Stability of grain zinc concentrations across lowland rice environments favors zinc biofortification breeding.

Introduction: One-third of the human population consumes insufficient zinc (Zn) to sustain a healthy life. Zn deficiency can be relieved by increasing the Zn concentration ([Zn]) in staple food crops through biofortification breeding. Rice is a poor source of Zn, and in countries predominantly relying on rice without sufficient dietary diversification, such as Madagascar, Zn biofortification is a priority. Methods: Multi-environmental trials were performed in Madagascar over two years, 2019 and 2020, to screen a total of 28 genotypes including local and imported germplasm. The trials were conducted in the highlands of Ankazomiriotra, Anjiro, and Behenji and in Morovoay, a location representative of the coastal ecosystem. Contributions of genotype (G), environment (E), and G by E interactions (GEIs) were investigated. Result: The grain [Zn] of local Malagasy rice varieties was similar to the internationally established grain [Zn] baseline of 18-20 µg/g for brown rice. While several imported breeding lines reached 50% of our breeding target set at +12 µg/g, only few met farmers' appreciation criteria. Levels of grain [Zn] were stable across E. The G effects accounted for a main fraction of the variation, 76% to 83% of the variation for year 1 and year 2 trials, respectively, while GEI effects were comparatively small, contributing 23% to 9%. This contrasted with dominant E and GEI effects for grain yield. Our results indicate that local varieties tested contained insufficient Zn to alleviate Zn malnutrition, and developing new Zn-biofortified varieties should therefore be a priority. GGE analysis did not distinguish mega-environments for grain [Zn], whereas at least three mega-environments existed for grain yield, differentiated by the presence of limiting environmental conditions and responsiveness to improved soil fertility. Discussion: Our main conclusion reveals that grain [Zn] seems to be under strong genetic control in the agro-climatic conditions of Madagascar. We could identify several interesting genotypes as potential donors for the breeding program, among those BF156, with a relatively stable grain [Zn] (AMMI stability value (ASV) = 0.89) reaching our target (>26 µg/g). While selection for grain yield, general adaptation, and farmers' appreciation would have to rely on multi-environment testing, selection for grain [Zn] could be centralized in earlier generations.

Quantitative trait loci and genomic prediction for grain sugar and mineral concentrations of cowpea [Vigna unguiculata (L.) Walp.].

Development of high yielding cowpea varieties coupled with good taste and rich in essential minerals can promote consumption and thus nutrition and profitability. The sweet taste of cowpea grain is determined by its sugar content, which comprises mainly sucrose and galacto-oligosaccharides (GOS) including raffinose and stachyose. However, GOS are indigestible and their fermentation in the colon can produce excess intestinal gas, causing undesirable bloating and flatulence. In this study, we aimed to examine variation in grain sugar and mineral concentrations, then map quantitative trait loci (QTLs) and estimate genomic-prediction (GP) accuracies for possible application in breeding. Grain samples were collected from a multi-parent advanced generation intercross (MAGIC) population grown in California during 2016-2017. Grain sugars were assayed using high-performance liquid chromatography. Grain minerals were determined by inductively coupled plasma-optical emission spectrometry and combustion. Considerable variation was observed for sucrose (0.6-6.9%) and stachyose (2.3-8.4%). Major QTLs for sucrose (QSuc.vu-1.1), stachyose (QSta.vu-7.1), copper (QCu.vu-1.1) and manganese (QMn.vu-5.1) were identified. Allelic effects of major sugar QTLs were validated using the MAGIC grain samples grown in West Africa in 2017. GP accuracies for minerals were moderate (0.4-0.58). These findings help guide future breeding efforts to develop mineral-rich cowpea varieties with desirable sugar content.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Dietary Zn deficiency, the current situation and potential solutions.

Zinc (Zn) deficiency is a worldwide problem, and this review presents an overview of the magnitude of Zn deficiency with a particular emphasis on present global challenges, current recommendations for Zn intake, and factors that affect dietary requirements. The challenges of monitoring Zn status are clarified together with the discussion of relevant Zn bioaccessibility and bioavailability issues. Modern lifestyle factors that may exacerbate Zn deficiency and new strategies of reducing its effects are presented. Biofortification, as a potentially useful strategy for improving Zn status in sensitive populations, is discussed. The review proposes potential actions that could deliver promising results both in terms of monitoring dietary and physiological Zn status as well as in alleviating dietary Zn deficiency in affected populations.

