Selection Mapping Identifies Loci Underpinning Autumn Dormancy in Alfalfa (Medicago sativa).

Autumn dormancy in alfalfa (Medicago sativa) is associated with agronomically important traits including regrowth rate, maturity, and winter survival. Historical recurrent selection experiments have been able to manipulate the dormancy response. We hypothesized that artificial selection for dormancy phenotypes in these experiments had altered allele frequencies of dormancy-related genes. Here, we follow this hypothesis and analyze allele frequency changes using genome-wide polymorphisms in the pre- and postselection populations from one historical selection experiment. We screened the nondormant cultivar CUF 101 and populations developed by three cycles of recurrent phenotypic selection for taller and shorter plants in autumn with markers derived from genotyping-by-sequencing (GBS). We validated the robustness of our GBS-derived allele frequency estimates using an empirical approach. Our results suggest that selection mapping is a powerful means of identifying genomic regions associated with traits, and that it can be exploited to provide regions on which to focus further mapping and cloning projects.

A method for distinctly marking honey bees, Apis mellifera, originating from multiple apiary locations.

Inexpensive and non-intrusive marking methods are essential to track natural behavior of insects for biological experiments. An inexpensive, easy to construct, and easy to install bee marking device is described in this paper. The device is mounted at the entrance of a standard honey bee Apis mellifera L. (Hymenoptera: Apidae) hive and is fitted with a removable tube that dispenses a powdered marker. Marking devices were installed on 80 honey bee colonies distributed in nine separate apiaries. Each device held a tube containing one of five colored fluorescent powders, or a combination of a fluorescent powder (either green or magenta) plus one of two protein powders, resulting in nine unique marks. The powdered protein markers included egg albumin from dry chicken egg whites and casein from dry powdered milk. The efficacy of the marking procedure for each of the unique markers was assessed on honey bees exiting each apiary. Each bee was examined, first by visual inspection for the presence of colored fluorescent powder and then by egg albumin and milk casein specific enzyme-linked immunosorbent assays (ELISA). Data indicated that all five of the colored fluorescent powders and both of the protein powders were effective honey bee markers. However, the fluorescent powders consistently yielded more reliable marks than the protein powders. In general, there was less than a 1% chance of obtaining a false positive colored or protein-marked bee, but the chance of obtaining a false negative marked bee was higher for "protein-marked" bees.

Foraging range of honey bees, Apis mellifera, in alfalfa seed production fields.

A study was conducted in 2006 and 2007 designed to examine the foraging range of honey bees, Apis mellifera (Hymenoptera: Apidae), in a 15.2 km(2) area dominated by a 128.9 ha glyphosate-resistant Roundup Ready® alfalfa seed production field and several non-Roundup Ready alfalfa seed production fields (totaling 120.2 ha). Each year, honey bee self-marking devices were placed on 112 selected honey bee colonies originating from nine different apiary locations. The foraging bees exiting each apiary location were uniquely marked so that the apiary of origin and the distance traveled by the marked (field-collected) bees into each of the alfalfa fields could be pinpointed. Honey bee self-marking devices were installed on 14.4 and 11.2% of the total hives located within the research area in 2006 and 2007, respectively. The frequency of field-collected bees possessing a distinct mark was similar, averaging 14.0% in 2006 and 12.6% in 2007. A grand total of 12,266 bees were collected from the various alfalfa fields on seven sampling dates over the course of the study. The distances traveled by marked bees ranged from a minimum of 45 m to a maximum of 5983 m. On average, marked bees were recovered ~ 800 m from their apiary of origin and the recovery rate of marked bees decreased exponentially as the distance from the apiary of origin increased. Ultimately, these data will be used to identify the extent of pollen-mediated gene flow from Roundup Ready to conventional alfalfa.

Identification of endo- and exo-polygalacturonase activity in Lygus hesperus (Knight) salivary glands.

Polygalacturonase (PG) activity found in the salivary gland apparatus of the western tarnished plant bug (WTPB, Lygus hesperus Knight) has been thought to be the main chemical cause of the damage inflicted by this mirid when feeding on its plant hosts. Early viscosity and thermal stability studies of the PG activity in L. hesperus protein extracts were difficult to interpret. Thus, it has been suggested that one or more PG protein(s) with different hydrolytic modes of action are produced by this mirid. In order to understand the quantitative complexity of the WTPB salivary PG activity, PG purification from a protein extract from salivary glands excised from L. hesperus insects was performed using affinity and ion exchange chromatography. To elucidate the qualitative complexity of the purified PGs, the digestion products generated by the PGs were separated using high performance anion exchange chromatography with pulsed amperometric detection. At least five PG proteins were detected; these differing in terms of their glycosylation, mass-to-charge ratios, and/or molecular mass. The characterization of the products generated by these PGs showed that endo- and exo-acting PGs are produced by WTPB. Although none of the PGs was purified to homogeneity, the present work provides biochemical evidence of a multiplicity of PGs that degrade the pectin component of the plant tissue in different fashions. The implications of these findings affect the understanding of WTPB feeding damage and, potentially, help identify ways to control this important crop pest. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.

Micro-injection of Lygus salivary gland proteins to simulate feeding damage in alfalfa and cotton flowers.

Alfalfa and cotton flowers were pierced with small glass capillaries of an overall size and shape similar to that of Lygus stylets, and injected with small quantities (6 to 100 nL) of solutions that contained Lygus salivary enzymes. Crude and partially purified protein solutions from Lygus heads and isolated salivary glands showed substantial polygalacturonase (PG) activity, as has been previously reported. Following injection with both crude and partially purified protein solutions, as well as with pure fungal and bacterial PGs, flowers of both alfalfa and cotton exhibited damage similar to that caused by Lygus feeding. Injection with the same volume of a buffer control as well as a buffer control containing BSA at a comparable protein concentration (approximately 6 microg/mL) showed no symptoms. These results are consistent with a previously suggested hypothesis that the extensive tissue damage caused by Lygus feeding is primarily due to the action of the PG enzyme on the host tissue, rather than to mechanical damage caused by the insect stylet. Substantial genotypic variation for a PG inhibiting protein (PGIP) exists in alfalfa and cotton. We, therefore, suggest that breeding and selection for increased native PGIP levels, or transformation with genes encoding PGIP from other plant species, may be of value in obtaining alfalfa and cotton varieties that are more resistant to Lygus feeding damage.

Microbial products trigger amino acid exudation from plant roots.

Plants naturally cycle amino acids across root cell plasma membranes, and any net efflux is termed exudation. The dominant ecological view is that microorganisms and roots passively compete for amino acids in the soil solution, yet the innate capacity of roots to recover amino acids present in ecologically relevant concentrations is unknown. We find that, in the absence of culturable microorganisms, the influx rates of 16 amino acids (each supplied at 2.5 microm) exceed efflux rates by 5% to 545% in roots of alfalfa (Medicago sativa), Medicago truncatula, maize (Zea mays), and wheat (Triticum aestivum). Several microbial products, which are produced by common soil microorganisms such as Pseudomonas bacteria and Fusarium fungi, significantly enhanced the net efflux (i.e. exudation) of amino acids from roots of these four plant species. In alfalfa, treating roots with 200 microm phenazine, 2,4-diacetylphloroglucinol, or zearalenone increased total net efflux of 16 amino acids 200% to 2,600% in 3 h. Data from (15)N tests suggest that 2,4-diacetylphloroglucinol blocks amino acid uptake, whereas zearalenone enhances efflux. Thus, amino acid exudation under normal conditions is a phenomenon that probably reflects both active manipulation and passive uptake by microorganisms, as well as diffusion and adsorption to soil, all of which help overcome the innate capacity of plant roots to reabsorb amino acids. The importance of identifying potential enhancers of root exudation lies in understanding that such compounds may represent regulatory linkages between the larger soil food web and the internal carbon metabolism of the plant.