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Background: Seed morphology and color are critical agronomic traits in Medicago spp., reflecting adaptations to diverse environments and influencing seedling establishment and vigor. Understanding the interplay between seed traits, geographic origin, and genetic diversity is crucial for effective germplasm conservation and breeding. This study presents a comprehensive analysis of these factors in a diverse collection of Medicago accessions, leveraging machine learning to illuminate these complex relationships. Results: We analyzed seed size, shape, and color data from 318 Medicago accessions representing 29 species/subspecies from 31 countries. Machine learning models, including Neural Boost, Bootstrap Forest, and Support Vector Machines, effectively classified accessions based on seed traits and geographic origin, achieving up to 80% accuracy. Seed size was accurately predicted (R-squared > 0.80) using a combination of species, geographic origin, and shape descriptors. Hierarchical clustering of 189 M. sativa accessions based on 8,565 SNP markers revealed 20 distinct genetic clusters, indicating substantial population structure. A machine learning-based genome-wide association (GWA) analysis identified SNPs on chromosomes 1, 6, and 8 with high importance for predicting geographic origin. Notably, the most significant SNPs were located in or near genes involved in stress response and genome stability, suggesting their potential role in local adaptation. Finally, we successfully imputed missing M. sativa SNP genotypes using multiple machine learning approaches, achieving over 70% accuracy overall and over 80% for individual nucleotides (A, T, C, G), enhancing the utility of genomic datasets with missing data. Conclusions: Our integrated analysis of phenotypic, genetic, and geographic data, coupled with a machine learning-based GWAS approach, provides valuable insights into the diverse patterns within Medicago spp. We demonstrate the power of machine learning for germplasm characterization, trait prediction, and imputation of missing genomic data. These findings have significant implications for seed trait improvement, germplasm management, and understanding adaptation in Medicago and other diverse crop species. The identified candidate genes associated with geographic origin provide a foundation for future investigations into the functional mechanisms of local adaptation. Furthermore, our imputation method offers a valuable data for maximizing the utility of genomic resources in Medicago and other species. [ABSTRACT FROM AUTHOR]

Background: Seed morphology and color are critical agronomic traits in Medicago spp., reflecting adaptations to diverse environments and influencing seedling establishment and vigor. Understanding the interplay between seed traits, geographic origin, and genetic diversity is crucial for effective germplasm conservation and breeding. This study presents a comprehensive analysis of these factors in a diverse collection of Medicago accessions, leveraging machine learning to illuminate these complex relationships. Results: We analyzed seed size, shape, and color data from 318 Medicago accessions representing 29 species/subspecies from 31 countries. Machine learning models, including Neural Boost, Bootstrap Forest, and Support Vector Machines, effectively classified accessions based on seed traits and geographic origin, achieving up to 80% accuracy. Seed size was accurately predicted (R-squared > 0.80) using a combination of species, geographic origin, and shape descriptors. Hierarchical clustering of 189 M. sativa accessions based on 8,565 SNP markers revealed 20 distinct genetic clusters, indicating substantial population structure. A machine learning-based genome-wide association (GWA) analysis identified SNPs on chromosomes 1, 6, and 8 with high importance for predicting geographic origin. Notably, the most significant SNPs were located in or near genes involved in stress response and genome stability, suggesting their potential role in local adaptation. Finally, we successfully imputed missing M. sativa SNP genotypes using multiple machine learning approaches, achieving over 70% accuracy overall and over 80% for individual nucleotides (A, T, C, G), enhancing the utility of genomic datasets with missing data. Conclusions: Our integrated analysis of phenotypic, genetic, and geographic data, coupled with a machine learning-based GWAS approach, provides valuable insights into the diverse patterns within Medicago spp. We demonstrate the power of machine learning for germplasm characterization, trait prediction, and imputation of missing genomic data. These findings have significant implications for seed trait improvement, germplasm management, and understanding adaptation in Medicago and other diverse crop species. The identified candidate genes associated with geographic origin provide a foundation for future investigations into the functional mechanisms of local adaptation. Furthermore, our imputation method offers a valuable data for maximizing the utility of genomic resources in Medicago and other species. [ABSTRACT FROM AUTHOR]

