteurization (90-95°C for 5min) was effective in preventing microbial growth. Although pH remained constant, decrease in protein solubility and color changes were observed over the storage time in all the three flavored beverages. Cloudiness in beverage C increased over the storage period while beverages T and MB were very stable. Overall, T and MB flavored beverages have the potential for commercial application.

Identification of discrepancies in grain quality and grain protein composition through avenin proteins of oat after an effort to increase protein content

Oat prolamin (avenin) is a family of proteins that contain several polymorphic components. The high interspecific variability of avenin among cultivars, electrophoretic patterns have been used for grain protein comparison and species relationships. Therefore, the present study was carried out to evaluate the effect of environment on avenin constitution following an effort to increase protein content of oat using different approaches. The avenin electrophoretic patterns of 25 samples resulting from 20 oat cultivars and 5 mutated oat lines grown or developed in different fields and greenhouses were compared. Quantification of total proteins and SDS-PAGE analysis of avenin protein extracts were carried out. Different pattern of avenin allele in electrophoretic gel allowed the comparison within and between groups. This analysis shows that the cultivars obtained from Swedish fields had differences in the number of alleles compared with the parental cultivar, so were not stable across sites. The study revealed that oats treated with nitrogen and the selected mutated lines showed increase in protein concentration, with consistent avenin loci to the parental line, so these lines are now being used as potential candidates for developing high-protein oat lines in the future. Our results provide useful information, on the stability of modifications, for breeders aiming to increasing the content and nutritional value of oat protein. The study will increase our knowledge in the stability of different modifications and thus to accordingly select oat lines with stable and improved nutritional values.

Identification and Fine Mapping of a Soybean Seed Protein QTL from PI 407788A on Chromosome 15

ABSTRACTSoybean [Glycine max (L.) Merr.] is grown primarily as a source of protein and oil. The objectives of this study were to identify soybean seed protein quantitative trait loci (QTL) from the high protein line PI 407788A and to fine map an important QTL for protein on chromosome (chr) 15. The mapping was done in a population of inbred lines developed with one backcross (BC1) using Williams 82 as a recurrent parent and the high protein source PI 407788A as a donor parent. The population was evaluated with genetic markers and for seed protein and oil concentrations in three Illinois locations in 2005 and 2006. The location of the major protein QTL on chr 15 was fine mapped by evaluating six families of lines segregating for sections of the QTL interval in Illinois during 2008. Genetic marker analysis of the BC1 population revealed the locations of significant protein and oil QTL on chr 20 and 15 within regions where QTL for these traits were previously mapped. Fine mapping of the chr 15 QTL placed it in a 535 kb interval between BARCSOYSSR_15_0161 and BARCSOYSSR_15_0194 based on the Glyma.Wm82.a2 assembly. This fine mapping information will be useful in marker‐assisted selection and efforts to clone the QTL.

Molecular cytogenetic identification of a novel hexaploid wheat–Thinopyrum intermedium partial amphiploid with high protein content

Genomic in situ hybridization and fluorescence in situ hybridization were used to define the cytogenetic constitution of the intergeneric introgression line 29(1-57) obtained from the wheat × Thinopyrum intermedium (wheatgrass) cross. This line is a high-protein line resistant to leaf rust, yellow rust, and powdery mildew under field conditions and has 42 chromosomes. GISH analysis using a total genomic DNA probe from Thinopyrum intermedium resulted in the identification of 14 chromosomes of wheatgrass origin. The sequential GISH patterns obtained using St-genomic and J-genomic DNA as probes and ABD-genomic DNA from wheat as a blocker led to a better determination of the genomic type of wheatgrass chromosomes. Rearrangements involving Thinopyrum chromosomes and the A, B, and D genomes of wheat were not observed. FISH using the repetitive DNA probes pSc119.2, Afa-family, and pTa71 allowed for identification of all the wheat chromosomes present.

Amino Acid Profiles of 44 Soybean Lines and ACE‐I Inhibitory Activities of Peptide Fractions from Selected Lines

AbstractSoybeans are cultivated in the United States chiefly for cooking oil, while the residue after oil extraction (soybean meal) is mostly used in animal feed formulations. High protein content in the defatted soybean meals led to the extraction of pure protein and its application in food products. We selected 44 soybean lines to determine their moisture and protein contents, and their amino acid composition was investigated. Soybean lines with high protein content, one high yielding (R95‐1705), and two high oleic acid (N98‐4445A, S03‐543CR), were selected for protein isolate preparation, hydrolysis using alcalase and gastro‐intestinal (GI) resistance. Furthermore, the GI resistant hydrolysates were fractionated and tested for angiotensin‐I‐converting enzyme (ACE‐I) inhibition activity. The amino acid analysis showed high methionine in the high protein and fatty acid lines (R05‐4494 and R05‐5491), and high cysteine content in one of the high oleic acid soybean line CRR05‐188 in comparison to the check lines (UA‐4805 and 5601‐T). The protein isolate with the highest purity (90–93 %) was derived from the selected lines N98‐4445A and S03‐543CR, and hydrolyzed using alcalase enzyme. The protein hydrolysates (500 µg/mL) showed inhibition of the ACE‐I by 49 %. The results from this study will promote the use of high oleic acid soybeans as a source of protein and peptides with functional activities.

