Development of a STS marker assay for detecting loss of heterozygosity in rice hybrids.

The RAPD (random amplified polymorphic DNA) markers OPE15750 and OPE15300 were affected by loss of heterozygosity (LOH) in rice hybrids AMR x 'M202' and AMR x 'L202'. The markers were mapped to the same locus at or near the centromere of rice chromosome 2. The two RAPD products were cloned, sequenced, and found to have lengths of 734 bp and 297 bp, respectively. The 297-bp sequence shares a 98% homology with one end of the 734-bp sequence, accounting for the cross-hybridization previously observed between the two clones. Based on the DNA sequence of the 734-bp fragment, a pair of STS (sequence-tagged site) primers was designed and tested. Rice plants homozygous for either OPE15734 or OPE15297 all produced PCR fragments of the same length, 482 bp. However, the two PCR products were discernible by digestion with the restriction enzyme XbaI prior to gel electrophoresis. The STS product from plants homozygous for OPE15734 was cut into two fragments of 239 and 240 bp, which appeared as one single band in an agarose gel; whereas the STS product from plants homozygous for OPE15297 was not cut by XbaI and was characterized by a 482-bp band in the agarose gel. These STS primers were tested in rice hybrids and F2 progenies derived from the hybrids of AMR x 'M202' and AMR x 'L202'. Homozygosity for OPE15297 was confirmed for all F2 panicle rows derived from AMR x 'M202'. In contrast, F2 panicle rows of AMR x 'L202' exhibited two different segregation patterns (genotypes), i.e., either uniformly homozygous for the 240-bp marker (OPE15734/OPE15734) or segregating for the 482- and 240-bp markers (OPE15734/OPE15297). This STS-marker system provides a robust assay for detecting the occurrence of LOH in rice hybrid progenies.

Linkage mapping and nucleotide polymorphisms of the 6-SFT gene of cool-season grasses.

Fructan plays an important role as an alternate carbohydrate and may contribute to drought and cold-stress tolerances in various plant species. The gene coding for sucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4.1.10), an enzyme that catalyzes the formation and extension of beta-2,6-linked fructans (levans), is important to fructan synthesis in many cool-season grasses, including cereal species. In this study, we compared a conserved sequence from the 6-SFT gene in barley with comparable sequences in 20 other cool-season grasses. We detected several DNA length polymorphisms, including variations in one simple-sequence repeat (SSR) in a 6-SFT intron of the barley cultivars Steptoe and Morex. Using the 'Steptoe' x 'Morex' doubled-haploid mapping population, the 6-SFT gene was genetically mapped to the distal region in the short arm of barley chromosome 1 (7H), where it is closely linked with trait locus Rpg1. Primers designed from other conserved regions of the barley 6-SFT gene successfully amplified 351- or 354-bp sequences of this gene from diverse cool season grass species. Sequence identities of the PCR products were greater than 80% among the 21 species. Phylogeny, as determined using these DNA sequences, is similar to that obtained from rDNA ITS sequences, and congruent with our current knowledge of genome relationships.

Elevated CO2 enhances resprouting of a tropical savanna tree.

The savannas (cerrado) of south-central Brazil are currently subjected to frequent anthropogenic burning, causing widespread reduction in tree density. Increasing concentrations of atmospheric CO2 could reduce the impact of such frequent burning by increasing the availability of nonstructural carbohydrate, which is necessary for resprouting. We tested the hypotheses that elevated CO2 stimulates resprouting and accelerates replenishment of carbohydrate reserves. Using a factorial experiment, seedlings of a common Brazilian savanna tree, Keilmeyera coriacea, were grown at 350 ppm and 700 ppm CO2 and at two nutrient levels. To simulate burning, the plants were either clipped at 15 weeks or were left unclipped. Among unclipped plants, CO2 and nutrients both stimulated growth, with no significant interaction between nutrient and CO2 effects. Among clipped plants, both CO2 and nutrients stimulated resprouting. However, there was a strong interaction between CO2 and nutrient effects, with CO2 having a significant effect only in the presence of high nutrient availability. Under elevated CO2, carbohydrate reserves remained at higher levels following clipping. Root total nonstructural carbohydrate remained above 36% in all treatments, so carbohydrate reserves did not limit regrowth. These results indicate that under elevated CO2 this species may be better able to endure the high frequency of anthropogenic burning in the Brazilian savannas.

