The effect of induced polyploidy on phytochemistry, cellular organelles and the expression of genes involved in thymol and carvacrol biosynthetic pathway in thyme (Thymus vulgaris).

Induced polyploidy usually results in larger vegetative and reproductive plant organs. In order to study the effect of chromosome doubling on Thymus vulgaris, three levels of colchicine concentration including 0.1, 0.3 and 0.5% (w/v) were applied for 6, 12 and 24 hours on apical meristem of 2- and 4-leaf seedlings. Ploidy level was evaluated by flow cytometry and microscopic chromosome counting. Chemical composition of essential oils extracted by hydro-distillation was analyzed by gas Chromatography/mass spectrometry (GC/MS) and gas Chromatography (GC). The application of 0.3% colchicine at 4-leaf seedling for 6 hours resulted in the highest survival rate and the highest number of tetraploid plants. Cytogenetic and flow cytometry analyses confirmed the increase of chromosome number from 2n=2x=30 in diploids to 2n=4x=60 in induced tetraploids. Tetraploid plants had larger leaves, taller and thicker stems, dense branching, longer trichome, larger stomata, larger guard cells, and decreased number of stomata. The number of chloroplasts and mitochondria increased significantly in tetraploid plants by 1.66 and 1.63 times, respectively. The expression of CYP71D178, CYP71D180 and CYP71D181 increased in tetraploids by 3.27, 7.39 and 2.15 times, respectively, probably resulting in higher essential oil compounds, as tetraploids outyielded the diploid plants by 64.7% in essential oil, 40.9% in thymol and 18.6% in carvacrol content.

Analysis of genetic diversity and genome-wide association study for drought tolerance related traits in Iranian bread wheat.

BACKGROUND: Drought is most likely the most significant abiotic stress affecting wheat yield. The discovery of drought-tolerant genotypes is a promising strategy for dealing with the world's rapidly diminishing water resources and growing population. A genome-wide association study (GWAS) was conducted on 298 Iranian bread wheat landraces and cultivars to investigate the genetic basis of yield, yield components, and drought tolerance indices in two cropping seasons (2018-2019 and 2019-2020) under rainfed and well-watered environments. RESULTS: A heatmap display of hierarchical clustering divided cultivars and landraces into four categories, with high-yielding and drought-tolerant genotypes clustering in the same group. The results of the principal component analysis (PCA) demonstrated that selecting genotypes based on the mean productivity (MP), geometric mean productivity (GMP), harmonic mean (HM), and stress tolerance index (STI) can help achieve high-yield genotypes in the environment. Genome B had the highest number of significant marker pairs in linkage disequilibrium (LD) for both landraces (427,017) and cultivars (370,359). Similar to cultivars, marker pairs on chromosome 4A represented the strongest LD (r2 = 0.32). However, the genomes D, A, and B have the highest LD, respectively. The single-locus mixed linear model (MLM) and multi-locus random-SNP-effect mixed linear model (mrMLM) identified 1711 and 1254 significant marker-trait association (MTAs) (-log10 P > 3) for all traits, respectively. A total of 874 common quantitative trait nucleotides (QTNs) were simultaneously discovered by both MLM and mrMLM methods. Gene ontology revealed that 11, 18, 6, and 11 MTAs were found in protein-coding regions (PCRs) for spike weight (SW), thousand kernel weight (TKW), grain number per spike (GN), and grain yield (GY), respectively. CONCLUSION: The results identified rich regions of quantitative trait loci (QTL) on Ch. 4A and 5A suggest that these chromosomes are important for drought tolerance and could be used in wheat breeding programs. Furthermore, the findings indicated that landraces studied in Iranian bread wheat germplasm possess valuable alleles, that are responsive to water-limited conditions. This GWAS experiment is one of the few types of research conducted on drought tolerance that can be exploited in the genome-mediated development of novel varieties of wheat.

Application of machine learning algorithms and feature selection in rapeseed (Brassica napus L.) breeding for seed yield.

