Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3.

Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere-mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N-terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T1 seedlings identified two T0 lines that produced five haploid individuals representing an induction rate of 0.5% and 1.4%. Eight different T0 lines produced aneuploids at rates ranging from 2.1 to 14.6%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms.

Environmentally responsive QTL controlling surface wax load in switchgrass.

KEY MESSAGE: Quantitation of leaf surface wax on a population of switchgrass identified three significant QTL present across six environments that contribute to leaf glaucousness and wax composition and that show complex genetic × environmental (G × E) interactions. The C4 perennial grass Panicum virgatum (switchgrass) is a native species of the North American tallgrass prairie. This adaptable plant can be grown on marginal lands and is useful for soil and water conservation, biomass production, and as a forage. Two major switchgrass ecotypes, lowland and upland, differ in a range of desirable traits, and the responsible underlying loci can be localized efficiently in a pseudotestcross design. An outbred four-way cross (4WCR) mapping population of 750 F2 lines was used to examine the genetic basis of differences in leaf surface wax load between two lowland (AP13 and WBC) and two upland (DAC and VS16) tetraploid cultivars. The objective of our experiments was to identify wax compositional variation among the population founders and to map underlying loci responsible for surface wax variation across environments. GCMS analyses of surface wax extracted from 4WCR F0 founders and F1 hybrids reveal higher levels of wax in lowland genotypes and show quantitative differences of ß-diketones, primary alcohols, and other wax constituents. The full mapping population was sampled over two seasons from four field sites with latitudes ranging from 30 to 42 °N, and leaf surface wax was measured. We identified three high-confidence QTL, of which two displayed significant G × E effects. Over 50 candidate genes underlying the QTL regions showed similarity to genes in either Arabidopsis or barley known to function in wax synthesis, modification, regulation, and transport.

Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii.

Swertia mussotii Franch. is an important traditional Tibetan medicinal plant with pharmacological properties effective in the treatment of various ailments including hepatitis. Secoiridoids are the major bioactive compounds in S. mussotii. To better understand the secoiridoid biosynthesis pathway, we generated transcriptome sequences from the root, leaf, stem, and flower tissues, and performed de novo sequence assembly, yielding 98,613 unique transcripts with an N50 of 1,085 bp. Putative functions could be assigned to 35,029 transcripts (35.52%) based on BLAST searches against annotation databases including GO and KEGG. The expression profiles of 39 candidate transcripts encoding the key enzymes for secoiridoid biosynthesis were examined in different S. mussotii tissues, validated by qRT-PCR, and compared with the homologous genes from S. japonica, a species in the same family, unveiling the gene expression, regulation, and conservation of the pathway. The examination of the accumulated levels of three bioactive compounds, sweroside, swertiamarin, and gentiopicroside, revealed their considerable variations in different tissues, with no significant correlation with the expression profiles of key genes in the pathway, suggesting complex biological behaviours in the coordination of metabolite biosynthesis and accumulation. The genomic dataset and analyses presented here lay the foundation for further research on this important medicinal plant.