Fertility, segregation at a herbicide-resistance locus, and genome structure in BC hybrids from two important weedy Amaranthus species.

Field studies have established high potential for hybridization between two important and often coexisting weedy species, Amaranthus hybridus and Amaranthus tuberculatus. Prezygotic reproductive barriers between these species are believed to be limited to pollen competition and availability. A greenhouse study showed that a herbicide-resistance gene (ALS) from A. hybridus could be introgressed into an advanced A. tuberculatus background (BC2). However, evidence is lacking in support of such transfer in nature. Postzygotic reproductive barriers may minimize, if not preclude, natural introgression. Indeed, A. hybridus xA. tuberculatus hybrids are characterized by reduced fertility and even floral neuterism. The purpose of this study was to assess hybrid fertility in the BC1 generation and its relationship with genome structure and segregation at ALS. Fertility was assessed by measuring seed output and by pollen evaluation, and segregation at ALS was determined via a molecular marker system. The two parental species have the same ploidy (2n = 32) but differ in DNA content (2C) values, with A. tuberculatus chromosomes being on average 29% greater than those of A. hybridus. Given that most (98%) BC(1)s were homoploid, 2C values were used as indicators of relative genomic constitution. Fertility in the BC1 generation was greater than that of F1s, and 3% of BC1s had seed output similar to that of the parental species. Fertility in the BC1 did not correlate (in a strict way) with reconstitution of parental genomes. Hybrid sterility appeared to be controlled by relatively few loci. Heterozygosity at ALS was negatively correlated with fertility. Also, the A. tuberculatus ALS allele was not observed in the A. hybridus sexual condition, monoecism. Linkage of ALS to a locus associated (directly or via epistasis) with hybrid sterility may explain the fertility penalty observed with ALS introgression. Moreover, this linkage might explain why sequenced herbicide-resistance ALS alleles from sympatric A. tuberculatus and A. hybridus populations show independent evolution.

Amplified fragment length polymorphism-based genetic relationships among weedy Amaranthus species.

Weedy Amaranthus species frequently cause economically significant reductions in crop yields. Accurate identification of Amaranthus species is important for efficient weed control, but Amaranthus species can interbreed, which might cause difficulty when identifying hybrid-derived specimens. To determine which of several economically important weedy Amaranthus species are most genetically similar, and thus most likely to produce viable hybrids, we performed amplified fragment length polymorphism (AFLP)-based unweighted pair group method with arithmetic mean (UPGMA) analysis on 8 of these species, with 141 specimens representing 98 accessions. The analysis grouped the specimens into four principal clusters composed of Palmer amaranth (Amaranthus palmeri S. Wats.) and spiny amaranth (Amaranthus spinosus L.); Powell amaranth (Amaranthus powellii S. Wats.), redroot pigweed (Amaranthus retroflexus L.), and smooth pigweed (Amaranthus hybridus L.); waterhemp (Amaranthus tuberculatus (Moq.) Sauer) and sandhills amaranth (Amaranthus arenicola I.M. Johnst.); and tumble pigweed (Amaranthus albus L.). The cluster analysis provided evidence suggesting hybridization among Powell amaranth, redroot pigweed, and smooth pigweed. Further investigations using molecular analysis of the ribosomal internal transcribed spacer region from atypical plants supported this notion. Three species, Palmer amaranth, sandhills amaranth, and waterhemp, are dioecious; nevertheless, the Palmer amaranth and waterhemp-sandhills amaranth clusters were distinct from each other. The Palmer amaranth-spiny amaranth cluster included a cluster of Palmer amaranth and two clusters of spiny amaranth, a monoecious species. Thus the dioecious species Palmer amaranth and waterhemp may not necessarily hybridize with each other more readily than they would to one or more of the monoecious Amaranthus species.

Amaranthus hybridus can be pollinated frequently by A. tuberculatus under field conditions.