Introgression of leginsulin, a cysteine-rich protein, and high-protein trait from an Asian soybean plant introduction genotype into a North American experimental soybean line.

Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur-containing amino acids could enhance the nutritive value of soybean meal. Leginsulin, a cysteine-rich peptide, predominantly accumulates in Asian soybean accessions but not in most North American cultivars. By screening diverse soybean accessions from the USDA Soybean Germplasm Collection, we were able to identify one plant introduction, PI 427138, as a high-protein line with relatively high amounts of both elemental sulfur and leginsulin. We introgressed these desirable traits from PI 427138 into an experimental line with the aim of improving the overall protein content and quality of seed proteins. Biochemical characterization of inbred progenies from the cross of LD00-3309 with PI 427138 grown at six locations revealed stable ingression of high protein, high elemental sulfur, and high leginsulin accumulation. Comparison of soybean seed proteins resolved by high-resolution 2-D gel electrophoresis in combination with Delta2D image analysis software revealed preferential accumulation of a few glycinin subunits contributed to the increased protein content in the introgressed lines. Amino acid analysis revealed that even though the leginsulin introgressed lines had higher protein, leginsulin, and elemental sulfur, the overall concentration of sulfur-containing amino acids was not significantly altered when compared with the parental lines. The experimental soybean lines developed during this study (Leg-3, Leg-7, and Leg-8) lack A5, A4, and B3 glycinin subunits and could be utilized in breeding programs to develop high-quality tofu cultivars.

Break-Even Profitability for Food-Grade Specialty Soybeans

Cultivar selection for specialty soybeans is mainly based on seed-yield performance, disease resistance, and value-increasing seed attributes. However, adoption of food-grade specialty soybean cultivars by farmers for commercial production requires studies on profitability and economic factors. This research evaluated the profitability of small-seeded, large-seeded, and high-protein specialty soybeans using break-even (BE) analysis to establish guidelines for cultivar selection and adoption based on economic feasibility. Differential costs for seed and weed control were considered in the BE analysis of two different planting systems: conventional (Scenario I) and herbicide tolerant (Scenario II) soybeans. Average BE premiums were $2.74, $4.26, and $1.30 bu-¹ under Scenario I, and $2.02, $4.57, and $0.66 bu-¹ under Scenario II for small seeded, large seeded, and high-protein test lines, respectively. At current premium level of $3.50 bu-¹ for small seeded, $2.50 bu-¹ for large seeded, and $1.50bu-¹ for high-protein specialty soybean, BE yields for these three types of specialty soybean should be 76.46, 85.21, and 89.28% of the check’s yield when compared with conventional soybean; and 77.47, 92.92, and 90.71% of the check’s yield when compared with Roundup Ready soybean, respectively. Additional positive returns will be expected when the current premiums offered in the market are higher than the BE premium of a specialty soybean cultivar, or when the actual yields of this cultivar are higher than the BE yield at current premiums. Based on the economic feasibilities, the present study proposed a new model for the selection and adoption of specialty soybean cultivars, both in breeding programs and for commercial production.

Nitrogen Leaching Of Spring Wheat Genotypes (Triticum Aestivum L.) Varying In Nitrogen-Related Traits

Efficient use of nitrogen (N) by wheat crop and hence prevention of possible contamination of ground and surface waters by nitrates has aroused environmental concerns. The present study was conducted in drainage lysimeters for three years (1998–2000) to identify whether spring wheat genotypes (Triticum aestivum L.) that differ in N-related traits differ in N leaching and to relate parameters of N use efficiency (NUE) with parameters of N leaching. For this reason two spring wheat cultivars (‘Albis’ and ‘Toronit’) and an experimental line (‘L94491’) were grown under low (20 kg N ha−1) and ample N supply (270 kg N ha−1). The genotypes varied in parameters of NUE but not in N leaching. Grain yield of the high-protein line (‘L94491’) was, on average, 11% lower than that of ‘Toronit’ but among genotypes had significantly higher N in the grain (%), grain N yield, and N harvest index. Nitrogen lost through leaching was considerably low (0.42–0.52 g m−2) mainly due to low volume of percolating water or the ability of the genotypes to efficiently exploit soil mineral N. There were no clear relationships between N-related genotype traits and N leaching, but across all treatments significantly negative correlations between volume of leachate and the amount of N in the total biomass and grain N yield existed.