Coffea stenophylla is a rare Coffea species boasting a flavor profile comparable to Arabica coffee (Coffea arabica) and has a good adaptability to lowland tropical climates. This species faces increasing threats from climate change, deforestation, and habitat fragmentation in its West African homeland. Using 1037 novel SNP markers derived from Genotyping-by-Sequencing (GBS), we revealed the presence of three distinct natural populations (mean Fst = 0.176) in Sierra Leone. Evidence of recent bottlenecks and small effective population size (118–140) was found across all three populations, reflecting the impact of recent anthropogenic disturbances on this species. Using a model-flexible inference approach, we unveiled a strong ancient bottleneck approximately 23,000 years ago, coinciding with the last glacial maximum (LGM), followed by post-glacial expansion and divergence into distinct genetic clusters. A comparative analysis between ex situ genebanks and natural populations detected a significant gap in genetic diversity, with two out of three natural populations missing from the ex situ genebank collection. These findings highlight the urgent need to improve conservation practices for C. stenophylla in Sierra Leone. The novel SNP markers developed in this study provided valuable tools to support future efforts in conservation and utilization of C. stenophylla genetic resources in West Africa. [ABSTRACT FROM AUTHOR]

Anthracnose and grain mold are two of the most significant diseases of sorghum, a versatile crop that plays an important part in the daily lives of millions of inhabitants, especially in the drier tropical regions. The aim of this study was to determine the influence of four agronomic traits in selected sorghum germplasms on the two diseases using Spearman's ρ test to identify significant pairwise correlations. Both anthracnose and grain mold scores were significantly and negatively correlated with seed weight and germination rate. The grain mold infection score also demonstrated negative correlations with plant height (Spearman ρ = −0.61 and p-value = <0.0001) and panicle length (Spearman ρ = −0.27 and p-value = 0.0022). In this investigation, principal component analysis and clustering variables analysis revealed that seed weight and germination rate exhibited a directional alignment, suggesting a positive association. Similarly, panicle length and plant height clustered together, suggesting a shared variation pattern. Additionally, a support vector machine and random forest models effectively predicted the germination rate based on the studied traits, highlighting the potential of machine learning in understanding complex trait relationships in sorghum. This work provides insights into the relationship between agronomic traits and disease resistance, thus contributing to sorghum improvement efforts. [ABSTRACT FROM AUTHOR]

Sorghum (Sorghum bicolor L.) ranks fifth as the most crucial cereal crop globally, yet its seed morphology remains relatively unexplored. This study investigated seed morphology in sorghum based on 115 mini-core and 130 Senegalese germplasms. Eight seed morphology traits encompassing size, shape, and color parameters were assessed. Statistical analyses explored potential associations between these traits and resistance to three major sorghum diseases: anthracnose, head smut, and downy mildew. Furthermore, genome-wide association studies (GWAS) were conducted using phenotypic data from over 24,000 seeds and over 290,000 publicly available single nucleotide polymorphisms (SNPs) through the Genome Association and Prediction Integrated Tool (GAPIT) R package. Significant SNPs associated with various seed morphology traits were identified and mapped onto the reference sorghum genome to identify novel candidate defense genes. [ABSTRACT FROM AUTHOR]

A newly documented pathotype 5 of the soil-borne fungus Sporisorium reilianum, causing head smut in sorghum, was tested against 153 unexplored Senegalese sorghum accessions. Among the 153 sorghum accessions tested, 63 (41%) exhibited complete resistance, showing no signs of infection by the fungus. The remaining 90 accessions (59%) displayed varying degrees of susceptibility. Sorghum responses against S. reilianum were explored to analyze the potential link with previously known seed morphology-related traits and new phenotype data from 59 lines for seed weight. A genome-wide association study (GWAS) screened 297,876 SNPs and identified highly significant associations (p < 1 × 10−5) with head smut resistance in sorghum. By mapping these significant SNPs to the reference genome, this study revealed 35 novel candidate defense genes potentially involved in disease resistance. [ABSTRACT FROM AUTHOR]

