The micronutrient content in underutilized crops: the Lupinus mutabilis sweet case

Adequate intake of micronutrients is necessary to reduce widespread health issues linked to low intake of iron (Fe), zinc (Zn), boron (B), copper (Cu), and manganese (Mn). Because more than two billion people suffer from micronutrient deficiency globally, to address this problem, highly-nutritious ancestral Peruvian crops like tarwi can be an important component of food security. Thus, our work explores the tarwi micronutrient variability to select biofortified genotypes without affecting seed size and weight. Tarwi is a biofortified food because of its seeds' Fe, Zn, and B content. Furthermore, Boron showed a positive correlation between seed size and weight. At the same time, copper showed a negative correlation. Finally, six accessions (P14, P16, P21, T05, T08, and T25) that are biofortified for Fe, Zn, and B with excellent seed size and weight and with adequate levels of Cu and Mn; adding value to Peruvian biodiversity at a low cost is a starting point for a breeding program to prevent micronutrient disorders.


Results and discussions
Micronutrient profile of Lupinus mutabilis. It is critical to have high variability among micronutrients to identify biofortified genotypes for Fe, Zn 26 , B 27 , Cu, and Mn 28 . Thus, Fig. 1A shows greater Fe, Zn, B, Cu, and Mn variability in L. mutabilis. Moreover, there was a higher Fe, B, Cu, and Mn content variability in the early-flowering accessions, while Zn variability was higher in late-flowering genotypes ( Fig. 1A and Table S2). Additionally, there were statistically significant differences in the accessions' micronutrient concentrations of Fe, Zn, B, Cu, and Mn (Tables 1 and 2). For early-flowering accessions, the Fe concentration was between 46.67 and 88 mg kg −1 , whereas the range for late-flowering accessions was 44.67-70.33 mg kg −1 . Without employing a plant breeding strategy, the seed iron concentration in L. mutabilis exceeds 40 mg kg −1 of Fe 29 ; it can be considered a biofortified food. The highest Fe concentration observed in genotypes P03 (Table 1) and T05 (Table 2) was higher than that reported by Sanca 30 , Rodriguez 31 , and Villacres et al. 32 . In contrast, the P03 accession was only higher than Villacres et al. 33 . However, the iron concentrations reported by Ortega-David et al. 34 were superior to this study.
The Zn concentration in early-flowering accessions was between 35.67 and 52.67 mg kg −1 and for lateflowering accessions between 35.33 and 48.67 mg kg −1 , being considered as Zn-biofortified 29,35 as well. P07 and T15 genotypes showed the highest Zn concentrations (Tables 1 and 2). The Zn content reported in L. mutabilis by Villacres et al. 33 , Rodriguez 31 , and Ortega-David et al. 34 were lower than those of P07 and T15. However, the higher Zn concentrations over P07 and T15 were reported by Sanca 30 and Villacres et al. 32 .
The high levels of Fe and Zn observed in tarwi (Fig. 1A) could be explained by Zhao et al. 36 work. He suggests a possible link between grain protein and the levels of the two trace elements under the control of a NAC  www.nature.com/scientificreports/ transcription factor (NAM-B1) that accelerates senescence and increases the remobilization of nutrients (N, Fe, and Zn) from leaves to developing grains 37 . Though Zhao's findings need to be validated in tarwi, they are relevant to this study because the tarwi seed protein content is higher than 40% 6 . Due to massive reports on B-deficiency in cropping systems 38 , biofortification is a sustainable option to increase the yield and quality of crops 39 . Boron levels in food are critical because it is involved in the formation and hardness of bone structure 40 . Under this consideration, Boron concentration in the late and early-flowering accessions was between 20.33 and 29.33 mg kg −1 and 16.67 and 34 mg kg −1 , respectively (Tables 1 and 2), where T15 and P12 are unique genotypes to generate B biofortified cultivars. Although the T15 genotype showed high levels of B and Zn, its iron level was 55.33 mg kg −1 . Since the interaction between B and Zn affects Fe transport and Fe content in organs 41 .
Despite the significant variability in Cu content, genetic selection should focus on a balanced intake of this micronutrient (≤ 10 mg kg −1 ) because consuming foods rich in Cu may increase the incidence of neurodegenerative pathologies like Alzheimer's 42 . According to Schilsky 43 , the adequate concentration of Cu within a balanced diet should not be higher than 11 mg kg −1 . Thus, Cu concentration in the late and early-flowering groups of tarwi is between 7 and 11 mg kg −1 and 5 and 10.67 mg kg −1 , respectively (Table 1 and 2). According to the Schilsky 43 criterion, T08, T12, T14, P03, and P07 genotypes have an adequate Cu concentration for a balanced diet, unlike tarwi genotypes studied by Villacres et al. 33 , Rodriguez 31 and Ortega-David et al. 34 .
