Genetic diversity and population structure in cultivated sunflower and a comparison to its wild progenitor, Helianthus annuus L.

Crop germplasm collections are valuable resources for ongoing plant breeding efforts. To fully utilize such collections, however, researchers need detailed information about the amount and distribution of genetic diversity present within collections. Here, we report the results of a population genetic analysis of the primary gene pool of sunflower (Helianthus annuus L.) based on a broad sampling of 433 cultivated accessions from North America and Europe, as well as a range-wide collection of 24 wild sunflower populations. Gene diversity across the cultivars was 0.47, as compared with 0.70 in the wilds, indicating that cultivated sunflower harbors roughly two-thirds of the total genetic diversity present in wild sunflower. Population structure analyses revealed that wild sunflower can be subdivided into four genetically distinct population clusters throughout its North American range, whereas the cultivated sunflower gene pool could be split into two main clusters separating restorer lines from the balance of the gene pool. Use of a maximum likelihood method to estimate the contribution of the wild gene pool to the cultivated sunflower germplasm revealed that the bulk of the cultivar diversity is derived from two wild sunflower population genetic clusters that are primarily composed of individuals from the east-central United States, the same general region in which sunflower domestication is believed to have occurred. We also identified a nested subset of accessions that capture as much of the allelic diversity present within the sampled cultivated sunflower germplasm collection as possible. At the high end, a core set of 288 captured nearly 90% of the alleles present in the full set of 433, whereas a core set of just 12 accessions was sufficient to capture nearly 50% of the total allelic diversity present within this sample of cultivated sunflower.

A new integrated genetic linkage map of the soybean.

A total of 391 simple sequence repeat (SSR) markers designed from genomic DNA libraries, 24 derived from existing GenBank genes or ESTs, and five derived from bacterial artificial chromosome (BAC) end sequences were developed. In contrast to SSRs derived from EST sequences, those derived from genomic libraries were a superior source of polymorphic markers, given that the mean number of tandem repeats in the former was significantly less than that of the latter ( P<0.01). The 420 newly developed SSRs were mapped in one or more of five soybean mapping populations: "Minsoy" x "Noir 1", "Minsoy" x "Archer", "Archer" x "Noir 1", "Clark" x "Harosoy", and A81-356022 x PI468916. The JoinMap software package was used to combine the five maps into an integrated genetic map spanning 2,523.6 cM of Kosambi map distance across 20 linkage groups that contained 1,849 markers, including 1,015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, six AFLPs, ten isozymes, and 12 others. The number of new SSR markers added to each linkage group ranged from 12 to 29. In the integrated map, the ratio of SSR marker number to linkage group map distance did not differ among 18 of the 20 linkage groups; however, the SSRs were not uniformly spaced over a linkage group, clusters of SSRs with very limited recombination were frequently present. These clusters of SSRs may be indicative of gene-rich regions of soybean, as has been suggested by a number of recent studies, indicating the significant association of genes and SSRs. Development of SSR markers from map-referenced BAC clones was a very effective means of targeting markers to marker-scarce positions in the genome.

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.

Resistance gene analogs are conserved and clustered in soybean.

Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.

Photosynthesis and photorespiration in presenescent, senescent, and rejuvenated soybean cotyledons.

Various growth and physiological parameters were measured in germinating, presenescent, and senescing soybean (Glycine max [L.] Merr.) cotyledons and in cotyledons rejuvenated by epicotyl removal 18 days after planting. The maximal measured carbon dioxide exchange rates (CER) in the cotyledons were in the range of those reported for field-grown soybean leaves. Rejuvenated cotyledons accumulated total chlorophyll in excess of the maximum observed in presenescent cotyledons. When photosynthetic rates were expressed per cotyledon, the CER in rejuvenated tissue recovered to the maximal rates observed in presenescent cotyledons. Ribulose-1,5-bisphosphate carboxylase/oxygenase in rejuvenated cotyledons also recovered to the maximal amount seen in presenescent cotyledons so that CER appeared to be a function of ribulose-1,5-bisphosphate carboxylase/oxygenase content during most of the period studied. Observations of the postillumination outburst of CO(2) and (14)C label in glycine indicated that photorespiration was occurring in the cotyledons and that photorespiration relative to photosynthesis was different in rejuvenated compared with presenescent cotyledons.

Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii.

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO(2)-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO(2). Adaptation to air levels of CO(2) by Chlamydomonas involves induction of a CO(2)-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO(2)-enriched cells. Unlike WT, the high-CO(2)-requiring mutant, cia-5, does not respond to limiting CO(2) conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO(2) which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.

A Photorespiratory Mutant of Chlamydomonas reinhardtii.