A quantitative histochemical procedure for measurement of starch in apple fruits.

Measurements of starch (e.g. amyloplasts in stomatal guard cells, sieve elements, root tips or the starch sheath) is often very difficult using most analytical methods. An evaluation was made of interactive computer image analysis of starch measurements in apple fruits. The results obtained indicate that quantitative histochemistry can be an appropriate method to quantify starch. Correlations for starch values between the image analysis system and a colorimetric system were quantified. The thickness of plastic-embedded slices had no influence on the accuracy of the area occupied by image-quantified starch (starch/slice) or on its variance. The magnification of the objective also had no effect on measured starch-occupied areas (starch/slice), but there were big differences in variance. The number of replications required to establish statistically significant differences were calculated.

Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae), inferred from nuclear rDNA (internal transcribed spacer) sequences.

Phylogenetic relationships of 30 diploid species of Triticeae (Poaceae) representing 19 genomes were estimated from the sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The ITS sequence phylogeny indicated that: (i) each genome group of species is monophyletic, concordant with cytogenetic evidence; (ii) Hordeum (I) and Critesion (H) are basal; (iii) Australopyrum (W) is closely related to Agropyron (P); (iv) Peridictyon (G), Heteranthelium (Q), and Dasypyrum (V) are closely related to Pseudoroegneria (S); (v) most of the annuals, Triticum s.l. (A, B, D), Crithopsis (K), Taeniatherum (T), Eremopyrum (F), Henrardia (O), Secale (R), and two perennials, Thinopyrum (J) and Lophopyrum (E), all of Mediterranean origin, are a monophyletic group. However, phylogenetic trees based on morphology group these Mediteranean species with various perennial lineages of the Arctic-temperate region. The molecular data and biogeography of the tribe suggest that the Mediterranean lineage is derived from the Arctic-temperate lineage and that the two lineages have evolved in parallel. Extensive morphological parallelism apparently obscures the true genealogical history of the tribe when only morphology is considered.

Molecular phylogeny of the Pooideae (Poaceae) based on nuclear rDNA (ITS) sequences.

Phylogenetic relationships of the Poaceae subfamily, Pooideae, were estimated from the sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The entire ITS region of 25 species belonging to 19 genera representing seven tribes was directly sequenced from polymerase chain reaction (PCR)-amplified DNA fragments. The published sequence of rice, Oryza saliva, was used as the outgroup. Sequences of these taxa were analyzed with maximum parsimony (PAUP) and the neighbor-joining distance method (NJ). Among the tribes, the Stipeae, Meliceae and Brachypodieae, all with small chromosomes and a basic number more than x=7, diverged in succession. The Poeae, Aveneae, Bromeae and Triticeae, with large chromosomes and a basic number of x=7, form a monophyletic clade. The Poeae and Aveneae are the sister group of the Bromeae and Triticeae. On the ITS tree, the Brachypodieae is distantly related to the Triticeae and Bromeae, which differs from the phylogenies based on restriction-site variation of cpDNA and morphological characters. The phylogenetic relationships of the seven pooid tribes inferred from the ITS sequences are highly concordant with the cytogenetic evidence that the reduction in chromosome number and the increase in chromosome size evolved only once in the pooids and pre-dated the divergence of the Poeae, Aveneae, Bromeae and Triticeae.This paper reports factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

Phylogenetic relationships of 10 grass species: an assessment of phylogenetic utility of the internal transcribed spacer region in nuclear ribosomal DNA in monocots.

