Transformation and gene editing in the bioenergy grass Miscanthus.

BACKGROUND: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. RESULTS: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. CONCLUSIONS: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

Population Genomics of the Neotropical Brown Stink Bug, Euschistus heros: The Most Important Emerging Insect Pest to Soybean in Brazil.

Recent changes in soybean management like the adoption of transgenic crops and no-till farming, in addition to the expansion of cultivated areas into new virgin frontiers, are some of the hypotheses that can explain the rise of secondary pests, such as the Neotropical brown stink bug, Euschistus heros, in Brazil. To better access the risk of increased pests like E. heros and to determine probabilities for insecticide resistance spreading, it is necessary first to access the levels of the genetic diversity, how the genetic diversity is distributed, and how natural selection is acting upon the natural variation. Using the genotyping by sequencing (GBS) technique, we generated ~60,000 single-nucleotide polymorphisms (SNPs) distributed across the E. heros genome to answer some of those questions. The SNP data was used to investigate the pattern of genetic structure, hybridization and natural selection of this emerging pest. We found that E. heros populations presented similar levels of genetic diversity with slightly higher values at several central locations in Brazil. Our results also showed strong genetic structure separating northern and southern Brazilian regions (FST = 0.22; p-value = 0.000) with a very distinct hybrid zone at the central region. The analyses also suggest the possibility that GABA channels and odorant receptors might play a role in the process of natural selection. At least one marker was associated with soybean and beans crops, but no association between allele frequency and cotton was found. We discuss the implications of these findings in the management of emerging pests in agriculture, particularly in the context of large areas of monoculture such as soybean and cotton.

Patterns of Genome-Wide Variation, Population Differentiation and SNP Discovery of the Red Banded Stink Bug (Piezodorus guildinii).

Unravelling the details of range expansion and ecological dominance shifts of insect pests has been challenging due to the lack of basic knowledge about population structure, gene flow, and most importantly, how natural selection is affecting the adaptive process. Piezodous guildinii is an emerging pest of soybean in the southern region of the United States, and increasingly important in Brazil in recent years. However, the reasons P. guildinii is gradually becoming more of a problem are questions still mostly unanswered. Here, we have genotyped P. guildinii samples and discovered 1,337 loci containing 4,083 variant sites SNPs that were used to estimate genetic structure and to identify gene candidates under natural selection. Our results revealed the existence of a significant genetic structure separating populations according to their broad geographic origin, i.e., U.S. and Brazil, supported by AMOVA (FGT = 0.26), STRUCTURE, PCA, and FST analyses. High levels of gene flow or coancestry within groups (i.e., within countries) can be inferred from the data, and no spatial pattern was apparent at the finer scale in Brazil. Samples from different seasons show more heterogeneous compositions suggesting mixed ancestry and a more complex dynamic. Lastly, we were able to detect and successfully annotated 123 GBS loci (10.5%) under positive selection. The gene ontology (GO) analysis implicated candidate genes under selection with genome reorganization, neuropeptides, and energy mobilization. We discuss how these findings could be related to recent outbreaks and suggest how new efforts directed to better understand P. guildinii population dynamics.

Genome-wide association mapping of resistance to a Brazilian isolate of Sclerotinia sclerotiorum in soybean genotypes mostly from Brazil.

BACKGROUND: Sclerotinia Stem Rot (SSR), caused by the fungal pathogen Sclerotinia sclerotiorum, is ubiquitous in cooler climates where soybean crops are grown. Breeding for resistance to SSR remains challenging in crops like soybean, where no single gene provides strong resistance, but instead, multiple genes work together to provide partial resistance. In this study, a genome-wide association study (GWAS) was performed to dissect the complex genetic architecture of soybean quantitative resistance to SSR and to provide effective molecular markers that could be used in breeding programs. A collection of 420 soybean genotypes were selected based on either reports of resistance, or from one of three different breeding programs in Brazil, two commercial, one public. Plant genotype sensitivity to SSR was evaluated by the cut stem inoculation method, and lesion lengths were measured at 4 days post inoculation. RESULTS: Genotyping-by-sequencing was conducted to genotype the 420 soybean lines. The TASSEL 5 GBSv2 pipeline was used to call SNPs under optimized parameters, and with the extra step of trimming adapter sequences. After filtering missing data, heterozygosity, and minor allele frequency, a total of 11,811 SNPs and 275 soybean genotypes were obtained for association analyses. Using a threshold of FDR-adjusted p-values <0.1, the Compressed Mixed Linear Model (CMLM) with Genome Association and Prediction Integrated Tool (GAPIT), and the Fixed and Random Model Circulating Probability Unification (FarmCPU) methods, both approaches identified SNPs with significant association to disease response on chromosomes 1, 11, and 18. The CMLM also found significance on chromosome 19, whereas FarmCPU also identified significance on chromosomes 4, 9, and 16. CONCLUSIONS: These similar and yet different results show that the computational methods used can impact SNP associations in soybean, a plant with a high degree of linkage disequilibrium, and in SSR resistance, a trait that has a complex genetic basis. A total of 125 genes were located within linkage disequilibrium of the three loci shared between the two models. Their annotations and gene expressions in previous studies of soybean infected with S. sclerotiorum were examined to narrow down the candidates.