Dataset Information

Isolation and characterization of microsatellite loci for a bioenergy grass, Miscanthus sacchariflorus (Poaceae).

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Microsatellite loci were developed for the biomass C4 grass, Miscanthus sacchariflorus, and proved to be suitable markers for population genetic studies and germplasm management of this species. • METHODS AND RESULTS:Twenty-three polymorphic microsatellite loci were identified from an enriched genomic library of M. sacchariflorus. The polymorphism was assessed in 50 individuals from two populations in China. The number of alleles per locus varied from two to 18, with a mean of 8.13. The observed and expected heterozygosities ranged from 0.2 to 1.0 and from 0.198 to 0.898, respectively. • CONCLUSIONS:These new markers will be useful for further investigation of genetic diversity and population genetic structure as well as molecular breeding of Miscanthus species.

SUBMITTER: Zhou HF

PROVIDER: S-EPMC4105351 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Isolation and characterization of microsatellite loci for a bioenergy grass, Miscanthus sacchariflorus (Poaceae).

Zhou Hai-Fei HF Li Shan-Shan SS Ge Song S

Applications in plant sciences 20130102 1

<h4>Unlabelled</h4><h4>Premise of the study</h4>Microsatellite loci were developed for the biomass C4 grass, Miscanthus sacchariflorus, and proved to be suitable markers for population genetic studies and germplasm management of this species. •<h4>Methods and results</h4>Twenty-three polymorphic microsatellite loci were identified from an enriched genomic library of M. sacchariflorus. The polymorphism was assessed in 50 individuals from two populations in China. The number of alleles per locus v ...[more]

PMID: 25202478

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Project description:BackgroundMiscanthus, 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.ResultsWe 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.ConclusionsWe 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.

Project description:BackgroundMiscanthus is a commercial lignocellulosic biomass crop owing to its high biomass productivity, resilience and photosynthetic capacity at low temperature. These qualities make Miscanthus a particularly good candidate for temperate marginal land, where yields can be limited by insufficient or excessive water supply. Differences in response to water stress have been observed among Miscanthus species, which correlated to origin. In this study, we compared the physiological and molecular responses among Miscanthus species under excessive (flooded) and insufficient (drought) water supply in glasshouse conditions.ResultsA significant biomass loss was observed under drought conditions in all genotypes. M. x giganteus showed a lower reduction in biomass yield under drought conditions compared to the control than the other species. Under flooded conditions, biomass yield was as good as or better than control conditions in all species. 4389 of the 67,789 genes (6.4%) in the reference genome were differentially expressed during drought among four Miscanthus genotypes from different species. We observed the same biological processes were regulated across Miscanthus species during drought stress despite the DEGs being not similar. Upregulated differentially expressed genes were significantly involved in sucrose and starch metabolism, redox, and water and glycerol homeostasis and channel activity. Multiple copies of the starch metabolic enzymes BAM and waxy GBSS-I were strongly up-regulated in drought stress in all Miscanthus genotypes, and 12 aquaporins (PIP1, PIP2 and NIP2) were also up-regulated in drought stress across genotypes.ConclusionsDifferent phenotypic responses were observed during drought stress among Miscanthus genotypes from different species, supporting differences in genetic adaption. The low number of DEGs and higher biomass yield in flooded conditions supported Miscanthus use in flooded land. The molecular processes regulated during drought were shared among Miscanthus species and consistent with functional categories known to be critical during drought stress in model organisms. However, differences in the regulated genes, likely associated with ploidy and heterosis, highlighted the value of exploring its diversity for breeding.

Dataset Information

Isolation and characterization of microsatellite loci for a bioenergy grass, Miscanthus sacchariflorus (Poaceae).

Publications

Isolation and characterization of microsatellite loci for a bioenergy grass, Miscanthus sacchariflorus (Poaceae).

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