Sporisorium reilianum, the causal agent of sorghum (Sorghum bicolor (L.) Moench) head smut, is present in most sorghum-producing regions. This seed replacement fungal disease can reduce yield by up to 80% in severely infected fields. Management of this disease can be challenging due to the appearance of different pathotypes within the pathogenic population. In this research, the genetic variability and pathogenicity of isolates collected from five Texas Counties was conducted. Due to the lack of available space, 21 out of 32 sequenced isolates were selected and evaluated for virulence patterns on the six sorghum differentials, Tx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), Tx414, and BTx643. The results reveal the occurrence of a new pathotype, 1A, and four previously documented US pathotypes when the 21 isolates were evaluated for virulence patterns on the differentials. The most prevalent was pathotype 5, which was recovered from Brazos, Hidalgo, Nueces, and Willacy Counties, Texas. This pathotype was followed by 1A and 6 in frequency of recovery. Pathotype 4 was identified only from isolates collected from Hidalgo County, while pathotype 1 was from Burleson County, Texas. It appeared that the previous US head smut pathotypes (2 and 3) are no longer common, and the new pathotypes, 1A, 5, and 6, are now predominant. The phylogenetic tree constructed from the single-nucleotide polymorphism (SNP) data through the neighbor-joining method showed high genetic diversity among the tested isolates. Some of the diverse clades among the tested isolates were independent of their sampled locations. Notably, HS37, HS49, and HS65 formed a clade and were classified as 1A in the virulence study, while HS 61 and HS 66, which were collected from Nueces County, were grouped and identified as pathotype 5. [ABSTRACT FROM AUTHOR]

Anthracnose, incited by Colletotrichum sublineola, is the most destructive foliar disease of sorghum and, under severe conditions, yield losses can exceed 80% on susceptible cultivars. The hyper-variable nature of the pathogen makes its management challenging despite the occurrence of several resistant sources. In this study, the genetic variability and pathogenicity of 140 isolates of C. sublineola, which were sequenced using restriction site-associated sequencing (RAD-Seq), resulted in 1244 quality SNPs. The genetic relationship based on the SNP data showed low to high genetic diversity based on isolates' origin. Isolates from Georgia and North Carolina were grouped into multiple clusters with some level of genetic relationships to each other. Even though some isolates from Texas formed a cluster, others clustered with isolates from Puerto Rico. The isolates from Puerto Rico showed scattered distribution, indicating the diverse nature of these isolates. A population structure and cluster analysis revealed that the genetic variation was stratified into eight populations and one admixture group. The virulence pattern of 30 sequenced isolates on 18 sorghum differential lines revealed 27 new pathotypes. SC748-5, SC112-14, and Brandes were resistant to all the tested isolates, while BTx623 was susceptible to all. Line TAM428 was susceptible to all the pathotypes, except for pathotype 26. Future use of the 18 differentials employed in this study, which contains cultivars/lines which have been used in the Americas, Asia, and Africa, could allow for better characterization of C. sublineola pathotypes at a global level, thus accelerating the development of sorghum lines with stable resistance to the anthracnose pathogen. [ABSTRACT FROM AUTHOR]

In Senegal, sorghum ranks third after millet and maize among dryland cereal production and plays a critical role in the daily lives of millions of inhabitants. Yet, the crop's productivity and profitability are hampered by biotic stresses, including Exserohilum turcicum, causing leaf blight. A total of 101 sorghum accessions collected from Niger and Senegal, SC748-5 and BTx623, were evaluated in three different environments (Kaymor, Kolda, and Ndiaganiao) in Senegal for their reactions against the leaf blight pathogen. The results showed that 11 out of the 101 accessions evaluated exhibited 100% incidence, and the overall mean incidence was 88.4%. Accession N15 had the lowest incidence of 50%. The overall mean severity was 31.6%, while accessions N15, N43, N38, N46, N30, N28, and N23 from Niger recorded the lowest severity levels, ranging from 15.5% to 25.5%. Accession N15 exhibited both low leaf blight incidence and severity, indicating that it may possess genes for resistance to E. turcicum. Also, the accessions evaluated in this study were sequenced. A GWAS identified six novel single-nucleotide polymorphisms (SNPs) associated with an average leaf blight incidence rate. The candidate genes were found in chromosomes 2, 3, 5, 8, and 9. Except for SNP locus S05_48064154, all five SNPs associated with the leaf blight incidence rate were associated with the plant defense and stress responses. In conclusion, the candidate genes identified could offer additional options for enhancing plant resistance against E. turcicum through plant breeding or gene editing. [ABSTRACT FROM AUTHOR]

