First Report of Pantoea ananatis causing leaf streak disease on wheat (Triticum aestivum) in the United States of America.

Wheat (Triticum aestivum) loses 21.5% yield to pests and diseases annually (Savary et al. 2019). Among the wheat diseases, bacterial leaf streak (BLS) is a growing problem, costing $78.5 million in losses (https://cropprotectionnetwork.org/). In July 2022, we sampled winter wheat leaf samples at Volga (44.30, -96.92), South Dakota, USA with an estimated disease incidence of 40% (n=100). The typical symptoms were water-soaking with large necrotic and chlorotic streaks extending the length of the leaves and were strikingly similar to BLS. To isolate the pathogen, leaves were cut lengthwise into 1 cm pieces and surface-sterilized using a 10% NaOCl solution for 3 min, followed by 70% ethanol for 3 min, and then rinsed with sterile distilled water and placed in 500 ul of sterile distilled water for 5 min and using a sterile loop the water was streaked over a plate of Nutrient Agar (NA). Following Duveiller et al. (1997), the streaked plate was incubated in the dark at 28℃ for 48 h. Observed single colonies were sub-cultured thrice onto fresh NA plates to obtain a pure culture. We named the culture SD101. Bacteria were found to be gram-negative with a colony morphology initially raised, smooth, and white that later turned yellow. DNA was extracted using the Wizard HMW DNA Extraction Kit (Promega, Madison, WI) following the manufacturer's protocol, and sequenced using Nanopore MinION R9.4 (Oxford Nanopore Technology). We used the Rapid Annotation Using Subsystems Technology approach (Aziz eal. 2008) to extract the 16S rDNA, DNA gyrase subunit B (gyrB), and translation initiation factor IF-2 (infB) gene sequences that were deposited in GenBank under accession numbers PP329908.1 for 16S rDNA, PP496481 for infB, and PP328920.1 for gyrB. Homology analysis using CLC Genomics Workbench 22.0.2 (QIAGEN) and BLASTn against the GenBank nucleotide database resulted in a 99.74% match (1543/ 1547 bp) of the 16S sequence, 99.59% match (2674/ 2685 bp) of the infB sequence, and 99.42% match (2396/ 2410 bp) of the gyrB sequence with Pantoea ananatis strain AJ13355 (AP012032). To test pathogenicity, seeds of spring wheat breeding line SD4892 were planted in 30 cm × 30 cm pots in a greenhouse under a 16 h light photoperiod. The inoculum was prepared from 48-h-old NA plates of SD101 rinsed with 1X Phosphate Buffer Saline (PBS buffer), adjusted to an OD600 = 1.0, and amended with two drops of Tween 20 (polyoxyethylene sorbitol ester, Millipore Sigma). PBS with Tween 20 was used as a negative control. The inoculum was sprayed on 15 replicates of 15-day-old seedlings, kept at 95% relative humidity for 48 h, then moved to the greenhouse at 23 to 25°C. The symptoms appeared as water soaking that later turned to necrotic streaks with surrounding chlorosis on all 15 inoculated plants while control plants remained healthy. The pathogen was re-isolated from the leaves as described above. The 16S rDNA, infB, and gyrB sequences amplified and sequenced were identical to the gene sequences from the whole genome sequencing. The experiment was repeated with the same results, completing Koch's postulates. Although P. ananatis is pathogenic on corn, rice, and other plant species in the United States (Coutinho et al. 2009), and was reported pathogenic on wheat in Poland (Krawczyk et al. 2020), this is the first report of its pathogenicity on wheat in the United States. The prevalence, and incidence of BLS disease caused by P. ananatis in wheat is needed to estimate its threat to wheat production and to develop management strategies.

The genetic architecture of genome-wide recombination rate variation in allopolyploid wheat revealed by nested association mapping.