Proof of concept and early development stage of market-oriented high iron and zinc rice expressing dicot ferritin and rice nicotianamine synthase genes.

Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 (OsNAS2) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 µg g-1 and up to 37 µg g-1, respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector.

Genomic prediction of zinc-biofortification potential in rice gene bank accessions.

KEY MESSAGE: A genomic prediction model successfully predicted grain Zn concentrations in 3000 gene bank accessions and this was verified experimentally with selected potential donors having high on-farm grain-Zn in Madagascar. Increasing zinc (Zn) concentrations in edible parts of food crops, an approach termed Zn-biofortification, is a global breeding objective to alleviate micro-nutrient malnutrition. In particular, infants in countries like Madagascar are at risk of Zn deficiency because their dominant food source, rice, contains insufficient Zn. Biofortified rice varieties with increased grain Zn concentrations would offer a solution and our objective is to explore the genotypic variation present among rice gene bank accessions and to possibly identify underlying genetic factors through genomic prediction and genome-wide association studies (GWAS). A training set of 253 rice accessions was grown at two field sites in Madagascar to determine grain Zn concentrations and grain yield. A multi-locus GWAS analysis identified eight loci. Among these, QTN_11.3 had the largest effect and a rare allele increased grain Zn concentrations by 15%. A genomic prediction model was developed from the above training set to predict Zn concentrations of 3000 sequenced rice accessions. Predicted concentrations ranged from 17.1 to 40.2 ppm with a prediction accuracy of 0.51. An independent confirmation with 61 gene bank seed samples provided high correlations (r = 0.74) between measured and predicted values. Accessions from the aus sub-species had the highest predicted grain Zn concentrations and these were confirmed in additional field experiments, with one potential donor having more than twice the grain Zn compared to a local check variety. We conclude utilizing donors from the aus sub-species and employing genomic selection during the breeding process is the most promising approach to raise grain Zn concentrations in rice.

Identification of genomic regions conferring rust resistance and enhanced mineral accumulation in a HarvestPlus Association Mapping Panel of wheat.

KEY MESSAGE: New genomic regions for high accumulation of 10 minerals were identified. The 1B:1R and 2NS translocations enhanced concentrations of four and two minerals, respectively, in addition to disease resistance. Puccinia species, the causal agents of rust diseases of wheat, have the potential to cause total crop failures due their high evolutionary ability to acquire virulence for resistance genes deployed in commercial cultivars. Hence, the discovery of genetically diverse sources of rust resistance is essential. On the other hand, biofortification of wheat for essential nutrients, such as zinc (Zn) and iron (Fe), is also an objective in wheat improvement programs to tackle micronutrient deficiency. The development of rust-resistant and nutrient-concentrated wheat cultivars would be important for sustainable production and the fight against malnutrition. The HarvestPlus association mapping panel (HPAMP) that included nutrient-dense sources from diverse genetic backgrounds was genotyped using a 90 K Infinium SNP array and 13 markers linked with rust resistance genes. The HPAMP was used for genome-wide association mapping to identify genomic regions underpinning rust resistance and mineral accumulation. Twelve QTL for rust resistance and 53 for concentrations of 10 minerals were identified. Comparison of results from this study with the published QTL information revealed the detection of already known and some putatively new genes/QTL underpinning stripe rust and leaf rust resistance in this panel. Thirty-six new QTL for mineral concentration were identified on 17 chromosomes. Accessions carrying the 1B:1R translocation accumulated higher concentrations of Zn, Fe, Copper (Cu) and sulphur (S). The 2NS segment showed enhanced accumulation of grain Fe and Cu. Fifteen rust-resistant and biofortified accessions were identified for use as donor sources in breeding programs.

A high-resolution genome-wide association study of the grain ionome and agronomic traits in rice Oryza sativa subsp. indica.

This study presents a comprehensive study of the genetic bases controlling variation in the rice ionome employing genome-wide association studies (GWAS) with a diverse panel of indica accessions, each genotyped with 5.2 million markers. GWAS was performed for twelve elements including B, Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, P, and Zn and four agronomic traits including days to 50% flowering, grain yield, plant height and thousand grain weight. GWAS identified 128 loci associated with the grain elements and 57 associated with the agronomic traits. There were sixteen co-localization regions containing QTL for two or more traits. Fourteen grain element quantitative trait loci were stable across growing environments, which can be strong candidates to be used in marker-assisted selection to improve the concentrations of nutritive elements in rice grain. Potential candidate genes were revealed including OsNAS3 linked to the locus that controls the variation of Zn and Co concentrations. The effects of starch synthesis and grain filling on multiple grain elements were elucidated through the likely involvement of OsSUS1 and OsGSSB1 genes. Overall, our study provides crucial insights into the genetic basis of ionomic variations in rice and will facilitate improvement in breeding for trace mineral content.

