TagDigger: user-friendly extraction of read counts from GBS and RAD-seq data

Background: In genotyping-by-sequencing (GBS) and restriction site-associated DNA sequencing (RAD-seq), read depth is important for assessing the quality of genotype calls and estimating allele dosage in polyploids. However, existing pipelines for GBS and RAD-seq do not provide read counts in formats that are both accurate and easy to access. Additionally, although existing pipelines allow previously-mined SNPs to be genotyped on new samples, they do not allow the user to manually specify a subset of loci to examine. Pipelines that do not use a reference genome assign arbitrary names to SNPs, making meta-analysis across projects difficult. Results: We created the software TagDigger, which includes three programs for analyzing GBS and RAD-seq data. The first script, tagdigger_interactive.py, rapidly extracts read counts and genotypes from FASTQ files using user-supplied sets of barcodes and tags. Input and output is in CSV format so that it can be opened by spreadsheet software. Tag sequences can also be imported from the Stacks, TASSEL-GBSv2, TASSEL-UNEAK, or pyRAD pipelines, and a separate file can be imported listing the names of markers to retain. A second script, tag_manager.py, consolidates marker names and sequences across multiple projects. A third script, barcode_splitter.py, assists with preparing FASTQ data for deposit in a public archive by splitting FASTQ files by barcode and generating MD5 checksums for the resulting files. Conclusions: TagDigger is open-source and freely available software written in Python 3. It uses a scalable, rapid search algorithm that can process over 100 million FASTQ reads per hour. TagDigger will run on a laptop with any operating system, does not consume hard drive space with intermediate files, and does not require programming skill to use.DOE Office of Science, Office of Biological and Environmental Research (grant number DE-SC0012379)Ope

Can miscanthus C4 photosynthesis compete with festulolium C3 photosynthesis in a temperate climate?

Miscanthus, a perennial grass with C4 photosynthesis, is regarded as a promising energy crop due to its high biomass productivity. Compared with other C4 species, most miscanthus genotypes have high cold tolerances at 14ºC. However, in temperate climates, temperatures below 14ºC are common and our aim was to elucidate cold tolerances of different miscanthus genotypes and compare with a C3 perennial grass - festulolium. Eleven genotypes of M. sacchariflorus, M. sinensis, M. tinctorius, M. x giganteus as well as festulolium were grown under warm (24/20ºC, day/night) and three under cold (14/10ºC, 10/8ºC and 6/4ºC) conditions in a controlled environment. Measurements of photosynthetic light response curves, operating quantum yield of photosystem II (ΦPSII), net photosynthetic rate at a PAR of 1000 lmol m-2 s-1 (A1000) and dark-adapted chlorophyll fluorescence (Fv/Fm) were made at each temperature. In addition, temperature response curves were measured after the plants had been grown at 6/4ºC. The results showed that two tetraploid M. sacchariflorus and the standard triploid M. x giganteus cv. Hornum retained a significantly higher photosynthetic capacity than other miscanthus genotypes at each temperature level and still maintained photosynthesis after growing for a longer period at 6/4ºC. Only two of five measured miscanthus genotypes increased photosynthesis immediately after the temperature was raised again. The photosynthetic capacity of festulolium was significantly higher at 10/8ºC and 6/4ºC than of miscanthus genotypes. This indicates that festulolium may be more productive than the currently investigated miscanthus genotypes in cool, maritime climates. Within miscanthus, only one M. sacchariflorus genotype exhibited the same photosynthetic capacity as Hornum at both cold conditions and when the temperature was raised again. Therefore, this genotype could be useful for breeding new varieties with an improved cold tolerance vis-a-vis Hornum, and be valuable in broadening the genetic diversity of miscanthus for more widespread cultivation in temperate climates

Can chilling tolerance of C4 photosynthesis in \u3ci\u3eMiscanthus\u3c/i\u3e be transferred to sugarcane?