BACKGROUND: Studying the relationships between rapeseed seed yield (SY) and its yield-related traits can assist rapeseed breeders in the efficient indirect selection of high-yielding varieties. However, since the conventional and linear methods cannot interpret the complicated relations between SY and other traits, employing advanced machine learning algorithms is inevitable. Our main goal was to find the best combination of machine learning algorithms and feature selection methods to maximize the efficiency of indirect selection for rapeseed SY. RESULTS: To achieve that, twenty-five regression-based machine learning algorithms and six feature selection methods were employed. SY and yield-related data from twenty rapeseed genotypes were collected from field experiments over a period of 2 years (2019-2021). Root mean square error (RMSE), mean absolute error (MAE), and determination coefficient (R2) were used to evaluate the performance of the algorithms. The best performance with all fifteen measured traits as inputs was achieved by the Nu-support vector regression algorithm with quadratic polynomial kernel function (R2 = 0.860, RMSE = 0.266, MAE = 0.210). The multilayer perceptron neural network algorithm with identity activation function (MLPNN-Identity) using three traits obtained from stepwise and backward selection methods appeared to be the most efficient combination of algorithms and feature selection methods (R2 = 0.843, RMSE = 0.283, MAE = 0.224). Feature selection suggested that the set of pods per plant and days to physiological maturity along with plant height or first pod height from the ground are the most influential traits in predicting rapeseed SY. CONCLUSION: The results of this study showed that MLPNN-Identity along with stepwise and backward selection methods can provide a robust combination to accurately predict the SY using fewer traits and therefore help optimize and accelerate SY breeding programs of rapeseed.

Genome-wide association mapping and genomic prediction of agronomical traits and breeding values in Iranian wheat under rain-fed and well-watered conditions.

BACKGROUND: The markers detected by genome-wide association study (GWAS) make it possible to dissect genetic structure and diversity at many loci. This can enable a wheat breeder to reveal and used genomic loci controlling drought tolerance. This study was focused on determining the population structure of Iranian 208 wheat landraces and 90 cultivars via genotyping-by-sequencing (GBS) and also on detecting marker-trait associations (MTAs) by GWAS and genomic prediction (GS) of wheat agronomic traits for drought-tolerance breeding. GWASs were conducted using both the original phenotypes (pGWAS) and estimated breeding values (eGWAS). The bayesian ridge regression (BRR), genomic best linear unbiased prediction (gBLUP), and ridge regression-best linear unbiased prediction (rrBLUP) approaches were used to estimate breeding values and estimate prediction accuracies in genomic selection. RESULTS: Population structure analysis using 2,174,975 SNPs revealed four genetically distinct sub-populations from wheat accessions. D-Genome harbored the lowest number of significant marker pairs and the highest linkage disequilibrium (LD), reflecting different evolutionary histories of wheat genomes. From pGWAS, BRR, gBLUP, and rrBLUP, 284, 363, 359 and 295 significant MTAs were found under normal and 195, 365, 362 and 302 under stress conditions, respectively. The gBLUP with the most similarity (80.98 and 71.28% in well-watered and rain-fed environments, correspondingly) with the pGWAS method in the terms of discovered significant SNPs, suggesting the potential of gBLUP in uncovering SNPs. Results from gene ontology revealed that 29 and 30 SNPs in the imputed dataset were located in protein-coding regions for well-watered and rain-fed conditions, respectively. gBLUP model revealed genetic effects better than other models, suggesting a suitable tool for genome selection in wheat. CONCLUSION: We illustrate that Iranian landraces of bread wheat contain novel alleles that are adaptive to drought stress environments. gBLUP model can be helpful for fine mapping and cloning of the relevant QTLs and genes, and for carrying out trait introgression and marker-assisted selection in both normal and drought environments in wheat collections.

Genome-wide association mapping for wheat morphometric seed traits in Iranian landraces and cultivars under rain-fed and well-watered conditions.

