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Switchgrass (Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10, encoding a rice BAHD acyltransferase and QsuB, encoding dehydroshikimate dehydratase from Corynebacterium glutamicum, to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p-coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions.

BackgroundBreeding for genes controlling key agronomic traits is an important goal of rice genetic improvement. To gain insight into genes controlling grain morphology, we screened M3 plants derived from 1,000 whole-genome sequenced (WGS) M2 Kitaake mutants to identify lines with altered grain size.ResultsIn this study, we isolated a mutant, named fast-neutron (FN) 60-4, which exhibits a significant reduction in grain size. We crossed FN60-4 with the parental line Kitaake and analyzed the resulting backcross population. Segregation analysis of 113 lines from the BC2F2 population revealed that the mutant phenotype is controlled by a single semi-dominant locus. Mutant FN60-4 is reduced 20% in plant height and 8.8% in 1000-grain weight compared with Kitaake. FN60-4 also exhibits an 8% reduction in cell number and a 9% reduction in cell length along the vertical axis of the glume. We carried out whole-genome sequencing of DNA pools extracted from segregants with long grains or short grains, and revealed that one gene, LOC_Os09g02650, cosegregated with the grain size phenotype in the BC1F2 and BC2F2 populations. This mutant allele was named grain shape 9-1 (gs9-1). gs9-1 carries a 3-bp deletion that affects two amino acids. This locus is a new allele of the BC12/GDD1/MTD1 gene that encodes a kinesin-like protein involved in cell-cycle progression, cellulose microfibril deposition and gibberellic acid (GA) biosynthesis. The GA biosynthesis-related gene KO2 is down-regulated in gs9-1. The dwarf phenotype of gs9-1 can be rescued by adding exogenous GA3. In contrast to the phenotypes for the other alleles, the gs9-1 is less severe, consistent with the nature of the mutation, which does not disrupt the open reading frame as observed for the other alleles.ConclusionsIn this study, we isolated a mutant, which exhibits altered grain shape and identified the mutated gene, gs9-1. Our study reveals that gs9-1 is a semi-dominant gene that carries a two-amino acid mutation. gs9-1 is allelic to the BC12/GDD1/MTD1 gene involved in GA biosynthesis. These results demonstrate the efficiency and convenience of cloning genes from the whole-genome sequenced Kitaake mutant population to advance investigations into genes controlling key agronomic traits in rice.

BackgroundThe availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species.ResultsHere, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics.ConclusionsThe high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

BackgroundSwitchgrass (Panicum virgatum L.) is a promising bioenergy feedstock because it can be grown on marginal land and produces abundant biomass. Recalcitrance of the lignocellulosic components of the switchgrass cell wall to enzymatic degradation into simple sugars impedes efficient biofuel production. We previously demonstrated that overexpression of OsAT10, a BAHD acyltransferase gene, enhances saccharification efficiency in rice.ResultsHere we show that overexpression of the rice OsAT10 gene in switchgrass decreased the levels of cell wall-bound ferulic acid (FA) in green leaf tissues and to a lesser extent in senesced tissues, and significantly increased levels of cell wall-bound p-coumaric acid (p-CA) in green leaves but decreased its level in senesced tissues of the T0 plants under greenhouse conditions. The engineered switchgrass lines exhibit an approximate 40% increase in saccharification efficiency in green tissues and a 30% increase in senesced tissues.ConclusionOur study demonstrates that overexpression of OsAT10, a rice BAHD acyltransferase gene, enhances saccharification of lignocellulosic biomass in switchgrass.

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.