Transcriptome and metabolome analyses reveal regulatory networks associated with nutrition synthesis in sorghum seeds.

Cereal seeds are vital for food, feed, and agricultural sustainability because they store and provide essential nutrients to human and animal food and feed systems. Unraveling molecular processes in seed development is crucial for enhancing cereal grain yield and quality. We analyze spatiotemporal transcriptome and metabolome profiles during sorghum seed development in the inbred line 'BTx623'. Morphological and molecular analyses identify the key stages of seed maturation, specifying starch biosynthesis onset at 5 days post-anthesis (dpa) and protein at 10 dpa. Transcriptome profiling from 1 to 25 dpa reveal dynamic gene expression pathways, shifting from cellular growth and embryo development (1-5 dpa) to cell division, fatty acid biosynthesis (5-25 dpa), and seed storage compounds synthesis in the endosperm (5-25 dpa). Network analysis identifies 361 and 207 hub genes linked to starch and protein synthesis in the endosperm, respectively, which will help breeders enhance sorghum grain quality. The availability of this data in the sorghum reference genome line establishes a baseline for future studies as new pangenomes emerge, which will consider copy number and presence-absence variation in functional food traits.

Identification and characterization of a temperature sensitive chlorotic soybean mutant.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "vir1" phenotype results in smaller stature, weaker stems, and a smaller root system with smaller nodules. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells and RNA sequencing data indicates it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice homolog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean vir1 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild type Glyma.07G102300 in the knockout mutant of the Arabidopsis homolog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the vir1 phenotype.

Modeling the evolution of herbicide resistance in weed species with a complex life cycle.

A growing number of weed species have evolved resistance to herbicides in recent years, which causes an immense financial burden to farmers. An increasingly popular method of weed control is the adoption of crops that are resistant to specific herbicides, which allows farmers to apply the herbicide during the growing season without harming the crop. If such crops are planted in the presence of closely related weed species, it is possible that resistance genes could transfer from the crop species to feral populations of the wild species via gene flow and become stably introgressed under ongoing selective pressure by the herbicide. We use a density-dependent matrix model to evaluate the effect of planting such crops on the evolution of herbicide resistance under a range of management scenarios. Our model expands on previous simulation studies by considering weed species with a more complex life cycle (perennial, rhizomatous weed species), studying the effect of environmental variation in herbicide effectiveness, and evaluating the role of common simplifying genetic assumptions on resistance evolution. Our model predictions are qualitatively similar to previous modeling studies using species with a simpler life cycle, which is, crop rotation in combination with rotation of herbicide site of action effectively controls weed populations and slows the evolution of herbicide resistance. We find that ignoring the effect of environmental variation can lead to an over- or under-prediction of the speed of resistance evolution. The effect of environmental variation in herbicide effectiveness depends on the resistance allele frequency in the weed population at the beginning of the simulation. Finally, we find that degree of dominance and ploidy level have a much larger effect on the predicted speed of resistance evolution compared to the rate of gene flow.

De novo identification and targeted sequencing of SSRs efficiently fingerprints Sorghum bicolor sub-population identity.

