FIELD MEASUREMENTS OF NATURAL AND SEXUAL SELECTION IN THE FUNGUS BEETLE,BOLITOTHERUS CORNUTUS

Selection on three phenotypic traits was estimated in a natural population of a fungus beetle, Bolitotherus cornutus. Lifetime fitness of a group of males in this population was estimated, and partitioned into five components: lifespan, attendance at the mating area, number of females courted, number of copulations attempted, and number of females inseminated. Three phenotypic characters were measured—elytral length, horn length, and weight; there were strong positive correlations among the three characters. Selection was estimated by regressing each component of fitness on the phenotypic traits. Of the three traits, only horn length was under significant direct selection. This selection was for longer horns and was due mainly to differences in lifespan and access to females. The positive selection on horn length combined with the positive correlations between horn length and the other two characters resulted in positive total selection on all three characters.     

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.     

Genetic analysis and QTL mapping of the seed hardness trait in a black common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) recombinant inbred line (RIL) population

Seed hardness trait has a profound impact on cooking time and canning quality in dry beans. This study aims to identify the unknown genetic factors and associated molecular markers to better understand and tag this trait. An F_2:7 recombinant inbred line (RIL) population was derived from a cross between the hard and soft seeded black bean parents (H68-4 and BK04-001). Eighty-five RILs and the parental lines were grown at two locations in southern Manitoba during years 2014–2016. Seed samples were harvested manually at maturity to test for seed hardness traits. The hydration capacity and stone seed count were estimated by soaking the seeds overnight at room temperature following AACC method 56-35.01. Seed samples from 2016 tests were also cooked to determine effect of seed hardness on cooking quality. For mapping of genomic regions contributing to the traits, the RIL population was genotyped using the genotype by sequencing (GBS) approach. The QTL mapping revealed that in addition to the major QTL on chromosome 7 at a genomic location previously reported to affect seed-hydration, two novel QTL with significant effects were also detected on chromosomes 1 and 2. In addition, a major QTL affecting the visual appeal of cooked bean was mapped on chromosome 4. This multi-year-site study shows that despite large environmental effects, seed hardness is an oligo-genic and highly heritable trait, which is inherited independently of the cooking quality scored as visual appeal of cooked beans. The identification of the QTLs and development of SNP markers associated with seed hardness can be applied for common bean variety improvement and genetic exploitation of these traits.     

MEASUREMENTS OF NATURAL SELECTION ON FLORAL TRAITS IN WILD RADISH (RAPHANUS RAPHANISTRUM). I. SELECTION THROUGH LIFETIME FEMALE FITNESS

Although the role of natural selection in the evolution of floral traits has been of great interest to biologists since Darwin, studies of selection on floral traits through differences in lifetime fitness have been rare. We measured selection acting on flower number, flower size, stigma exsertion, and ovule number per flower using field data on lifetime female fitness (seed production) in wild radish, Raphanus raphanistrum. The patterns of selection were reasonably consistent across three field seasons, with strong directional selection for increased flower production in all three years, weaker selection for increased ovule number per flower in two years, and selection for increased flower size in one year. The causes of the selection were investigated using path analysis combined with multiplicative fitness components. Increased flower production increased fruit production directly, and increased numbers of ovules per flower increased the number of seeds per fruit in all three years; pollinator visitation did not influence either of these fitness components. Increased flower size was associated with increases in both the number of fruit and the number of seeds per fruit in one year, with the latter relationship being stronger. Total lifetime seed production was affected more strongly by differences in fruit production than by differences in either the number of seeds per fruit or the proportion of fertilized seeds that were viable, but all three fitness components were positively correlated with total seed production.     

Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M_r value 56 000 (80–90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10–20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M_r value of 52 000 which was close to the M_r value of 52 614±9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1–2% of total E3 activity. The specific activity of the chloroplast enzyme was three-to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD⁺ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP⁺ as cofactor nor was it inhibited by the presence of high concentrations of NADP⁺ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - PDC pyruvate dehydrogenase complex - OGDC 2-oxoglutarate dehydrogenase complex - GDC glycine decarboxylase complex - SDS-PAGE sodium dodecyl sulphate/polyacrylamide gel electrophoresis - TDP thiamine diphosphate - M_r relative molecular mass J.G.L. is grateful to the Biotechnology and Biological Sciences Research Council (BBSRC), U.K. for continuing financial support. M.C. is the holder of a BBSRC-funded earmarked Ph.D. studentship.     