Sorghum is considered the fifth most important crop in the world. Despite the potential value of Senegalese germplasm for various traits, such as resistance to fungal diseases, there is limited information on the study of sorghum seed morphology. In this study, 162 Senegalese germplasms were evaluated for seed area size, length, width, length-to-width ratio, perimeter, circularity, the distance between the intersection of length & width (IS) and center of gravity (CG), and seed darkness and brightness by scanning and analyzing morphology-related traits with SmartGrain software at the USDA-ARS Plant Science Research Unit. Correlations between seed morphology-related traits and traits associated with anthracnose and head smut resistance were analyzed. Lastly, genome-wide association studies were performed on phenotypic data collected from over 16,000 seeds and 193,727 publicly available single nucleotide polymorphisms (SNPs). Several significant SNPs were found and mapped to the reference sorghum genome to uncover multiple candidate genes potentially associated with seed morphology. The results indicate clear correlations among seed morphology-related traits and potential associations between seed morphology and the defense response of sorghum. GWAS analysis listed candidate genes associated with seed morphologies that can be used for sorghum breeding in the future. [ABSTRACT FROM AUTHOR]

Multivariate linear mixed models (mvLMMs) are widely applied for genome-wide association studies (GWAS) to detect genetic variants affecting multiple traits with correlations and/or different plant growth stages. Subsets of multiple sorghum populations, including the Sorghum Association Panel (SAP), the Sorghum Mini Core Collection and the Senegalese sorghum population, have been screened against various sorghum diseases such as anthracnose, downy mildew, grain mold and head smut. Still, these studies were generally performed in a univariate framework. In this study, we performed GWAS based on the principal components of defense-related multi-traits against the fungal diseases, identifying new potential SNPs (S04_51771351, S02_66200847, S09_47938177, S08_7370058, S03_72625166, S07_17951013, S04_66666642 and S08_51886715) associated with sorghum's defense against these diseases. [ABSTRACT FROM AUTHOR]

Sorghum is the fifth most important crop globally. Researching interactions between sorghum and fungal pathogens is essential to further elucidate plant defense mechanisms to biotic stress, which allows breeders to employ genetic resistance to disease. A variety of creative and useful inoculation and screening methods have been developed by sorghum pathologists to study major fungal diseases. As inoculation and screening methods can be keys for successfully conducting experiments, it is necessary to summarize the techniques developed by this research community. Among many fungal pathogens of sorghum, here we summarize inoculation and screening methods for five important fungal pathogens of sorghum: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina. The methods described within will be useful for researchers who are interested in exploring sorghum-fungal pathogen interactions. Finally, we discuss the latest biotechnologies and methods for studying plant-fungal pathogen interactions and their applicability to sorghum pathology. [ABSTRACT FROM AUTHOR]

Sporisorium reilianum is a fungal pathogen that causes head smut in sorghum. In addition to pathotypes (P) 1-4, P5 and P6 were identified recently. In this study, seedlings of Senegalese sorghum, comprising 163 accessions, were evaluated for response to Sporisorium reilianum. Teliospores of pathotype P5 of the pathogen in dilute agar were pipetted onto seedling shoots while still in soil, and inoculated seedlings were submerged under water at 4 days post-inoculation. Signs of infection (noticeable spots) on the first leaf were checked daily up to 6 days post submergence. A genome-wide association study (GWAS) was conducted using 193,727 single-nucleotide polymorphisms (SNPs) throughout the genome based on two types of phenotypic data: whether noticeable spots were shown or not and the average time for an observation of the spots across 163 accessions. When mapped back to the reference sorghum genome, most of the top candidate SNP loci were associated with plant defense or plant stress response-related genes. The identified SNP loci were associated with spot appearance in sorghum seedlings under flooding following inoculation with P5 of Sporisorium reilianum. [ABSTRACT FROM AUTHOR]

