Marker-assisted selection for complex trait in common bean (Phaseolus vulgaris L.) using QTL-based index

A procedure was developed for marker-assisted selection of complex traits for common bean (Phaseolus vulgarisL.) using an index based on QTL-linked markers and ultrametric genetic distances between lines and a target parent. A comparison of the mean seed yields of the top five lines selected by different schemes demonstrated that the highest yielding group was selected on the basis of a combination of phenotypic performance and a high QTL-based index,followed by groups identified by a high QTL-based-index, conventional selection,and a low QTL-based-index. This study demonstrated a simple way to use information obtained from QTL studies to make selection decisions. The study also showed that the use of the QTL-based-index in conjunction with the ultrametric genetic distance to the target parent would enablea plant breeder to select lines that retain important QTL in a desirable genetic background. Therefore, this type of MAS would be expected to be superior to the phenotypic selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis

Roundup Ready (RR) genetically modified (GM) corn and soybean comprise a large portion of the annual planted acreage of GM crops. Plant growth and subsequent plant decomposition introduce the recombinant DNA (rDNA) into the soil environment, where its fate has not been completely researched. Little is known of the temporal and spatial distribution of plant-derived rDNA in the soil environment and in situ transport of plant DNA by leachate water has not been studied before. The objectives of this study were to determine whether sufficient quantities of plant rDNA were released by roots during growth and early decomposition to be detected in water collected after percolating through a soil profile and to determine the influence of temperature on DNA persistence in the leachate water. Individual plants of RR corn and RR soybean were grown in modified cylinders in a growth room, and the cylinders were flushed with rain water weekly. Immediately after collection, the leachate was subjected to DNA purification followed by rDNA quantification using real-time Polymerase Chain Reaction (PCR) analysis. To test the effects of temperature on plant DNA persistence in leachate water, water samples were spiked with known quantities of RR soybean or RR corn genomic DNA and DNA persistence was examined at 5, 15, and 25 degrees C. Differences in the amounts and temporal distributions of root-derived rDNA were observed between corn and soybean plants. The results suggest that rainfall events may distribute plant DNA throughout the soil and into leachate water. Half-lives of plant DNA in leachate water ranged from 1.2 to 26.7 h, and persistence was greater at colder temperatures (5 and 15 degrees C).     

Amplification of Bovine Viral Diarrhoea Virus Introduced into an In Vitro Embryo Production System Via Oocytes from Persistently Infected Cattle

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus. Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2) oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus. Virus was not isolated from any samples in treatment group 1. As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.     

An empirical approach to target DNA quantification in environmental samples using real-time polymerase chain reactions

Recent advances in molecular techniques have allowed for the routine examination of nucleic acids in environmental samples. Although current methodologies are very sensitive, accurate target DNA quantification from environmental samples remains challenging. To facilitate high-throughput DNA quantification from environmental samples, we developed a novel DNA quantification method based on a non-linear curve-fitting approach to extract additional information from quantitative PCR amplification curves and used the fitted parameters to develop multiple regression standard equations for target DNA quantification. A 3-parameter sigmoidal function performed superior to a 4-parameter Weibull function for generating the multiple regression standard equations. In a verification experiment, target DNA was quantified in a series of ‘unknown’ samples in three soils using this approach and the results were compared to target DNA values determined using corrected and uncorrected Ct-based (threshold cycle) methods. For each method, the deviations from the expected target DNA content were determined. Results clearly showed that over all DNA concentrations, target DNA content determined by the non-linear curve-fitting method was more accurate and more precise than values predicted by all other methods. Analysis of variance conducted on the predicted DNA contents also revealed fewer statistical artifacts with the non-linear curve fitting method compared to the conventional Ct-based methods. The novel approach described here is accurate, inexpensive, and very amenable for automation and high-throughput applications.     

Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation

The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.     

Effect of glyphosate on the tripartite symbiosis formed by Glomus intraradices,Bradyrhizobium japonicum,and genetically modified soybean

Most soybeans grown in North America are genetically modified (GM) to tolerate applications of the broad-spectrum herbicide glyphosate; as a result, glyphosate is now extensively used in soybean cropping systems. Soybean roots form both arbuscular mycorrhizal (AM) and rhizobial symbioses. In addition to individually improving host plant fitness, these symbioses also interact to influence the functioning of each symbiosis, thereby establishing a tripartite symbiosis. The objectives of this study were to (1) estimate the effects of glyphosate on the establishment and functioning of AM and rhizobial symbioses with GM soybean, and (2) to estimate the interdependence of the symbioses in determining the response of each symbiosis to glyphosate. These objectives were addressed in two experiments; the first investigated the importance of the timing of glyphosate application in determining the responses of the symbionts and the second varied the rate of glyphosate application. Glyphosate applied at recommended field rates had no effect on Glomus intraradices or Bradyrhizobium japonicum colonization of soybean roots, or on soybean foliar tissue [P]. N₂-fixation was greater for glyphosate-treated soybean plants than for untreated-plants in both experiments, but only when glyphosate was applied at the first trifoliate soybean growth stage. These data deviate from previous studies estimating the effect of glyphosate on the rhizobial symbiosis, some of which observed negative effects on rhizobial colonization and/or N₂-fixation. We did observe evidence of the response of one symbiont (stimulation of N₂-fixation following glyphosate) being dependent on co-inoculation with the other; however, this interactive response appeared to be contextually dependent as it was not consistent between experiments. Future research needs to consider the role of environmental factors and other biota when evaluating rhizobial responses to herbicide applications.     

Sequence variants in SLITRK1 are associated with Tourette's syndrome

Tourette's syndrome (TS) is a genetically influenced developmental neuropsychiatric disorder characterized by chronic vocal and motor tics. We studied Slit and Trk-like 1 (SLITRK1) as a candidate gene on chromosome 13q31.1 because of its proximity to a de novo chromosomal inversion in a child with TS. Among 174 unrelated probands, we identified a frameshift mutation and two independent occurrences of the identical variant in the binding site for microRNA hsa-miR-189. These variants were absent from 3600 control chromosomes. SLITRK1 mRNA and hsa-miR-189 showed an overlapping expression pattern in brain regions previously implicated in TS. Wild-type SLITRK1, but not the frameshift mutant, enhanced dendritic growth in primary neuronal cultures. Collectively, these findings support the association of rare SLITRK1 sequence variants with TS.     

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.