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO(2) concentration for photoautrophic growth in spite of the apparent induction of a functional CO(2) concentrating mechanism in air-adapted cells. In 2% O(2) the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O(2), photosynthetic O(2) evolution is severely inhibited in the mutant by preillumination in limiting CO(2), although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O(2) evolution measurements. Net CO(2) uptake is also inhibited when the cells are exposed to air (21% O(2), 0.035% CO(2), balance N(2)) for longer than a few minutes. [(14)C]Phosphoglycolate accumulates within 5 minutes of photosynthetic (14)CO(2) fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO(2) requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.

Reduction of filamin in late passage human diploid fibroblasts (IMR-90).

Progressive subcultivation of IMR-90 cells results in non-proliferative, heterogeneous cultures which may reflect aging of the diploid line (Hayflick, Exp. Cell Res., 37 (1965) 614). We have observed that late passage cells exhibit different rates of spreading and morphogenesis when compared to early passage groups, phenomena which we attribute to altered reassembly of the cytoskeleton in senescent cells (Kelley et al. Mech. Ageing Dev., 13 (1980) 127). To determine whether potential differences in cytoskeletal proteins develop with progressive subcultivation, early and late passage cultures were extracted with 0.5% Triton X-100 for 1 min followed by 1.0% sodium dodecyl sulfate (SDS) prior to separation and characterization of extracted proteins by electrophoresis on 7.5-15% gradient SDS gels. Extractions were made of both culture groups 3, 6 and 24 h after reseeding. Cytoskeletal ultrastructure at each stage of spreading was examined either in replicas of extracted cells or directly by scanning electron microscopy. Although considerable variation in cytoskeletal organization was observed, qualitative differences in gel banding patterns of actin, myosin and tubulin were not apparent at selected time points. However, late passage cells at 6 h and 24 h did not exhibit filamin associated with the Triton insoluble fraction as did early passage cells. Since it has been demonstrated that filamin is capable of cross-linking actin microfilaments into bundles or sheets, we suggest that it is a principal element for the variant cell shape and cytoskeletal morphology observed during altered spreading behavior of late passage human diploid fibroblasts.

Murine Sertoli cells: major histocompatibility antigens and glycoconjugates.

Sertoli cells, cultured from testes of 2-3-week-old Balb/c mice, contain tripartite nucleoli, exhibit phagocytic function, and have the typical morphologic appearance of Sertoli cells by light microscopy, transmission and scanning electron microscopy. Fluorescence-activated cell sorter analysis indicated the presence on mouse. Sertoli cells of H-2 but not Ia antigens. Alpha-D-mannose and N-acetyl-D-glucosamine determinants are detected on Sertoli cell surface by inhibition of lectin binding using appropriate sugars. Interaction of Sertoli cells with concanavalin A (Con A) or wheatgerm agglutinin (WGA) results in rapid patching of the labeled lectin, which become internalized as perinuclear vesicles. These changes are accompanied by rounding of the Sertoli cell, mimicking cellular changes known to occur when fat Sertoli cells are stimulated in vitro by FSH or cyclic AMP. Thus, Sertoli cells have surface alloantigens that permit them to serve as target to cytotoxic T lymphocytes, but not as antigen presenting cells.

A simple technique for the visualization of whole mount cytoskeletons with transmission electron microscopy.

Examination of whole mount cells in the transmission electron microscope has been useful in studies of cellular architecture. The common technique is to grow cells directly on formvar-coated, gold grids for direct observation through a cell. We report a technique for obtaining whole mount preparations which requires neither fragile formvar films nor expensive, gold grids. Cells are grown on palladium-coated coverslips and processed for electron microscopy. The cells and the palladium substrate are separated from the coverslip. The cell-palladium complex is then picked up on copper grids as in thin section processing. We compare images of the cytoskeleton using our technique with images using previously described techniques and present preliminary observations of contracting cell models. Such contractions would tear formvar films if attempted on cells grown in the conventional manner for whole mount examination. Our technique allows cells to contract without tearing the underlying substrate.

Photorespiratory glycine metabolism in corn leaf discs.

The total glycine pool in Zea mays L. Mo17xB73 leaf discs was measured after steady state photosynthesis in 50%, 21% and 1% O(2). The glycine pool was a function of O(2) concentration; it was largest in 50% O(2) and smallest in 1% O(2). Incubation of discs with methyl hydroxybutynoic acid in 21% O(2) in the light caused an accumulation of carbon in glycolate. This accumulation was O(2) sensitive, as subsequent photosynthetic periods in 50%, 21%, and 1% O(2) resulted in the largest glycolate pool in 50% O(2) and the smallest in 1% O(2). At the same time, the O(2)-dependent increase in the glycine pool was eliminated. After untreated leaf discs reached steady state photosynthesis in 21% O(2), measurements made subsequently in darkness, or in 1% O(2) in the light, showed that the glycine pool decreased. On the basis of these results, we conclude that a major portion of the total glycine pool in corn is an intermediate in the photorespiratory glycolate pathway. Considering both the rate of decay of the glycine pool in the dark and the rate of decay of the glycine pool after changing from 21% to 1% O(2), we conclude that this glycine pool is turning over slowly.