In contrast, Rodriguez 31 reported accessions with lower amounts of Mn for this species. On the other hand, Villacres et al. 33 affirm that the Mn content can be reduced by up to 50% during the debittering process of seeds, turning them into safe foods 45 . In this sense, the maximum Mn concentration in seeds without debittering should not exceed 36 mg.kg −1 to be included in a balanced diet. Furthermore, more than 90% of absorbed manganese is mainly excreted via the bile into the feces 46 . Table 1. Genotype by trait data for 20 early-flowering accessions of L. mutabilis for eight traits. Data are expressed as mean ± SD. Significant differences between treatments (P < 0.05) are indicated by different letters (ns: no significant differences; *p < 0.05; ***p < 0.001 and ****p < 0.0001).  47 . Yield components include seed traits such as seed size (SL and SW) and 100-SW 48 ; these characteristics showed considerable variability in early-flowering accessions than in late-flowering accessions (Fig. 1B,C) and were statistically significant differences for each group (Table 1 and 2). In tarwi, there is a high correlation between 100-SW and yield 49 . The 100-SW ranged from 20.80 to 30.64 g for the early-flowering genotypes (  60 . Seed size is an important agronomic trait because of its importance for consumers and industry 61 . Thus the genotypes with large seed sizes (AS and LS) were P14, P16, T02, and T22 (Tables 1 and 2). This pattern was also observed in eight genotypes by Cayo 54 . Additionally, P14 and P16 genotypes displayed excellent seed morphological characteristics (AS, LS, and 100-WS) with a high content of micronutrients (Fig. 1A), demonstrating the crop's excellent potential for food and feed industries.
Selection of tarwi accessions. The first three components in the principal component analysis (PCA) account for 72.2% of the total variability ( Table 3). The weight of 100 seeds (100-SW), seed length (SL), seed width (SW), and Boron content (B) are related to PC1 (41.2%). The Mn and Zn contents are closely related to PC2 (17.4%), and the iron content (Fe) is closely related to PC3 (13.6%). In PC1, the morphological characteristics showed a high correlation with B (Table 3, Fig. 2, and Table S3), which can be attributed to B's role in seed development and size 62 , which can boost crop yields 63 . The high B concentration in L. mutabilis may be due to the high efficiency of B transport to the seeds. Furthermore, B can modulate the absorption and translocation of nutrients to the seed 64 , allowing a higher absorption of Fe, Zn, and Cu 65 but less in Mn 66 . Table 2. Genotype by trait data for 25 late-flowering accessions of L. mutabilis for eight traits. Data are expressed as mean ± SD. Significant differences between treatments (P < 0.05) are indicated by different letters (ns: no significant differences; ***p < 0.001 and ****p < 0.0001). www.nature.com/scientificreports/ However, the relationship between Cu and seed size was inverse and significant; because, in high concentrations, copper reduces the seed size, impacting grain yield and quality 67 . However, the only two micronutrients with a significant and positive correlation were Mn and Zn (Table S3), and Biplot-PCA corroborates it (Fig. 2). This may show a synergistic effect between these two micronutrients 68 , suggesting a cotransport of Mn and Zn.
Since 100-WS is highly associated with yield in L. mutabilis 49 and seed size can affect it, the Mahalanobis cluster analysis was used to select the best genotypes. Thus, the late-flowering accessions were divided into 12 accessions with 100-SW > 23 g and 13 accessions with 100-SW < 23 g (Fig. 3A). Simultaneously, 20 early-flowering accessions were divided into two subgroups, 2 genotypes with 100-SW < 21 g and 18 genotypes with 100-SW > 21 g (Fig. 3C). To narrow down the selection, results based on the micronutrient criteria (Fe > 40 mg kg −1 , Zn > 28 mg kg −1 , B > 13 mg kg −1 , Cu < 9 mg kg −1 and Mn < 30 mg kg −1 ) and a mean coefficient of variation of micronutrients (MCV micronutrient ≤ 10%) were used and allowed to select three late-flowering accessions (T05, T08 and T25) and three early-flowering accessions (P14, P16 and P21) that were biofortified for Fe, Zn and B with adequate nutritional requirements in Cu and Mn and excellent seed size and weight (Fig. 3C,D and Table S4), unlike than Villacres et al. 33 , Rodríguez 31 and Ortega-David et al. 34 . Furthermore, a MCV micronutrient value of ≤ 10% among the selected accessions could be due to a high specificity of the proteins associated with the absorption, translocation, remobilization and/or storage of these micronutrients. Therefore, a better understanding of these processes is required to explain the micronutrient homeostasis of tarwi. Moreover, our data demonstrated that the selection of micronutrient-rich tarwi genotypes was achieved without affecting the morphological traits of the seeds (Fig. 3 and Table S4). Finally, its unique micronutrient levels make it a suitable grain to be included in a healthy diet.   (Table S1). All the measurements for this study were done between 2019 and 2020 at Universidad Nacional Agraria La Molina.