Recent plant breeding studies of several species have demonstrated the utility of combining molecular assessments of genetic distance into trait-linked SNP genotyping during the development of parent lines to maximize yield gains due to heterosis. SSRs (Short Sequence Repeats) are the molecular marker of choice to determine genetic diversity, but the methods historically used to sequence them have been burdensome. The ability to analyze SSRs in a higher-throughput manner independent of laboratory conditions would increase their utility in molecular ecology, germplasm curation, and plant breeding programs worldwide. This project reports simple bioinformatics methods that can be used to generate genome-wide de novo SSRs in silico followed by targeted Next Generation Sequencing (NGS) validation of those that provide the most information about sub-population identity of a breeding line, which influences heterotic group selection. While these methods were optimized in sorghum [Sorghum bicolor (L.) Moench], they were developed to be applied to any species with a reference genome and high-coverage whole-genome sequencing data of individuals from the sub-populations to be characterized. An analysis of published sorghum genomes selected to represent its five main races (bicolor, caudatum, durra, kafir, and guinea; 75 accessions total) identified 130,120 SSR motifs. Average lengths were 23.8 bp and 95% were between 10 and 92 bp, making them suitable for NGS. Validation through targeted sequencing amplified 188 of 192 assayed SSR loci. Results highlighted the distinctness of accessions from the guinea sub-group margaritiferum from all other sorghum accessions, consistent with previous studies of nuclear and mitochondrial DNA. SSRs that efficiently fingerprinted margaritiferum individuals (Xgma1 -Xgma6) are presented. Developing similar fingerprints of other sub-populations (Xunr1 -Xunr182) was not possible due to the extensive admixture between them in the data set analyzed. In summary, these methods were able to fingerprint specific sub-populations when rates of admixture between them are low.

Physiological and molecular analysis of glyphosate resistance in non-rapid response Ambrosia trifida from Wisconsin.

BACKGROUND: We previously identified a glyphosate-resistant A. trifida phenotype from Wisconsin USA that showed a non-rapid response to glyphosate. The mechanism of glyphosate resistance in this phenotype has yet to be elucidated. We conducted experiments to investigate non-target-site resistance and target-site resistance mechanisms. The roles of glyphosate absorption, translocation, and metabolism in resistance of this phenotype have not been reported previously, nor have EPSPS protein abundance or mutations to the full-length sequence of EPSPS. RESULTS: Whole-plant dose-response results confirmed a 6.5-level of glyphosate resistance for the resistant (R) phenotype compared to a susceptible (S) phenotype. Absorption and translocation of 14 C-glyphosate were similar between R and S phenotypes over 72 h. Glyphosate and AMPA concentrations in leaf tissue did not differ between R and S phenotypes over 96 h. In vivo shikimate leaf disc assays confirmed that glyphosate EC50 values were 4.6- to 5.4-fold greater for the R than S phenotype. Shikimate accumulation was similar between phenotypes at high glyphosate concentrations (>1000 µM), suggesting that glyphosate entered chloroplasts and inhibited EPSPS. This finding was supported by results showing that EPSPS copy number and EPSPS protein abundance did not differ between R and S phenotypes, nor did EPSPS sequence at Gly101, Thr102, and Pro106 positions. Comparison of full-length EPSPS sequences found five nonsynonymous polymorphisms that differed between R and S phenotypes. However, their locations were distant from the glyphosate target site and, therefore, not likely to affect enzyme-glyphosate interaction. CONCLUSION: The results suggest that a novel mechanism confers glyphosate resistance in this A. trifida phenotype. © 2019 Society of Chemical Industry.

Response of Sweet Sorghum Lines to Stalk Pathogens Fusarium thapsinum and Macrophomina phaseolina.

Sweet sorghum (Sorghum bicolor (L.) Moench) has potential for bioenergy. It is adapted to a variety of U.S. locations and the extracted juice can be directly fermented into ethanol. However, little research on fungal stalk rots, diseases that pose serious constraints for yield and quality of juice and biomass, has been reported. A greenhouse bioassay was designed to assess charcoal rot (Macrophomina phaseolina) and Fusarium stalk rot (Fusarium thapsinum) in plants at maturity, the developmental stage at which these diseases are manifested. Multiple plantings of a susceptible grain line, RTx430, were used as a control for variation in flowering times among sweet sorghum lines. Lesion length measurements in inoculated peduncles were used to quantify disease severity. Sweet sorghum lines 'Rio' and 'M81E' exhibited resistance to F. thapsinum and M. phaseolina, respectively; and, in contrast, 'Colman' sorghum exhibited susceptibility to both pathogens. Lesion development over time in Colman was monitored. These results will enhance molecular and biochemical analyses of responses to pathogens, and breeding stalk-rot-resistant sweet sorghum lines.