Calcium Biofortification of Crops-Challenges and Projected Benefits.

Despite Calcium (Ca) being an essential nutrient for humans, deficiency of Ca is becoming an ensuing public health problem worldwide. Breeding staple crops with higher Ca concentrations is a sustainable long-term strategy for alleviating Ca deficiency, and particular criteria for a successful breeding initiative need to be in place. This paper discusses current challenges and projected benefits of Ca-biofortified crops. The most important features of Ca nutrition in plants are presented along with explicit recommendations for additional exploration of this important issue. In order for Ca-biofortified crops to be successfully developed, tested, and effectively implemented in most vulnerable populations, further research is required.

Genomic selection can accelerate the biofortification of spring wheat.

KEY MESSAGE: Genomic selection enabled accurate prediction for the concentration of 13 nutritional element traits in wheat. Wheat biofortification is one of the most sustainable strategies to alleviate mineral deficiency in human diets. Here, we investigated the potential of genomic selection using BayesR and Bayesian ridge regression (BRR) models to predict grain yield (YLD) and the concentration of 13 nutritional elements in grains (B, Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P and Zn) using a population of 1470 spring wheat lines. The lines were grown in replicated field trials with two times of sowing (TOS) at 3 locations (Narrabri-NSW, all lines; Merredin-WA and Horsham-VIC, 200 core lines). Narrow-sense heritability across environments (locations/TOS) ranged from 0.09 to 0.45. Co, K, Na and Ca showed low to negative genetic correlations with other traits including YLD, while the remaining traits were negatively correlated with YLD. When all environments were included in the reference population, medium to high prediction accuracy was observed for the different traits across environments. BayesR had higher average prediction accuracy for mineral concentrations (r = 0.55) compared to BRR (r = 0.48) across all traits and environments but both methods had comparable accuracies for YLD. We also investigated the utility of one or two locations (reference locations) to predict the remaining location(s), as well as the ability of one TOS to predict the other. Under these scenarios, BayesR and BRR showed comparable performance but with lower prediction accuracy compared to the scenario of predicting reference environments for new lines. Our study demonstrates the potential of genomic selection for enriching wheat grain with nutritional elements in biofortification breeding.

High-resolution genome-wide association study pinpoints metal transporter and chelator genes involved in the genetic control of element levels in maize grain.

Despite its importance to plant function and human health, the genetics underpinning element levels in maize grain remain largely unknown. Through a genome-wide association study in the maize Ames panel of nearly 2,000 inbred lines that was imputed with ∼7.7 million SNP markers, we investigated the genetic basis of natural variation for the concentration of 11 elements in grain. Novel associations were detected for the metal transporter genes rte2 (rotten ear2) and irt1 (iron-regulated transporter1) with boron and nickel, respectively. We also further resolved loci that were previously found to be associated with one or more of five elements (copper, iron, manganese, molybdenum, and/or zinc), with two metal chelator and five metal transporter candidate causal genes identified. The nas5 (nicotianamine synthase5) gene involved in the synthesis of nicotianamine, a metal chelator, was found associated with both zinc and iron and suggests a common genetic basis controlling the accumulation of these two metals in the grain. Furthermore, moderate predictive abilities were obtained for the 11 elemental grain phenotypes with two whole-genome prediction models: Bayesian Ridge Regression (0.33-0.51) and BayesB (0.33-0.53). Of the two models, BayesB, with its greater emphasis on large-effect loci, showed ∼4-10% higher predictive abilities for nickel, molybdenum, and copper. Altogether, our findings contribute to an improved genotype-phenotype map for grain element accumulation in maize.

Higher Photochemical Quenching and Better Maintenance of Carbon Dioxide Fixation Are Key Traits for Phosphorus Use Efficiency in the Wheat Breeding Line, RAC875.