Recent studies have confirmed that weedy Amaranthus species are capable of interspecific hybridization, and such hybridization may foster the evolution of herbicide resistance. However, the extent to which hybridization among these species occurs in nature is unknown. The purpose of this study was to determine the frequency under field conditions at which A. hybridus, a monoecious and predominantly self-pollinated species, would be pollinated by A. tuberculatus, a dioecious species. To do this, parents carrying different alleles at the ALS locus, which encodes a herbicide target site, were used. Male A. tuberculatus parents were homozygous for a dominant herbicide-insensitive allele, while A. hybridus parents were homozygous for a sensitive form. Hybrid progeny therefore could be detected via herbicide selection. Mean hybridization frequencies between 0.4 and 2.3% were obtained, depending on the proximity between parents (P=0.02). The robustness of the hybrid selection assay was verified using a molecular marker and DNA content analyses. Using these techniques, more than 99% of the progeny that survived the herbicide were confirmed to be hybrids. Frequencies obtained in this study were many times higher than the generally expected rate of mutation. Therefore, even minimal fertility in hybrid progeny would support the view that hybridization could play a role in adaptive evolution of weedy Amaranthus species.

The clonal structure of Quercus geminata revealed by conserved microsatellite loci.

The scrub oak communities of the southeastern USA may have existed at their present locations for thousands of years. These oaks form suckers, and excavations of root systems suggest that clones may occupy very large areas. Resolution of the clonal nature of scrub oaks is important both to manage the tracts of this ecosystem that remain, and in conducting long-term ecological studies, where the study area must substantially exceed the area occupied by any single clone. Microsatellites were used to determine the genetic diversity of a dominant oak species within a 2-ha long-term experimental site on Merritt Island at the Kennedy Space Center. This area contains a long-term study of the effects of elevated CO2 on the ecosystem. Conservation of seven microsatellite loci, previously identified in the sessile oak, Quercus petraea, was tested in two Florida scrub oak species, Q. geminata and Q. myrtifolia. Sequence analysis revealed that all seven microsatellite loci were conserved in Q. geminata and five loci were conserved in Q. myrtifolia. Six microsatellite loci were polymorphic in Q. geminata and these were subsequently used to investigate the clonal structure of the Q. geminata population. Twenty-one unique combinations of microsatellites, or haplotypes, occurred only once, whereas the remaining 26 individuals belonged to a total of seven different haplotypes. Trees with identical haplotypes were in close proximity, supporting the interpretation that they were clones. The results showed that there is significant genetic diversity within the 2-ha experimental site. Microsatellites provided a powerful and noninvasive tool for distinguishing individual genotypes and determining an adequate area for long-term ecosystem studies.

A novel, bipartite transit peptide targets OEP75 to the outer membrane of the chloroplastic envelope.

OEP75 is an outer envelope membrane component of the chloroplastic protein import apparatus and is synthesized in the cytoplasm as a higher molecular weight precursor (prOEP75). During its own import, prOEP75 is processed first to an intermediate (iOEP75) and subsequently to the mature form (mOEP75). Experiments conducted with stromal extracts indicated that iOEP75 was generated from prOEP75 by the activity of the stromal processing peptidase. The specific processing site was determined and used to divide the prOEP75 transit peptide into N- and C-terminal domains. To determine the targeting functions of the two domains of the transit peptide and of the mature region of prOEP75, we created a deletion mutant construct from prOEP75 and chimeric constructs between domains of prOEP75 and the precursor to a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Analysis of these constructs by in vitro chloroplastic protein import assays revealed that the transit peptide of prOEP75 is bipartite in that the N- and C-terminal portions contain chloroplastic and intraorganellar targeting information, respectively.

A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

A chloroplastic outer envelope membrane protein of 75 kDa (OEP75) was identified previously as a component of the protein import machinery. Here we provide additional evidence that OEP75 is a component of protein import, present the isolation of a cDNA clone encoding this protein, briefly describe its developmental expression and tissue specificity, and characterize its insertion into the outer envelope membrane. OEP75 was synthesized as a higher molecular weight precursor (prOEP75) which bound to isolated chloroplasts in an in vitro import assay and subsequently was processed to the mature form (mOEP75). During this import assay, two proteins intermediate in size between prOEP75 and mOEP75 were detected. One of these intermediates was also detected in chloroplast envelopes isolated from young pea leaves. Binding and processing of prOEP75 required ATP and one or more surface-exposed proteinaceous components, and was competed by prSSU, a stromal-targeted protein. We propose that the N-terminus of the prOEP75 transit peptide acts as a stromal-targeting domain and a central, hydrophobic region of this transit peptide acts as a stop-transfer domain. A complex route of insertion and processing of prOEP75 may exist to ensure high fidelity targeting of this import component.