Viridis Accessions Research Articles

Comparative transcriptome analysis and genetic dissection of vegetative branching traits in foxtail millet (Setaria italica).

Two major genetic loci, qTN5.1 and qAB9.1, were identified and finely mapped to the 255Kb region with one potential candidate gene for tiller number and the 521Kb region with eight candidate genes for axillary branch number, respectively. Vegetative branching including tillering and axillary branching are vital traits affecting both the plant architecture and the biomass in cereal crops. However, the mechanism underlying the formation of vegetative branching in foxtail millet is largely unknown. Here, a foxtail millet cultivar and its bushy wild relative Setaria viridis accession were used to construct segregating populations to identify candidate genes regulating tiller number and axillary branch number. Transcriptome analysis using vegetative branching bud samples of parental accessions was performed, and key differentially expressed genes and pathways regulating vegetative branching were pointed out. Bulk segregant analysis on their F2:3 segregating population was carried out, and a major QTL for tiller number (qTN5.1) and two major QTLs for axillary branch number (qAB2.1 and qAB9.1) were detected. Fine-mapping strategy was further performed on F2:4 segregate population, and Seita.5G356600 encoding β-glucosidase 11 was identified as the promising candidate gene for qTN5.1, and eight genes, especially Seita.9G125300 and Seita.9G125400 annotated as B-S glucosidase 44, were finally identified as candidate genes for regulating axillary branching. Findings in this study will help to elucidate the genetic basis of the vegetative branching formation of foxtail millet and lay a foundation for breeding foxtail millet varieties with ideal vegetative branching numbers.

Evaluation of the Genetic Stability of Amaranthus viridis L. Species in Selected Regions of Western Ghats using Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA is cheap and fast molecular technique to identify the genetic resemblances or variations in DNA in various plants. The proposed research work wasintendedto compare the genetic diversification of Amaranthus viridis species in selected regions of Western Ghats in Tamilnadu and Kerala by RAPD analysis.The biometric morphological data among the five A. viridis accessions were compared. Their genomic DNA was isolated and finger prints were obtained using three RAPD markers. The percentage polymorphism, polymorphism information content, effective multiplex ration and resolving power were calculated. UPGMA dendrogram was constructed and their genetic relatedness was compared using Jaccard coefficient.Upon finger printing, 66 bands were counted for the three RAPD primers used,among which 46 bands were polymorphic band numbers from 8 to 21. Percentage of polymorphic bands ranged between 44.44% and 80.77%. EMRvalues for 66 polymorphic loci ranged between 3.56 and 17.01. The UPGMA dendrogram comparing the genomic profiles of the 5 A. viridis accessions using Jaccard coefficient revealed their genetic distances where the maximum Jaccard’s coefficient value observed was 0.96 between AVKATN and AVNEK. Among the 5 selections the peak similarity index (0.9565) was witnessed between AVKATN and AVNEK. The cuurent study revealed that AVKATN and AVNEK had the highest their genetic distances, whereas AVKATN and AVNEK were genetically similar among the 5 A. viridis accessions. Several polymorphic bands which ranged up to 80.77% have proved their genetic variations.

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway.

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.

Agrobacterium-Mediated Transformation of Setaria viridis, a Model System for Cereals and Bioenergy Crops.

Setaria viridis is an emerging model system for the genetic and molecular characterization of cereals and bioenergy crops. Here, we describe a detailed procedure for genetic transformation of the S. viridis accession line ME034V-1. This method utilizes callus generated from mature seeds for infection with Agrobacterium tumefaciens strain AGL1 to regenerate hygromycin-resistant stable transgenic plants. It takes approximately 7 weeks to generate callus from mature seeds, 11-17 weeks from infection to the regeneration of transgenic lines, and an additional 3-4 weeks for plant growth in the greenhouse for seed collection. The protocol as presented consistently results in transformation frequency of approximately 25% for the generation of transgenic plants, with fewer escapes and higher survivability in soil for optimal seed collection. © 2021 Donald Danforth Plant Science Center. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of S. viridis (Accession ME034V-1) callus from mature seeds Basic Protocol 2: Agrobacterium-mediated transformation of callus to generate transgenic plants Basic Protocol 3: Plantlet transplantation in soil, plant growth in greenhouse, and seed collection Support Protocol: Preparation of Agrobacterium culture for infection.

Heterologous Overexpression of Arabidopsis cel1 Enhances Grain Yield, Biomass and Early Maturity in Setaria viridis.