The goal of this study was to investigate whether chilling tolerance of C4 photosynthesis in Miscanthus can be transferred to sugarcane by hybridization. Net leaf CO2 uptake (Asat) and the maximum operating efficiency of photosystem II (ФPSII) were measured in warm conditions (25 °C/20 °C), and then during and following a chilling treatment of 10 °C/5 °C for 11 day in controlled environment chambers. Two of three hybrids (miscanes), ‘US 84-1058’ and ‘US 87-1019’, did not differ significantly from the chilling tolerant M. xgiganteus ‘Illinois’ (Mxg), for Asat, and ΦPSII measured during chilling. For Mxg grown at 10 °C/5 °C for 11 days, Asat was 4.4 μmol m-2 s-1, while for miscane ‘US 84-1058’ and ‘US 87-1019’, Asat was 5.7 and 3.5 μmol m-2 s-1, respectively. Miscanes ‘US 84-1058’ and ‘US 87-1019’ and Mxg had significantly higher rates of Asat during chilling than three tested sugarcanes. A third miscane showed lower rates than Mxg during chilling, but recovered to higher rates than sugarcane upon return to warm conditions. Chilling tolerance of ‘US 84-1058’ was further confirmed under autumn field conditions in southern Illinois. The selected chilling tolerant miscanes have particular value for biomass feedstock and biofuel production and at the same time they can be a starting point for extending sugarcane’s range to colder climates

Genetic variation in \u3ci\u3eMiscanthus\u3c/i\u3e X \u3ci\u3egiganteus\u3c/i\u3e and the importance of estimating genetic distance thresholds for differentiating clones

Miscanthus x giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among legacy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type-specimen. A subset of accessions was also evaluated by restriction-site associated DNA sequencing (RAD-seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD-seq, the former is currently more cost-effective for determining if two accessions are genetically identical. We concluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In contrast with the Mxg legacy cultivars, genetic similarity to the type-specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much-needed variation to growers

Transformation and gene editing in the bioenergy grass \u3ci\u3eMiscanthus\u3c/i\u3e

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. sacchariforus 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. sacchariforus, 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 goo

Winter hardiness of \u3ci\u3eMiscanthus\u3c/i\u3e (III): Genome‐wide association and genomic prediction for overwintering ability in \u3ci\u3eMiscanthus sinensis\u3c/i\u3e

Overwintering ability is an important selection criterion for Miscanthus breeding in temperate regions. Insufficient overwintering ability of the currently leading Miscanthus biomass cultivar, M. ×giganteus (M×g) ‘1993–1780’, in regions where average annual minimum temperatures are −26.1°C (USDA hardiness zone 5) or lower poses a pressing need to develop new cultivars with superior cold tolerance. To facilitate breeding of Miscanthus, this study characterized phenotypic and genetic variation of overwintering ability in an M. sinensis germplasm panel consisting of 564 accessions, evaluated in field trials at three locations in North America and two in Asia. Genome‐wide association (GWA) and genomic prediction analyses were performed. The Korea/N China M. sinensis genetic group is a valuable gene pool for cold tolerance. The Yangtze‐Qinling, Southern Japan, and Northern Japan genetic groups were also potential sources of cold tolerance. A total of 73 marker–trait associations were detected for overwintering ability. Estimated breeding value for overwintering ability based on these 73 markers could explain 55% of the variation for first winter overwintering ability among M. sinensis. Average genomic prediction ability for overwintering ability across 50 fivefold cross‐validations was high (~0.73) after accounting for population structure. Common genomic regions for overwintering ability were detected by GWA analyses and a previous parallel QTL mapping study using three interconnected biparental F1 populations. One QTL on Miscanthus LG 8 encompassed five GWA hits and a known cold‐responsive gene, COR47. The other overwintering ability QTL on Miscanthus LG 11 contained two GWA hits and three known cold stress‐related genes, carboxylesterase 13 (CEX13), WRKY2 transcription factor, and cold shock domain (CSDP1). Miscanthus accessions collected from high latitude locations with cold winters had higher rates of overwintering, and more alleles for overwintering, than accessions collected from southern locations with mild winters

Genetic structure of Miscanthus sinensis and Miscanthus sacchariflorus in Japan indicates a gradient of bidirectional but asymmetric introgression

