Genetic Parameter and Hyper-Parameter Estimation Underlie Nitrogen Use Efficiency in Bread Wheat.

Estimation and prediction play a key role in breeding programs. Currently, phenotyping of complex traits such as nitrogen use efficiency (NUE) in wheat is still expensive, requires high-throughput technologies and is very time consuming compared to genotyping. Therefore, researchers are trying to predict phenotypes based on marker information. Genetic parameters such as population structure, genomic relationship matrix, marker density and sample size are major factors that increase the performance and accuracy of a model. However, they play an important role in adjusting the statistically significant false discovery rate (FDR) threshold in estimation. In parallel, there are many genetic hyper-parameters that are hidden and not represented in the given genomic selection (GS) model but have significant effects on the results, such as panel size, number of markers, minor allele frequency, number of call rates for each marker, number of cross validations and batch size in the training set of the genomic file. The main challenge is to ensure the reliability and accuracy of predicted breeding values (BVs) as results. Our study has confirmed the results of bias-variance tradeoff and adaptive prediction error for the ensemble-learning-based model STACK, which has the highest performance when estimating genetic parameters and hyper-parameters in a given GS model compared to other models.

Genetic dissection of root architectural plasticity and identification of candidate loci in response to drought stress in bread wheat.

BACKGROUND: The frequency of droughts has dramatically increased over the last 50 years, causing yield declines in cereals, including wheat. Crop varieties with efficient root systems show great potential for plant adaptation to drought stress, however; genetic control of root systems in wheat under field conditions is not yet well understood. RESULTS: Natural variation in root architecture plasticity (phenotypic alteration due to changing environments) was dissected under field-based control (well-irrigated) and drought (rain-out shelter) conditions by a genome-wide association study using 200 diverse wheat cultivars. Our results revealed root architecture and plasticity traits were differentially responded to drought stress. A total of 25 marker-trait associations (MTAs) underlying natural variations in root architectural plasticity were identified in response to drought stress. They were abundantly distributed on chromosomes 1 A, 1B, 2 A, 2B, 3 A, 3B, 4B, 5 A, 5D, 7 A and 7B of the wheat genome. Gene ontology annotation showed that many candidate genes associated with plasticity were involved in water-transport and water channel activity, cellular response to water deprivation, scavenging reactive oxygen species, root growth and development and hormone-activated signaling pathway-transmembrane transport, indicating their response to drought stress. Further, in silico transcript abundance analysis demonstrated that root plasticity-associated candidate genes were highly expressed in roots across different root growth stages and under drought treatments. CONCLUSION: Our results suggest that root phenotypic plasticity is highly quantitative, and the corresponding loci are associated with drought stress that may provide novel ways to enable root trait breeding.

Chromosome 3A harbors several pleiotropic and stable drought-responsive alleles for photosynthetic efficiency selected through wheat breeding.

Water deficit is the most severe stress factor in crop production threatening global food security. In this study, we evaluated the genetic variation in photosynthetic traits among 200 wheat cultivars evaluated under drought and rainfed conditions. Significant genotypic, treatments, and their interaction effects were detected for chlorophyll content and chlorophyll fluorescence parameters. Drought stress reduced the effective quantum yield of photosystem II (YII) from the anthesis growth stage on. Leaf chlorophyll content measured at anthesis growth stages was significantly correlated with YII and non-photochemical quenching under drought conditions, suggesting that high throughput chlorophyll content screening can serve as a good indicator of plant drought tolerance status in wheat. Breeding significantly increased the photosynthetic efficiency as newer released genotypes had higher YII and chlorophyll content than the older ones. GWAS identified a stable drought-responsive QTL on chromosome 3A for YII, while under rainfed conditions, it detected another QTL on chromosome 7A for chlorophyll content across both growing seasons. Molecular analysis revealed that the associated alleles of AX-158576783 (515.889 Mbp) on 3A co-segregates with the NADH-ubiquinone oxidoreductase (TraesCS3A02G287600) gene involved in ATP synthesis coupled electron transport and is proximal to WKRY transcription factor locus. This allele on 3A has been positively selected through breeding and has contributed to increasing the grain yield.

Identification of QTLs for wheat heading time across multiple-environments.

