Identifying similar transcripts in a related organism from de Bruijn graphs of RNA-Seq data, with applications to the study of salt and waterlogging tolerance in Melilotus.

BACKGROUND: A popular strategy to study alternative splicing in non-model organisms starts from sequencing the entire transcriptome, then assembling the reads by using de novo transcriptome assembly algorithms to obtain predicted transcripts. A similarity search algorithm is then applied to a related organism to infer possible function of these predicted transcripts. While some of these predictions may be inaccurate and transcripts with low coverage are often missed, we observe that it is possible to obtain a more complete set of transcripts to facilitate possible functional assignments by starting the search from the intermediate de Bruijn graph that contains all branching possibilities. RESULTS: We develop an algorithm to extract similar transcripts in a related organism by starting the search from the de Bruijn graph that represents the transcriptome instead of from predicted transcripts. We show that our algorithm is able to recover more similar transcripts than existing algorithms, with large improvements in obtaining longer transcripts and a finer resolution of isoforms. We apply our algorithm to study salt and waterlogging tolerance in two Melilotus species by constructing new RNA-Seq libraries. CONCLUSIONS: We have developed an algorithm to identify paths in the de Bruijn graph that correspond to similar transcripts in a related organism directly. Our strategy bypasses the transcript prediction step in RNA-Seq data and makes use of support from evolutionary information.

Aerenchymatous phellem in hypocotyl and roots enables O2 transport in Melilotus siculus.

• Aerenchymatous phellem (secondary aerenchyma) has rarely been studied in roots. Its formation and role in internal aeration were evaluated for Melilotus siculus, an annual legume of wet saline land. • Plants were grown for 21 d in aerated or stagnant (deoxygenated) agar solutions. Root porosity and maximum diameters were measured after 0, 7, 14 and 21 d of treatment. Phellem anatomy was studied and oxygen (O(2)) transport properties examined using methylene blue dye and root-sleeving O(2) electrodes. • Interconnecting aerenchymatous phellem developed in hypocotyl, tap root and older laterals (but not in aerial shoots), with radial intercellular connections to steles. Porosity of main roots containing phellem was c. 25%; cross-sectional areas of this phellem were threefold greater for stagnant than for aerated treatments. Root radial O(2) loss was significantly reduced by complete hypocotyl submergence; values approached zero after disruption of hypocotyl phellem below the waterline or, after shoot excision, by covering hypocotyl phellem in nontoxic cream. • Aerenchymatous phellem enables hypocotyl-to-root O(2) transport in M. siculus. Phellem increases radially under stagnant conditions, and will contribute to waterlogging tolerance by enhancing root aeration. It seems likely that with hypocotyl submerged, O(2) will diffuse via surface gas-films and internally from the shoot system.

Mechanisms of Cl(-) transport contributing to salt tolerance.

Mechanisms of Cl(-) transport in plants are poorly understood, despite the importance of minimizing Cl(-) toxicity for salt tolerance. This review summarizes Cl(-) transport processes in plants that contribute to genotypic differences in salt tolerance, identifying key traits from the cellular to whole-plant level. Key aspects of Cl(-) transport that contribute to salt tolerance in some species include reduced net xylem loading, intracellular compartmentation and greater efflux of Cl(-) from roots. We also provide an update on the biophysics of anion transport in plant cells and address issues of charge balance, selectivity and energy expenditure relevant to Cl(-) transport mechanisms. Examples are given of anion transport systems where electrophysiology has revealed possible interactions with salinity. Finally, candidate genes for anion transporters are identified that may be contributing to Cl(-) movement within plants during salinity. This review integrates current knowledge of Cl(-) transport mechanisms to identify future pathways for improving salt tolerance.

Lotus tenuis tolerates combined salinity and waterlogging: maintaining O2 transport to roots and expression of an NHX1-like gene contribute to regulation of Na+ transport.

