GWAS analysis of sorghum association panel lines identifies SNPs associated with disease response to Texas isolates of Colletotrichum sublineola.

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.

Genome-Wide Association Analysis Uncovers Genes Associated with Resistance to Head Smut Pathotype 5 in Senegalese Sorghum Accessions.

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.

Genetic and Pathogenic Variability among Isolates of Sporisorium reilianum Causing Sorghum Head Smut.

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.

Analyzing Medicago spp. seed morphology using GWAS and machine learning.

Alfalfa is widely recognized as an important forage crop. To understand the morphological characteristics and genetic basis of seed morphology in alfalfa, we screened 318 Medicago spp., including 244 Medicago sativa subsp. sativa (alfalfa) and 23 other Medicago spp., 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 & red-green-blue (RGB) intensities. The results revealed phenotypic diversity and correlations among the tested accessions. Based on the phenotypic data of M. sativa subsp. sativa, a genome-wide association study (GWAS) was conducted using single nucleotide polymorphisms (SNPs) called against the Medicago truncatula genome. Genes in proximity to associated markers were detected, including CPR1, MON1, a PPR protein, and Wun1(threshold of 1E-04). Machine learning models were utilized to validate GWAS, and identify additional marker-trait associations for potentially complex traits. Marker S7_33375673, upstream of Wun1, was the most important predictor variable for red color intensity and highly important for brightness. Fifty-two markers were identified in coding regions. Along with strong correlations observed between seed morphology traits, these genes will facilitate the process of understanding the genetic basis of seed morphology in Medicago spp.

Multi-Trait Genome-Wide Association Studies of Sorghum bicolor Regarding Resistance to Anthracnose, Downy Mildew, Grain Mold and Head Smut.

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.

Ideotype breeding and genome engineering for legume crop improvement.

Ideotype breeding is a strategy whereby traits are modeled a priori and then introduced into a model or crop species to assess their impact on yield. Thus, knowledge about the connection between genotype and phenotype is required for ideotype breeding to be deployed successfully. The growing understanding of the genetic basis of yield-related traits, combined with increasingly efficient genome engineering tools, improved transformation efficiency, and high-throughput genotyping of regenerants paves the way for the widespread adoption of ideotype breeding as a complement to conventional breeding. We briefly discuss how ideotype breeding, coupled with such state-of-the-art biotechnological tools, could contribute to knowledge-based legume breeding and accelerate yield gains to ensure food security in the coming decades.

Inoculation and Screening Methods for Major Sorghum Diseases Caused by Fungal Pathogens: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina.

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.

Genome-Wide Association Study of Seed Morphology Traits in Senegalese Sorghum Cultivars.

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.

A Genome-Wide Association Study of Nigerien and Senegalese Sorghum Germplasm of Exserohilum turcicum, the Causal Agent of Leaf Blight.

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.

Genetic Diversity and Classification of Colletotrichum sublineola Pathotypes Using a Standard Set of Sorghum Differentials.

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.

Genome-wide association study of Senegalese sorghum seedlings responding to a Texas isolate of Colletotrichum sublineola.

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.

A Genome-Wide Association Study of Senegalese Sorghum Seedlings Responding to Pathotype 5 of Sporisorium reilianum.

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.

Genome-wide association analysis for response of Senegalese sorghum accessions to Texas isolates of anthracnose.

Anthracnose disease of sorghum is caused by Colletotrichum sublineola, a filamentous fungus. The genetic basis of resistance to anthracnose in sorghum is largely unclear, especially in Senegalese sorghum germplasm. In this study, 163 Senegalese sorghum accessions were evaluated for response to C. sublineola, and a genome-wide association study (GWAS) was performed to identify genetic variation associated with response to C. sublineola using 193,727 single nucleotide polymorphisms (SNPs) throughout the genome. Germplasm diversity analysis showed low genetic diversity and slow linkage disequilibrium (LD) decay among the Senegalese accessions. Phenotypic analysis resulted in relatively low differences to C. sublineola among the tested population. Genome-wide association study did not identify any significant association based on a strict threshold for the number of SNPs available. However, individual analysis of the top eight SNPs associated with relative susceptibility and resistance identified candidate genes that have been shown to play important roles in plant stress tolerance in previous studies. This study identifies sorghum genes whose annotated properties have known roles in host defense and thus identify them as candidates for use in breeding for resistance to anthracnose.

RNA-Sequencing in Resistant (QL3) and Susceptible (Theis) Sorghum Cultivars Inoculated With Johnsongrass Isolates of Colletotrichum sublineola.

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.

Leaf angle distribution in Johnsongrass, leaf thickness in sorghum and Johnsongrass, and association with response to Colletotrichum sublineola.

Basal leaf angle distribution was surveyed in twenty-one Johnsongrass cultivars near the end of the vegetative stage. The angles increased from the top to the bottom leaves, and compared to cultivated grain sorghums, the average angle was larger in Johnsongrass. When basal leaf angle distribution data were correlated with pathogenicity test data from excised-leaf assays for three isolates of Colletotrichum sublineola, the results showed a weak positive correlation between basal leaf angle and pathogenicity level in Johnsongrass. In order to investigate a protective role of leaf thickness to C. sublineola, leaf thickness was measured in three sorghum cultivars and one Johnsongrass cultivar at the 8-leaf-stage. Leaf thickness near the apex, near the base, and half-way between the two points were measured in the top four leaves of each plant. Thickness of leaf blade and midrib were recorded separately. Using an excised-leaf-assay, the three points were inoculated with C. sublineola, and pathogenicity level was recorded 4-days-post-inoculation. Results showed strong negative correlations between leaf midrib thickness and pathogenicity level in sorghum and Johnsongrass but not in leaf blades.

Genome wide association analysis of sorghum mini core lines regarding anthracnose, downy mildew, and head smut.

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.

Distribution and population structure of endobacteria in arbuscular mycorrhizal fungi at North Atlantic dunes.

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.