KEY MESSAGE: The genetic response to changing climatic factors selects consistent across the tested environments and location-specific thermo-sensitive and photoperiod susceptible alleles in lower and higher altitudes, respectively, for starting flowering in winter wheat. Wheat breeders select heading date to match the most favorable conditions for their target environments and this is favored by the extensive genetic variation for this trait that has the potential to be further explored. In this study, we used a germplasm with broad geographic distribution and tested it in multi-location field trials across Germany over three years. The genotypic response to the variation in the climatic parameters depending on location and year uncovered the effect of photoperiod and spring temperatures in accelerating heading date in higher and lower latitudes, respectively. Spring temperature dominates other factors in inducing heading, whereas the higher amount of solar radiation delays it. A genome-wide scan of marker-trait associations with heading date detected two QTL: an adapted allele at locus TaHd102 on chromosome 5A that has a consistent effect on HD in German cultivars in multiple environments and a non-adapted allele at locus TaHd044 on chromosome 3A that accelerates flowering by 5.6 days. TaHd102 and TaHd044 explain 13.8% and 33% of the genetic variance, respectively. The interplay of the climatic variables led to the detection of environment specific association responding to temperature in lower latitudes and photoperiod in higher ones. Another locus TaHd098 on chromosome 5A showed epistatic interactions with 15 known regulators of flowering time when non-adapted cultivars from outside Germany were included in the analysis.

Importance of correcting genomic relationships in single-locus QTL mapping model with an advanced backcross population.

Advanced backcross (AB) populations have been widely used to identify and utilize beneficial alleles in various crops such as rice, tomato, wheat, and barley. For the development of an AB population, a controlled crossing scheme is used and this controlled crossing along with the selection (both natural and artificial) of agronomically adapted alleles during the development of AB population may lead to unbalanced allele frequencies in the population. However, it is commonly believed that interval mapping of traits in experimental crosses such as AB populations is immune to the deviations from the expected frequencies under Mendelian segregation. Using two AB populations and simulated data sets as examples, we describe the severity of the problem caused by unbalanced allele frequencies in quantitative trait loci mapping and demonstrate how it can be corrected using the linear mixed model having a polygenic effect with the covariance structure (genomic relationship matrix) calculated from molecular markers.

Detection of breeding signatures in wheat using a linkage disequilibrium-corrected mapping approach.

Marker assisted breeding, facilitated by reference genome assemblies, can help to produce cultivars adapted to changing environmental conditions. However, anomalous linkage disequilibrium (LD), where single markers show high LD with markers on other chromosomes but low LD with adjacent markers, is a serious impediment for genetic studies. We used a LD-correction approach to overcome these drawbacks, correcting the physical position of markers derived from 15 and 135 K arrays in a diversity panel of bread wheat representing 50 years of breeding history. We detected putative mismapping of 11.7% markers and improved the physical alignment of 5.4% markers. Population analysis indicated reduced genetic diversity over time as a result of breeding efforts. By analysis of outlier loci and allele frequency change over time we traced back the 2NS/2AS translocation of Aegilops ventricosa to one cultivar, "Cardos" (registered in 1998) which was the first among the panel to contain this translocation. A "selective sweep" for this important translocation region on chromosome 2AS was found, putatively linked to plant response to biotic stress factors. Our approach helps in overcoming the drawbacks of incorrectly anchored markers on the wheat reference assembly and facilitates detection of selective sweeps for important agronomic traits.

Transcriptome profiling at osmotic and ionic phases of salt stress response in bread wheat uncovers trait-specific candidate genes.

BACKGROUND: Bread wheat is one of the most important crops for the human diet, but the increasing soil salinization is causing yield reductions worldwide. Improving salt stress tolerance in wheat requires the elucidation of the mechanistic basis of plant response to this abiotic stress factor. Although several studies have been performed to analyze wheat adaptation to salt stress, there are still some gaps to fully understand the molecular mechanisms from initial signal perception to the onset of responsive tolerance pathways. The main objective of this study is to exploit the dynamic salt stress transcriptome in underlying QTL regions to uncover candidate genes controlling salt stress tolerance in bread wheat. The massive analysis of 3'-ends sequencing protocol was used to analyze leave samples at osmotic and ionic phases. Afterward, stress-responsive genes overlapping QTL for salt stress-related traits in two mapping populations were identified. RESULTS: Among the over-represented salt-responsive gene categories, the early up-regulation of calcium-binding and cell wall synthesis genes found in the tolerant genotype are presumably strategies to cope with the salt-related osmotic stress. On the other hand, the down-regulation of photosynthesis-related and calcium-binding genes, and the increased oxidative stress response in the susceptible genotype are linked with the greater photosynthesis inhibition at the osmotic phase. The specific up-regulation of some ABC transporters and Na+/Ca2+ exchangers in the tolerant genotype at the ionic stage indicates their involvement in mechanisms of sodium exclusion and homeostasis. Moreover, genes related to protein synthesis and breakdown were identified at both stress phases. Based on the linkage disequilibrium blocks, salt-responsive genes within QTL intervals were identified as potential components operating in pathways leading to salt stress tolerance. Furthermore, this study conferred evidence of novel regions with transcription in bread wheat. CONCLUSION: The dynamic transcriptome analysis allowed the comparison of osmotic and ionic phases of the salt stress response and gave insights into key molecular mechanisms involved in the salt stress adaptation of contrasting bread wheat genotypes. The leveraging of the highly contiguous chromosome-level reference genome sequence assembly facilitated the QTL dissection by targeting novel candidate genes for salt tolerance.

