Investigation of morpho-physiolgical traits and gene expression in barley under nitrogen deficiency.

Nitrogen (N) is an essential element for plant growth, and its deficiency influences plants at several physiological and gene expression levels. Barley (Hordeum vulgare) is one of the most important food grains from the Poaceae family and one of the most important staple food crops. However, the seed yield is limited by a number of stresses, the most important of which is the insufficient use of N. Thus, there is a need to develop N-use effective cultivars. In this study, comparative physiological and molecular analyses were performed using leaf and root tissues from 10 locally grown barley cultivars. The expression levels of nitrate transporters, HvNRT2 genes, were analyzed in the leaf and root tissues of N-deficient (ND) treatments of barley cultivars after 7 and 14 days following ND treatment as compared to the normal condition. Based on the correlation between the traits, root length (RL) had a positive and highly significant correlation with fresh leaf weight (FLW) and ascorbate peroxidase (APX) concentration in roots, indicating a direct root and leaf relationship with the plant development under ND. From the physiological aspects, ND enhanced carotenoids, chlorophylls a/b (Chla/b), total chlorophyll (TCH), leaf antioxidant enzymes such as ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT), and root antioxidant enzymes (APX and POD) in the Sahra cultivar. The expression levels of HvNRT2.1, HvNRT2.2, and HvNRT2.4 genes were up-regulated under ND conditions. For the morphological traits, ND maintained root dry weight among the cultivars, except for Sahra. Among the studied cultivars, Sahra responded well to ND stress, making it a suitable candidate for barely improvement programs. These findings may help to better understand the mechanism of ND tolerance and thus lead to the development of cultivars with improved nitrogen use efficiency (NUE) in barley.

Comparative analysis of physiological traits and gene expression patterns in nitrogen deficiency among barley cultivars.

BACKGROUND: Nitrogen is one of the most important mineral nutrients for plants and is absorbed by the root system mainly in the inorganic form (NH+4 and NO-3). Plants absorb nitrogen as a food source for growth, biomass production, and development. Nitrogen is mainly absorbed as nitrate, which is the most common source of nitrogen available to higher plants. One of the unique features of nitrate transport is that NO-3 is both a substrate for transport and an inducer of NO-3 transport systems in genes and at physiological levels. METHODS: In the present study, morphological and physiological traits (chlorophyll a/b, total chlorophyll, and carotenoid, antioxidant enzymes, and protein content), correlation between traits and gene expression, and principle component analysis of traits among five barley cultivars were measured in response to nitrogen deficiency (ND). The starved plants were transferred to a nutrient solution containing 0.2 mM and 2 mM NO-3 up to 7 and 14 days after ND application and non-stressed conditions, respectively. RESULTS: Gene expression analysis revealed that the 10 HvNRT2 genes were induced in the leaf and root tissues at 7 and 14 days after ND treatments in five barley cultivars. Expression of NRT2 genes by relative quantitative qRT-PCR analysis for 10 HvNRT2 genes were determined. Based on the gene expression, HvNRT2.1, HvNRT2.2, and HvNRT2.4 were strongly induced by NO-3, peaking at 7 and 14 days after ND treatment. In contrast, the HvNRT2.4 showed only moderate induction in both leaves and roots. From our results, the Reyhan cultivar showed a significant increase in root fresh weight (RFW), protein content, and antioxidant enzyme activity in roots at 7 and 14 days after ND treatment as compared to the non-stressed condition. A highly positive correlation was observed between root catalase (CATr) and HvNRT2.2/2.5/2.6 leaves. CONCLUSION: The expression of HvNRT2.4 is increased during long-term nitrogen starvation, while the expression of HvNRT2.1 and HvNRT2.2 are transiently increased by ND. Based on physiological and morphological traits and molecular mechanisms, the Reyhan is considered a tolerant cultivar under ND condition.

Selection of High-Yielding and Stable Genotypes of Barley for the Cold Climate in Iran.