Entire sequences of the internal transcribed spacers (ITSs) and 5.8S subunit of nuclear ribosomal DNA (nrDNA) were obtained from nine grass species by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified DNA fragments. These sequences from subfamily Pooideae (Triticum aestivum, Crithodium monococcum, Sitopsis speltoides, Hordeum vulgare, Secale montanum, Avena longiglumis, Bromus inermis, Brachypodium distachyon) and subfamily Panicoideae (Sorghum bicolor) together with published ITS sequence of rice (Oryza sativa, Bambusoideae) were analyzed using Wagner parsimony (PAUP) and the neighbor-joining distance method to assess the phylogenetic utility of ITS sequences at various taxonomic levels. Among the aligned sequences that ranged from 588 to 603 nucleotides in length, 118 of 269 variable sites contained potential phylogenetic information. A member of Bromus, B. inermis, was the sister taxon to the Triticeae species. Brachypodium was more distantly related to Triticeae than was Bromus or Avena. These data, with Oryza sativa as the outgroup, indicate monophyly of the Pooideae species and of the members of the tribe Triticeae within Pooideae. Phylogenetic trees of the 10 grass species generated from the ITS sequence data were in general agreement with phylogenies based on molecular data from ribosomal RNA (rRNA) and chloroplast DNA (cpDNA) of similar grass taxa. This study reaffirms that sequences of the ITS region are useful for phylogenetic inference among closely related monocot species.

The influence of elevated CO2 on non-structural carbohydrate distribution and fructan accumulation in wheat canopies.

We grew 2.4 m2 wheat canopies in a large growth chamber under high photosynthetic photon flux (1000 micromoles m-2 s-1) and using two CO2 concentrations, 360 and 1200 micromoles mol-1. Photosynthetically active radiation (400-700 nm) was attenuated slightly faster through canopies grown in 360 micromoles mol-1 than through canopies grown in 1200 micromoles mol-1, even though high-CO2 canopies attained larger leaf area indices. Tissue fractions were sampled from each 5-cm layer of the canopies. Leaf tissue sampled from the tops of canopies grown in 1200 micromoles mol-1 accumulated significantly more total non-structural carbohydrate, starch, fructan, sucrose, and glucose (p < 0.05) than for canopies grown in 360 micromoles mol-1. Non-structural carbohydrate did not significantly increase in the lower canopy layers of the elevated CO2 treatment. Elevated CO2 induced fructan synthesis in all leaf tissue fractions, but fructan formation was greatest in the uppermost leaf area. A moderate temperature reduction of 10 degrees C over 5 d increased starch, fructan and glucose levels in canopies grown in 1200 micromoles mol-1, but concentrations of sucrose and fructose decreased slightly or remained unchanged. Those results may correspond with the use of fructosyl-residues and release of glucose when sucrose is consumed in fructan synthesis.

Proton and carbon NMR chemical-shift assignments for [beta-D-Fru f-(2-->1)]3-(2<==>1)-alpha-D-Glc p (nystose) and [beta-D-Fru f-(2-->1)]4-(2<==>1)-alpha-D-Glc p (1,1,1-kestopentaose) from two-dimensional NMR spectral measurements.

The proton chemical-shift assignment of nystose (1) [beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-(2-->1)-beta-D- fructofuranosyl-(2<==>1)-alpha-D-glucopyranoside], was determined by using two-dimensional (2D) NMR spectral methods, and corrections of, and additions to the previous 13C chemical-shift assignments were made. The 1H peak of H-1 of the D-glucosyl group was determined by its chemical shift. Signals from fructose-1 were distinguished by the observation of long-range C-H coupling between H-1 of the D-glucosyl group and C-2 of fructose-1. The distinction between fructose-2 and fructose-3 was made by the different 1JCH coupling patterns between C-1 and H-1. Assignments of 13C and 1H chemical shifts of the related dp 5 compound, beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-(2-->1)-beta-D- fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-(2<==>1)-alpha-D-glucopyr anoside (1,1,1-kestopentaose, 2) are also reported here with comparisons of its spectral data with the data from 1-kestose, nystose and inulin. Based on differences in 13C chemical shifts, it appears that the chemical environment of inulin is not attained in nystose, and only partially attained in 1,1,1-kestopentaose.

Proton and carbon chemical-shift assignments for 6-kestose and neokestose from two-dimensional n.m.r. measurements.