Salinity and waterlogging interact to reduce growth for most crop and pasture species. The combination of these stresses often cause a large increase in the rate of Na(+) and Cl(-) transport to shoots; however, the mechanisms responsible for this are largely unknown. To identify mechanisms contributing to the adverse interaction between salinity and waterlogging, we compared two Lotus species with contrasting tolerances when grown under saline (200 mM NaCl) and O(2)-deficient (stagnant) treatments. Measurements of radial O(2) loss (ROL) under stagnant conditions indicated that more O(2) reaches root tips of Lotus tenuis, compared with Lotus corniculatus. Better internal aeration would contribute to maintaining Na(+) and Cl(-) transport processes in roots of L. tenuis exposed to stagnant-plus-NaCl treatments. L. tenuis root Na(+) concentrations after stagnant-plus-NaCl treatment (200 mM) were 17% higher than L. corniculatus, with 55% of the total plant Na(+) being accumulated in roots, compared with only 39% for L. corniculatus. L. tenuis accumulated more Na(+) in roots, presumably in vacuoles, thereby reducing transport to the shoot (25% lower than L. corniculatus). A candidate gene for vacuole Na(+) accumulation, an NHX1-like gene, was cloned from L. tenuis and identity established via sequencing and yeast complementation. Transcript levels of NHX1 in L. tenuis roots under stagnant-plus-NaCl treatment were the same as for aerated NaCl, whereas L. corniculatus roots had reduced transcript levels. Enhanced O(2) transport to roots enables regulation of Na(+) transport processes in L. tenuis roots, contributing to tolerance to combined salinity and waterlogging stresses.

Improvement of salt and waterlogging tolerance in wheat: comparative physiology of Hordeum marinum-Triticum aestivum amphiploids with their H. marinum and wheat parents.

Hordeum marinum Huds. is a waterlogging-tolerant halophyte that has been hybridised with bread wheat (Triticum aestivum L.) to produce an amphiploid containing both genomes. This study tested the hypothesis that traits associated with waterlogging and salinity tolerances would be expressed in H. marinum-wheat amphiploids. Four H. marinum accessions were used as parents to produce amphiploids with Chinese Spring wheat, and their responses to hypoxic and 200mM NaCl were evaluated. Relative growth rate (RGR) in the hypoxic-saline treatment was better maintained in the amphiploids (58-71% of controls) than in wheat (56% of control), but the amphiploids were more affected than H. marinum (68-97% of controls). In hypoxic-saline conditions, leaf Na+ concentrations in the amphiploids were lower than in wheat (30-41% lower) but were 39-47% higher than in the H. marinum parents. A strong barrier to radial oxygen loss formed in basal root zones under hypoxic conditions in two H. marinum accessions; this barrier was moderate in the amphiploids, absent in wheat, and was weaker for the hypoxic-saline treatment. Porosity of adventitious roots increased with the hypoxic treatments; values were 24-38% in H. marinum, 16-27% in the amphiploids and 16% in wheat. Overall, the amphiploids showed greater salt and waterlogging tolerances than wheat, demonstrating the expression of relevant traits from H. marinum in the amphiploids.

Measuring soluble ion concentrations (Na(+), K(+), Cl(-)) in salt-treated plants.

The control of Na(+) and Cl(-) uptake from soils, and the partitioning of these ions within plants, is an essential component of salinity tolerance. Genetic variation in the ability of roots to exclude Na(+) and Cl(-) from the transpiration stream flowing to the shoot has been associated with salinity tolerance in many species. The maintenance of a high uptake of K(+) is also essential, so measurements of Na(+), K(+) or Cl(-) are frequently used to screen for genetic variation in salinity tolerance. As these ions are not bound covalently to compounds in cells, they can be readily extracted with dilute acid. Na(+) and K(+) can be measured in a dilute nitric acid extract using a flame photometer, by atomic absorption spectrometry or by inductively coupled plasma (ICP)-atomic emission spectrometry. Cl(-) can be measured in the same acid extract with a chloridometer or colorimetrically using a spectrophotometer.

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.