Unilateral introgression from diploids to tetraploids has been hypothesized to be an important evolutionary mechanism in plants. However, few examples have been definitively identified, perhaps because data of sufficient depth and breadth were difficult to obtain prior to the advent of affordable high-density genotyping. Throughout Japan, tetraploid Miscanthus sacchariflorus and diploid M. sinensis are common, and occasionally hybridize. In this study, we characterized 667 M. sinensis and 78 M. sacchariflorus genotypes from Japan using 20,704 SNPs and ten plastid microsatellites. Similarity of SNP genotypes between diploid and tetraploid M. sacchariflorus indicated that the tetraploids originated via autopolyploidy. Structure analysis indicated a gradient of introgression from diploid M. sinensis into tetraploid M. sacchariflorus throughout Japan; most tetraploids had some M. sinensis DNA. Among phenotypically M. sacchariflorus tetraploids, M. sinensis ancestry averaged 7% and ranged from 1-39%, with introgression greatest in southern Japan. Unexpectedly, rare (~1%) diploid M. sinensis individuals from northern Japan were found with 6-27% M. sacchariflorus ancestry. Population structure of M. sinensis in Japan included three groups, and was driven primarily by distance, and secondarily by geographic barriers such as mountains and straits. Miscanthus speciation is a complex and dynamic process. In contrast to limited introgression between diploid M. sacchariflorus and M. sinensis in northern China, selection for adaptation to a moderate maritime climate likely favored cross-ploidy introgressants in southern Japan. Our results will help guide the selection of Miscanthus accessions for the breeding of biomass cultivars.Office of Science - Biological and Environmental Research, U.S. Department of Energy [Project ID 0017582]Energy Biosciences InstituteOpe

Identification and genomic location of a reniform nematode (Rotylenchulus reniformis) resistance locus (Renari) introgressed from Gossypium aridum into upland cotton (G. hirsutum)

In this association mapping study, a tri-species hybrid, [Gossypium arboreum × (G. hirsutum × G. aridum)2], was crossed with MD51ne (G. hirsutum) and progeny from the cross were used to identify and map SSR markers associated with reniform nematode (Rotylenchulus reniformis) resistance. Seventy-six progeny (the 50 most resistant and 26 most susceptible) plants were genotyped with 104 markers. Twenty-five markers were associated with a resistance locus that we designated Renari and two markers, BNL3279_132 and BNL2662_090, mapped within 1 cM of Renari. Because the SSR fragments associated with resistance were found in G. aridum and the bridging line G 371, G. aridum is the likely source of this resistance. The resistance is simply inherited, possibly controlled by a single dominant gene. The markers identified in this project are a valuable resource to breeders and geneticists in the quest to produce cotton cultivars with a high level of resistance to reniform nematode

Chronic Consumption of Farmed Salmon Containing Persistent Organic Pollutants Causes Insulin Resistance and Obesity in Mice

Background: Dietary interventions are critical in the prevention of metabolic diseases. Yet, the effects of fatty fish consumption on type 2 diabetes remain unclear. The aim of this study was to investigate whether a diet containing farmed salmon prevents or contributes to insulin resistance in mice. Methodology/Principal Findings: Adult male C57BL/6J mice were fed control diet (C), a very high-fat diet without or with farmed Atlantic salmon fillet (VHF and VHF/S, respectively), and Western diet without or with farmed Atlantic salmon fillet (WD and WD/S, respectively). Other mice were fed VHF containing farmed salmon fillet with reduced concentrations of persistent organic pollutants (VHF/S-POPs). We assessed body weight gain, fat mass, insulin sensitivity, glucose tolerance, ex vivo muscle glucose uptake, performed histology and immunohistochemistry analysis, and investigated gene and protein expression. In comparison with animals fed VHF and WD, consumption of both VHF/S and WD/S exaggerated insulin resistance, visceral obesity, and glucose intolerance. In addition, the ability of insulin to stimulate Akt phosphorylation and muscle glucose uptake was impaired in mice fed farmed salmon. Relative to VHF/S-fed mice, animals fed VHF/S-POPs had less body burdens of POPs, accumulated less visceral fat, and had reduced mRNA levels of TNFa as well as macrophage infiltration in adipose tissue. VHF/S-POPs-fed mice further exhibited better insulin sensitivity and glucose tolerance than mice fed VHF/S. Conclusions/Significance: Our data indicate that intake of farmed salmon fillet contributes to several metabolic disorders linked to type 2 diabetes and obesity, and suggest a role of POPs in these deleterious effects. Overall, these findings may participate to improve nutritional strategies for the prevention and therapy of insulin resistance