The mitochondrial genome of the hexactinellid sponge Aphrocallistes vastus: Evidence for programmed translational frameshifting

<p>Abstract</p> <p>Background</p> <p>Mitochondrial genomes (mtDNA) of numerous sponges have been sequenced as part of an ongoing effort to resolve the class-level phylogeny of the Porifera, as well as to place the various lower metazoan groups on the animal-kingdom tree. Most recently, the partial mtDNA of two glass sponges, class Hexactinellida, were reported. While previous phylogenetic estimations based on these data remain uncertain due to insufficient taxon sampling and accelerated rates of evolution, the mtDNA molecules themselves reveal interesting traits that may be unique to hexactinellids. Here we determined the first complete mitochondrial genome of a hexactinellid sponge, <it>Aphrocallistes vastus</it>, and compared it to published poriferan mtDNAs to further describe characteristics specific to hexactinellid and other sponge mitochondrial genomes.</p> <p>Results</p> <p>The <it>A. vastus </it>mtDNA consisted of a 17,427 base pair circular molecule containing thirteen protein-coding genes, divergent large and small subunit ribosomal RNAs, and a reduced set of 18 tRNAs. The <it>A. vastus </it>mtDNA showed a typical hexactinellid nucleotide composition and shared a large synteny with the other sequenced glass sponge mtDNAs. It also contained an unidentified open reading frame and large intergenic space region. Two frameshifts, in the <it>cox3 </it>and <it>nad6 </it>genes, were not corrected by RNA editing, but rather possessed identical shift sites marked by the extremely rare tryptophan codon (UGG) followed by the common glycine codon (GGA) in the +1 frame.</p> <p>Conclusion</p> <p>Hexactinellid mtDNAs have shown similar trends in gene content, nucleotide composition, and codon usage, and have retained a large gene syntenty. Analysis of the mtDNA of <it>A. vastus </it>has provided evidence diagnostic for +1 programmed translational frameshifting, a phenomenon disparately reported throughout the animal kingdom, but present in the hexactinellid mtDNAs that have been sequenced to date.</p

Pollen Sterility—A Promising Approach to Gene Confinement and Breeding for Genetically Modified Bioenergy Crops

Advanced genetic and biotechnology tools will be required to realize the full potential of food and bioenergy crops. Given current regulatory concerns, many transgenic traits might never be deregulated for commercial release without a robust gene confinement strategy in place. The potential for transgene flow from genetically modified (GM) crops is widely known. Pollen-mediated transfer is a major component of gene flow in flowering plants and therefore a potential avenue for the escape of transgenes from GM crops. One approach for preventing and/or mitigating transgene flow is the production of trait linked pollen sterility. To evaluate the feasibility of generating pollen sterility lines for gene confinement and breeding purposes we tested the utility of a promoter (Zm13Pro) from a maize pollen-specific gene (Zm13) for driving expression of the reporter gene GUS and the cytotoxic gene barnase in transgenic rice (Oryza sativa ssp. Japonica cv. Nipponbare) as a monocot proxy for bioenergy grasses. This study demonstrates that the Zm13 promoter can drive pollen-specific expression in stably transformed rice and may be useful for gametophytic transgene confinement and breeding strategies by pollen sterility in food and bioenergy crops

\u3cem\u3eIn situ\u3c/em\u3e embryo rescue for generation of wide intra- and interspecific hybrids of \u3cem\u3ePanicum virgatum\u3c/em\u3e L.

Wide crosses have been used for decades as a method for transferring novel genetic material and traits in plant breeding. Historically, many products of wide crosses require tedious and inefficient surgical embryo rescue prior to embryo abortion to recover single plantlets. We have utilized transgenic switchgrass (Panicum virgatum L. cv Alamo) as a pollen donor in conjunction with antibiotic or herbicide selection for recovery of intra-and interspecific F1 crosses by using developing ovules from the female parent and selecting for embryogenic cultures derived from the in situ immature embryo. Using this approach, several intravarietial crosses were generated between transgenic Alamo and the switchgrass varieties Kanlow, Blackwell and Cave-in-Rock as well as an interspecific cross with Atlantic coastal panicgrass. This procedure selected F1 embryogenic callus produced from the developing embryo contained within isolated immature ovules. Several clonal plants were successfully regenerated from each cross. Southern blot, PCR, phenotypic analyses and genomic analysis confirmed F1 hybrids. Using genotyping-bysequencing shows the hybridization of the recovered plants by determining the ratio of transgressive markers to total compared markers between parents and their potential offspring. The ratio of transgressive markers to total compared markers was significantly lower between parents and their predicted offspring than between parents and offspring unrelated to them. This approach provides the possibility to move useful transgenes into varieties that are recalcitrant to direct transformation which can be optionally segregated thus useful to create new hybrids, as well as recovery of wide crosses that are either difficult or impossible using traditional techniques