Heterologous overexpression of Arabidopsis cellulase 1 (Atcel1) results in enhanced yield, early maturity, and increased biomass in dicotyledonous species like poplar and eucalyptus but has not been demonstrated in monocots. We produced transgenic Setaria viridis accession A10.1 plants overexpressing a monocotyledonous codon optimized (MCO) Atcel1. Agronomic characterization of the transgenic events showed that heterologous overexpression of MCOAtcel1 caused enhanced grain yield, shoot biomass, and accelerated maturation rate in the model grass species S. viridis under growth chamber conditions. The agronomic trait differences observed were consistent with previous reports in dicots but are here described in a monocot species and associated with increased seed yield. Overexpression of Atcel1 in S. viridis was shown to increase the number of panicles and seeds by 24–30%, enhance overall grain yield by up to 26%, and lead to a shoot dry biomass increase of 16–19%. Overexpression also reduced time to plant maturation and senescence by 12.5%. Our findings in S. viridis suggest that manipulation of Atcel1 has potential for developing early-maturing and higher-yielding monocotyledonous biomass crops suitable for climate-smart agriculture.

Reference Genome for the Highly Transformable Setaria viridis ME034V.

Setaria viridis (green foxtail) is an important model system for improving cereal crops due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. viridis accession ME034V is exceptionally transformable, but the lack of a sequenced genome for this accession has limited its utility. We present a 397 Mb highly contiguous de novo assembly of ME034V using ultra-long nanopore sequencing technology (read N50 = 41kb). We estimate that this genome is largely complete based on our updated k-mer based genome size estimate of 401 Mb for S. viridis. Genome annotation identified 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the genome. We compared the ME034V assembly with two other previously sequenced Setaria genomes as well as to a diversity panel of 235 S. viridis accessions. We found the genome assemblies to be largely syntenic, but numerous unique polymorphic structural variants were discovered. Several ME034V deletions may be associated with recent retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low genotype frequencies in the sampled population. Lastly, we performed a phylogenomic analysis to identify gene families that have expanded in Setaria, including those involved in specialized metabolism and plant defense response. The high continuity of the ME034V genome assembly validates the utility of ultra-long DNA sequencing to improve genetic resources for emerging model organisms. Structural variation present in Setaria illustrates the importance of obtaining the proper genome reference for genetic experiments. Thus, we anticipate that the ME034V genome will be of significant utility for the Setaria research community.

Profiling of the Salt Stress Responsive MicroRNA Landscape of C4 Genetic Model Species Setaria viridis (L.) Beauv

Setaria viridis has recently emerged as an ideal model species to genetically characterize the C4 monocotyledonous grasses via a molecular modification approach. Soil salinization has become a compelling agricultural problem globally with salinity adversely impacting the yield potential of many of the major cereals. Small regulatory molecules of RNA, termed microRNAs (miRNAs), were originally demonstrated crucial for developmental gene expression regulation in plants, however, miRNAs have since been shown to additionally command a central regulatory role in abiotic stress adaptation. Therefore, a small RNA sequencing approach was employed to profile the salt stress responsive miRNA landscapes of the shoot and root tissues of two Setaria viridis accessions (A10 and ME034V) amenable to molecular modification. Small RNA sequencing-identified abundance alterations for miRNAs, miR169, miR395, miR396, miR397, miR398 and miR408, were experimentally validated via RT-qPCR. RT-qPCR was further applied to profile the molecular response of the miR160 and miR167 regulatory modules to salt stress. This analysis revealed accession- and tissue-specific responses for the miR160 and miR167 regulatory modules in A10 and ME034V shoot and root tissues exposed to salt stress. The findings reported here form the first crucial step in the identification of the miRNA regulatory modules to target for molecular manipulation to determine if such modification provides S. viridis with an improved tolerance to salt stress.

Identification and characterization of core abscisic acid (ABA) signaling components and their gene expression profile in response to abiotic stresses in Setaria viridis