Colletotrichum sublineola is a destructive fungal pathogen that causes anthracnose in sorghum. Senegalese sorghum germplasm is currently being considered as an option of sources for genetic resistance. In a recent study, Senegalese sorghum accessions were evaluated for response to a mixture of Texas isolates of C. sublineola at the 8-leaf stage in the greenhouse. As a comparison, 159 Senegalese sorghum accessions at the 1-leaf developmental stage were evaluated against a single Texas isolate of C. sublineola (FSP53) using an excised-leaf assay. A genome-wide association study (GWAS) was conducted based on the phenotypic data acquired to discover genetic variation associated with response to C. sublineola using 193,727 single nucleotide polymorphisms (SNPs) throughout the genome. Sorghum seedlings tended to be more resistant when compared with sorghum plants inoculated at the 8-leaf stage in the greenhouse in previous experiments. Based on the highest score evaluated in the 1-leaf developmental stage excised leaf assay for each accession, 16 accessions were labeled as susceptible. GWAS identified the SNP locus S01_72868925 that is associated with protein kinase domain // Leucine rich repeat N-terminal domain at a level of confidence that surpassed Bonferroni correction. Along with the SNP locus S01_72868925, other top SNP loci were also associated with genes that are known to play critical roles in plant defense or plant stress responses. [ABSTRACT FROM AUTHOR]

Sporisorium reilianum causes head smut in sorghum. A total of 36 Senegalese sorghum accessions comprised of sorghum lines that have not been explored with response to pathotype 5 of S. reilianum were evaluated with 3 different treatments. First, seedling shoots were inoculated while still in soil with teliospores in agar, and then submerged under water at 4 days post inoculation. Signs of infection (noticeable spots) on the first leaf were observed up to 6 days post submergence. Second, seedlings at the same stage were inoculated by placing the teliospore impregnated agar around the stem in pots, moved to a greenhouse and grown to full panicle development stage. Third, seedings were inoculated via syringe inoculation in the greenhouse. Although soil inoculated seedlings grown in the greenhouse did not result in systemic infection as determined by lack of symptoms at panicle exsertion, 88.9% of tested cultivars showed systemic infections when syringe inoculated in the greenhouse. Inoculation of seedlings maintained under water led to broad range of noticeable spots that are assumed to be potential infection sites based on a previous study. In addition, seedling inoculation led to slightly upregulated expression of chitinase and PR10, genes that are associated with defense in aerial parts of plants. [ABSTRACT FROM AUTHOR]

Gene expression was analyzed at 0- and 24-h post-inoculation of two inbred sorghum cultivars known to differ in response to inoculation with Colletotrichum sublineola , the fungal pathogen that causes anthracnose. QL3 is reported to have quantitative resistance, while Theis is susceptible to most pathotypes of the pathogen; RNASeq identified over 3,000 specific genes in both cultivars as showing significant changes in expression following inoculation; in all but one gene, the changes in QL3 and Thies were in the same direction. Many other genes showed significant changes in only one of the two cultivars. Overall, more genes were downregulated than upregulated. Differences in changes in expression levels of a few genes suggested potential roles for the difference in disease response between QL3 and Theis, but did not identify known resistance genes. Gene ontology (GO) and pathway enrichment analysis identified upregulation of 23 transcription factor encoding genes as well as genes involved in the production of secondary metabolites, which are part of a typical host defense reaction. [ABSTRACT FROM AUTHOR]