Organization of the cytoskeleton in square fibroblasts.

The relationship between the cytoskeleton, stress fiber formation, and cell shape has been difficult to determine in fibroblasts grown in tissue culture. Vagaries in cell shape are complicated, as well, by stochastic cell movements. We dictated the attachment sites and shape of fibroblasts by growing them on square adhesive substrates surrounded by nonadhesive substrates. Cytoskeletal models were made by treating the cells with buffered Triton X-100 and glycerol. The residues were then examined by scanning electron microscopy followed by light microscopy of the same cells. The cytoskeletons of randomly moving cells were examined with whole mount transmission microscopy to confirm images seen with scanning microscopy. The cells thus examined demonstrated definite relationships between ruffling activity and stress fiber terminations, which were limited to the more adhesive, palladium substrate. No stress fibers were seen to end on the lesser adhesive substrate, agarose, and ruffling did not occur across the agarose. Cells too small to fill an entire square tended to extend across one diagonal of the square, and the stress fibers ran parallel to the longest axis of these cells. Larger cells were able to completely fill their squares. The cytoskeletons of these cells were organized in a spatial relation to the square shape of the cells. The cortical meshwork was aligned circularly and diagonally within the cells. Stress fibers appeared to form from the microfilaments of the meshwork and were aligned diagonally across the cells. We conclude that the diagonal arrangement of the stress fibers and cortical meshwork is caused by the same mechanism by which smaller cells spread over the longest axis of a square. Regions of cells where the meshwork was absent or where stress fibers were tightly bundled were occupied by more randomly arranged cytoskeletal components. Regions of tightly bundled stress fibers did not seem to coincide with regions of cortical meshwork as seen by either whole mount transmission or scanning electron microscopy. Stress fibers were revealed in the light microscope to course beneath more randomly oriented cytoskeletal elements. These "lacework-like" elements were found frequently in square cells. Conspicuous structures in this random lacework were focal points of radially arranged filaments. Our observations suggest a continuity between stress fibers and the cortical microfilaments. The orientation of fibers and filaments was, in turn, dependent on cell shape for organization within the cell.

Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90).

The cytoskeletons of early and late passage IMR-90 human diploid fibroblasts have been directly imaged in replicas of Triton X-100 extracted cells during spreading following reseeding. All cells from both young and sensescent cultures exhibit a cytoskeletal network of actin microfilaments, intermediate (10 nm) filaments, microtubules, and interconnecting thin filaments (6-8 nm in diameter) which do not interact with heavy meromyosin. Early passage cells assemble linear aggregates of actin filaments within 1 h of spreading. By 4 h of incubation, these bundles establish a structural bond with the cell membrane which results in resistance by the plasmalemma to detergent extraction at these sites. Furthermore, these membrane regions are associated with developing stress fibers of well-spread cells. In contrast, late passage cells exhibit slower spreading which correlates with a retarded assembly of actin bundles. In addition, by 8 h of spreading, cells of older cultures do not exhibit the regions of membrane-actin interaction which impart detergent resistance to the plasmalemma. We conclude that the ability to reassemble actin-actin and actin-membrane association during cell spreading is reduced with increased serial subcultivation of cells.

Wounding Stimulates Cyanide-sensitive Respiration in the Highly Cyanide-resistant Leaves of Bryophyllum tubiflorum Harv.

The rate of respiration in sectioned leaves of Bryophyllum tubiflorum Harv. increases with decreasing section thickness. The rates of uninhibited respiration in 2- and 8-millimeter-thick sections are 74 and 46 microliters of O(2) per gram fresh weight of unruptured tissue per hour at 20 C, whereas the rate in the presence of cyanide is 31 microliters of O(2) in each case. The rates are unaffected by salicylhydroxamic acid, but cyanide and salicylhydroxamic acid together completely eliminate O(2) uptake. The capacity of the alternative respiratory pathway is thus initially high (estimated at 84% of the uninhibited respiratory rate in whole leaves) and remains constant but probably unexpressed subsequent to the rapid induction of wound respiration.

Loss of division potential in vitro: aging or differentiation?

We have examined the hypothesis that diploid cells grown in vitro age, and propose that only proliferative potential and not life-span is telescoped. We suggest that explanted or transplanted diploid cells are driven to divide by the process of subculturing in vitro or in vivo and, in response to this pressure, also complete their differentiation and become refractory to further mitotic stimulation. We conclude that differentiation rather than "mortality" distinguishes diploid from transformed cells and that the former may not age in vitro, but are lost because culture methods are selective for cycling cells.