Abscisic acid (ABA) is an essential phytohormone that regulates growth, development and adaptation of plants to environmental stresses. In Arabidopsis and other higher plants, ABA signal transduction involves three core components namely PYR/PYL/RCAR ABA receptors (PYLs), type 2C protein phosphatases (PP2Cs) and class III SNF-1-related protein kinase 2 (SnRK2s). In the present study, we reported the identification and characterization of the core ABA signaling components in Setaria viridis, an emerging model plant for cereals and feedstock crops presenting C4 metabolism, leading to the identification of eight PYL (SvPYL1 to 8), twelve PP2C (SvPP2C1 to 12) and eleven SnRK2 (SvSnRK2.1 through SvSnRK2.11) genes. In order to study the expression profiles of these genes, two different S. viridis accessions (A10.1 and Ast-1) were submitted to drought, salinity and cold stresses, in addition to application of exogenous ABA. Differential gene expression profiles were observed in each treatment and plant genotype, demonstrating variations of ABA stress responses within the same species. These differential responses to stresses were also assessed by physiological measurements such as photosynthesis, stomatal conductance and transpiration rate. This study allows a detailed analysis of gene expression of the core ABA signaling components in Setaria viridis submitted to different treatments and provides suitable targets for genetic engineering of C4 plants aiming tolerance to abiotic stresses.

QTL Mapping Combined With Comparative Analyses Identified Candidate Genes for Reduced Shattering in Setaria italica.

Setaria (L.) P. Beauv is a genus of grasses that belongs to the Poaceae (grass) family, subfamily Panicoideae. Two members of the Setaria genus, Setaria italica (foxtail millet) and S. viridis (green foxtail), have been studied extensively over the past few years as model species for C4-photosynthesis and to facilitate genome studies in complex Panicoid bioenergy grasses. We exploited the available genetic and genomic resources for S. italica and its wild progenitor, S. viridis, to study the genetic basis of seed shattering. Reduced shattering is a key trait that underwent positive selection during domestication. Phenotyping of F2:3 and recombinant inbred line (RIL) populations generated from a cross between S. italica accession B100 and S. viridis accession A10 identified the presence of additive main effect quantitative trait loci (QTL) on chromosomes V and IX. As expected, enhanced seed shattering was contributed by the wild S. viridis. Comparative analyses pinpointed Sh1 and qSH1, two shattering genes previously identified in sorghum and rice, as potentially underlying the QTL on Setaria chromosomes IX and V, respectively. The Sh1 allele in S. italica was shown to carry a PIF/Harbinger MITE in exon 2, which gave rise to an alternatively spliced transcript that lacked exon 2. This MITE was universally present in S. italica accessions around the world and absent from the S. viridis germplasm tested, strongly suggesting a single origin of foxtail millet domestication. The qSH1 gene carried two MITEs in the 5′UTR. Presence of one or both MITEs was strongly associated with cultivated germplasm. If the MITE insertion(s) in qSH1 played a role in reducing shattering in S. italica accessions, selection for the variants likely occurred after the domestication of foxtail millet.

Effects of abiotic stress on physiological plasticity and water use of Setaria viridis (L.)

The emerging model Setaria viridis with its C4 photosynthesis and adaptation to hot and dry locations is a promising system to investigate water use and abiotic stress tolerance. We investigated the physiological plasticity of six S. viridis natural accessions that originated from different regions of the world under normal conditions and conditions of water-deficit stress and high temperatures. Accessions Zha-1, A10.1 and Ula-1 showed significantly higher leaf water potential (Ψleaf), photosynthesis (A), transpiration (E), and stomatal conductance (gs) rates compared to Ast-1, Aba-1 and Sha-1 when grown under stress conditions. Expression analysis of genes associated with C4 photosynthesis, aquaporins, ABA biosynthesis and signaling including genes involved in stress revealed an increased sensitivity of Ast-1, Aba-1 and Sha-1 to stresses. Correlation analysis of gene expression data with physiological and biochemical changes characterized A10.1 and Ast-1 as two extreme tolerant and sensitive accessions originated from United States and Azerbaijan under water-deficit and heat stress, respectively. Although preliminary, our study demonstrated the plasticity of S. viridis accessions under stress, and allows the identification of tolerant and sensitive accessions that could be use to study the mechanisms associated with stress tolerance and to characterize of the regulatory networks involved in C4 grasses.

Population genetics of Setaria viridis, a new model system.

An extensive survey of the standing genetic variation in natural populations is among the priority steps in developing a species into a model system. In recent years, green foxtail (Setaria viridis), along with its domesticated form foxtail millet (S. italica), has rapidly become a promising new model system for C4 grasses and bioenergy crops, due to its rapid life cycle, large amount of seed production and small diploid genome, among other characters. However, remarkably little is known about the genetic diversity in natural populations of this species. In this study, we survey the genetic diversity of a worldwide sample of more than 200 S. viridis accessions, using the genotyping-by-sequencing technique. Two distinct genetic groups in S. viridis and a third group resembling S. italica were identified, with considerable admixture among the three groups. We find the genetic variation of North American S. viridis correlates with both geography and climate and is representative of the total genetic diversity in this species. This pattern may reflect several introduction/dispersal events of S. viridis into North America. We also modelled demographic history and show signal of recent population decline in one subgroup. Finally, we show linkage disequilibrium decay is rapid (<45 kb) in our total sample and slow in genetic subgroups. These results together provide an in-depth understanding of the pattern of genetic diversity of this new model species on a broad geographic scale. They also provide key guidelines for on-going and future work including germplasm preservation, local adaptation, crossing designs and genomewide association studies.

