Reviewing the essential roles of remote phenotyping, GWAS and explainable AI in practical marker-assisted selection for drought-tolerant winter wheat breeding.

Marker-assisted selection (MAS) plays a crucial role in crop breeding improving the speed and precision of conventional breeding programmes by quickly and reliably identifying and selecting plants with desired traits. However, the efficacy of MAS depends on several prerequisites, with precise phenotyping being a key aspect of any plant breeding programme. Recent advancements in high-throughput remote phenotyping, facilitated by unmanned aerial vehicles coupled to machine learning, offer a non-destructive and efficient alternative to traditional, time-consuming, and labour-intensive methods. Furthermore, MAS relies on knowledge of marker-trait associations, commonly obtained through genome-wide association studies (GWAS), to understand complex traits such as drought tolerance, including yield components and phenology. However, GWAS has limitations that artificial intelligence (AI) has been shown to partially overcome. Additionally, AI and its explainable variants, which ensure transparency and interpretability, are increasingly being used as recognised problem-solving tools throughout the breeding process. Given these rapid technological advancements, this review provides an overview of state-of-the-art methods and processes underlying each MAS, from phenotyping, genotyping and association analyses to the integration of explainable AI along the entire workflow. In this context, we specifically address the challenges and importance of breeding winter wheat for greater drought tolerance with stable yields, as regional droughts during critical developmental stages pose a threat to winter wheat production. Finally, we explore the transition from scientific progress to practical implementation and discuss ways to bridge the gap between cutting-edge developments and breeders, expediting MAS-based winter wheat breeding for drought tolerance.

Impact of conservation tillage on wheat performance and its microbiome.

Winter wheat is an important cereal consumed worldwide. However, current management practices involving chemical fertilizers, irrigation, and intensive tillage may have negative impacts on the environment. Conservation agriculture is often presented as a sustainable alternative to maintain wheat production, favoring the beneficial microbiome. Here, we evaluated the impact of different water regimes (rainfed and irrigated), fertilization levels (half and full fertilization), and tillage practices (occasional tillage and no-tillage) on wheat performance, microbial activity, and rhizosphere- and root-associated microbial communities of four winter wheat genotypes (Antequera, Allez-y, Apache, and Cellule) grown in a field experiment. Wheat performance (i.e., yield, plant nitrogen concentrations, and total nitrogen uptake) was mainly affected by irrigation, fertilization, and genotype, whereas microbial activity (i.e., protease and alkaline phosphatase activities) was affected by irrigation. Amplicon sequencing data revealed that habitat (rhizosphere vs. root) was the main factor shaping microbial communities and confirmed that the selection of endophytic microbial communities takes place thanks to specific plant-microbiome interactions. Among the experimental factors applied, the interaction of irrigation and tillage influenced rhizosphere- and root-associated microbiomes. The findings presented in this work make it possible to link agricultural practices to microbial communities, paving the way for better monitoring of these microorganisms in the context of agroecosystem sustainability.

Potato root-associated microbiomes adapt to combined water and nutrient limitation and have a plant genotype-specific role for plant stress mitigation.

BACKGROUND: Due to climate change and reduced use of fertilizers combined stress scenarios are becoming increasingly frequent in crop production. In a field experiment we tested the effect of combined water and phosphorus limitation on the growth performance and plant traits of eight tetraploid and two diploid potato varieties as well as on root-associated microbiome diversity and functional potential. Microbiome and metagenome analysis targeted the diversity and potential functions of prokaryotes, fungi, plasmids, and bacteriophages and was linked to plant traits like tuber yield or timing of canopy closure. RESULTS: The different potato genotypes responded differently to the combined stress and hosted distinct microbiota in the rhizosphere and the root endosphere. Proximity to the root, stress and potato genotype had significant effects on bacteria, whereas fungi were only mildly affected. To address the involvement of microbial functions, we investigated well and poorly performing potato genotypes (Stirling and Desirée, respectively) under stress conditions and executed a metagenome analysis of rhizosphere microbiomes subjected to stress and no stress conditions. Functions like ROS detoxification, aromatic amino acid and terpene metabolism were enriched and in synchrony with the metabolism of stressed plants. In Desirée, Pseudonocardiales had the genetic potential to take up assimilates produced in the fast-growing canopy and to reduce plant stress-sensing by degrading ethylene, but overall yield losses were high. In Stirling, Xanthomonadales had the genetic potential to reduce oxidative stress and to produce biofilms, potentially around roots. Biofilm formation could be involved in drought resilience and nutrient accessibility of Stirling and explain the recorded low yield losses. In the rhizosphere exposed to combined stress, the relative abundance of plasmids was reduced, and the diversity of phages was enriched. Moreover, mobile elements like plasmids and phages were affected by combined stresses in a genotype-specific manner. CONCLUSION: Our study gives new insights into the interconnectedness of root-associated microbiota and plant stress responses in the field. Functional genes in the metagenome, phylogenetic composition and mobile elements play a role in potato stress adaption. In a poor and a well performing potato genotype grown under stress conditions, distinct functional genes pinpoint to a distinct stress sensing, water availability and compounds in the rhizospheres.

A compendium of genome-wide sequence reads from NBS (nucleotide binding site) domains of resistance genes in the common potato.

SolariX is a compendium of DNA sequence tags from the nucleotide binding site (NBS) domain of disease resistance genes of the common potato, Solanum tuberosum Group Tuberosum. The sequences, which we call NBS tags, for nearly all NBS domains from 91 genomes-representing a wide range of historical and contemporary potato cultivars, 24 breeding programs and 200 years-were generated using just 16 amplification primers and high-throughput sequencing. The NBS tags were mapped to 587 NBS domains on the draft potato genome DM, where we detected an average, over all the samples, of 26 nucleotide polymorphisms on each locus. The total number of NBS domains observed, differed between potato cultivars. However, both modern and old cultivars possessed comparable levels of variability, and neither the individual breeder or country nor the generation or time appeared to correlate with the NBS domain frequencies. Our attempts to detect haplotypes (i.e., sets of linked nucleotide polymorphisms) frequently yielded more than the possible 4 alleles per domain indicating potential locus intermixing during the mapping of NBS tags to the DM reference genome. Mapping inaccuracies were likely a consequence of the differences of each cultivar to the reference genome used, coupled with high levels of NBS domain sequence similarity. We illustrate that the SolariX database is useful to search for polymorphism linked with NBS-LRR R gene alleles conferring specific disease resistance and to develop molecular markers for selection.

Roots and Panicles of the C4 Model Grasses Setaria viridis (L). and S. pumila Host Distinct Bacterial Assemblages With Core Taxa Conserved Across Host Genotypes and Sampling Sites.

Virtually all studied plant tissues are internally inhabited by endophytes. Due to their relevance for plant growth and health, bacterial microbiota of crop plants have been broadly studied. In plant microbiome research the root is the most frequently addressed environment, whereas the ecology of microbiota associated with reproductive organs still demands investigation. In this work, we chose the model grasses Setaria viridis and Setaria pumila to better understand the drivers shaping bacterial communities associated with panicles (representing a reproductive organ) as compared to those associated with roots. We collected wild individuals of both grass species from 20 different locations across Austria and investigated the bacterial assemblages within roots and ripe grain-harboring panicles by 16S rRNA gene-based Illumina sequencing. Furthermore, plant samples were subjected to genotyping by genetic diversity-focused Genotyping by Sequencing. Overall, roots hosted more diverse microbiota than panicles. Both the plant organ and sampling site significantly shaped the root and panicle-associated microbiota, whereas the host genotype only affected root communities. In terms of community structure, root-specific assemblages were highly diverse and consisted of conserved bacterial taxa. In contrast, panicle-specific communities were governed by Gammaproteobacteria, were less diverse and highly origin-dependent. Among OTUs found in both plant tissues, relative abundances of Gammaproteobacteria were higher in panicles, whereas Rhizobiales dominated root communities. We further identified core and non-core taxa within samples of both Setaria species. Non-core taxa included members of the Saccharibacteria and Legionelalles, while core communities encompassed eleven OTUs of seven bacterial orders, together with a set of ten panicle-enriched OTUs. These communities were widespread across root and panicle samples from all locations, hinting toward an evolved form of mutualism through potential vertical transmission of these taxa within Setaria species.

Comparative genome analysis of the vineyard weed endophyte Pseudomonas viridiflava CDRTc14 showing selective herbicidal activity.

Microbes produce a variety of secondary metabolites to be explored for herbicidal activities. We investigated an endophyte Pseudomonas viridiflava CDRTc14, which impacted growth of its host Lepidium draba L., to better understand the possible genetic determinants for herbicidal and host-interaction traits. Inoculation tests with a variety of target plants revealed that CDRTc14 shows plant-specific effects ranging from beneficial to negative. Its herbicidal effect appeared to be dose-dependent and resembled phenotypically the germination arrest factor of Pseudomonas fluorescens WH6. CDRTc14 shares 183 genes with the herbicidal strain WH6 but the formylaminooxyvinylglycine (FVG) biosynthetic genes responsible for germination arrest of WH6 was not detected. CDRTc14 showed phosphate solubilizing ability, indole acetic acid and siderophores production in vitro and harbors genes for these functions. Moreover, genes for quorum sensing, hydrogen cyanide and ACC deaminase production were also found in this strain. Although, CDRTc14 is related to plant pathogens, we neither found a complete pathogenicity island in the genome, nor pathogenicity symptoms on susceptible plant species upon CDRTc14 inoculation. Comparison with other related genomes showed several unique genes involved in abiotic stress tolerance in CDRTc14 like genes responsible for heavy metal and herbicide resistance indicating recent adaptation to plant protection measures applied in vineyards.

Ecology and Genomic Insights into Plant-Pathogenic and Plant-Nonpathogenic Endophytes.

Plants are colonized on their surfaces and in the rhizosphere and phyllosphere by a multitude of different microorganisms and are inhabited internally by endophytes. Most endophytes act as commensals without any known effect on their plant host, but multiple bacteria and fungi establish a mutualistic relationship with plants, and some act as pathogens. The outcome of these plant-microbe interactions depends on biotic and abiotic environmental factors and on the genotype of the host and the interacting microorganism. In addition, endophytic microbiota and the manifold interactions between members, including pathogens, have a profound influence on the function of the system plant and the development of pathobiomes. In this review, we elaborate on the differences and similarities between nonpathogenic and pathogenic endophytes in terms of host plant response, colonization strategy, and genome content. We furthermore discuss environmental effects and biotic interactions within plant microbiota that influence pathogenesis and the pathobiome.

Shared and host-specific microbiome diversity and functioning of grapevine and accompanying weed plants.

Weeds and crop plants select their microbiota from the same pool of soil microorganisms, however, the ecology of weed microbiomes is poorly understood. We analysed the microbiomes associated with roots and rhizospheres of grapevine and four weed species (Lamium amplexicaule L., Veronica arvensis L., Lepidium draba L. and Stellaria media L.) growing in proximity in the same vineyard using 16S rRNA gene sequencing. We also isolated and characterized 500 rhizobacteria and root endophytes from L. draba and grapevine. Microbiome data analysis revealed that all plants hosted significantly different microbiomes in the rhizosphere as well as in root compartment, however, differences were more pronounced in the root compartment. The shared microbiome of grapevine and the four weed species contained 145 OTUs (54.2%) in the rhizosphere, but only nine OTUs (13.2%) in the root compartment. Seven OTUs (12.3%) were shared in all plants and compartments. Approximately 56% of the major OTUs (>1%) showed more than 98% identity to bacteria isolated in this study. Moreover, weed-associated bacteria generally showed a higher species richness in the rhizosphere, whereas the root-associated bacteria were more diverse in the perennial plants grapevine and L. draba. Overall, weed isolates showed more plant growth-promoting characteristics compared with grapevine isolates.

High-Quality Draft Genome Sequence of an Endophytic Pseudomonas viridiflava Strain with Herbicidal Properties against Its Host, the Weed Lepidium draba L.

Here, we report the draft genome sequence of Pseudomonas viridiflava strain CDRTc14 a pectinolytic bacterium showing herbicidal activity, isolated from the root of Lepidium draba L. growing as a weed in an Austrian vineyard. The availability of this genome sequence allows us to investigate the genetic basis of plant-microbe interactions.

The role of plant-microbiome interactions in weed establishment and control.

The soil microbiome plays an important role in the establishment of weeds and invasive plants. They associate with microorganisms supporting their growth and health. Weed management strategies, like tillage and herbicide treatments, to control weeds generally alter soil structure going alongside with changes in the microbial community. Once a weed population establishes in the field, the plants build up a close relationship with the available microorganisms. Seeds or vegetative organs overwinter in soil and select early in the season their own microbiome before crop plants start to vegetate. Weed and crop plants compete for light, nutrition and water, but may differently interact with soil microorganisms. The development of new sequencing technologies for analyzing soil microbiomes has opened up the possibility for in depth analysis of the interaction between 'undesired' plants and crop plants under different management systems. These findings will help us to understand the functions of microorganisms involved in crop productivity and plant health, weed establishment and weed prevention. Exploitation of the knowledge offers the possibility to search for new biocontrol methods against weeds based on soil and plant-associated microorganisms. This review discusses the recent advances in understanding the functions of microbial communities for weed/invasive plant establishment and shows new ways to use plant-associated microorganisms to control weeds and invasive plants in different land management systems.

Transcriptome Profiling of the Endophyte Burkholderia phytofirmans PsJN Indicates Sensing of the Plant Environment and Drought Stress.