Recombination affects the fate of alleles in populations by imposing constraints on the reshuffling of genetic information. Understanding the genetic basis of these constraints is critical for manipulating the recombination process to improve the resolution of genetic mapping, and reducing the negative effects of linkage drag and deleterious genetic load in breeding. Using sequence-based genotyping of a wheat nested association mapping (NAM) population of 2,100 recombinant inbred lines created by crossing 29 diverse lines, we mapped QTL affecting the distribution and frequency of 102 000 crossovers (CO). Genome-wide recombination rate variation was mostly defined by rare alleles with small effects together explaining up to 48.6% of variation. Most QTL were additive and showed predominantly trans-acting effects. The QTL affecting the proximal COs also acted additively without increasing the frequency of distal COs. We showed that the regions with decreased recombination carry more single nucleotide polymorphisms (SNPs) with possible deleterious effects than the regions with a high recombination rate. Therefore, our study offers insights into the genetic basis of recombination rate variation in wheat and its effect on the distribution of deleterious SNPs across the genome. The identified trans-acting additive QTL can be utilized to manipulate CO frequency and distribution in the large polyploid wheat genome opening the possibility to improve the efficiency of gene pyramiding and reducing the deleterious genetic load in the low-recombining pericentromeric regions of chromosomes.

Genetic analyses using GGE model and a mixed linear model approach, and stability analyses using AMMI bi-plot for late-maturity alpha-amylase activity in bread wheat genotypes.

Low falling number and discounting grain when it is downgraded in class are the consequences of excessive late-maturity α-amylase activity (LMAA) in bread wheat (Triticum aestivum L.). Grain expressing high LMAA produces poorer quality bread products. To effectively breed for low LMAA, it is necessary to understand what genes control it and how they are expressed, particularly when genotypes are grown in different environments. In this study, an International Collection (IC) of 18 spring wheat genotypes and another set of 15 spring wheat cultivars adapted to South Dakota (SD), USA were assessed to characterize the genetic component of LMAA over 5 and 13 environments, respectively. The data were analysed using a GGE model with a mixed linear model approach and stability analysis was presented using an AMMI bi-plot on R software. All estimated variance components and their proportions to the total phenotypic variance were highly significant for both sets of genotypes, which were validated by the AMMI model analysis. Broad-sense heritability for LMAA was higher in SD adapted cultivars (53%) compared to that in IC (49%). Significant genetic effects and stability analyses showed some genotypes, e.g. 'Lancer', 'Chester' and 'LoSprout' from IC, and 'Alsen', 'Traverse' and 'Forefront' from SD cultivars could be used as parents to develop new cultivars expressing low levels of LMAA. Stability analysis using an AMMI bi-plot revealed that 'Chester', 'Lancer' and 'Advance' were the most stable across environments, while in contrast, 'Kinsman', 'Lerma52' and 'Traverse' exhibited the lowest stability for LMAA across environments.

Additive-dominance genetic model analyses for late-maturity alpha-amylase activity in a bread wheat factorial crossing population.

Elevated level of late maturity α-amylase activity (LMAA) can result in low falling number scores, reduced grain quality, and downgrade of wheat (Triticum aestivum L.) class. A mating population was developed by crossing parents with different levels of LMAA. The F2 and F3 hybrids and their parents were evaluated for LMAA, and data were analyzed using the R software package 'qgtools' integrated with an additive-dominance genetic model and a mixed linear model approach. Simulated results showed high testing powers for additive and additive × environment variances, and comparatively low powers for dominance and dominance × environment variances. All variance components and their proportions to the phenotypic variance for the parents and hybrids were significant except for the dominance × environment variance. The estimated narrow-sense heritability and broad-sense heritability for LMAA were 14 and 54%, respectively. High significant negative additive effects for parents suggest that spring wheat cultivars 'Lancer' and 'Chester' can serve as good general combiners, and that 'Kinsman' and 'Seri-82' had negative specific combining ability in some hybrids despite of their own significant positive additive effects, suggesting they can be used as parents to reduce LMAA levels. Seri-82 showed very good general combining ability effect when used as a male parent, indicating the importance of reciprocal effects. High significant negative dominance effects and high-parent heterosis for hybrids demonstrated that the specific hybrid combinations; Chester × Kinsman, 'Lerma52' × Lancer, Lerma52 × 'LoSprout' and 'Janz' × Seri-82 could be generated to produce cultivars with significantly reduced LMAA level.