Methods to promote germination of dormant Setaria viridis seeds.

Setaria viridis has recently emerged as a promising genetic model system to study diverse aspects of monocot biology. While the post-germination life cycle of S. viridis is approximately 8 weeks long, the prolonged dormancy of freshly harvested seeds can more than double the total time required between successive generations. Here we describe methods that promote seed germination in S. viridis. Our results demonstrate that treating S. viridis seeds with liquid smoke or a GA3 and KNO3 solution improves germination rates to 90% or higher even in seeds that are 6 days post-harvest with similar results obtained whether seeds are planted in soil or on gel-based media. Importantly, we show that these treatments have no significant effect on the growth of the adult plant. We have tested these treatments on diverse S. viridis accessions and show variation in their response. The methods described here will help advance research using this model grass species by increasing the pace at which successive generations of plants can be analyzed.

A haplotype map of genomic variations and genome-wide association studies of agronomic traits in foxtail millet (Setaria italica)

Foxtail millet (Setaria italica) is an important grain crop that is grown in arid regions. Here we sequenced 916 diverse foxtail millet varieties, identified 2.58 million SNPs and used 0.8 million common SNPs to construct a haplotype map of the foxtail millet genome. We classified the foxtail millet varieties into two divergent groups that are strongly correlated with early and late flowering times. We phenotyped the 916 varieties under five different environments and identified 512 loci associated with 47 agronomic traits by genome-wide association studies. We performed a de novo assembly of deeply sequenced genomes of a Setaria viridis accession (the wild progenitor of S. italica) and an S. italica variety and identified complex interspecies and intraspecies variants. We also identified 36 selective sweeps that seem to have occurred during modern breeding. This study provides fundamental resources for genetics research and genetic improvement in foxtail millet.

Interspecific Hybridization of Fragaria vesca subspecies with F. nilgerrensis, F. nubicola, F. pentaphylla, and F. viridis

The potential of using Fragaria vesca L. as a bridge species for interspecific hybridization to F. nilgerrensis Schlect, F. nubicola Lindl., F. pentaphylla Losinsk, and F. viridis Duch. was investigated using a wide germplasm base of 40 F. vesca accessions. This study was successful in producing many hybrids between F. vesca and other diploid species indicating its value as a bridge species. Of the species used as males, F. nubicola, F. pentaphylla, and F. viridis accessions were more successful, averaging 8 to 16 fruit and 16 to 25 seeds/fruit. It was most difficult to obtain hybrids with F. nilgerrensis, which had only three seeds per fruit. Differences among pollen donors were minimal when hybrid seeds were germinated in vitro. For different species combinations, 75% to 99% of seeds had embryos and 77% to 89% of these embryos germinated. The lack of significant differences in crossability variables among the four F. vesca subspecies [i.e., ssp. americana (Porter) Staudt, ssp. bracteata (Heller) Staudt, ssp. vesca L., and ssp. vesca var. semper-florens L.] demonstrated the similarity between these species and the strong potential for gene flow between F. vesca and other diploid species. As European and North American F. vesca subspecies are not sufficiently divergent to differ in interspecific hybridization, F. vesca may be a younger species rather than an older progenitor species.

Development and characterization of polymorphic microsatellite markers from Fragaria viridis, a wild diploid strawberry

AbstractTo date, the development of microsatellite (SSR) markers in the genus Fragaria has focused on F. vesca. However, further species are thought to have contributed to the complex allo‐octoploid genome of the cultivated strawberry, F.×ananassa. Here, we present 22 new SSR markers developed from the diploid species F. viridis. Twenty‐one of the primer pairs amplified polymorphisms in six F. viridis accessions, with an average of 4.95 alleles per primer pair and an average expected heterozygosity of 0.68. Fourteen of these primer pairs, and a locus monomorphic in F. viridis, amplified polymorphic alleles in the parents of a F. vesca mapping population.

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes

We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata.