Use of genetic markers for the detection of off-types for DUS phenotypic traits in the inbreeding crop, barley

Detection of crop off-types is of interest for multiple uses, including the assessment of uniformity for new plant variety applications during distinctness, uniformity and stability (DUS) testing for the awarding of plant breeders’ rights (PBR). Here, we investigate whether genetic markers, in this case Kompetitive Allele-Specific PCR (KASP), can be used for the identification off-types for phenotypes assessed for DUS in the inbreeding cereal crop, barley (Hordeum vulgare). To demonstrate proof of principle, KASP markers diagnostic for phenotypic expression of nine DUS phenotypes, and DNA from two barley varieties (‘Pelican’ and ‘Felicie’) carrying contrasting alleles at each marker were used. We found that for the majority of markers, it was possible to robustly call alleles down to template DNA concentrations of 2 ng, but not ≤ 0.2 ng. When used in mixtures of DNA consisting of ‘Felicie’ DNA spiked with different concentrations of ‘Pelican’ DNA, robust allele calling was possible in DNA mixtures down to 18 ng:2 ng. Collectively, this demonstrates that where diagnostic markers are available, molecular identification of a single off-type for a given DUS trait within a bulk of ten individuals should be possible. We validated this assumption, with all of the diagnostic genetic markers investigated found to robustly detect DUS off-types at a frequency of 10% in DNA extracted from tissue collected from pools of 10 individuals. Ultimately, this work demonstrates that, where diagnostic polymorphisms are known for DUS traits, KASP markers should be able to robustly detect off-types or cross-contamination within DNA samples from a diploid inbred species down to 10%. While just two varieties that contrasted for the eight DUS targeted were investigated in this study, as the markers used are diagnostic for their relevant phenotype (or a proportion of the variation observed for that phenotype), in theory the approach should be valid for any variety studied— although the introduction of novel alleles via spontaneous mutation or more exotic germplasm pools may mean that marker sets would need to be periodically added to or updated. However, we nevertheless demonstrate the principle that, for a subset of DUS traits, molecular markers can now be robustly used as a tool towards determining all three components of the DUS testing process in barley. These results are relevant for the assessment of varietal uniformity by crop breeders, crop testing authorities and germplasm maintenance, as well as highlighting the potential use of bulk samples rather than individual plant samples for assessment of distinctness by molecular methods

Long-term vegetation studies in the southwest

In this paper, we describe several long-term studies in the ponderosa pine (Pinus ponderosa) forests of the American Southwest, focusing on the unique insights and contributions of these studies. Many of the studies that we discuss were established by staff from the Fort Valley Experiment Station (FVES; http://www.rmrs.nau.edu/fortvalley/)

Continuing fire regimes in remote forest of Grand Canyon National Park

Ponderosa pine forests in which frequent fire regimes continue up to the present would be invaluable points of reference for assessing natural ecological attributes. A few remote forests on the North Rim of Grand Canyon National Park come close to this ideal: never-harvested, distant from human communities and fire suppression resources, and with several low-intensity fires in the past century—a highly unusual recent fire regime in the Southwest. Recent fires appear to have played a crucial role in preventing the increases in forest density that characterize most southwestern pine forests. The study sites are not unaffected by the ecological changes associated with settlement, but they do present an important reference resource for study and management of ponderosa pine ecosystems

Seeding versus natural regeneration: a comparison of vegetation change following thinning and burning in ponderosa pine

The decision whether to seed with native species following restoration treatments should be based on existing vegetation, species present in or absent from the soil seed bank, past management history, microclimate conditions and soils. We installed three permanent monitoring plots in two areas (total 18.6 ha) at Mt. Trumbull, AZ. Trees were thinned and the sites burned in 1996 and 1997. A 5 ha area was seeded with native shrub, grass and forb species; the remaining 13.6 ha were unseeded. Pretreatment species richness ranged from none to five species per plot. We recorded 13 graminoid and eight shrub species in the seeded area, and four graminoid and four shrub species in the unseeded area. The greatest increase in species richness in both seeded and unseeded plots occurred approximately 1.8 years posttreatment. Perennial native species dominated plant cover by 2.8 years, although annual native forbs dominate the soil seed bank. Perennial grasses are nearly absent from the seed bank. The seeded area had the highest diversity, but it also had twice as many nonnative species (14 versus 7 in the unseeded plots). By August 1999, maximum species richness reached 51 species on the seeded plot. Of these species, 80 percent were native. Although seeding increases diversity, it may also have the long-term tradeoff of introducing new genotypes and species, both native and nonnative

Ponderosa pine forest reconstruction: Comparisons with historical data

Dendroecological forest reconstruction techniques are used to estimate presettlement structure of northern Arizona ponderosa pine forests. To test the accuracy of these techniques, we remeasured 10 of the oldest forest plots in Arizona, a subset of 51 historical plots established throughout the region from 1909 to 1913, and compared reconstruction outputs to historical data collected. Results of this analysis revealed several distinct sources of error: (1) After about 90 years, 94 percent of the recorded trees were relocated and remeasured, but approximately three trees/ha were missing in the field due to obliteration by fire or decay; (2) sizes of trees living in 1909 were overestimated by an average of 11.9 percent; (3) snag and log decomposition models tended to underestimate time since tree death by an undetermined amount; and (4) historical sizes of cut trees were difficult to estimate due to uncertainties concerning harvest dates. The aggregate effect of these errors was to overestimate the number of trees occurring in 1909-1913. Sensitivity analysis applied to decomposition equations showed variations in reconstructed sizes of snags and logs by +/- 7 percent and stand density estimates by 7 percent. Results suggest that these reconstruction techniques are robust but tend to overestimate tree size and forest density

A sex-specific microRNA-96/5HT1B Axis influences development of pulmonary hypertension

Rationale: Females are predisposed to pulmonary arterial hypertension (PAH); evidence suggests that serotonin, mutations in the bone morphogenetic protein receptor (BMPR) II gene, and estrogens influence development of PAH. The 5-hydroxytryptamine 1B receptor (5-HT1BR) mediates human pulmonary artery smooth muscle cell (hPASMC) proliferation. Objectives: We aimed to determine whether selected microRNAs (miRNAs) expressed in PASMCs are influenced by sex, BMPR-II mutations, and estrogens, and contribute to PASMC proliferation in PAH. Methods: Expression levels of miRNAs targeting genes related to PAH, estrogen, and serotonin were determined by quantitative RT-PCR in hPASMCs and mouse PASMCs harboring a heterozygous mutation in BMPR-II (BMPR-IIR899X+/− PASMCs). miRNA-96 targets 5-HT1BR and was selected for further investigation. miRNA target validation was confirmed by luciferase reporter assay. Precursor miRNA-96 was transfected into hPASMCs to examine effects on proliferation and 5-HT1BR expression. The effect of a miRNA-96 mimic on the development of hypoxic pulmonary hypertension in mice was also assessed. Measurements and Main Results: miRNA-96 expression was reduced in BMPR-IIR899X+/− PASMCs from female mice and hPASMCs from female patients with PAH; this was associated with increased 5-HT1BR expression and serotonin-mediated proliferation. 5-HT1BR was validated as a target for miRNA-96. Transfection of precursor miRNA-96 into hPASMCs reduced 5-HT1BR expression and inhibited serotonin-induced proliferation. Restoration of miRNA-96 expression in pulmonary arteries in vivo via administration of an miRNA-96 mimic reduced the development of hypoxia-induced pulmonary hypertension in the mouse. Conclusions: Increased 5-HT1BR expression may be a consequence of decreased miRNA-96 expression in female patient PASMCs, and this may contribute to the development of PAH

Initial carbon, nitrogen, and phosphorus fluxes following ponderosa pine restoration treatments

Southwestern ponderosa pine forests were dramatically altered by fire regime disruption that accompanied Euro-American settlement in the 1800s. Major changes include increased tree density, diminished herbaceous cover, and a shift from a frequent lowintensity fire regime to a stand-replacing fire regime. Ecological restoration via thinning and prescribed burning is being widely applied to return forests to the pre-settlement condition, but the effects of restoration on ecosystem function are unknown. We measured carbon (C), nitrogen (N), and phosphorus (P) fluxes during the first two years after the implementation of a replicated field experiment comparing thinning and composite (thinning, forest floor fuel reduction, and prescribed burning) restoration treatments to untreated controls in a ponderosa pine forest in northern Arizona, USA. Total net primary productivity (260 g Cm22yr21) was similar among treatments because a 3050(percent) decrease in pine foliage and fine-root production in restored ecosystems was balanced by greater wood, coarse root, and herbaceous production. Herbaceous plants accounted for ,20(percent) of total plant C, N, and P uptake in the controls but from 25(percent) to 70(percent) in restored plots. Total plant N uptake was ;3 g Nm22yr21 in all treatments, but net N mineralization was just one-half and twothirds of this value in the control and composite restoration, respectively. Element flux rates in controls generally declined more in a drought year than rates in restoration treatments. In this ponderosa pine forest, ecological restoration that emulated pre-settlement stand structure and fire characteristics had a small effect on plant C, N, and P fluxes at the whole ecosystem level because lower pine foliage and fine-root fluxes in treated plots (compared to controls) were approximately balanced by higher fluxes in wood and herbaceous plants

Quantifying forest reference conditions for ecological restoration: The Woolsey Plots

Sixty-six of the approximately 140 original historical plots (or 47percent) have been relocated on eight National Forests thus far. Of these 66 relocated plots 0 (0/13) are spruce-fir, 13 (13/29) are mixed conifer, and the remainder 53 (53/98) are dominated by ponderosa pine (at least historically pine dominated). This study focused on the ponderosa pine-dominated plots, of which we have relocated over 54 percent. NOTE: This total does NOT include those historical plots located on the Long Valley Experimental Forest near Clints Well, AZ

Modeling genetic imprinting effects of DNA sequences with multilocus polymorphism data

Single nucleotide polymorphisms (SNPs) represent the most widespread type of DNA sequence variation in the human genome and they have recently emerged as valuable genetic markers for revealing the genetic architecture of complex traits in terms of nucleotide combination and sequence. Here, we extend an algorithmic model for the haplotype analysis of SNPs to estimate the effects of genetic imprinting expressed at the DNA sequence level. The model provides a general procedure for identifying the number and types of optimal DNA sequence variants that are expressed differently due to their parental origin. The model is used to analyze a genetic data set collected from a pain genetics project. We find that DNA haplotype GAC from three SNPs, OPRKG36T (with two alleles G and T), OPRKA843G (with alleles A and G), and OPRKC846T (with alleles C and T), at the kappa-opioid receptor, triggers a significant effect on pain sensitivity, but with expression significantly depending on the parent from which it is inherited (p = 0.008). With a tremendous advance in SNP identification and automated screening, the model founded on haplotype discovery and statistical inference may provide a useful tool for genetic analysis of any quantitative trait with complex inheritance