Seed traits in bread wheat are valuable to breeders and farmers, thus it is important exploring putative QTLs responsible for key traits to be used in breeding programs. GWAS was carried out using 298 bread wheat landraces and cultivars from Iran to uncover the genetic basis of seed characteristics in both rain-fed and well-watered environments. The analyses of linkage disequilibrium (LD) between marker pairs showed that the largest number of significant LDs in landraces (427,017) and cultivars (370,359) was recorded in genome B, and the strongest LD was identified on chromosome 4A (0.318). LD decay was higher in the B and A genomes, compared to the D genome. Mapping by using mrMLM (LOD > 3) and MLM (0.05/m, Bonferroni) led to 246 and 67 marker-trait associations (MTAs) under rain-fed, as well as 257 and 74 MTAs under well-watered conditions, respectively. The study found that 3VmrMLM correctly detected all types of loci and estimated their effects in an unbiased manner, with high power and accuracy and a low false positive rate, which led to the identification of 140 MTAs (LOD > 3) in all environments. Gene ontology revealed that 10 and 10 MTAs were found in protein-coding regions for rain-fed and well-watered conditions, respectively. The findings suggest that landraces studied in Iranian bread wheat germplasm possess valuable alleles, which are responsive to water-limited conditions. MTAs uncovered in this study can be exploited in the genome-mediated development of novel wheat cultivars.

Genome-wide association mapping and genomic prediction for pre­harvest sprouting resistance, low α-amylase and seed color in Iranian bread wheat.

BACKGROUND: Pre-harvest sprouting (PHS) refers to a phenomenon, in which the physiologically mature seeds are germinated on the spike before or during the harvesting practice owing to high humidity or prolonged period of rainfall. Pre-harvest sprouting (PHS) remarkably decreases seed quality and yield in wheat; hence it is imperative to uncover genomic regions responsible for PHS tolerance to be used in wheat breeding. A genome-wide association study (GWAS) was carried out using 298 bread wheat landraces and varieties from Iran to dissect the genomic regions of PHS tolerance in a well-irrigated environment. Three different approaches (RRBLUP, GBLUP and BRR) were followed to estimate prediction accuracies in wheat genomic selection. RESULTS: Genomes B, A, and D harbored the largest number of significant marker pairs (MPs) in both landraces (427,017, 328,006, 92,702 MPs) and varieties (370,359, 266,708, 63,924 MPs), respectively. However, the LD levels were found the opposite, i.e., genomes D, A, and B have the highest LD, respectively. Association mapping by using GLM and MLM models resulted in 572 and 598 marker-trait associations (MTAs) for imputed SNPs (- log10 P > 3), respectively. Gene ontology exhibited that the pleitropic MPs located on 1A control seed color, α-Amy activity, and PHS. RRBLUP model indicated genetic effects better than GBLUP and BRR, offering a favorable tool for wheat genomic selection. CONCLUSIONS: Gene ontology exhibited that the pleitropic MPs located on 1A can control seed color, α-Amy activity, and PHS. The verified markers in the current work can provide an opportunity to clone the underlying QTLs/genes, fine mapping, and genome-assisted selection.Our observations uncovered key MTAs related to seed color, α-Amy activity, and PHS that can be exploited in the genome-mediated development of novel varieties in wheat.

Imputation accuracy of wheat genotyping-by-sequencing (GBS) data using barley and wheat genome references.

Genotyping-by-sequencing (GBS) provides high SNP coverage and has recently emerged as a popular technology for genetic and breeding applications in bread wheat (Triticum aestivum L.) and many other plant species. Although GBS can discover millions of SNPs, a high rate of missing data is a major concern for many applications. Accurate imputation of those missing data can significantly improve the utility of GBS data. This study compared imputation accuracies among four genome references including three wheat references (Chinese Spring survey sequence, W7984, and IWGSC RefSeq v1.0) and one barley reference genome by comparing imputed data derived from low-depth sequencing to actual data from high-depth sequencing. After imputation, the average number of imputed data points was the highest in the B genome (~48.99%). The D genome had the lowest imputed data points (~15.02%) but the highest imputation accuracy. Among the four reference genomes, IWGSC RefSeq v1.0 reference provided the most imputed data points, but the lowest imputation accuracy for the SNPs with < 10% minor allele frequency (MAF). The W7984 reference, however, provided the highest imputation accuracy for the SNPs with < 10% MAF.