Reconsideration of pollen dispersal data from field trials of transgenic potatoes

During the initial field evaluation of transgenic plants, it is usual to isolate them genetically from other plants of the same species. Several field experiments on potatoes, using transgenes as markers, have shown that transgene dispersal by pollen to other potato plants is limited and very unlikely at distances over 10 m. In a recent study in Sweden, a frequency of transgene-containing progeny of over 30% is reported from non-transgenic potato plants grown at distances of 10–1000 m from transgenic plants containing nptII and gus marker genes. Data from the Swedish study is discussed along with other relevant observations, and it is concluded that the high frequency of gene dispersal in that study results from a high frequency of false positives during PCR analysis of the nptII gene. From the data available in potato, it is concluded that a distance of 20 m is generally adequate for the initial field evaluation of transgenic potatoes containing novel gene constructs.     

The significance of antibiotic production by Pseudomonas aureofaciens PA 147-2 for biological control of Phytophthora megasperma root rot of asparagus

Pseudomonas aureofaciens PA147-2 produces an antibiotic (Af⁺) which inhibits the growth of fungal phytopathogens on phosphate buffered potato dextrose agar (PBPDA). To determine the role of the antibiotic in disease suppression in vivo, PA147-2 and an antibiotic-deficient Tn5 mutant (Af^-) PA109, were tested for their ability to suppress root rot of Asparagus officinalis seedlings caused by Phytophthora megasperma var sojae, in the glasshouse. Seedlings coinoculated with the pathogen and the wildtype strain PA147-2, showed a significantly reduced level of infection and disease severity compared to seedlings inoculated with the pathogen alone. However, 100% of seedlings treated with Af^- mutant PA109 were diseased. Furthermore, a strain derived from mutant PA109, restored to Af⁺ through allele replacement of Tn5 by homologous recombination, gave similar levels of disease suppression as the wildtype. This suggests the antibiotic produced by PA147-2 is important for the control of P. megasperma in vitro as well as in planta. Treatment of seedlings with PA147-2 and derived strains including the Tn5 mutant (Af^-) strain improved plant weight by 40–100% in the presence and absence of the pathogen suggesting PA147-2 also has a direct growth stimulatory effect independent of antibiotic production.     

The effect of wild radish floral morphology on pollination efficiency by four taxa of pollinators

The effects of floral morphology on rates of pollen removal and deposition by different pollinators in generalist plant species are not well known. We studied pollination dynamics in wild radish, Raphanus raphanistrum, a plant visited by four groups of pollinators: honey bees, small native bees, butterflies, and syrphyd flies. The effects of anther position and other factors on pollen removal during single visits by all four pollinator taxa were measured. Flowers with high anther exsertion (i.e., anthers placed higher above the opening of the corolla tube) tended to have the highest numbers of pollen grains removed, but this effect was strongest for honey bees and butterflies. For all pollinator taxa, pollen removal increased with the number of pollen grains available on a flower and whowed a positive, decelerating relationship with the duration of the visit. The effects of stigma position and other factors on pollen deposition during single visits by honey bees and butterflies were also studied. The nectar-feeding butterflies had a higher pollination efficiency (percentage of pollen grains removed from anthers that were subsequently deposited on a stigma) than the nectar- and pollen-feeding honey bees. Flowers with intermediate stigma exsertion had the highest numbers of pollen grains deposited on their stigmas by butterflies, but stigma exsertion had no effect on deposition by honey bees. For both butterflies and honey bees, pollen deposition on the recipient flower increased with the amount of pollen removed from the donor flower, and there was a positive, decelerating relationship between deposition and time spent at the flower; these results are analogous to those for pollen removal. The effects of anther and stigma exsertion on pollen removal and denosition did not fit predictions based on patterns of floral correlations, but results for morphology, pollen availability, time spent per visit, and pollinator efficiency are in broad agreement with previous studies, suggesting the possible emergence of some general rules of pollen transfer.