Impact of Solid and Hollow Varieties of Winter and Spring Wheat on Severity of Wheat Stem Sawfly (Hymenoptera: Cephidae) Infestations and Yield and Quality of Grain.

Wheat stem sawfly (WSS), Cephus cinctus Norton (Hymenoptera: Cephidae), has recently emerged as a key pest of wheat (Triticum aestivum L.) in the Great Plains and Canadian provinces. The expanding impact of WSS has caused considerable economic losses to wheat production. Solid-stem varieties of wheat remain the only effective measure of suppression of WSS, and the goal of this research was to test whether five solid- and hollow-stem varieties of winter and spring wheat reduce survival of WSS in South Dakota. We reported that solid-stem varieties had significantly lower numbers of WSS larvae, and this effect was especially evident when WSS infestation rates exceeded 15%. We also observed that the yield of solid-stem varieties was significantly lower than hollow-stem varieties when the abundance of WSS was low, but not when populations of WSS were relatively high. We did not observe consistent differences in grain quality between solid- and hollow-stem varieties, however, and in case of protein levels of grain, solid-stem wheat varieties performed better than hollow-stem wheat. We conclude that solid-stem varieties of wheat appear to effectively suppress WSS survival, and reduced yield of these varieties is less apparent when populations of C. cinctus are high enough to affect the yield of hollow-stem wheat. This is the first report to describe the effectiveness of solid-stem varieties of wheat on WSS in South Dakota. More research in the state is necessary before more robust conclusions can be drawn.

Enhancing the potential of phenomic and genomic prediction in winter wheat breeding using high-throughput phenotyping and deep learning.

Integrating high-throughput phenotyping (HTP) based traits into phenomic and genomic selection (GS) can accelerate the breeding of high-yielding and climate-resilient wheat cultivars. In this study, we explored the applicability of Unmanned Aerial Vehicles (UAV)-assisted HTP combined with deep learning (DL) for the phenomic or multi-trait (MT) genomic prediction of grain yield (GY), test weight (TW), and grain protein content (GPC) in winter wheat. Significant correlations were observed between agronomic traits and HTP-based traits across different growth stages of winter wheat. Using a deep neural network (DNN) model, HTP-based phenomic predictions showed robust prediction accuracies for GY, TW, and GPC for a single location with R2 of 0.71, 0.62, and 0.49, respectively. Further prediction accuracies increased (R2 of 0.76, 0.64, and 0.75) for GY, TW, and GPC, respectively when advanced breeding lines from multi-locations were used in the DNN model. Prediction accuracies for GY varied across growth stages, with the highest accuracy at the Feekes 11 (Milky ripe) stage. Furthermore, forward prediction of GY in preliminary breeding lines using DNN trained on multi-location data from advanced breeding lines improved the prediction accuracy by 32% compared to single-location data. Next, we evaluated the potential of incorporating HTP-based traits in multi-trait genomic selection (MT-GS) models in the prediction of GY, TW, and GPC. MT-GS, models including UAV data-based anthocyanin reflectance index (ARI), green chlorophyll index (GCI), and ratio vegetation index 2 (RVI_2) as covariates demonstrated higher predictive ability (0.40, 0.40, and 0.37, respectively) as compared to single-trait model (0.23) for GY. Overall, this study demonstrates the potential of integrating HTP traits into DL-based phenomic or MT-GS models for enhancing breeding efficiency.

Multi-trait genomic selection improves the prediction accuracy of end-use quality traits in hard winter wheat.

