Phenotyping and Exploitation of Kompetitive Allele-Specific PCR Assays for Genes Underpinning Leaf Rust Resistance in New Spring Wheat Mutant Lines.

Leaf rust (Puccinia triticina Eriks) is a wheat disease causing substantial yield losses in wheat production globally. The identification of genetic resources with permanently effective resistance genes and the generation of mutant lines showing increased levels of resistance allow the efficient incorporation of these target genes into germplasm pools by marker-assisted breeding. In this study, new mutant (M3 generation) lines generated from the rust-resistant variety Kazakhstanskaya-19 were developed using gamma-induced mutagenesis through 300-, 350-, and 400-Gy doses. In field trials after leaf rust inoculation, 75 mutant lines showed adult plant resistance. These lines were evaluated for resistance at the seedling stage via microscopy in greenhouse experiments. Most of these lines (89.33%) were characterized as resistant at both developmental stages. Hyperspectral imaging analysis indicated that infected leaves of wheat genotypes showed increased relative reflectance in visible and near-infrared light compared to the non-infected genotypes, with peak means at 462 and 644 nm, and 1936 and 2392 nm, respectively. Five spectral indexes, including red edge normalized difference vegetation index (RNDVI), structure-insensitive pigment index (SIPI), ratio vegetation index (RVSI), water index (WI), and normalized difference water index (NDWI), demonstrated significant potential for determining disease severity at the seedling stage. The most significant differences in reflectance between susceptible and resistant mutant lines appeared at 694.57 and 987.51 nm. The mutant lines developed were also used for the development and validation of KASP markers for leaf rust resistance genes Lr1, Lr2a, Lr3, Lr9, Lr10, and Lr17. The mutant lines had high frequencies of "a" resistance alleles (0.88) in all six Lr genes, which were significantly associated with seedling resistance and suggest the potential of favorable haplotype introgression through functional markers. Nine mutant lines characterized by the presence of "b" alleles in Lr9 and Lr10-except for one line with allele "a" in Lr9 and three mutant lines with allele "a" in Lr10-showed the progressive development of fungal haustorial mother cells 72 h after inoculation. One line from 300-Gy-dosed mutant germplasm with "b" alleles in Lr1, Lr2a, Lr10, and Lr17 and "a" alleles in Lr3 and Lr9 was characterized as resistant based on the low number of haustorial mother cells, suggesting the contribution of the "a" alleles of Lr3 and Lr9.

Organ-specific expression of genes involved in iron homeostasis in wheat mutant lines with increased grain iron and zinc content.

Background: Iron deficiency is a well-known nutritional disorder, and the imbalance of trace-elements, specifically iron, is the most common nutrient deficiency of foods across the world, including in Kazakhstan. Wheat has significant nutritional relevance, especially in the provision of iron, however many bread wheat varieties have low iron despite the need for human nourishment. In this study, the expression profiles of wheat homologous genes related to iron homeostasis were investigated. The work resulted in the development of two new M5 mutant lines of spring bread wheat through gamma-irradiation (200 Gy) with higher grain iron and zinc content, lower phytic acid content, and enhanced iron bioavailability compared to the parent variety. Mutant lines were also characterized by higher means of yield associated traits such as grain number per main spike, grain weight per main spike, grain weight per plant, and thousand-grain weight. Methods: The homologous genes of bread wheat from several groups were selected for gene expression studies exploring the tight control of iron uptake, translocation rate and accumulation in leaves and roots, and comprised the following: (1) S-adenosylmethionine synthase (SAMS), nicotianamine synthase (NAS1), nicotianamine aminotransferase (NAAT), deoxymugineic acid synthetase (DMAS), involved in the synthesis and release of phytosiderophores; (2) transcription factor basic helix-loop-helix (bHLH); (3) transporters of mugineic acid (TOM), involved in long-distance iron transport; (4) yellow stripe-like (YSlA), and the vacuolar transporter (VIT2), involved in intracellular iron transport and storage; and lastly (5) natural resistance-associated macrophage protein (NRAMP) and ferritin (Fer1A). Results: The wheat homologous genes TaSAMS, TaNAS1, and TaDMAS, were significantly up-regulated in the roots of both mutant lines by 2.1-4.7-fold compared to the parent variety. The combined over-expression of TaYSlA and TaVIT2 was also revealed in the roots of mutant lines by 1.3-2.7-fold. In one of the mutant lines, genes encoding intracellular iron transport and storage genes TaNRAMP and TaFer1A-D showed significant up-regulation in roots and leaves (by 1.4- and 3.5-fold, respectively). The highest expression was recorded in the transcription factor TabHLH, which was expressed 13.1- and 30.2-fold in the roots of mutant lines. Our research revealed that genotype-dependent and organ-specific gene expression profiles can provide new insights into iron uptake, translocation rate, storage, and regulation in wheat which aid the prioritization of gene targets for iron biofortification and bioavailability.