The interaction between genotypes and environments plays an important role in selecting superior genotypes for target locations. The main objectives of the present study were to analyze the effect of the genotype-by-environment interaction (GEI) and identify superior, newly developed, and promising barley genotypes for cold regions in Iran. For these purposes, a set of genotypes obtained from breeding programs for cold climates in Iran, along with two reference genotypes, were investigated at eight research stations (Tabriz, Ardabil, Arak, Miandoab, Mashhad, Jolge Rokh, Karaj, and Hamadan) during two consecutive growing seasons (2019-2020 and 2020-2021). The results of the freezing test (LT50) showed that most of the tested genotypes had significant cold tolerance at the seedling stage. Based on the additive main effect and multiplicative interaction (AMMI) analysis, environment (E) and GEI effects explained 49.44% and 16.55% of the total variation in grain yield, respectively. Using AMMI1 and AMMI2 models, G2 and G20 were found to be superior genotypes in terms of grain yield and stability. Moreover, AMMI-based stability parameters considered the G20 genotype to be the ideal genotype. A two-plot analysis of the genotype-by-environment interaction (GGE) biplot showed that the 16 experimental environments were grouped into 2 mega-environments. Of the test environments, ARK1 and KAJ2 had the highest discriminating power and representativeness ability, and these were identified as ideal environments for testing advanced genotypes for yield and stability performance during early barley breeding practices in cold areas in Iran. In conclusion, both AMMI and GGE biplot models identified several superior genotypes, among which G20, with a high average yield relative to the overall average yield and the lowest IPC1 score, was found to have high yield stability and is recommended for inclusion in breeding programs for cold climates in Iran.

Origin and genetic analysis of stem rust resistance in wheat line Tr129.

Wheat line Tr129 is resistant to stem rust, caused by Puccinia graminis f. sp. tritici (Pgt). The resistance in Tr129 was reportedly derived from Aegilops triuncialis, but the origin and genetics of resistance have not been confirmed. Here, genomic in situ hybridization (GISH) showed that no Ae. triuncialis chromatin was present in Tr129. Genetic and phenotypic analysis was conducted on F2 and DH populations from the cross RL6071/Tr129. Seedlings were tested with six Pgt races and were genotyped using an Illumina iSelect 90 K SNP array and kompetitive allele specific PCR (KASP) markers. Mapping and phenotyping showed that Tr129 carried four stem rust resistance (Sr) genes on chromosome arms 2BL (Sr9b), 4AL (Sr7b), 6AS (Sr8a), and 6DS (SrTr129). SrTr129 co-segregated with markers for SrCad, however Tr129 has a unique haplotype suggesting the resistance could be new. Analysis of a RL6071/Peace population revealed that like SrTr129, SrCad is ineffective against three North American races. This new understanding of SrCad will guide its use in breeding. Tr129 and the DNA markers reported here are useful resources for improving stem rust resistance in cultivars.

Characterization of specific DNA markers at VRN-H1 and VRN-H2 loci for growth habit of barley genotypes.

Requirement of vernalization is an important factor which plays a crucial role in cereals to transit from vegetative to reproductive phase. There are three types of growth habit in barley: winter, spring and facultative types; in which spring type does not require vernalization but winter and facultative genotypes require full and partial vernalization, respectively. Combination of two loci, Vrn-h1 and Vrn-h2, regulates vernalization in barley genotypes. Specific DNA markers have been identified for growth habit regulator genes in barley. In this study, we examined 24 barley genotypes using specific primers for detecting Vrn-h1and Vrn-h2 loci. Results showed that among all differently suggested primer combinations, a few markers were precisely correlated with seasonal growth habit in barley. The specific markers of 600, 600 and 200 bps were verified for ZCCT-Ha, ZCCT-Hb and ZCCT-Hc loci, respectively. Our field growth habit test showed that cultivar Bahman as a winter growth habit, where all the others genotypes exhibited spring growth habit. By using specific primers for Vrn-h1, only Bahman cultivar produced 616 bp and 830 bp fragments and spring genotypes showed 574 bp or 616 bp alleles without any amplification for 830 bp fragments. Therefore, presence of 616 bp and 830 bp alleles together in each genotype can be considered as an informative marker for winter growth habit in barley. These informative markers can be used easily in barley breeding programmes for detection of growth habit types in the seedling stage.