UNLABELLED: It is widely accepted that bacterial endophytes actively colonize plants, interact with their host, and frequently show beneficial effects on plant growth and health. However, the mechanisms of plant-endophyte communication and bacterial adaption to the plant environment are still poorly understood. Here, whole-transcriptome sequencing of B. phytofirmans PsJN colonizing potato (Solanum tuberosum L.) plants was used to analyze in planta gene activity and the response of strain PsJN to plant stress. The transcriptome of PsJN colonizing in vitro potato plants showed a broad array of functionalities encoded in the genome of strain PsJN. Transcripts upregulated in response to plant drought stress were mainly involved in transcriptional regulation, cellular homeostasis, and the detoxification of reactive oxygen species, indicating an oxidative stress response in PsJN. Genes with modulated expression included genes for extracytoplasmatic function (ECF) group IV sigma factors. These cell surface signaling elements allow bacteria to sense changing environmental conditions and to adjust their metabolism accordingly. TaqMan quantitative PCR (TaqMan-qPCR) was performed to identify ECF sigma factors in PsJN that were activated in response to plant stress. Six ECF sigma factor genes were expressed in PsJN colonizing potato plants. The expression of one ECF sigma factor was upregulated whereas that of another one was downregulated in a plant genotype-specific manner when the plants were stressed. Collectively, our study results indicate that endophytic B. phytofirmans PsJN cells are active inside plants. Moreover, the activity of strain PsJN is affected by plant drought stress; it senses plant stress signals and adjusts its gene expression accordingly. IMPORTANCE: In recent years, plant growth-promoting endophytes have received steadily growing interest as an inexpensive alternative to resource-consuming agrochemicals in sustainable agriculture. Even though promising effects are recurrently observed under controlled conditions, these are rarely reproducible in the field or show undesirably strong variations. Obviously, a better understanding of endophyte activities in plants and the influence of plant physiology on these activities is needed to develop more-successful application strategies. So far, research has focused mainly on analyzing the plant response to bacterial inoculants. This prompted us to study the gene expression of the endophyte Burkholderia phytofirmans PsJN in potato plants. We found that endophytic PsJN cells express a wide array of genes and pathways, pointing to high metabolic activity inside plants. Moreover, the strain senses changes in the plant physiology due to plant stress and adjusts its gene expression pattern to cope with and adapt to the altered conditions.

Multi-laboratory evaluation of the rapid genoserotyping array (SGSA) for the identification of Salmonella serovars.

Salmonella serotyping is an essential first step for identification of isolates associated with disease outbreaks. The Salmonella genoserotyping array (SGSA) is a microarray-based alternative to standard serotyping designed to rapidly identify 57 of the most commonly reported serovars through detection of the genes encoding surface O and H antigens and reporting the corresponding serovar in accordance with the existing White-Kaufmann-Le Minor serotyping scheme. In this study, we evaluated the SGSA at 4 laboratories in 3 countries by testing 1874 isolates from human and non-human sources. The SGSA correctly identified 96.7% of isolates from the target 57 serovars. For the prevalent and clinically important Salmonella serovars Enteritidis and Typhimurium, test specificity and sensitivity were greater than 98% and 99%, respectively. Due to its high-throughput nature, the SGSA is a rapid and cost-effective alternative to standard serotyping for identifying the most prevalent serovars of Salmonella.

Metabolic potential of endophytic bacteria.

The bacterial endophytic microbiome promotes plant growth and health and beneficial effects are in many cases mediated and characterized by metabolic interactions. Recent advances have been made in regard to metabolite production by plant microsymbionts showing that they may produce a range of different types of metabolites. These substances play a role in defense and competition, but may also be needed for specific interaction and communication with the plant host. Furthermore, few examples of bilateral metabolite production are known and endophytes may modulate plant metabolite synthesis as well. We have just started to understand such metabolic interactions between plants and endophytes, however, further research is needed to more efficiently make use of beneficial plant-microbe interactions and to reduce pathogen infestation as well as to reveal novel bioactive substances of commercial interest.

Organ-specific defence strategies of pepper (Capsicum annuum L.) during early phase of water deficit.

