Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa

<p>Abstract</p> <p>Background</p> <p>Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (<it>Lolium perenne</it>). We report on the exploitation of a comprehensive EST collection in <it>L. perenne </it>for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of <it>L. perenne</it>, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of <it>L. perenne</it>, <it>Festuca arundinacea, Brachypodium distachyon</it>, and <it>O. sativa</it>, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding.</p> <p>Results</p> <p>From 25,744 ESTs, representing 8.53 megabases of nucleotide information from three genotypes of <it>L. perenne</it>, 1,458 ESTs (5.7%) contained one or more SSRs. Of these SSRs, 955 (3.7%) were non-redundant. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. The EST-SSRs from the three genotypes were analysed for allelic- and/or genotypic SSR motif polymorphisms. Most of the SSR motifs (97.7%) showed no polymorphisms, whereas 22 EST-SSRs showed allelic- and/or genotypic polymorphisms. All polymorphisms identified were changes in the number of repeat units. Comparative analysis of the <it>L. perenne </it>EST-SSRs with sequences of <it>Festuca arundinacea</it>, <it>Brachypodium distachyon</it>, and <it>Oryza sativa </it>identified 19 clusters of orthologous sequences between these four species. Analysis of the clusters showed that the SSR motif generally is conserved in the closely related species <it>F. arundinacea</it>, but often differs in length of the SSR motif. In contrast, SSR motifs are often lost in the more distant related species <it>B. distachyon </it>and <it>O. sativa</it>.</p> <p>Conclusion</p> <p>The results indicate that the <it>L. perenne </it>EST-SSR markers are a valuable resource for genetic mapping, as well as evaluation of co-location between QTLs and functionally associated markers.</p

Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha

Dihydroxyacetone synthase (DAS) and methanol oxidase (MOX) are the major enzyme constituents of the peroxisomal matrix in the methylotrophic yeast Hansenula polymorpha when grown on methanol as a sole carbon source. In order to characterize their topogenic signals the localization of truncated polypeptides and hybrid proteins was analysed in transformed yeast cells by subcellular fractionation and electron microscopy. The C-terminal part of DAS, when fused to the bacterial β-lactamase or mouse dihydrofolate reductase, directed these hybrid polypeptides to the peroxisome compartment. The targeting signal was further delimited to the extreme C-terminus, comprising the sequence N-K-L-COOH, similar to the recently identified and widely distributed peroxisomal targeting signal (PTS) S-K-L-COOH in firefly luciferase. By an identical approach, the extreme C-terminus of MOX, comprising the tripeptide A-R-F-COOH, was shown to be the PTS of this protein. Furthermore, on fusion of a C-terminal sequence from firefly luciferase including the PTS, β-lactamase was also imported into the peroxisomes of H. polymorpha. We conclude that, besides the conserved PTS (or described variants), other amino acid sequences with this function have evolved in nature

Ensiling and hydrothermal pretreatment of grass: Consequences for enzymatic biomass conversion and total monosaccharide yields

BACKGROUND: Ensiling may act as a pretreatment of fresh grass biomass and increase the enzymatic conversion of structural carbohydrates to fermentable sugars. However, ensiling does not provide sufficient severity to be a standalone pretreatment method. Here, ensiling of grass is combined with hydrothermal treatment (HTT) with the aim of improving the enzymatic biomass convertibility and decrease the required temperature of the HTT. RESULTS: Grass silage (Festulolium Hykor) was hydrothermally treated at temperatures of 170, 180, and 190°C for 10 minutes. Relative to HTT treated dry grass, ensiling increased the solubilization of dry matter (DM) during HTT and gave increased glucan content, but lower lignin in the insoluble fiber fraction. Ensiling improved glucose yields in the enzymatic hydrolysis of the washed solid fiber fraction at the lower HTT temperatures. At 170°C glucose yield improved from 17 to 24 (w/w)% (45 to 57% cellulose convertibility), and at 180°C glucose yield improved from 22 to 29 (w/w)% (54 to 69% cellulose convertibility). Direct HTT of grass at 190°C gave the same high glucose yield as for grass silage (35 (w/w)% (77% cellulose convertibility)) and improved xylan yields (27% xylan convertibility). The effect of ensiling of grass prior to HTT improved the enzymatic conversion of cellulose for HTT at 170 and 180°C, but the increased glucose release did not make up for the loss of water soluble carbohydrates (WSC) during ensiling. Overall, sugar yields (C6 + C5) were similar for HTT of grass and grass silage at both 170 and 180°C, but at 190°C the overall sugar yield was better for HTT of dry grass. CONCLUSIONS: This study unequivocally establishes that ensiling of grass as a biomass pretreatment method comes with a loss of WSC. The loss of WSC by ensiling is not necessarily compensated for by providing a lower temperature requirement for HTT for high enzymatic monosaccharide release. However, ensiling can be an advantageous storage method prior to grass processing

Transcriptional and Metabolomic Analyses Indicate that Cell Wall Properties are Associated with Drought Tolerance in Brachypodium distachyon

Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for “regulated stress response”, “plant cell wall” and “oxidative stress” associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops

Genome Report: Whole Genome Sequence of Two Wild-Derived Mus musculus domesticus Inbred Strains, LEWES/EiJ and ZALENDE/EiJ, with Different Diploid Numbers

Wild-derived mouse inbred strains are becoming increasingly popular for complex traits analysis, evolutionary studies, and systems genetics. Here, we report the whole-genome sequencing of two wild-derived mouse inbred strains, LEWES/EiJ and ZALENDE/EiJ, of Mus musculus domesticus origin. These two inbred strains were selected based on their geographic origin, karyotype, and use in ongoing research. We generated 14× and 18× coverage sequence, respectively, and discovered over 1.1 million novel variants, most of which are private to one of these strains. This report expands the number of wild-derived inbred genomes in the Mus genus from six to eight. The sequence variation can be accessed via an online query tool; variant calls (VCF format) and alignments (BAM format) are available for download from a dedicated ftp site. Finally, the sequencing data have also been stored in a lossless, compressed, and indexed format using the multi-string Burrows-Wheeler transform. All data can be used without restriction