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.

Distribution and ecology of Frankliniella occidentalis (Thysanoptera: Thripidae) bacterial symbionts.

Bacterial populations in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) collected in diverse California environments consisted of two bacterial symbionts: BFo-1 and BFo-2 (B = bacteria, Fo = Frankliniella occidentalis, numbers reflect different types). Dual infections of BFo-1 and BFo-2 were found in 50% of the thrips, 18% had neither bacterium, and 24 and 8% were infected solely with BFo-1 and BFo-2, respectively. No other bacteria consistently infected F. occidentalis. Dual infections occurred more often in male thrips and in thrips of both sexes from southern mountain and valley sites. As average collection year or month minimum temperature decreased, infections of BFo-1, alone or in dual infections, increased significantly. As yearly precipitation increased, infection with BFo-1 alone also increased. F. occidentalis color morphology did not affect bacterial infection. BFo-1 created weak biofilms at 25 and 32 degrees C; BFo-2 made strong biofilms at 25 degrees C and no biofilms at 32 degrees C. When the bacteria were grown in culture together, weak biofilms formed at both temperatures studied, although there was no way to determine what each bacterium contributed to the biofilm. BFo-1 and BFo-2 grew at similar rates at 25 and 30 degrees C. Our data show BFo-1 and BFo-2 occur in natural populations of F. occidentalis and support the hypothesis BFo have a symbiotic relationship with F. occidentalis. Regional differences in bacterial prevalence suggest bacterial infection is associated with environmental conditions, and altitude, temperature, and precipitation may be important factors.

Characterization of bacterial symbionts in Frankliniella occidentalis (Pergande), Western flower thrips.

Many insects have associations with bacteria, although it is often difficult to determine the intricacies of the relationships. In one such case, facultative bacteria have been discovered in a major crop pest and virus vector, the Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Several bacterial isolates have been studied in Netherlands greenhouse thrips populations, with molecular data indicating that these bacteria were similar to Escherichia coli, although biochemical properties suggested these microbes might actually be most similar to plant pathogenic bacteria in the genus Erwinia. We focused on the bacterial flora of the Hawaiian Islands thrips population where these gut bacteria were first reported in 1989. We also analyzed a German population and a 1965 California population preserved in ethanol. Culture and culture-independent techniques revealed a consistent microflora that was similar to the Netherlands isolates studied. The similarity among thrips microbes from multiple populations and environments suggested these bacteria and their hosts share a widespread association. Molecular phylogeny based on the 16S rRNA gene and biochemical analysis of thrips bacteria suggested two distinctive groups of microbes are present in thrips. Phylogenetic analysis also revealed support for one thrips bacterial group having a shared ancestry with Erwinia, whereas the second group of thrips bacteria fell out with E. coli, but without support. Although species-specific relationships were indeterminable due to the conservative nature of 16S, there is strong indication that thrips symbionts belong to two different genera and originated from environmental microbes.