Development of a multiple bulked segregant analysis (MBSA) method used to locate a new stem rust resistance gene (Sr54) in the winter wheat cultivar Norin 40.

An important aspect of studying putative new genes in wheat is determining their position on the wheat genetic map. The primary difficulty in mapping genes is determining which chromosome carries the gene of interest. Several approaches have been developed to address this problem, each with advantages and disadvantages. Here we describe a new approach called multiple bulked segregant analysis (MBSA). A set of 423 simple sequence repeat (SSR) markers were selected based on profile simplicity, frequency of polymorphism, and distribution across the wheat genome. SSR primers were preloaded in 384-well PCR plates with each primer occupying 16 wells. In practice, 14 wells are reserved for "mini-bulks" that are equivalent to four gametes (e.g. two F(2) individuals) comprised of individuals from a segregated population that have a known homozygous genotype for the gene of interest. The remaining two wells are reserved for the parents of the population. Each well containing a mini-bulk can have one of three allele compositions for each SSR: only the allele from one parent, only the allele from the other parent, or both alleles. Simulation experiments were performed to determine the pattern of mini-bulk allele composition that would indicate putative linkage between the SSR in question and the gene of interest. As a test case, MBSA was employed to locate an unidentified stem rust resistance (Sr) gene in the winter wheat cultivar Norin 40. A doubled haploid (DH) population (n = 267) was produced from hybrids of the cross LMPG-6S/Norin 40. The DH population segregated for a single gene (χ (1:1) (2) = 0.093, p = 0.76) for resistance to Puccinia graminis f.sp. tritici race LCBN. Four resistant DH lines were included in each of the 14 mini-bulks for screening. The Sr gene was successfully located to the long arm of chromosome 2D using MBSA. Further mapping confirmed the chromosome location and revealed that the Sr gene was located in a linkage block that may represent an alien translocation. The new Sr gene was designated as Sr54.

Inheritance of resistance to Ug99 stem rust in wheat cultivar Norin 40 and genetic mapping of Sr42.

Stem rust, caused by Puccinia graminis f. sp. tritici, is a devastating disease of wheat. The emergence of race TTKSK (Ug99) and new variants in Africa threatens wheat production worldwide. The best method of controlling stem rust is to deploy effective resistance genes in wheat cultivars. Few stem rust resistance (Sr) genes derived from the primary gene pool of wheat confer resistance to TTKSK. Norin 40, which carries Sr42, is resistant to TTKSK and variants TTKST and TTTSK. The goal of this study was to elucidate the inheritance of resistance to Ug99 in Norin 40 and map the Sr gene(s). A doubled haploid (DH) population of LMPG-6/Norin 40 was evaluated for resistance to the race TTKST. Segregation of 248 DH lines fitted a 1:1 ratio (χ (2) 1:1= 0.58, p = 0.45), indicating a single gene in Norin 40 conditioned resistance to Ug99. This was confirmed by an independent F(2:3) population also derived from the cross LMPG-6/Norin 40 where a 1:2:1 ratio (χ (2)1:2:1 = 0.69, p = 0.71) was observed following the inoculation with race TTKSK. Mapping with DNA markers located this gene to chromosome 6DS, the known location of Sr42. PCR marker FSD_RSA co-segregated with Sr42, and simple sequence repeat (SSR) marker BARC183 was closely linked (0.5 cM) to Sr42. A previous study found close linkage between FSD_RSA and SrCad, a temporarily designated gene that also confers resistance to Ug99, thus Sr42 may be the same gene or allelic. Marker FSD_RSA is suitable for marker-assisted selection (MAS) in wheat breeding programs to improve stem rust resistance, including Ug99.