Genetic dissection of bread wheat diversity and identification of adaptive loci in response to elevated tropospheric ozone.

Rising tropospheric ozone affects the performance of important cereal crops thus threatening global food security. In this study, genetic variation of wheat regarding its physiological and yield responses to ozone was explored by exposing a diversity panel of 150 wheat genotypes to elevated ozone and control conditions throughout the growing season. Differential responses to ozone were observed for foliar symptom formation quantified as leaf bronzing score (LBS), vegetation indices and yield components. Vegetation indices representing the carotenoid to chlorophyll pigment ratio (such as Lic2) were particularly ozone-responsive and were thus considered suitable for the non-invasive diagnosing of ozone stress. Genetic variation in ozone-responsive traits was dissected by a genome-wide association study (GWAS). Significant marker-trait associations were identified for LBS on chromosome 5A and for vegetation indices (NDVI and Lic2) on chromosomes 6B and 6D. Analysis of linkage disequilibrium (LD) in these chromosomal regions revealed distinct LD blocks containing genes with a putative function in plant redox biology such as cytochrome P450 proteins and peroxidases. This study gives novel insight into the natural genetic variation in wheat ozone response, and lays the foundation for the molecular breeding of tolerant wheat varieties.

Dissecting the Genetic Complexity of Fusarium Crown Rot Resistance in Wheat.

Fusarium crown rot (FCR) is one of the most important diseases of wheat (Triticum aestivum L.). FCR is mainly caused by the fungal pathogens Fusarium culmorum and F. pseudograminearum. In order to identify new sources of resistance to FCR and to dissect the complexity of FCR resistance, a panel of 161 wheat accessions was phenotyped under growth room (GR) and greenhouse conditions (GH). Analysis of variance showed significant differences in crown rot development among wheat accessions and high heritability of genotype-environment interactions for GR (0.96) and GH (0.91). Mixed linear model analysis revealed seven novel quantitative trait loci (QTLs) linked to F. culmorum on chromosomes 2AL, 3AS, 4BS, 5BS, 5DS, 5DL and 6DS for GR and eight QTLs on chromosomes on 3AS, 3BS, 3DL, 4BS (2), 5BS, 6BS and 6BL for GH. Total phenotypic variances (R²) explained by the QTLs linked to GR and GH were 48% and 59%, respectively. In addition, five favorable epistasis interactions among the QTLs were detected for both GR and GH with and without main effects. Epistatic interaction contributed additional variation up to 21% under GR and 7% under GH indicating strong effects of environment on the expression of QTLs. Our results revealed FCR resistance responses in wheat to be complex and controlled by multiple QTLs.

Multi-dimensional evaluation of response to salt stress in wheat.

Soil salinity is a major threat to crop production worldwide. The global climate change is further accelerating the process of soil salinization, particularly in dry areas of the world. Increasing genetic variability of currently used wheat varieties by introgression of exotic alleles/genes from related progenitors' species in breeding programs is an efficient approach to overcome limitations due to the absence of valuable genetic diversity in elite cultivars. Synthetic hexaploid wheat (SHW) is widely regarded as donor of favourable exotic alleles to improve tolerance against biotic and abiotic stresses such as salinity stress. In this study, synthetic backcross lines (SBLs) winter wheat population "Z86", derived from crosses involving synthetic hexaploid wheat Syn86L with German elite winter wheat cultivar Zentos, was evaluated for salinity tolerance at different developmental stages under controlled and field conditions in three growing seasons. High genetic variability was detected across the SBLs and their parents at various growth stages under controlled as well as under salt stress field trials. Greater performance of Zentos over Syn86L was detected at germination stage across all salt treatments and with respect to shoot dry weight (SDW) and root dry weight (RDW) at seedling stage. Whereas for the root length (RL) and the shoot length (SL) Syn86L surpassed the elite cultivar and most of the progenies. Our experiments revealed for almost all traits that some genotypes among the SBLs showed higher performance than their parents. Furthermore, positive transgressive segregations were detected among the SBLs for germination at high salinity levels, as well as for RDW and SDW at seedling stage. Therefore, the studied Z86 population is a suitable population for assessment of salinity stress on morphological and physiological traits at different plant growth stages. The identified SBLs provide a valuable source for genetic gain through recombination of superior alleles that can be directly applied in breeding programs for efficiently breeding cultivars with improved salinity tolerance and desired agronomic traits.

Genetic Analysis and Transfer of Favorable Exotic QTL Alleles for Grain Yield Across D Genome Using Two Advanced Backcross Wheat Populations.

