Population genetics structure of glyphosate-resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance.
ABSTRACT: Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.
Project description:Six Johnsongrass populations suspected of being glyphosate resistant were collected from railways and freeways near Cordoba (SW Spain), where glyphosate is the main weed control tool. The 50% reduction in shoot fresh weight (GR50) values obtained for these six populations ranged from 550.4 to 1169 g ae ha-1, which were 4.2 to 9 times greater than the value obtained for the susceptible population. Glyphosate was equally metabolized to the same extent in both resistant and susceptible populations, with no significant differences in either 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibition or basal activity. No amino acid substitutions were observed in any of the resistant populations. Slight but significant differences in glyphosate penetration were observed among some but not all of the resistant populations and for the times of incubation assayed, although these differences were not considered further. The proposed primary mechanism of resistance in these six glyphosate-resistant Johnsongrass populations is reduced herbicide translocation, because the amount of glyphosate that translocated from treated leaves to shoots and roots in the susceptible population was double that observed in the resistant populations. As glyphosate multiple resistance due to more than one mechanism is not uncommon, this is the first time that glyphosate-resistant Johnsongrass populations have been fully described for all known mechanisms.
Project description:From noble beginnings as a prospective forage, polyploid Sorghum halepense ('Johnsongrass') is both an invasive species and one of the world's worst agricultural weeds. Formed by S. bicolor x S. propinquum hybridization, we show S. halepense to have S. bicolor-enriched allele composition and striking mutations in 5,957 genes that differentiate it from representatives of its progenitor species and an outgroup. The spread of S. halepense may have been facilitated by introgression from closely-related cultivated sorghum near genetic loci affecting rhizome development, seed size, and levels of lutein, a photochemical protectant and abscisic acid precursor. Rhizomes, subterranean stems that store carbohydrates and spawn clonal propagules, have growth correlated with reproductive rather than other vegetative tissues, and increase survival of both temperate cold seasons and tropical dry seasons. Rhizomes of S. halepense are more extensive than those of its rhizomatous progenitor S. propinquum, with gene expression including many alleles from its non-rhizomatous S. bicolor progenitor. The first surviving polyploid in its lineage in ?96 million years, its post-Columbian spread across six continents carried rich genetic diversity that in the United States has facilitated transition from agricultural to non-agricultural niches. Projected to spread another 200-600 km northward in the coming century, despite its drawbacks S. halepense may offer novel alleles and traits of value to improvement of sorghum.
Project description:Weeds are one of the main causes of the decrease in crop yields, with Johnsongrass (<i>Sorghum halepense</i> L.) being one of the most significant. Weeds can be controlled by herbicides, but some have developed resistance. Quantitative PCR is the technique of choice for studying gene expression related to herbicide resistance because of its high sensitivity and specificity, although its quantitative accuracy is highly dependent on the stability of the reference genes. Thus, in this study we evaluated the stability of different reference genes of glyphosate-resistant <i>S. halepense</i>. Nine genes frequently used as reference genes were selected: MDH, ADP, PP2A, EIF4α, ACT, ARI8, DnaJ, Hsp70, and ALS1, and their expression analyzed in susceptible and resistant biotypes at 0, 24 and 72 h post-application of glyphosate. The stability was analyzed with the geNorm, NormFinder, and BestKeeper software programs and using the ΔCt method. RefFinder was used to generate a comprehensive stability ranking. The results showed that PP2A and ARI8 were the most stable genes under the test conditions. EPSPS expression was also verified against the best two and the worst two reference genes. This study provides useful information for gene expression analysis under glyphosate stress and will facilitate resistance mechanism studies in this weed species.
Project description: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.