The proton and carbon chemical shift assignments of the simplest levan, 6-kestose, [O-beta-D-fructofuranosyl-(2----6)-beta-D-fructofuranosyl-(2 in equilibrium with 1)-alpha-D-glucopyranoside], along with another trisaccharide, neokestose, [O-beta-D-fructofuranosyl-(2----6)-alpha-D-glucopyranosyl-(1 in equilibrium with 2)-beta-D-fructofuranoside], were determined using two-dimensional (2D) homonuclear and heteronuclear n.m.r. methods. The 1H peak of H-1 of the glucose residue was determined by its chemical shift. Using H-H coupling information to this proton, the chemical shifts of most of the proton signals of the glucose moiety were determined. Though the signals from the two fructose residues were very close, a NOESY experiment and long-range C-H correlation experiments allowed their complete carbon and proton assignment. This work completes and corrects literature assignments.

Photosynthate Partitioning into Starch in Soybean Leaves: II. IRRADIANCE LEVEL AND DAILY PHOTOSYNTHETIC PERIOD DURATION EFFECTS.

Two photosynthetic periods and photosynthetic photon flux densities (PPFD) were used to study the relationship between the rate of photosynthesis and starch accumulation in vegetative soybean leaves (Merr. cv Amsoy 71). Plants grown in short daily photosynthetic periods (7 hours) had higher rates of CO(2) fixation per unit leaf dry weight and of leaf starch accumulation than plants grown in long daily photosynthetic periods (14 hours) irrespective of PPFD. CO(2) fixation rates per unit leaf area were similar in 7-hour and 14-hour plants grown at low PPFD but were highest in 14-hour plants at the high PPFD. When single leaves of 14-hour plants were given 7-hour photosynthetic periods, their rates of starch accumulation remained unchanged. The programming of starch accumulation rate and possibly of photosynthetic rate by the length of the daily photosynthetic period is apparently a whole-plant, not an individual leaf, phenomenon. Programming of chloroplast starch accumulation rate by length of the daily photosynthetic and/or dark periods was independent of PPFD within the ranges used in this experiment.

Photosynthate Partitioning into Starch in Soybean Leaves: I. Effects of Photoperiod versus Photosynthetic Period Duration.

Photosynthesis, photosynthate partitioning into foliar starch, and translocation were investigated in soybean plants (Glycine max (L.) Merr. cv. Amsoy 71), grown under different photoperiods and photosynthetic periods to determine the controls of leaf starch accumulation. Starch accumulation rates in soybean leaves were inversely related to the length of the daily photosynthetic period under which the plants were grown. Photosynthetic period and not photoperiod per se appears to be the important factor. Plants grown in a 14-hour photosynthetic period partitioned approximately 60% of the daily foliar accumulation into starch whereas 7-hour plants partitioned about 90% of their daily foliar accumulation into starch. The difference in starch accumulation resulted from a change in photosynthate partitioning between starch and leaf residual dry weight. Residual dry weight is defined as leaf dry weight minus the weight of total nonstructural carbohydrates. Differences in photosynthate partitioning into starch were also associated with changes in photosynthetic and translocation rates, as well as with leaf and whole plant morphology. It is concluded that leaf starch accumulation is a programmed process and not simply the result of a limitation in translocation.

Photosynthate partitioning in soybean leaves at two irradiance levels: comparative responses of acclimated and unacclimated leaves.

High irradiance-acclimated soybean leaves had the same CO(2) exchange rates, but lower starch accumulation rates and correspondingly higher translocation rates than unacclimated leaves. Increased translocation rates were associated with increased sucrose phosphate synthetase (EC 2.4.1.14) activity. Foliar sucrose levels and adenosine diphosphate-glucose pyrophosphorylase (EC 2.7.7.9) activity were unaffected. Carbon assimilation, partitioning, and enzyme activity of unacclimated leaves were unaltered even after a second day's exposure to high irradiance. Results are consistent with the hypothesis that photosynthate partitioning between starch synthesis and sucrose translocation are controlled in part by the rate of sucrose synthesis.