Sample preparation. All samples were weighted with a Henkel balance (± 0.01) and dried until constant weight in an oven at 70 °C. Dry samples were ground with a Thomas Model 4 Wiley® Mill (Thomas Scientific) until a homogeneous mass was obtained. All samples were kept in sealed containers to avoid contact with atmos- www.nature.com/scientificreports/ pheric humidity.1 g of ground sample was weighed with an OHAUS PA313 balance (± 0.001 g) and used for sample digestion.
Analysis of the micronutrients profile (Fe, Zn, B, Cu, and Mn) and seed size and weight. In L.
mutabilis seeds, Fe, Zn, Cu, and Mn concentrations were measured by the wet digestion method 70 . The samples were digested in a nitroperchloric mixture and measured by atomic absorption spectrometry (Perkin Elmer Analyst 200). The curcumin-acetic acid method 72 determined boron (B) concentration. The micronutrient concentration was expressed in mg kg −1 . However, seed length (SL), seed width (SW), and the weight of 100 seeds (100-SW) were determined according to Pereira et al. 71 . Seed size was expressed in millimeters (mm), and the weight of 100 seeds in grams (g). All experiments were done in triplicate. The Pearson correlation analysis was performed in Excel to determine the relationship between variables. Finally, boxplots, bar-graphs, mean, standard deviation, and coefficient of variation (CV %) were calculated using the statistical InfoStat analysis system (www. infos tat. com. ar) software. Duncan's multiple range test was used to separate the differences in the mean scores at a significance level of P < 0.05.
Selection of tarwi accessions. The data in early and late-flowering genotypes of L. mutabilis (Table S2) were standardized to estimate the genetic distance matrix using the Euclidean distance 72 . The analysis was carried out using InfoStat software (www. infos tat. com. ar), and PCA and Biplot of PCA consistency were verified by cophenetic correlation coefficient 73 .
To identify the best traits, the cluster analysis was done first, where Mahalanobis distance was used as the distance matrix 74 with InfoStat software (www. infos tat. com. ar); and to narrow the selection results based on healthy eating, the micronutrient criteria: Fe > 40 mg kg −1 29 , Zn > 28 mg kg −1 29 , B > 13 mg kg −1 75 , Cu < 9 mg kg −1 76 , and Mn < 30 mg kg −1 77 and mean-coefficient of variation for micronutrient (MCV micronutrient ) of less or equal than 10% 78 were used.

Conclusions
Without employing a plant breeding strategy, Fe, Zn, and B content in tarwi seeds exceed the 40 mg kg −1 , 28 mg kg −1 , and 13 mg kg −1 , respectively, and it can be considered biofortified food. Moreover, Boron stands out as an important micronutrient because of its positive correlation with seed size and seed weight because B is involved in the development and size of the seeds. However, Cu and seed size showed a negative correlation, helping the selection of genotypes with good seed morphological traits and adequate Cu concentration (≤ 10 mg kg −1 ). These results do provide partial validation of the hypothesis.
100-WS, along with micronutrient criteria for healthy eating and MCV micronutrient ≤ 10% allowed to select six accessions (P14, P16, P21, T05, T08, and T25) that were biofortified for Fe, Zn and B with excellent seed size and weight and adequate levels of Cu and Mn. These genotypes will serve as the basis for a breeding program to boost micronutrient content, which will help avoid micronutrient problems and reduce healthcare expenditures by promoting healthy eating. Lastly, fostering well-being and ensuring healthy eating/living is crucial for sustainable development. Biofortification has a crucial role in reducing health costs by promoting healthy eating. However, it has limitations because the technology does not address the high-cost analysis to evaluate large seed banks in megadiverse countries. Thus, other technologies such as Micro-XRF and benchtop TXRF spectrometers could be an alternative to reduce analysis costs and build a high-throughput mineral-nutrient phenotyping platform.

Data availability
All relevant data are within the paper and its supporting Information files.