Lipoxygenase activities during development of root and nodule of soybean

The objective of this work was to evaluate root and nodule soybean lipoxygenases in Doko cultivar and in a near isogenic line lacking seed lipoxygenases, inoculated and uninoculated with Bradyrhizobium elkanii. The lipoxygenase activities from roots collected at 3, 5, 9, 13, 18 and 28 days post-inoculation and from nodules collected at 13, 18 and 28 days post-inoculation were measured. The pH-activity profiles from root and nodules suggested that the lipoxygenases pool expressed in these organs from Doko cultivar and triple-null near isogenic lines are similar. The root lipoxygenase activity of Doko and triple-null lines, inoculated and uninoculated, reduced over time. The highest lipoxygenase activity observed at the beginning of root formation suggests the involvement of this enzyme in growth and development of this organ. However, for nodules an expressive increase of lipoxygenase activity was noticed 28 days post-inoculation. Root and nodule showed, at least, two mobility groups for lipoxygenases in immunoblottings, with approximately 94 and 97 kDa.

Identification of quantitative trait loci for resistance against soybean sudden death syndrome caused by Fusarium tucumaniae

The objective of this work was to identify genomic regions that underlie resistance to Fusarium tucumaniae sp. nov., the causing agent of sudden death syndrome (SDS) in soybean in South America, using a population with a genetic background different from that previously reported for Fusarium virguliforme sp. nov. (F. solani f. sp. glycines), also responsible for SDS in soybean. Although major genes and quantitative trait loci (QTL) for SDS resistance have been identified, little is known about the same disease caused by Fusarium tucumaniae sp. nov., in South America. To identify genetic factors related to resistance to F. tucumaniae and DNA markers associated with them, a QTL analysis was performed using recombinant inbred lines. The map locations of the four loci, here identified, differed from those SDS resistance QTL previously described. It was screened a residual heterozygous line (RHL), which was heterozygous around the most effective QTL, RSDS1, and homozygous for the other genomic regions. The genetic effect of RSDS1 was confirmed using near-isogenic lines (NIL) derived from the RHL. The line which was homozygous for the Misuzudaizu genotype showed resistance levels comparable with that of the line homozygous for the Moshidou Gong 503 genotype.

Natural genetic variation in Arabidopsis: tools, traits and prospects for evolutionary ecology.

BACKGROUND: The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years. SCOPE: Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants. CONCLUSIONS: As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.

Post-market environmental monitoring of Bt maize in Spain: Non-target effects of varieties derived from the event MON810 on predatory fauna

The Spanish Government has established post-market environmental monitoring (PMEM) as mandatory for genetically modified (GM) crop varieties cultivated in Spain. In order to comply with this regulation, effects of Bt maize varieties derived from the event MON810 on the predatory fauna were monitored for two years in northeast and central Spain. The study was carried out with a randomized block design in maize fields of 3-4 ha on which the abundance of plant-dwelling predators and the activity-density of soil-dwelling predators in Bt vs. non-Bt near-isogenic varieties were compared. To this end, the plots were sampled by visual inspection of a certain number of plants and pitfall traps 6 or 7 times throughout two seasons. No significant differences in predator densities on plants were found between Bt and non-Bt varieties. In the pitfall traps, significant differences between the two types of maize were found only in Staphylinidae, in which trap catches in non-Bt maize were higher than in Bt maize in central Spain. Based on the statistical power of the assays, surrogate arthropods for PMEM purposes are proposed; Orius spp. and Araneae for visual sampling and Carabidae, Araneae, and Staphylinidae for pitfall trapping. The other predator groups recorded in the study, Nabis sp. and Coccinellidae in visual sampling and Dermaptera in pitfall trapping, gave very poor power results. To help to establish a standardized protocol for PMEM of genetically modified crops, the effect-detecting capacity with a power of 0.8 of each predator group is given.

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE)

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event

Reduced height (Rht) and photoperiod insensitivity (Ppd) allele associations with establishment and early growth of wheat in contrasting production systems