Improvement of end-use quality remains one of the most important goals in hard winter wheat (HWW) breeding. Nevertheless, the evaluation of end-use quality traits is confined to later development generations owing to resource-intensive phenotyping. Genomic selection (GS) has shown promise in facilitating selection for end-use quality; however, lower prediction accuracy (PA) for complex traits remains a challenge in GS implementation. Multi-trait genomic prediction (MTGP) models can improve PA for complex traits by incorporating information on correlated secondary traits, but these models remain to be optimized in HWW. A set of advanced breeding lines from 2015 to 2021 were genotyped with 8725 single-nucleotide polymorphisms and was used to evaluate MTGP to predict various end-use quality traits that are otherwise difficult to phenotype in earlier generations. The MTGP model outperformed the ST model with up to a twofold increase in PA. For instance, PA was improved from 0.38 to 0.75 for bake absorption and from 0.32 to 0.52 for loaf volume. Further, we compared MTGP models by including different combinations of easy-to-score traits as covariates to predict end-use quality traits. Incorporation of simple traits, such as flour protein (FLRPRO) and sedimentation weight value (FLRSDS), substantially improved the PA of MT models. Thus, the rapid low-cost measurement of traits like FLRPRO and FLRSDS can facilitate the use of GP to predict mixograph and baking traits in earlier generations and provide breeders an opportunity for selection on end-use quality traits by culling inferior lines to increase selection accuracy and genetic gains.

Evaluation of Spring Wheat Germplasm for Resistance to Bacterial Leaf Streak Caused by Xanthomonas campestris pv. translucens.

Bacterial leaf streak (caused by Xanthomonas campestris pv. translucens) has reemerged as a potential threat in spring wheat (Triticum aestivum) production areas in the northern Great Plains. As with other foliar bacterial diseases, chemical control under field situations is neither economical nor practical. Development of resistant genotypes will be needed for adequate management of the disease. There is currently limited information on sources of resistance in hard spring wheat germplasm. The main objective was to develop and apply a robust screening tool for evaluating germplasm against bacterial leaf streak, and to identify resistance sources for this disease. Inoculated field experiments were conducted in Brookings and Codington Counties, SD in 2009 and 2010 using a virulent local isolate (XctSD-017) inoculated after tillering stage. Forty-five hard red spring wheat genotypes with diverse genetic backgrounds were evaluated for disease severity, with ratings made at 7-day intervals from heading through dough stage. Results of this study showed clear differences in level of resistance among the 45 genotypes, with no immunity expressed. SD4205 was found resistant to the disease with the lowest area under the disease progress curve across location-years. Other genotypes showing moderate levels of resistance included SD4148, SD4176, 'Alsen', SD4023, 'Faller', SD4024, 'Knudson', and SD4199. Grain weight was negatively correlated to disease, as was days to heading. The resistance identified appeared to be quantitative in nature and was expressed through slower disease progress and reduced severity.

Population- and genome-specific patterns of linkage disequilibrium and SNP variation in spring and winter wheat (Triticum aestivum L.).