Maintaining carbohydrate biosynthesis and C assimilation is critical under phosphorus (P) deficiency as inorganic P (Pi) is essential for ATP synthesis. Low available P in agricultural soils occurs worldwide and fertilizer P sources are being depleted. Thus, identifying biosynthetic traits that are favorable for P use efficiency (PUE) in crops is crucial. This study characterized agronomic traits, gas exchange, and chlorophyll traits of two wheat genotypes that differ in PUE. RAC875 was a P efficient genotype and Wyalkatchem was a P inefficient genotype. The plants were grown in pots under growth room conditions at two P levels; 10 mg P kg-1 soil (low P) and 30 mg P kg-1 soil (adequate P) and gas exchange and chlorophyll fluorescence were measured at the vegetative and booting stages using a portable photosynthesis system (LI-6800, LI-COR, United States). Results showed significant differences in some agronomic traits between the two wheat genotypes, i.e., greater leaf size and area, and a higher ratio of productive tillers to total tillers in RC875 when compared with Wyalkatchem. The CO2 response curve showed Wyalkatchem was more severely affected by low P than RAC875 at the booting stage. The relative ratio of the photosynthetic rate at low P to adequate P was also higher in RAC875 at the booting stage. Photochemical quenching (qP) in RAC875 was significantly higher when compared with Wyalkatchem at the booting stage. Maintaining CO2 fixation capacity under low P and higher qP would be associated with P efficiency in RAC875 and measuring qP could be a potential method to screen for P efficient wheat.

Genetic dissection of zinc, iron, copper, manganese and phosphorus in wheat (Triticum aestivum L.) grain and rachis at two developmental stages.

The development of high-yielding wheat genotypes containing micronutrient-dense grains are the main priorities of biofortification programs. At the International Maize and Wheat Improvement Center, breeders have successfully crossed high zinc progenitors including synthetic hexaploid wheat, T. dicoccum, T. spelta and landraces to generate high-zinc varieties. In this study, we report a genome-wide association using a wheat diversity panel to dissect the genetics controlling zinc, iron, copper, manganese and phosphorus concentrations in the grain and rachis during grain development and at physiological maturity. Significant marker-trait associations (MTAs) were identified for each nutrient using multi-locus mixed model methodologies. For mature grain, markers that showed significant pleiotropic effects were found on chromosomes 1A, 3B and 5B, of which those on chromosome 5B at ∼95.5 cM were consistent over two growing seasons. Co-located MTAs were identified for the nutrient concentrations in developing grain, rachis and mature grain on multiple chromosomes. The identified genomic regions included putative candidate genes involved in metal uptake and transport and storage protein processing. These findings add to our understanding of the genetics of the five important nutrients in wheat grain and provide information on genetic markers for selecting high micronutrient genotypes.

Genotypic Variation in the Root and Shoot Metabolite Profiles of Wheat (Triticum aestivum L.) Indicate Sustained, Preferential Carbon Allocation as a Potential Mechanism in Phosphorus Efficiency.

Changes in the levels of plant metabolites in response to nutrient deficiency is indicative of how plants utilize scarce resources. In this study, changes in the metabolite profile of roots and shoots of wheat genotypes differing in phosphorus use efficiency (PUE) was investigated. Under low P supply and at 28 days after sowing (DAS), the wheat breeding line, RAC875 (P efficient) produced 42% more shoot biomass than the wheat variety, and Wyalkatchem (P inefficient). Significant changes in the metabolite profile in leaves and roots were observed under low P supply and significant genotypic variation was evident. Under low P supply, an increase in raffinose and 1-kestose was evident in roots of both wheat genotypes, with RAC875 accumulating more when compared to Wyalkatchem. There was no significant increase in raffinose and 1-kestose in leaves when plants were grown under P deficiency. P deficiency had no significant impact on the levels of sucrose, maltose, glucose and fructose in both genotypes, and while phosphorylated sugars (glucose-6-P and fructose-6-P) remained unchanged in RAC875, in Wyalkatchem, glucose-6-P significantly decreased in roots, and fructose-6-P significantly decreased in both leaves and roots. Glycerol-3-P decreased twofold in roots of both wheat genotypes in response to low P. In roots, RAC875 exhibited significantly lower levels of fumarate, malate, maleate and itaconate than Wyalkatchem, while low P enhanced organic acid exudation in RAC875 but not in Wyalkatchem. RAC875 showed greater accumulation of aspartate, glutamine and ß-alanine in leaves than Wyalkatchem under low P supply. Greater accumulation of raffinose and 1-kestose in roots and aspartate, glutamine and ß-alanine in leaves appears to be associated with enhanced PUE in RAC875. Glucose-6-P and fructose-6-P are important for glycolysis, thus maintaining these metabolites would enable RAC875 to maintain carbohydrate metabolism and shoot biomass under P deficiency. The work presented here provides evidence that differences in metabolite profiles can be observed between wheat varieties that differ in PUE and key metabolic pathways are maintained in the efficient genotype to ensure carbon supply under P deficiency.

