Genetic fortification strategies are highly suitable for developing barley genotypes with high mineral element contents. Therefore, this study focused on investigating the natural genetic variation in 216 spring barley accessions and on identifying candidate genes contributing to mineral accumulation in barley grains. Phenotypic analysis for mineral concentrations including Zn, Fe and Se, showed a wide variation between the genotypes based on BLUEs that equaled 38.37, 35.56 and 39.45 µg g-1 DW, respectively. Our findings agree with Herzig, et al. (2019) for grain elements in a wild barley. High heritability equaled 75.65% for Fe across the two environments indicating that the major part of the variability was due to genotypic effects, which is in agreement with previous studies (Garcia-Oliveira, et al. 2009; Peleg, et al. 2009; VREUGDENHIL, et al. 2004). However, Zn and Se showed moderate heritability (30.81% and 58.52%), whereby a lot of the phenotypic variance was represented by the year and error variance components. Similar heritabilities were found by Herzig, et al. (2019) for grain elements in a wild barley NAM population. Very strong significant Pearson’s correlation was detected between the two seasons indicating that the phenotypic measurements were quite stable in the different years for all the studied traits. Similar results were reported by (Alomari, et al. 2018; Alomari, et al. 2019) for Zn and Fe in wheat cultivars. The ANOVA results indicated that genotypes and environmental factors have a significant effect on mineral concentration including Zn, Fe and Se in barley grains. A similar conclusion was reached by (Alomari, et al. 2018; Alomari, et al. 2019) for grain Zn and Fe accumulation in wheat cultivars.
Candidate Genes
Candidate genes were identified for the most effective markers that had associations with more than one trait. Based on GWAS analysis, 222 significant associations were underlying grain Zn, Fe and Se which distributed on all chromosomes. Exclusively, 394 potential candidate genes were discovered on chromosome 3H, 5H and 7H that found to be highly associated with Zn, Fe and Se among the growing environments and BLUEs. The first region located on chromosome 3H and harbors two important candidate genes that explained the variation of Zn and Se_BLUEs. The first candidate at 3H is HORVU.MOREX.r2.3HG0258450 that annotated as Selenium-binding protein, is a ubiquitously expressed protein that can bind selenium (Se) specifically. SBP also plays essential roles in senescence, the stress response, cellular differentiation, protein transport, and the ubiquitinating/deubiquitinating pathways (Zhao and Castonguay 2015).
The second gene is HORVU.MOREX.r2.3HG0258460 that encodes 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein (2-ODDs), are non-heme proteins that are ubiquitously distributed throughout nature (de Carolis and de Luca 1994; Martens, et al. 2010). 2-ODDs are dependent on ferrous iron as a co-factor for the binding of molecular oxygen and subsequent oxidative reactions in plant metabolism. The landmark discovery of the 2-ODDs involved in epigenetic regulation, and others catalyzing several characteristic steps in specialized metabolic pathways (Farrow and Facchini 2014). Furthermore, 2-ODDs catalyses numerous oxidative reactions including hydroxylations, halogenations, desaturations, ring closure, ring cleavage, epimerization, rearrangements, halogenation, re-arrangements, demethylations, and demethylenations (Clifton, et al. 2006; Flashman and Schofield 2007; Loenarz and Schofield 2008; Tarhonskaya, et al. 2014). This impressive list of oxidative reactions reveals that 2-ODDs are among the most versatile and important oxidizing biological catalysts (Flashman and Schofield 2007).
The second important region located on chromosome 5H harbors four candidate genes that regulated the variation of all the studied minerals including Zn, Fe and Se in barley grains. The most prominent multifunctional gene HORVU.MOREX.r2.5HG0411320 at (500,037,722-500,044,141bp) annotated as An ATP-dependent zinc metalloprotease, FtsH, is the major thylakoid membrane protease. FtsHs in the thylakoid membranes of Arabidopsis thaliana form a hetero-hexameric complex consisting of two types of FtsH subunits: type A (FtsH1 and FtsH5) and type B (FtsH2 and FtsH8) (Sakamoto, et al. 2003; Yu, et al. 2005; Yu, et al. 2004). Aplethora of studies have identified the critical roles of FtsH in the biogenesis of thylakoid membranes and quality control in the photosystem II repair cycle. Furthermore, the involvement of FtsH in the degradation and assembly of several protein complexes in the photosynthetic electron-transport pathways (Kato and Sakamoto 2018). Substantial proportions of the cellular quota of the micronutrient metals copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn), are allocated to proteins acting in plant photosynthesis as well as the mechanisms involved in their homeostasis within chloroplasts, which places these metals at the core of plant energy metabolism and highlights their importance for plant‐specific biochemistry (Yruela 2013).