BACKGROUND: Single nucleotide polymorphisms (SNPs) are ideally suited for the construction of high-resolution genetic maps, studying population evolutionary history and performing genome-wide association mapping experiments. Here, we used a genome-wide set of 1536 SNPs to study linkage disequilibrium (LD) and population structure in a panel of 478 spring and winter wheat cultivars (Triticum aestivum) from 17 populations across the United States and Mexico. RESULTS: Most of the wheat oligo pool assay (OPA) SNPs that were polymorphic within the complete set of 478 cultivars were also polymorphic in all subpopulations. Higher levels of genetic differentiation were observed among wheat lines within populations than among populations. A total of nine genetically distinct clusters were identified, suggesting that some of the pre-defined populations shared significant proportion of genetic ancestry. Estimates of population structure (F(ST)) at individual loci showed a high level of heterogeneity across the genome. In addition, seven genomic regions with elevated F(ST) were detected between the spring and winter wheat populations. Some of these regions overlapped with previously mapped flowering time QTL. Across all populations, the highest extent of significant LD was observed in the wheat D-genome, followed by lower LD in the A- and B-genomes. The differences in the extent of LD among populations and genomes were mostly driven by differences in long-range LD ( > 10 cM). CONCLUSIONS: Genome- and population-specific patterns of genetic differentiation and LD were discovered in the populations of wheat cultivars from different geographic regions. Our study demonstrated that the estimates of population structure between spring and winter wheat lines can identify genomic regions harboring candidate genes involved in the regulation of growth habit. Variation in LD suggests that breeding and selection had a different impact on each wheat genome both within and among populations. The higher extent of LD in the wheat D-genome versus the A- and B-genomes likely reflects the episodes of recent introgression and population bottleneck accompanying the origin of hexaploid wheat. The assessment of LD and population structure in this assembled panel of diverse lines provides critical information for the development of genetic resources for genome-wide association mapping of agronomically important traits in wheat.

Use of near-isogenic wheat lines to determine the glutenin composition and functionality requirements for flour tortillas.

In wheat ( Triticum aestivum L), the synthesis of high molecular weight (HMW) glutenins (GS) is controlled by three heterologous genetic loci present on the long arms of group 1 wheat chromosomes. The loci Glu-A1, Glu-B1, and Glu-D1 and their allelic variants play important roles in the functional properties of wheat flour. This study focused on understanding the functionality of these protein subunits on tortilla quality. Near-isogenic wheat lines in which one or more of these loci were absent or deleted were used. Tortillas were prepared from each deletion line and the parent lines. The elimination of certain HMW-GS alleles alter distinct but critical aspects of tortilla quality such as diameter, shelf stability, and overall quality. Two deletion lines possessing HMW-GS 17 + 18 at Glu-B1 and deletions in Glu-A1 and Glu-D1 had significantly larger tortilla diameters, yet tortilla shelf life was compromised or unchanged from the parent lines used to develop the deletion lines or the commercial tortilla flour used as a control. Alternatively, a deletion line possessing Glu-A1 and Glu-D1 (HMW-GS 1, 5 + 10) and a deletion in Glu-B1 also significantly improved tortilla diameters. Whereas the increase in diameter was less than the line possessing only HMW-GS 17 + 18 at Glu-B1, the stability of the tortillas were, however, maintained and improved as compared to the parent lines containing a full compliment of HMW-GS. Thus, the presence of subunits 5 + 10 at Glu-D1 alone or in combination with subunit 1 at Glu-A1 appears to provide a compromise of improvement in dough extensibility for improved tortilla diameters while also providing sufficient gluten strength to maintain ideal shelf stability.

A Quantitative Proteomics View on the Function of Qfhb1, a Major QTL for Fusarium Head Blight Resistance in Wheat.

Fusarium head blight (FHB) is a highly detrimental disease of wheat. A quantitative trait locus for FHB resistance, Qfhb1, is the most utilized source of resistance in wheat-breeding programs, but very little is known about its resistance mechanism. In this study, we elucidated a prospective FHB resistance mechanism by investigating the proteomic signatures of Qfhb1 in a pair of contrasting wheat near-isogenic lines (NIL) after 24 h of inoculation of wheat florets by Fusarium graminearum. Statistical comparisons of the abundances of protein spots on the 2D-DIGE gels of contrasting NILs (fhb1+ NIL = Qfhb1 present; fhb1- NIL = Qfhb1 absent) enabled us to select 80 high-ranking differentially accumulated protein (DAP) spots. An additional evaluation confirmed that the DAP spots were specific to the spikelet from fhb1- NIL (50 spots), and fhb1+ NIL (seven spots). The proteomic data also suggest that the absence of Qfhb1 makes the fhb1- NIL vulnerable to Fusarium attack by constitutively impairing several mechanisms including sucrose homeostasis by enhancing starch synthesis from sucrose. In the absence of Qfhb1, Fusarium inoculations severely damaged photosynthetic machinery; altered the metabolism of carbohydrates, nitrogen and phenylpropanoids; disrupted the balance of proton gradients across relevant membranes; disturbed the homeostasis of many important signaling molecules induced the mobility of cellular repair; and reduced translational activities. These changes in the fhb1- NIL led to strong defense responses centered on the hypersensitive response (HSR), resulting in infected cells suicide and the consequent initiation of FHB development. Therefore, the results of this study suggest that Qfhb1 largely functions to either alleviate HSR or to manipulate the host cells to not respond to Fusarium infection.