Changes in the Elemental and Metabolite Profile of Wheat Phloem Sap during Grain Filling Indicate a Dynamic between Plant Maturity and Time of Day.

The long distance transport of Fe and Zn in the phloem sap of wheat (Triticum aestivum L.) is the key route for seed supply, due to wheat having a xylem discontinuity. To date, our knowledge is limited on Fe and Zn homeostasis in the phloem sap during the reproductive and grain filling stages. With the use of aphid stylectomy to collect samples of phloem sap, we explored maturity and morning versus afternoon (within-day) changes in nutrient and metabolite profiles. Phloem exudate was collected from a wheat breeding line, SAMNYT16, at three times during the grain filling period and at both midday and mid-afternoon. There were significant changes in the concentration of Mg, K, Fe and Zn during the course of grain loading and there were also significant within-day differences for Fe and K concentrations in the phloem exudate during the early phases of grain development. We found that, for K and Fe, there was an increase of 1.1- and 1.4-fold, respectively, for samples taken prior to midday to those from mid-afternoon. There was also a significant decrease in K, Fe and Zn phloem sap concentration of 1.5-, 1.4- and 1.1-fold, respectively, from the start of peak grain loading to the end of grain loading. Of the 79 metabolites detected within samples of phloem exudate, 43 had significant maturity differences and 38 had significant within-day variability. Glutamine was found to increase by 3.3⁻5.9-fold from midday to mid-afternoon and citric acid was found to decrease by 1.6-fold from the start of grain loading to the end of grain loading. These two metabolites are of interest as they can complex metal ions and may play a role in long distance transport of metal ions. The work presented here gives further insight into the complex composition of the phloem sap and variability that can occur during the day and also with increasing maturity.

Genetic dissection of grain zinc concentration in spring wheat for mainstreaming biofortification in CIMMYT wheat breeding.

Wheat is an important staple that acts as a primary source of dietary energy, protein, and essential micronutrients such as iron (Fe) and zinc (Zn) for the world's population. Approximately two billion people suffer from micronutrient deficiency, thus breeders have crossed high Zn progenitors such as synthetic hexaploid wheat, T. dicoccum, T. spelta, and landraces to generate wheat varieties with competitive yield and enhanced grain Zn that are being adopted by farmers in South Asia. Here we report a genome-wide association study (GWAS) using the wheat Illumina iSelect 90 K Infinitum SNP array to characterize grain Zn concentrations in 330 bread wheat lines. Grain Zn phenotype of this HarvestPlus Association Mapping (HPAM) panel was evaluated across a range of environments in India and Mexico. GWAS analysis revealed 39 marker-trait associations for grain Zn. Two larger effect QTL regions were found on chromosomes 2 and 7. Candidate genes (among them zinc finger motif of transcription-factors and metal-ion binding genes) were associated with the QTL. The linked markers and associated candidate genes identified in this study are being validated in new biparental mapping populations for marker-assisted breeding.

Effects of Dietary Fibre from the Traditional Indonesian Food, Green Cincau (Premna oblongifolia Merr.) on Preneoplastic Lesions and Short Chain Fatty Acid Production in an Azoxymethane Rat Model of Colon Cancer.

Green cincau (Premna oblongifolia Merr.) is a traditional food of Indonesia and provides a natural source of dietary fibre and antioxidants. This study evaluated the ability of green cincau, and other dietary fibres with or without the addition of anti-oxidant, epigallocatechin-3-gallate (EGCG), to prevent colorectal cancer in a 12 week azoxymethane (AOM) rat model. While all dietary treatments stimulated short chain fatty acid production (SCFA) in the digesta and faeces, no one treatment was able to significantly protect against aberrant crypt formation (ACF), when compared to the control diet. However, feeding green cincau leaves or extracts did not result in an increase in ACF compared to the control diet. Unexpectedly, when the dietary fibre source was pectin, 0.1% EGCG increased proliferative activity and liver lipid peroxidation when compared to the control diet containing cellulose. Examination of faecal microbial communities identified the presence of short chain acid producing bacteria, but a distinct community profile was not observed from any individual diet group. Overall, this research implies that combining dietary fibre with an antioxidant does not automatically equate to a beneficial response. Further work is required to investigate the health-promoting properties of green cincau.