Hexaploid wheat evolved through a spontaneous hybridization of tetraploid wheat (Triticum turgidum, AABB) with diploid wild grass (Aegilops tauschii, DD). Recent genome sequencing found alarmingly low genetic diversity and abundance of repeated sequences across D genome as compared to AB genomes. This characteristic feature of D genome often results in a low recombination rate and abrupt changes in chromosome, which are the major hurdles to utilize the genetic potential of D genome in wheat breeding. In the present study, we evaluated two advanced backcross populations designated as B22 (250 BC2F3:6 lines) and Z86 (150 BC2F3:6 lines) to test their yield potential and to enrich the D genome diversity simultaneously. The populations B22 and Z86 were derived by crossing winter wheat cultivars Batis and Zentos with synthetic hexaploid wheat accessions Syn022L and Syn086L, respectively. These populations were genotyped using SNP markers and phenotyped for yield traits in ten environments in Germany. Marker analysis identified lower recombination rate across D genome as compared to A and B genomes in both populations. Further, we compared the genotype data with the trait grain yield to identify favorable exotic introgressions from synthetic wheat accessions. QTL analysis identified seven and 13 favorable exotic QTL alleles associated with enhancement or at least stable grain yield in populations B22 and Z86, respectively. These favorable introgressions were located on all chromosomes from 1D to 7D. The strongest exotic QTL allele on chromosome 1D at SNP marker RAC875_c51493_471 resulted in a relative increase of 8.6% in grain yield as compared to cultivated allele. The identified exotic introgressions will help to refine useful exotic chromosome segments for their incorporation for improving yield and increasing D genome diversity among cultivated varieties.

An Ancestral Allele of Pyrroline-5-carboxylate synthase1 Promotes Proline Accumulation and Drought Adaptation in Cultivated Barley.

Water scarcity is a critical threat to global crop production. Here, we used the natural diversity of barley (Hordeum vulgare) to dissect the genetic control of proline (Pro) mediated drought stress adaptation. Genetic mapping and positional cloning of a major drought-inducible quantitative trait locus (QPro.S42-1H) revealed unique allelic variation in pyrroline-5-carboxylate synthase (P5cs1) between the cultivated cultivar Scarlett (ssp. vulgare) and the wild barley accession ISR42-8 (ssp. spontaneum). The putative causative mutations were located in the promoter of P5cs1 across the DNA binding motifs for abscisic acid-responsive element binding transcription factors. Introgression line (IL) S42IL-143 carrying the wild allele of P5cs1 showed significant up-regulation of P5cs1 expression compared to Scarlett, which was consistent with variation in Pro accumulation under drought. Next, we transiently expressed promoter::reporter constructs of ISR42-8 and Scarlett alleles in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. GUS expression analysis showed a significantly higher activation of the ISR42-8 promoter compared to Scarlett upon abscisic acid treatment. Notably, the ISR42-8 promoter activity was impaired in protoplasts isolated from the loss-of-function abf1abf2abf3abf4 quadruple mutant. A series of phenotypic evaluations demonstrated that S42IL-143 maintained leaf water content and photosynthetic activity longer than Scarlett under drought. These findings suggest that the ancestral variant of P5cs1 has the potential for drought tolerance and understanding drought physiology of barley and related crops.

Non-invasive assessment of leaf water status using a dual-mode microwave resonator.

The water status in plant leaves is a good indicator for the water status in the whole plant revealing stress if the water supply is reduced. The analysis of dynamic aspects of water availability in plant tissues provides useful information for the understanding of the mechanistic basis of drought stress tolerance, which may lead to improved plant breeding and management practices. The determination of the water content in plant tissues during plant development has been a challenge and is currently feasible based on destructive analysis only. We present here the application of a non-invasive quantitative method to determine the volumetric water content of leaves and the ionic conductivity of the leaf juice from non-invasive microwave measurements at two different frequencies by one sensor device. A semi-open microwave cavity loaded with a ceramic dielectric resonator and a metallic lumped-element capacitor- and inductor structure was employed for non-invasive microwave measurements at 150 MHz and 2.4 Gigahertz on potato, maize, canola and wheat leaves. Three leaves detached from each plant were chosen, representing three developmental stages being representative for tissue of various age. Clear correlations between the leaf- induced resonance frequency shifts and changes of the inverse resonator quality factor at 2.4 GHz to the gravimetrically determined drying status of the leaves were found. Moreover, the ionic conductivity of Maize leaves, as determined from the ratio of the inverse quality factor and frequency shift at 150 MHz by use of cavity perturbation theory, was found to be in good agreement with direct measurements on plant juice. In conjunction with a compact battery- powered circuit board- microwave electronic module and a user-friendly software interface, this method enables rapid in-vivo water amount assessment of plants by a handheld device for potential use in the field.