Near isogenic lines (NILs) varying for genes for reduced height (Rht) and photoperiod insensitivity (Ppd-D1a) in a cv. Mercia background (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c + Ppd-D1a, Rht-D1c, Rht12) were compared at one field site but within contrasting ('organic' vs. 'conventional') rotational and agronomic contexts, in each of 3 years. In the final year, further NILs (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b + Rht-D1b, Rht-D1b + Rht-B1c) in both Maris Huntsman and Maris Widgeon backgrounds were added together with 64 lines of a doubled haploid (DH) population [Savannah (Rht-D1b) x Renesansa (Rht-8c + Ppd-D1a)]. Assessments included laboratory tests of germination and coleoptile length, and various field measurements of crop growth between emergence and pre jointing [plant population, tillering, leaf length, ground cover (GC), interception of photosynthetically active radiation (PAR), crop dry matter (DM) and nitrogen accumulation (N), far red: red reflectance ratio (FR:R), crop height, and weed dry matter]. All of the dwarfing alleles except Rht12 in the Mercia background and Rht8c in the DHs were associated with reduced coleoptile length. Most of the dwarfing alleles (depending on background) reduced seed viability. Severe dwarfing alleles (Rht-B1c, Rht-D1c and Rht12) were routinely associated with fewer plant numbers and reduced early crop growth (GC, PAR, DM, N, FR:R), and in 1 year, increased weed DM. In the Mercia background and the DHs the semi-dwarfing allele Rht-D1b was also sometimes associated with reductions in early crop growth; no such negative effects were associated with the marker for Rht8c. When significant interactions between cropping system and genotype did occur it was because differences between lines were more exaggerated in the organic system than in the conventional system. Ppd-D1a was associated positively with plant numbers surviving the winter and early crop growth (GC, FR:R, DM, N, PAR, height), and was the most significant locus in a QTL analysis. We conclude that, within these environmental and system contexts, genes moderating development are likely to be more important in influencing early resource capture than using Rht8c as an alternative semi-dwarfing gene to Rht-D1b.

Effects of corn silage derived from a genetically modified variety containing two Transgenes on feed intake, milk production, and composition, and the absence of detectable Transgenic deoxyribonucleic acid in milk in holstein dairy cows

The objectives were to compare the chemical composition, nutritive value, feed intake, milk production and composition, and presence in milk of transgenic DNA and the encoded protein Cry1Ab when corn silages containing 2 transgenes (2GM: herbicide tolerance: mepsps and insect resistance: cry1Ab) were fed as part of a standard total mixed ration (TMR) compared with a near isogenic corn silage ( C) to 8 multiparous lactating Holstein dairy cows in a single reversal design study. Cows were fed a TMR ration ad libitum and milked twice daily. Diets contained [ dry matter (DM) basis] 45% corn silage, 10% alfalfa hay, and 45% concentrate (1.66 Mcal of net energy for lactation/kg of DM, 15.8% crude protein, 35% neutral detergent fiber, and 4.1% fat). Each period was 28-d long. During the last 4 d of each period, feed intake and milk production data were recorded and milk samples taken for compositional analysis, including the presence of transgenic DNA and Cry1Ab protein. There was no significant difference in the chemical composition between C and 2GM silages, and both were within the expected range (37.6% DM, 1.51 Mcal of net energy for lactation/kg, 8.6% crude protein, 40% neutral detergent fiber, 19.6% acid detergent fiber, pH 3.76, and 62% in vitro DM digestibility). Cows fed the 2GM silage produced milk with slightly higher protein (3.09 vs. 3.00%), lactose ( 4.83 vs. 4.72%) and solids-not-fat (8.60 vs. 8.40%) compared with C. However, the yield (kg/d) of milk (36.5), 3.5% fat-corrected milk (34.4), fat (1.151), protein (1.106), lactose (1.738), and solids-not-fat ( 3.094), somatic cell count (log(10): 2.11), change in body weight (+ 7.8 kg), and condition score (+ 0.09) were not affected by type of silage, indicating no overall production difference. All milk samples were negative for the presence of transgenic DNA from either trait or the Cry1Ab protein. Results indicate that the 2GM silage modified with 2 transgenes did not affect nutrient composition of the silages and had no effect on animal performance and milk composition. No transgenic DNA and Cry1Ab protein were detected in milk.

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments (similar to 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.

Mutant alleles at the rugosus loci in pea affect seed moisture sorption isotherms and the relations between seed longevity and moisture content

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 degreesC, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. C-w was lower). The low-moisture-content limit (m(c)) to this relation also differed, being lower in the mutant near-isogenic lines (5.4-5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P >0.25) in the negative semilogarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity. was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below mc where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.