Genotyping-by-Sequencing (GBS) Revealed Molecular Genetic Diversity of Iranian Wheat Landraces and Cultivars.

Background: Genetic diversity is an essential resource for breeders to improve new cultivars with desirable characteristics. Recently, genotyping-by-sequencing (GBS), a next-generation sequencing (NGS) technology that can simplify complex genomes, has now be used as a high-throughput and cost-effective molecular tool for routine breeding and screening in many crop species, including the species with a large genome. Results: We genotyped a diversity panel of 369 Iranian hexaploid wheat accessions including 270 landraces collected between 1931 and 1968 in different climate zones and 99 cultivars released between 1942 to 2014 using 16,506 GBS-based single nucleotide polymorphism (GBS-SNP) markers. The B genome had the highest number of mapped SNPs while the D genome had the lowest on both the Chinese Spring and W7984 references. Structure and cluster analyses divided the panel into three groups with two landrace groups and one cultivar group, suggesting a high differentiation between landraces and cultivars and between landraces. The cultivar group can be further divided into four subgroups with one subgroup was mostly derived from Iranian ancestor(s). Similarly, landrace groups can be further divided based on years of collection and climate zones where the accessions were collected. Molecular analysis of variance indicated that the genetic variation was larger between groups than within group. Conclusion: Obvious genetic diversity in Iranian wheat was revealed by analysis of GBS-SNPs and thus breeders can select genetically distant parents for crossing in breeding. The diverse Iranian landraces provide rich genetic sources of tolerance to biotic and abiotic stresses, and they can be useful resources for the improvement of wheat production in Iran and other countries.

Overexpression of Arabidopsis Dehydration-Responsive Element-Binding protein 2A confers tolerance to salinity stress to transgenic canola.

Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants are the classical transcriptional regulators involved in plant responses to drought, salt and cold stresses. To elucidate the transcriptional mechanism associated with the DREB2A gene after removing PEST sequence, which acts as a signal peptide for protein degradation, 34 transgenic T0 canola plants overexpressing DREB2A were developed. The quantitative Real time PCR of transgenic plants showed higher expression of downstream stress-responsive genes including COR14, HSF3, HSP70, PEROX and RD20. The transgenic plants exhibited enhanced tolerance to salt stress. At the high concentration of NaCl the growth of non-transformed plants had been clearly diminished, whereas transgenic line was survived. These results indicated that transformed DREB2A gene might improve the plant response to salinity in transgenic canola plants.

Evaluation of screening techniques for heat tolerance in wheat.

Cell membrane thermal stability, antioxidant activity, phenolics content, Paraquat tolerance and kernel weight were compared for their ability to identify heat tolerant genotypes. Four wheat (Triticum aestivum L.) genotypes, Kauz, MTRWA116, Opata and W7984 were used in this study, Kauz and MTRWA116 being thermotolerant and thermosensitive, respectively. Plants were exposed to high temperatures of 39 and 35 degrees C and then their measurements form different techniques were compared to each other and to the controls. The experiments were run several times to measure the repeatability of the measurements. Although the amount of phenolic compounds increased under stress condition, there was no significant difference between tolerant and susceptible varieties. Membrane thermal stability, antioxidant activity, phenolics content and Paraquat tolerance did not provide repeatable data. Nor did they discriminate among tolerant and susceptible genotypes. Kernel weight, however, varied between tolerant and susceptible genotypes. The results indicate that kernel weight is more suited for heat stress screening than other physioloical techniques evaluated in this study.