Genetic Background and Allorecognition Phenotype in Hydractinia symbiolongicarpus

The Hydractinia allorecognition complex (ARC) was initially identified as a single chromosomal interval using inbred and congenic lines. The production of defined lines necessarily homogenizes genetic background and thus may be expected to obscure the effects of unlinked allorecognition loci should they exist. Here, we report the results of crosses in which inbred lines were out-crossed to wild-type animals in an attempt to identify dominant, codominant, or incompletely dominant modifiers of allorecognition. A claim for the existence of modifiers unlinked to ARC was rejected for three different genetic backgrounds. Estimates of the genetic map distance of ARC in two wild-type haplotypes differed markedly from one another and from that measured in congenic lines. These results suggest that additional allodeterminants exist in the Hydractinia ARC

Genomic Characterization of Interspecific Hybrids and an Admixture Population Derived from Panicum amarum × \u3cem\u3eP. virgatum\u3c/em\u3e

Switchgrass (Panicum virgatum L.) and its relatives are regarded as top bioenergy crop candidates; however, one critical barrier is the introduction of useful genetic diversity and the development of new cultivars and hybrids. Combining genomes from related cultivars and species provides an opportunity to introduce new traits. In switchgrass, a breeding advantage would be achieved by combining the genomes of intervarietal ecotypes or interspecific hybrids. The recovery of wide crosses, however, is often tedious and may involve complicated embryo rescue and numerous backcrosses. Here, we demonstrate a straightforward approach to wide crosses involving the use of a selectable transgene for recovery of interspecific [P. virgatum cv. Alamo × Panicum amarum Ell. var amarulum or Atlantic Coastal Panicgrass (ACP)] F1 hybrids followed by backcrossing to generate a nontransgenic admixture population. A nontransgenic herbicide-sensitive (HbS) admixture population of 83 F1BC1 progeny was analyzed by genotyping-by-sequencing (GBS) to characterize local ancestry, parental contribution, and patterns of recombination. These results demonstrate a widely applicable breeding strategy that makes use of transgenic selectable resistance to identify and recover true hybrids

Flexible and Scalable Genotyping-by-Sequencing Strategies for Population Studies

Background: Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels. Results: A panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross. Conclusions: This technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation

Identification of the Maize Gravitropism Gene \u3ci\u3elazy plant1\u3c/i\u3e by a Transposon-Tagging Genome Resequencing Strategy

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform

Control of sexuality by the \u3cem\u3esk1\u3c/em\u3e-encoded UDP-glycosyltransferase of maize

Sex determination in maize involves the production of staminate and pistillate florets from an initially bisexual floral meristem. Pistil elimination in staminate florets requires jasmonic acid signaling, and functional pistils are protected by the action of the silkless 1 (sk1) gene. The sk1 gene was identified and found to encode a previously uncharacterized family 1 uridine diphosphate glycosyltransferase that localized to the plant peroxisomes. Constitutive expression of an sk1 transgene protected all pistils in the plant, causing complete feminization, a gain-of-function phenotype that operates by blocking the accumulation of jasmonates. The segregation of an sk1 transgene was used to effectively control the production of pistillate and staminate inflorescences in maize plants

The Early ANTP Gene Repertoire: Insights from the Placozoan Genome

The evolution of ANTP genes in the Metazoa has been the subject of conflicting hypotheses derived from full or partial gene sequences and genomic organization in higher animals. Whole genome sequences have recently filled in some crucial gaps for the basal metazoan phyla Cnidaria and Porifera. Here we analyze the complete genome of Trichoplax adhaerens, representing the basal metazoan phylum Placozoa, for its set of ANTP class genes. The Trichoplax genome encodes representatives of Hox/ParaHox-like, NKL, and extended Hox genes. This repertoire possibly mirrors the condition of a hypothetical cnidarian-bilaterian ancestor. The evolution of the cnidarian and bilaterian ANTP gene repertoires can be deduced by a limited number of cis-duplications of NKL and “extended Hox” genes and the presence of a single ancestral “ProtoHox” gene