In the same genetic region, two candidate genes are coding transcription factors; HORVU.MOREX.r2.5HG0411850 that encodes Protein FAR1-RELATED SEQUENCE 5 and HORVU.MOREX.r2.5HG0412340 that encodes Basic helix loop helix (BHLH). Both FAR1 and BHLH were shown to explain the variation of Zn, Fe and Se, implying that they attributed to improving grain mineral accumulation. FAR-RED ELONGATED HYPOCOTYLS3 (FHY3) and its homologue FAR-RED IMPAIRED RESPONSE1 (FAR1) encode transposase-derived transcription factors (Lin, et al. 2007; Wang and Deng 2002). Recent studies have demonstrated that FHY3 and FAR1 play multiple roles in a wide range of cellular processes, including light signal transduction (Lin, et al. 2007), circadian clock and flowering time (Li, et al. 2011), floral development (Li, et al. 2016), chloroplast division (Ouyang, et al. 2011), chlorophyll biosynthesis (Tang, et al. 2012), starch synthesis (Ma, et al. 2017), abscisic acid responses (Tang, et al. 2013), and plant immunity (Wang, et al. 2016), indicating that FHY3 and FAR1 have crucial functions in plant growth and development.
Basic helix loop helix (BHLH) family transcription factor are a superfamily of transcription factors that are important regulatory components in transcriptional networks in plants (Tanabe, et al. 2019). The bHLH transcription factor FER, a crucial regulator of iron uptake responses in root, was first identified from the analysis of the tomato fer mutant (Ling, et al. 2002). Furthermore, Tanabe, et al. (2019) characterized bHLH11 as a negative regulator of Fe homeostasis. The expression of FIT, a master regulator of Fe deficiency responses, was suppressed in the transgenic plants, indicating that the transcriptional repressor bHLH11 functions as a negative regulator of FIT-dependent Fe uptake and modulates Fe levels in Arabidopsis. Zheng, et al. (2010) reported that the Fe-regulated bHLH transcription factor, OsIRO2 acts as a negative regulator of the Fe deficiency response in rice. Recent studies also reported that bHLH was found in promoter regions of all TaMTPs (metal tolerance proteins) in common wheat, which are involved in trace metal homeostasis and have a potential role in cereal grain biofortification with essential micronutrients including Zn (Menguer, et al. 2018; Vatansever, et al. 2017). These findings declared the potentiality of this cluster of genes in conferring mineral accumulation in barley. Besides, they are pleiotropic genes thus; a selection for the region harboring them can improve many traits at once.
The last important candidate is a homeobox-leucine zipper protein HOX4 that annotated as HORVU.MOREX.r2.5HG0413150 at (505,319,272- 505,325,405 bp) was found to be associated with grain Zn, Fe and Se concentrations in the used panel. Similar results were found by Alomari, et al. (2018) who reported that TaHDZIP1 was found to be associated with grain Zn concentrations in the wheat cultivars. A total of 187 TabZIP genes have been identified in wheat (Li, et al. 2015) and a specific group of TabZIP genes such as bZIP19 and bZIP23 were shown to regulate the adaption to Zn deficiency in roots (Assunção, et al. 2010; Inaba, et al. 2015).
Interestingly, on chromosome 7H (205,216,091-205,221,133 bp), Squamosa promoter binding-like protein (SPL) gene family is one of the plant-specific putative transcription factor related to plant development and each member shares a highly conserved 76 amino acid residue SBP-domain (Birkenbihl, et al. 2005; Klein, et al. 1996). The SBP domain consists of three functionally important motifs, including two Zn-finger like structures formed by conserved cysteine and histidine residues (Yamasaki, et al. 2004). In the current study SPL genes controlled the variation of Zn and Se content, this agrees with other studies, where Arabidopsis Squamosa promoter binding Protein‐Like genes act together in the regulation of transition metal homeostasis including Cu and Zn (Schulten, et al. 2019). Furthermore, in rice, OsSPL13 positively regulates cell size in the grain hull, resulting in enhanced grain length and yield (Si, et al. 2016). OsSPL14 enhances shoot and panicle branching, leading to increases in grain productivity (Jiao, et al. 2010). OsSPL16 promotes cell division and grain filling, with positive consequences for grain size, shape, and quality (Wang, et al. 2012). Cao, et al. (2019) hypothesized that the homologous gene of rice OsSPL16 in bread wheat had functions in regulation of wheat grain size and yield. Interestingly, Thabet, et al. (2021) identified many candidate genes that were found to be linked to salinity stress tolerance during seedling developmental phase as a first time such as Squamosa promoter-binding-like protein 6 at chromosome 5H for the same barley population. These studies have indicated that SPL genes play crucial roles in regulation of plant development and yield-related traits in cereal crops.
Altogether, our results indicate potential genomic regions controlling Zn, Fe and Se in barley that can be used in further genetic investigations.