Drought is one of the major factors that limits crop production and reduces yield. To understand the early response of plants under nearly natural conditions, pepper plants (Capsicum annuum L.) were grown in a greenhouse and stressed by withholding water for 1 week. Plants adapted to the decreasing water content of the soil by adjustment of their osmotic potential in root tissue. As a consequence of drought, strong accumulation of raffinose, glucose, galactinol and proline was detected in the roots. In contrast, in leaves the levels of fructose, sucrose and also galactinol increased. Due to the water deficit cadaverine, putrescine, spermidine and spermine accumulated in leaves, whereas the concentration of polyamines was reduced in roots. To study the molecular basis of these responses, a combined approach of suppression subtractive hybridisation and microarray technique was performed on the same material. A total of 109 unique ESTs were detected as responsive to drought, while additional 286 ESTs were selected from the bulk of rare transcripts on the array. The metabolic profiles of stressed pepper plants are discussed with respect to the transcriptomic changes detected, while attention is given to the differences between defence strategies of roots and leaves.

Components of the gene network associated with genotype-dependent response of wheat to the Fusarium mycotoxin deoxynivalenol.

The Fusarium mycotoxin deoxynivalenol (DON) facilitates fungal spread within wheat tissue and the development of Fusarium head blight disease. The ability of wheat spikelets to resist DON-induced bleaching is genotype-dependent. In wheat cultivar (cv.) CM82036 DON resistance is associated with a quantitative trait locus, Fhb1, located on the short arm of chromosome 3B. Gene expression profiling (microarray and real-time RT-PCR analyses) of DON-treated spikelets of progeny derived from a cross between cv. CM82036 and the DON-susceptible cv. Remus discriminated ten toxin-responsive transcripts associated with the inheritance of DON resistance and Fhb1. These genes do not exclusively map to Fhb1. Based on the putative function of the ten Fhb1-associated transcripts, we discuss how cascades involving classical metabolite biotransformation and sequestration processes, alleviation of oxidative stress and promotion of cell survival might contribute to the host response and defence against DON.

Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping.

BACKGROUND: The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs). This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge. RESULTS: We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes. CONCLUSION: The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques. We show that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles.

Survey of resistance gene analogs in Solanum caripense, a relative of potato and tomato, and update on R gene genealogy.

The resistance (R) proteins of the TIR- and non-TIR (or CC-) superfamilies possess a nucleotide binding site (NBS) domain. Within an R gene, the NBS is the region of highest conservation, suggesting an essential role in triggering R protein activity. We compared the NBS domain of functional R genes and resistance gene analogs (RGA) amplified from S. caripense genomic DNA via PCR using specific and degenerate primers with its counterpart from other plants. An overall high degree of sequence conservation was apparent throughout the P-loop, kinase-2 and kinase-3a motifs of NBS fragments from all plants. Within the non-TIR class of R genes a prominent sub-class similar to the potato R1 gene conferring resistance to late blight, was detected. All non-TIR-R1-like R gene fragments that were sequenced possessed an intact open reading frame, whereas 22% of all non-TIR-non-R1-like fragments and 59% of all TIR-NBS RGA fragments had an interrupted reading frame or contained transposon-specific sequence. The non-TIR-R1-like fragments had high similarity to Solanaceae R genes and low similarity to RGAs of other plant species including A. thaliana and the cereals. It is concluded that appearance of the non-TIR-R1-like NBS domain represents a relatively recent evolutionary development.

Plant defense genes associated with quantitative resistance to potato late blight in Solanum phureja x dihaploid S. tuberosum hybrids.

Markers corresponding to 27 plant defense genes were tested for linkage disequilibrium with quantitative resistance to late blight in a diploid potato population that had been used for mapping quantitative trait loci (QTLs) for late blight resistance. Markers were detected by using (i) hybridization probes for plant defense genes, (ii) primer pairs amplifying conserved domains of resistance (R) genes, (iii) primers for defense genes and genes encoding transcriptional regulatory factors, and (iv) primers allowing amplification of sequences flanking plant defense genes by the ligation-mediated polymerase chain reaction. Markers were initially screened by using the most resistant and susceptible individuals of the population, and those markers showing different allele frequencies between the two groups were mapped. Among the 308 segregating bands detected, 24 loci (8%) corresponding to six defense gene families were associated with resistance at chi2 > or = 13, the threshold established using the permutation test at P = 0.05. Loci corresponding to genes related to the phenylpropanoid pathway (phenylalanine ammonium lyase [PAL], chalcone isomerase [CHI], and chalcone synthase [CHS]), loci related to WRKY regulatory genes, and other -defense genes (osmotin and a Phytophthora infestans-induced cytochrome P450) were significantly associated with quantitative disease resistance. A subset of markers was tested on the mapping population of 94 individuals. Ten defense-related markers were clustered at a QTL on chromosome III, and three defense-related markers were located at a broad QTL on chromosome XII. The association of candidate genes with QTLs is a step toward understanding the molecular basis of quantitative resistance to an important plant disease.