Detection of transgenic DNA and protein, in rumen fluid, duodenal digesta, milk, blood and faeces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Effect of corn silage from an herbicide-tolerant genetically modified variety on milk production and absence of transgenic DNA in milk

Data from 60 multiparous Holstein cows were used in a 12-wk continuous design feeding trial. Cows were allocated to 1 of 4 experimental treatments (T1 to T4). In T1 and T2, the total mixed ration (TMR) contained either corn silage from the genetically modified (GM) variety Chardon Liberty Link, which is tolerant to the herbicide glufosinate ammonium, or its near isogenic nonGM counterpart, whereas the TMR used in T3 and T4 contained corn silage from the commercially available nonGM varieties Fabius and Antares, respectively. The objectives of the study were to determine if the inserted gene produced a marked effect on chemical composition, nutritive value, feed intake, and milk production, and to determine if transgenic DNA and the protein expressed by the inserted gene could be detected in bovine milk. The nutritive value, fermentation characteristics, mineral content, and amino acid composition of all 4 silages were similar. There were no significant treatment effects on milk yield, milk composition, and yield of milk constituents, and the dry matter (DM) intake of the GM variety was not significantly different from the 2 commercial varieties. However, although the DM intake noted for the nonGM near-isogenic variety was similar to the commercial varieties, it was significantly lower when compared with the GM variety. Polymerase chain reaction analyses of milk samples collected at wk 1, 6, and 12 of the study showed that none of the 90 milk samples tested positive, above a detection limit of 2.5 ng of total genomic DNA/mL of milk, for either tDNA (event T25) or the single-copy endogenous Zea mays gene, alcohol dehydrogenase. Using ELISA assays, the protein expressed by the T25 gene was not detected in milk.

The effects of dwarfing genes on seedling root growth of wheat

Most modern wheat cultivars contain major dwarfing genes, but their effects on root growth are unclear. Near-isogenic lines (NILs) containing Rht-B1b, Rht-D1b, Rht-B1c, Rht8c, Rht-D1c, and Rht12 were used to characterize the effects of semi-dwarfing and dwarfing alleles on root growth of 'Mercia' and 'Maris Widgeon' wheat cultivars. Wheat seedlings were grown in gel chambers, soil-filled columns, and in the field. Roots were extracted and length and dry mass measured. No significant differences in root length were found between semi-dwarfing lines and the control lines in any experiment, nor was there a significant difference between the root lengths of the two cultivars grown in the field. Total root length of the dwarf lines (Rht-B1c, Rht-D1c, and Rht12) was significantly different from that of the control although the effect was dependent on the experimental methodology; in gel chambers root length of dwarfing lines was increased by; 40% while in both soil media it was decreased (by 24-33%). Root dry mass was 22-30% of the total dry mass in the soil-filled column and field experiments. Root length increased proportionally with grain mass, which varied between NILs, so grain mass was a covariate for the analysis of variance. Although total root length was altered by dwarf lines, root architecture (average root diameter, lateral root: total root ratio) was not affected by reduced height alleles. A direct effect of dwarfing alleles on root growth during seedling establishment, rather than a secondary partitioning effect, was suggested by the present experiments.

Effect of corn silage from an herbicide-tolerant genetically modified variety on milk production and absence of transgenic DNA in milk

Data from 60 multiparous Holstein cows were used in a 12-wk continuous design feeding trial. Cows were allocated to 1 of 4 experimental treatments (T1 to T4). In T1 and T2, the total mixed ration (TMR) contained either corn silage from the genetically modified (GM) variety Chardon Liberty Link, which is tolerant to the herbicide glufosinate ammonium, or its near isogenic nonGM counterpart, whereas the TMR used in T3 and T4 contained corn silage from the commercially available nonGM varieties Fabius and Antares, respectively. The objectives of the study were to determine if the inserted gene produced a marked effect on chemical composition, nutritive value, feed intake, and milk production, and to determine if transgenic DNA and the protein expressed by the inserted gene could be detected in bovine milk. The nutritive value, fermentation characteristics, mineral content, and amino acid composition of all 4 silages were similar. There were no significant treatment effects on milk yield, milk composition, and yield of milk constituents, and the dry matter (DM) intake of the GM variety was not significantly different from the 2 commercial varieties. However, although the DM intake noted for the nonGM near-isogenic variety was similar to the commercial varieties, it was significantly lower when compared with the GM variety. Polymerase chain reaction analyses of milk samples collected at wk 1, 6, and 12 of the study showed that none of the 90 milk samples tested positive, above a detection limit of 2.5 ng of total genomic DNA/mL of milk, for either tDNA (event T25) or the single-copy endogenous Zea mays gene, alcohol dehydrogenase. Using ELISA assays, the protein expressed by the T25 gene was not detected in milk.

Mutant alleles at the rugosus loci in pea affect seed moisture sorption isotherms and the relations between seed longevity and moisture content

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 °C, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. CW was lower). The low-moisture-content limit (mc) to this relation also differed, being lower in the mutant near-isogenic lines (5.4–5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P >0.25) in the negative semi-logarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below mc where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.