Reaction of Global Collection of Rye (Secale cereale L.) to Tan Spot and Pyrenophora tritici-repentis Races in South Dakota.

Rye (Secale cereale L.) serves as an alternative host of Pyrenophora tritici-repentis (PTR) the cause of tan spot on wheat. Rye is cultivated as a forage or cover crop and overlaps with a significant portion of wheat acreage in the U.S. northern Great Plains; however, it is not known whether the rye crop influences the evolution of PTR races. We evaluated a global collection of 211 rye accessions against tan spot and assessed the diversity in PTR population on rye in South Dakota. All the rye genotypes were inoculated with PTR races 1 and 5, and infiltrated with Ptr ToxA and Ptr ToxB, at seedling stage. We observed 21% of the genotypes exhibited susceptibility to race 1, whereas, 39% were susceptible to race 5. All 211 accessions were insensitive to both the Ptr toxins. It indicates that though rye exhibits diversity in reaction to tan spot, it lacks Ptr ToxA and ToxB sensitivity genes. This suggests that unknown toxins or other factors can lead to PTR establishment in rye. We characterized the race structure of 103 PTR isolates recovered from rye in South Dakota. Only 22% of the isolates amplified Ptr ToxA gene and were identified as race 1 based on their phenotypic reaction on the differential set. The remaining 80 isolates were noted to be race 4. Our results show that races 1 and 4 are prevalent on rye in South Dakota with a higher frequency of race 4, suggesting a minimal role of rye in the disease epidemiology.

Functionality of gliadin proteins in wheat flour tortillas.

Gliadins are monomeric proteins that are encoded by the genes at the loci Gli 1 and Gli 2 present on the short arm of homologous wheat chromosomes 1 and 6, respectively. Studies have suggested that gliadins may play an important role in determining the functional properties of wheat flour. The main objective of this study was to understand the functionality of gliadins with respect to tortilla quality. The important tortilla quality attributes are diameter, opacity, and shelf stability, designated here as rollability or the ability to roll or fold the tortilla without cracking. In this study gliadin functionality in tortilla quality was studied using near-isogenic wheat lines that have deletions in either Gli A1, Gli D1, Gli A2, or Gli D2 gliadin loci. The deletion lines are designated by the same abbreviations. Dough and tortillas were prepared from the parent line used to derive these deletion lines, each individual deletion line, and a control commercial tortilla flour. Quantitative and qualitative evaluations were performed on the dough and tortillas derived from the flour from each of these lines. None of the deletions in the gliadin loci altered the shelf stability versus that found for the parent to the deletion lines or control tortilla flour. However, deletions in the Gli 2 loci, in particular Gli A2 reduced the relative proportion of alpha- and beta-gliadins with a greater cysteine amino acid content and gluten cross-link function versus the chain-terminating omega-gliadins in Gli 1, which were still present. As such, the dough and gluten matrix appeared to have greater extensibility, which improved the diameter and overall quality of the tortillas while not altering the rollability. Deletions in the Gli 1 loci had the opposite result with increased cross-linking of alpha- and beta-gliadins, polymeric protein content, and a stronger dough that decreased the diameter and overall quality of the tortillas. The data suggest that altering certain Gli 2 loci through null alleles could be a viable strategy to develop cultivars improved for the specific functionality requirements needed for the rapidly growing tortilla market.