Project description:Rhizomes facilitate the wintering and vegetative propagation of many perennial grasses. Sorghum halepense (johnsongrass) is an aggressive perennial grass that relies on a robust rhizome system to persist through winters and reproduce asexually from its rootstock nodes. This study aimed to sequence and assemble expressed transcripts within the johnsongrass rhizome. A de novo transcriptome assembly was generated from a single johnsongrass rhizome meristem tissue sample. A total of 141,176 probable protein-coding sequences from the assembly were identified and assigned gene ontology terms using Blast2GO. Estimated expression analysis and BLAST results were used to reduce the assembly to 64,447 high-confidence sequences. The johnsongrass assembly was compared to Sorghum bicolor, a related nonrhizomatous species, along with an assembly of similar rhizome tissue from the perennial grain crop Thinopyrum intermedium. The presence/absence analysis yielded a set of 98 expressed johnsongrass contigs that are likely associated with rhizome development.
Project description:The National Plant Germplasm System (NPGS) Ethiopian sorghum [Sorghum bicolor (L.) Moench] collection of the United States is an important genetic resource for sorghum improvement. Anthracnose (Colletotrichum sublineolum) is one of the most harmful fungal diseases in humid sorghum production regions. Although multiple resistance sources have been identified in temperate-adapted germplasm in the Sorghum Association Panel (SAP), these resistance loci explain a limited portion of the total variation, and sources of resistance from tropical germplasm are not available for breeding programs at temperate regions. Using a core set of 335 previously genotyped NPGS Ethiopian accessions, we identified 169 accessions resistant to anthracnose. To identify resistance loci, we merged the genotypic and anthracnose response data for both NPGS Ethiopian germplasm and the SAP and performed genome-wide association scans using 219,037 single nucleotide polymorphisms and 617 accessions. The integrated data set enabled the detection of a locus on chromosome 9 present in the SAP at a low frequency. The locus explains a limited portion of the observed phenotypic variation (r2 = 0.31), suggesting the presence of other resistance loci. The locus in chromosome 9 was constituted by three R genes clustered within a 47-kb region. The presence of multiple sources of resistance in NPGS Ethiopian germplasm and SAP requires the inclusion of other resistance response evaluation that could revealed others low frequency resistance alleles in the panel.
Project description:Invasive plant species often dominate native species in competition, augmenting other potential advantages such as release from natural enemies. Resource pre-emption may be a particularly important mechanism for establishing dominance over competitors of the same functional type. We hypothesized that competitive success of an exotic grass against native grasses is mediated by establishing an early size advantage. We tested this prediction among four perennial C4 warm-season grasses: the exotic weed Johnsongrass (Sorghum halepense), big bluestem (Andropogon gerardii), little bluestem (Schizachyrium scoparius) and switchgrass (Panicum virgatum). We predicted that a) the competitive effect of Johnsongrass on target species would be proportional to their initial biomass difference, b) competitive effect and response would be negatively correlated and c) soil fertility would have little effect on competitive relationships. In a greenhouse, plants of the four species were grown from seed either alone or with one Johnsongrass neighbor at two fertilizer levels and periodically harvested. The first two hypotheses were supported: The seedling biomass of single plants at first harvest (50 days after seeding) ranked the same way as the competitive effect of Johnsongrass on target species: Johnsongrass < big bluestem < little bluestem/switchgrass, while Johnsongrass responded more strongly to competition from Johnsongrass than from native species. At final harvest, native plants growing with Johnsongrass attained between 2-5% of their single-plant non-root biomass, while Johnsongrass growing with native species attained 89% of single-plant non-root biomass. Fertilization enhanced Johnsongrass' competitive effects on native species, but added little to the already severe competitive suppression. Accelerated early growth of Johnsongrass seedlings relative to native seedlings appeared to enable subsequent resource pre-emption. Size-asymmetric competition and resource-pre-emption may be a critical mechanism by which exotic invasive species displace functionally similar native species and alter the functional dynamics of native communities.