A total of 377 lines from Sorghum Association Panel (SAP) grouped into 10 subpopulations based on eight phenotypic traits, including flag leaf width, terminal branch length, flag leaf height, and flowering time and genetic data were evaluated for grain mold resistance by inoculating them with Alternaria alternata alone, a mixture of A. alternata, Fusarium thapsinum, and Curvularia lunata, and untreated water-sprayed control during 2010, 2013–2015 growing seasons at the Texas A&M AgriLife Research Farm, Burleson County, Texas. Each accession was evaluated at least twice. Eleven accessions exhibited grain mold severity ratings of 2 or less, indicating that they were resistant to highly resistant to the disease when challenged with A. alternata alone, 7 lines identified as such when inoculated with a mixture of A. alternata, F. thapsinum, and C. lunata, and 17 resistant lines were identified under the untreated control. Three lines PI533871, PI576130, and PI656036 exhibited resistant to highly resistant response to grain mold across treatments. When the 10 subpopulations were compared for disease response, accessions within subpopulation 4 had the lowest overall grain mold severity ratings, indicating that this subpopulation (zerazera/caudatum racial type) harbors genes that may contribute to grain mold resistance. A genome-wide association study with over 79,000 single-nucleotide polymorphic (SNP) loci from a publicly available genotype by sequencing dataset available for the (SAP) lines was conducted to identify genomic regions associated with grain mold resistance response based on the individual and combined treatments. In most cases, the top-scoring SNPs were mapped to the nearest or a nearby annotated gene with precedence for a role in host defense. [ABSTRACT FROM AUTHOR]

In previous studies, a sorghum mini core collection was scored over several years for response to Colletotrichum sublineola, Peronosclerospora sorghi, and Sporisorium reilianum, the causal agents of the disease anthracnose, downy mildew, and head smut, respectively. The screening results were combined with over 290,000 Single nucleotide polymorphic (SNP) loci from an updated version of a publicly available genotype by sequencing (GBS) dataset available for the mini core collection. GAPIT (Genome Association and Prediction Integrated Tool) R package was used to identify chromosomal locations that differ in disease response. When the top scoring SNPs were mapped to the most recent version of the published sorghum genome, in each case, a nearby and most often the closest annotated gene has precedence for a role in host defense. [ABSTRACT FROM AUTHOR]

Key message: SNPs identify prospective genes related to response to Colletotrichum sublineola (anthracnose) in the sorghum association panel lines. Sorghum association panel (SAP) lines were scored over several years for response to Colletotrichum sublineola, the causal agent of the disease anthracnose. Known resistant and susceptible lines were included each year to verify successful inoculation. Over 79,000 single-nucleotide polymorphic (SNP) loci from a publicly available genotype by sequencing dataset available for the SAP lines were used with TASSEL association mapping software to identify chromosomal locations associated with differences in disease response. When the top-scoring SNPs were mapped to the published sorghum genome, in each case, the nearest annotated gene has precedence for a role in host defense. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF, Glomeromycotina), in addition to forming symbioses with the majority of land plants, harbor vertically transmitted endosymbiotic bacteria ‘Candidatus Glomeribacter gigasporarum’ (CaGg) and ‘Candidatus Moeniiplasma glomeromycotorum’ (CaMg). CaGg is a nonessential mutualist of AMF, whereas the lifestyle of CaMg is unknown. To start unraveling the interactions between AMF and their endosymbionts in nature, we examined diversity and distribution of AMF-associated endobacteria in North Atlantic dunes at Cape Cod. Of nearly 500 foredune AMF isolates successfully genotyped during a systematic study, 94% were classified as Gigasporaceae. Two percent of all AMF spores harbored CaGg, and 88% contained CaMg. CaGg was found only in the Gigasporaceae, whereas CaMg was present in Gigasporaceae, Acaulosporaceae, and Diversisporaceae. Incidence of CaGg across AMF was not affected by any of the environmental parameters measured, whereas distribution of CaMg in one of the fungal hosts was impacted by plant density. CaMg populations associated with AMF individuals displayed high levels of genetic diversity but no evidence of gene flow, suggesting that host physical proximity is not sufficient to facilitate horizontal transmission of CaMg. Finally, in addition to a novel lineage of CaGg, we discovered that AMF likely harbor Burkholderia-related bacteria with close phylogenetic affinity to free-living Burkholderia and endobacteria of other Mucoromycota fungi. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]