A rapid DNA isolation procedure for small quantities of fresh leaf tissue

Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

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A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.

Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components

Although the chloroplast genome contains many noncoding regions, relatively few have been exploited for interspecific phylogenetic and intraspecific phylogeographic studies. In our recent evaluation of the phylogenetic utility of 21 noncoding chloroplast regions, we found the most widely used noncoding regions are among the least variable, but the more variable regions have rarely been employed. That study led us to conclude that there may be unexplored regions of the chloroplast genome that have even higher relative levels of variability. To explore the potential variability of previously unexplored regions, we compared three pairs of single-copy chloroplast genome sequences in three disparate angiosperm lineages: Atropa vs. Nicotiana (asterids); Lotus vs. Medicago (rosids); and Saccharum vs. Oryza (monocots). These three separate sequence alignments highlighted 13 mutational hotspots that may be more variable than the best regions of our former study. These 13 regions were then selected for a more detailed analysis. Here we show that nine of these newly explored regions (rpl32-trnL((UAG)), trnQ((UUG))-5'rps16, 3'trnV((UAC))-ndhC, ndhF-rpl32, psbD-trnT((GGU)), psbJ-petA, 3'rps16-5'trnK((UUU)), atpI-atpH, and petL-psbE) offer levels of variation better than the best regions identified in our earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.

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Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis

Tobacco (Nicotiana rustica) plants were stressed by addition of polyethylene glycol solution (−20 bar) to the growth medium. The proline contents and concentrations in total protoplasts, vacuoles, and extravacuolar fractions of these plants have been determined and compared with protoplasts and cell fractions of well-watered plants. As compared to the control, the stress treatment of intact plants results in a 7-fold increase of the proline content in the extravacuolar fraction while the vacuolar content was enriched only 2.6-fold. In protoplasts of control plants, a proline concentration ratio (extravacuole to vacuole) of 1 was measured. In protoplasts of stressed plants, this ratio was nearly 3. Thus, water stress seems to have an effect on a tonoplast-localized transport system for proline.

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Influence of Water Stress on the Vacuole/Extravacuole Distribution of Proline in Protoplasts of Nicotiana rustica

An enzyme has been purified to homogeneity from barley seedlings which has `proline dehydrogenase9 and the pyrroline-5-carboxylic acid reductase activities. The purification achieved is 39,000-fold as calculated from the proline dehydrogenase activity. The subunit molecular weight of the protein is 30 kilodaltons. The native enzyme has molecular weights up to 480 kilodaltons, depending on the buffer environment. From the pH profiles, the specific activities and thermodynamic considerations, it is concluded that the plant proline dehydrogenase functions in vivo as a pyrroline-5-carboxylate reductase.

Purification to homogeneity of pyrroline-5-carboxylate reductase of barley.

The multiple molecular forms of glutamate dehydrogenase from pea seedlings (Pisum sativum, var. Spath's Violetta) have been investigated. When protein preparations are subjected to electrophoresis on polyacrylamide gels, the glutamate dehydrogenase can be localized by substrate staining. Shoots show seven activity bands, whereas roots have one main zone and several faint ones. SO2-fumigation generates typical alterations of the shoot zymogram. The molecular weight of all the distinct enzyme components is identical and has been shown to be 210000. Urea denaturation with subsequent renaturation of the various glutamate dehydrogenase preparations from roots, shoots and SO2-fumigated shoots results in the formation of one identical activity band on polyacrylamide gels. The results discussed here give much evidence that the multiple molecular forms of glutamate dehydrogenase from pea seedlings are conformers.

Sind die multiplen Formen der Glutamatdehydrogenase aus Erbsenkeimlingen Conformer

The most active multiple molecular form of glutamate dehydrogenase from pea seeds has been enriched 30000-fold with a recovery of 60–70%. The preparation is homogenous as judged from gel electrophoresis of native and dodecylsulfate-denatured enzyme and from analytical ultracentrifugation. Specific activities were 530 U/mg in the reductive amination and 90 U/mg in the oxidative deamination reaction. A sedimentation coefficient of s20, w= 10.49 S was determined. The specific volume and the molecular weight of the native enzyme were found to be v2= 0.759 cm3/g and Mr= 260000, respectively, by equilibrium sedimentation in H2O and 90%2H2O buffers. Dodecylsulfate electrophoresis of the denatured enzyme yielded a molecular weight of Mr= 44000 for the polypeptide chain. From our data we propose glutamate dehydrogenase from peas to be a hexamer. The hexameric structure is confirmed by the appearance of six electrophoretic bands after crosslinking with diimidates. Enzyme which was treated with Dowex A1 chelating resin was found to be almost completely inactive in the absence of divalent metal ions. From several metals tested, calcium was most efficient in reactivating the enzyme; half-maximal activity was attained at about 5 μM calcium. In contrast to potassium ions, sodium ions were found to interfere with this regulatory mechanism by activating the enzyme at high concentrations.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1980.tb06116.x

Edwin Pahlich

Papers

A rapid DNA isolation procedure for small quantities of fresh leaf tissue

Influence of Water Stress on the Vacuole/Extravacuole Distribution of Proline in Protoplasts of Nicotiana rustica

Purification to homogeneity of pyrroline-5-carboxylate reductase of barley.

Sind die multiplen Formen der Glutamatdehydrogenase aus Erbsenkeimlingen Conformer

Glutamate Dehydrogenase from Peas: Isolation, Quaternary Structure, and Influence of Cations on Activity