Project description:Sclerotinia stem rot (SSR) caused by the necrotrophic fungus <i>Sclerotinia sclerotiorum</i> is a major disease in rapeseed (<i>Brassica napus</i>) worldwide. Breeding for SSR resistance in <i>B. napus</i>, as in other crops, relies only on germplasms with quantitative resistance genes. A better understanding of the genetic basis for SSR resistance in <i>B. napus</i> thus holds promise for the genetic improvement of disease resistance. In the present study, a genome-wide association study (GWAS) for SSR resistance in <i>B. napus</i> were performed using an association panel of 448 accessions genotyped with the <i>Brassica</i> 60K Infinium<sup>®</sup> single-nucleotide polymorphism (SNP) array. A total of 26 SNPs corresponding to three loci, <i>DSRC4</i>, <i>DSRC6</i>, and <i>DSRC8</i> were associated with SSR resistance. Haplotype analysis showed that the three favorable alleles for SSR resistance exhibited cumulative effects. After aligning SSR resistance quantitative trait loci (QTL) identified in the present and previous studies to the <i>B. napus</i> reference genome, one locus (<i>DSRC6</i>) was found to be located within the confidence interval of a QTL identified in previous QTL mapping studies and another two loci (<i>DSRC4</i> and <i>DSRC8</i>) were considered novel loci for SSR resistance. A total of 39 candidate genes were predicted for the three loci based on the GWAS combining with the differentially expressed genes identified in previous transcriptomics analyses.
Project description:Centipedegrass (Eremochloa ophiuroides (Munro) Hack.) is a perennial, warm-season C4 grass species that shows great potential for use as a low-maintenance turfgrass species in tropical and subtropical regions. However, limited genetic and genomic information is available for this species, which has impeded systematic studies on the enhancement of its turf quality and resistance against biotic and abiotic stress. In this study, Illumina HiSeq high-throughput sequencing technology was performed to generate centipedegrass transcriptome sequences. A total of 352,513 assembled sequences were used to search for simple sequence repeat (SSR) loci, and 64,470 SSR loci were detected in 47,638 SSR containing sequences. The tri-nucleotides were the most frequent repeat motif, followed by di-nucleotides, tetra-nucleotides hexnucleotides, and pentanucleotides. A total of 48,061 primer pairs were successfully designed in the flanking sequences of the SSRs, and 100 sets of primers were randomly selected for the initial validation in four centipedegrass accessions. In total, 56 (56.0%) of the 100 primer pairs tested successfully amplified alleles from all four centipedegrass accessions, while 50 were identified as polymorphic markers and were then used to assess the level of genetic diversity among 43 centipedegrass core collections. The genetic diversity analysis exhibited that the number of alleles (Na) per locus ranged from 3 to 13, and the observed heterozygosity (Ho) ranged from 0.17 to 0.83. The polymorphism information content (PIC) value of the markers ranged from 0.15 to 0.78, and the genetic distances (coefficient Nei72) between the accessions varied from 0.07 to 0.48. The UPGMA-based dendrogram clustered all 43 core collections into two main groups and six subgroups, which further validated the effectiveness of these newly developed SSR markers. Hence, these newly developed SSR markers will be valuable and potentially useful for future genetic and genomic studies of E. ophiuroides.
Project description:Coconut (<i>Cocos nucifera</i> L.) is an important economic crop in tropical countries. However, the lack of a complete reference genome and the limitations of usable DNA markers hinder genomic studies and the molecular breeding of coconut. Here, we present the results of simple sequence repeat (SSR) mining from a high-throughput genotyping-by-sequencing (GBS) study of a collection of 38 coconut accessions. A total of 22,748 SSRs with di-, tri-, tetra-, penta- and hexanucleotide repeats of five or more were identified, 2451 of which were defined as polymorphic loci based on locus clustering in 38 coconut accessions, and 315 loci were suitable for the development of SSR markers. One hundred loci were selected, and primer pairs for each SSR locus were designed and validated in 40 coconut accessions. The analysis of 74 polymorphic markers identified between 2 and 9 alleles per locus, with an average of 3.01 alleles. The assessment of the genetic diversity and genetic relationships among the 40 coconut varieties based on the analysis of population structure, principal coordinate analysis (PCoA), and phylogenetic tree analysis using the 74 polymorphic SSR markers revealed three main groups of coconuts in Thailand. The identified SSR loci and SSR markers developed in this study will be useful for the study of coconut diversity and molecular breeding. The SSR mining approach used in this study could be applied to other plant species with a complex genome regardless of the availability of reference genome.