Alterations in the Gut ( Gallus gallus) Microbiota Following the Consumption of Zinc Biofortified Wheat ( Triticum aestivum)-Based Diet.

The structure and function of cecal microbiota following the consumption of a zinc (Zn) biofortified wheat diet was evaluated in a well-studied animal model of human nutrition ( Gallus gallus) during a six-week efficacy trial. Using 16S rRNA gene sequencing, a significant increase in ß- but not α-microbial diversity was observed in the animals receiving the Zn biofortified wheat diet, relative to the control. No significant taxonomic differences were found between the two groups. Linear discriminant analysis revealed a group of metagenomic biomarkers that delineated the Zn replete versus Zn deficient phenotypes, such that enrichment of lactic acid bacteria and concomitant increases in Zn-dependent bacterial metabolic pathways were observed in the Zn biofortified group, and expansion of mucin-degraders and specific bacterial groups able to participate in maintaining host Zn homeostasis were observed in the control group. Additionally, the Ruminococcus genus appeared to be a key player in delineating the Zn replete microbiota from the control group, as it strongly predicts host Zn adequacy. Our data demonstrate that the gut microbiome associated with Zn biofortified wheat ingestion is unique and may influence host Zn status. Microbiota analysis in biofortification trials represents a crucial area for study as Zn biofortified diets are increasingly delivered on a population-wide scale.

Linoleic Acid:Dihomo-γ-Linolenic Acid Ratio Predicts the Efficacy of Zn-Biofortified Wheat in Chicken (Gallus gallus).

The amount of Zn absorbed from Zn-biofortified wheat material has been determined using an in vivo model of Zn absorption. The erythrocyte linoleic:dihomo -γ-linolenic acid (LA:DGLA) ratio was used as a biomarker of Zn status. Two groups of chickens (n = 15) were fed different diets: a high-Zn (46.5 µg Zn g-1) and a low-Zn wheat-based diet (32.8 µg Zn g-1). Dietary Zn intakes, body weight, serum Zn, and the erythrocyte fatty acid profile were measured, and tissues were taken for gene expression analysis. Serum Zn concentrations were greater in the high Zn group (p < 0.05). Duodenal mRNA expression of various Zn transporters demonstrated expression upregulation in the birds fed a low Zn diet (n = 15, p < 0.05). The LA:DGLA ratio was higher in the birds fed the low Zn diet (p < 0.05). The higher amount of Zn in the biofortified wheat resulted in a greater Zn uptake.

QTL Mapping of Grain Zn and Fe Concentrations in Two Hexaploid Wheat RIL Populations with Ample Transgressive Segregation.

More than 50% of undernourished children live in Asia and more than 25% live in Africa. Coupled with an inadequate food supply, mineral deficiencies are widespread in these populations; particularly zinc (Zn) and iron (Fe) deficiencies that lead to retarded growth, adverse effects on both the immune system and an individual's cognitive abilities. Biofortification is one solution aimed at reducing the incidence of these deficiencies. To efficiently breed a biofortified wheat variety, it is important to generate knowledge of the genomic regions associated with grain Zn (GZn) and Fe (GFe) concentration. This allows for the introgression of favorable alleles into elite germplasm. In this study we evaluated two bi-parental populations of 188 recombinant inbred lines (RILs) displaying a significant range of transgressive segregation for GZn and GFe during three crop cycles in CIMMYT, Mexico. Parents of the RILs were derived from Triticum spelta L. and synthetic hexaploid wheat crosses. QTL analysis identified a number of significant QTL with a region denominated as QGZn.cimmyt-7B_1P2 on chromosome 7B explaining the largest (32.7%) proportion of phenotypic variance (PVE) for GZn and leading to an average additive effect of -1.3. The QTL with the largest average additive effect for GFe (-0.161) was found on chromosome 4A (QGFe.cimmyt-4A_P2), with 21.14% of the PVE. The region QGZn.cimmyt-7B_1P2 co-localized closest to the region QGZn.cimmyt-7B_1P1 in a consensus map built from the linkage maps of both populations. Pleiotropic or tightly linked QTL were also found on chromosome 3B, however of minor effects and PVE between 4.3 and 10.9%. Further efforts are required to utilize the QTL information in marker assisted backcrossing schemes for wheat biofortification. A strategy to follow is to intercross the transgressive individuals from both populations and then utilize them as sources in biofortification breeding pipelines.