Rice Plants (Oryza sativa L.) under Cd Stress in Fe Deficiency Conditions.

Due to the environment pollution by cadmium (Cd) near industrial metallurgic factories and the widespread use of phosphorus fertilizers, the problem of toxic Cd effect on plants is well discussed by many authors, but the phytotoxicity of Cd under iron (Fe) deficiency stress has not been sufficiently studied. The aim of the work was to study comprehensively the effect of Cd under Fe deficiency conditions on physiological, biochemical, and anatomical parameters of rice varieties, to identify varietal differences in plant response to the effect of double stress. Relative resistance and sensitivity to the joint effect of Cd and Fe deficiency stress rice varieties have been identified. Double stress decreased a linear growth and biomass accumulation of roots and shoots (by 36-50% and 33-46% and 32-56% and 32-48%, accordingly), content of photosynthetic pigments (Chla, Chlb, and carotenoids by 36-51%, 32-47%, and 64-78%, accordingly), and relative water content (by 18-26%). Proline content increased by 28-103% in all rice varieties, but to a lesser extent in sensitive varieties. The thickness of the lower and upper epidermis and the diameter of vascular bundles of leaves decreased by 18-50%, 46-60%, and 13-48%, accordingly. The thickness of the root endodermis and exodermis and diameter of the central cylinder mainly decreased. The thickness of the exodermis increased slightly by 7%, and the diameter of the central cylinder remained at the control level in resistant Madina variety while in sensitive Chapsari variety, these indicators decreased significantly by 50 and 45%, accordingly. Thus, the aggravation of adverse effect of Cd under Fe deficiency conditions and the varietal specificity of plants' response to double stress were shown. It creates the need for further study of these rice varieties using Fe to identify mechanisms for reducing the toxic effect of Cd on plants as well as the study of Fe and Cd transporter genes at the molecular level.

Mutant Lines of Spring Wheat with Increased Iron, Zinc, and Micronutrients in Grains and Enhanced Bioavailability for Human Health.

Deficiency of metals, primarily Fe and Zn, affects over half of the world's population. Human diets dominated by cereal products cause micronutrient malnutrition, which is common in many developing countries where populations depend heavily on staple grain crops such as wheat, maize, and rice. Biofortification is one of the most effective approaches to alleviate malnutrition. Genetically stable mutant spring wheat lines (M7 generation) produced via 100 or 200 Gy gamma treatments to broaden genetic variation for grain nutrients were analyzed for nutritionally important minerals (Ca, Fe, and Zn), their bioavailability, and grain protein content (GPC). Variation was 172.3-883.0 mg/kg for Ca, 40.9-89.0 mg/kg for Fe, and 22.2-89.6 mg/kg for Zn. In mutant lines, among the investigated minerals, the highest increases in concentrations were observed in Fe, Zn, and Ca when compared to the parental cultivar Zhenis. Some mutant lines, mostly in the 100 Gy-derived germplasm, had more than two-fold higher Fe, Zn, and Ca concentrations, lower phytic acid concentration (1.4-2.1-fold), and 6.5-7% higher grain protein content compared to the parent. Variation was detected for the molar ratios of Ca:Phy, Phy:Fe, and Phy:Zn (1.27-10.41, 1.40-5.32, and 1.78-11.78, respectively). The results of this study show how genetic variation generated through radiation can be useful to achieve nutrient biofortification of crops to overcome human malnutrition.