Relationship between ploidy level and genome size in strawberries

This study investigated the pattern of variation in nuclear DNA content at different ploidy levels in Fragaria (Strawberry, Rosaceae) using flow cytometry based on mean fluorescent intensity (MFI) reflected by propidium-iodide-stained nuclei. On average, MFI values were 237 for diploids F. vesca, F. viridis, and F. nubicola, 416.5 for tetraploid F. orientalis, 621.5 for hexaploid F. moschata, and 798 for octoploids F. × ananassa, F. virginiana, and F. chiloensis. Within diploids MFI ranged from 225.9 in F. vesca ssp. vesca to 255.4 in F. nubicola, and within octoploids varied from 766 in F. × ananassa to 808 in F. virginiana. The nuclear DNA variation was significant among diploid species (N = 21, P < 0.008), but not across octoploid species (N = 17, P>0.386). MFI values were also variable among different genotypes of a given species though not significant. The values of mean basic genome DNA (MFI divided by ploidy level) were 118.5, 104, 103.5, and 99.8, respectively, for diploids, tetraploid, hexaploid, and octoploid species. This indicates that relative genomic size decreases by increasing ploidy level, and that there is no direct proportional relationship between DNA content and ploidy levels in Fragaria, supporting the idea of genome downsizing during polyploidization in plants.     

Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups

In the US, soybean genotypes are classified into maturity groups (MG; total of 13) that represent areas of adaptation generally correlated with latitude bands. To determine if one regeneration procedure could regenerate representatives from diverse areas of adaptation, 18 soybean genotypes representing nine MG were compared for organogenic adventitious regeneration and plant formation from hypocotyl explants following the procedure previously tested on representatives from only three MG. Responding explants were those capable of producing shoots on the acropetal end of the explant from either the outer edge plus central region or the central region only. This enabled determination of the contribution of cotyledonary nodal tissue (outer edge) to shoot regeneration and by discounting those explants, it also enabled estimates of true adventitious regeneration. All 18 genotypes were capable of producing meristemoids/shoots solely from the central region with responses ranging from 28.5 to 64.3% after 4 weeks in culture. All genotypes were also capable of producing elongated shoots that could be successfully rooted. No morphological differences were noted among regenerants, or between them and seed-initiated plants. All regenerants produced viable seed which germinated and produced morphologically normal plants. This study confirmed the genotype- and MG-independent nature of this hypocotyl-based organogenic regeneration procedure and provided conservative estimates for responses that were truly/solely adventitious in nature.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

Identification of quantitative trait loci for plant height, lodging, and maturity in a soybean population segregating for growth habit

The use of molecular markers to identify quantitative trait loci (QTLs) has the potential to enhance the efficiency of trait selection in plant breeding. The purpose of the present study was to identify additional QTLs for plant height, lodging, and maturity in a soybean, Glycine max (L.) Merr., population segregating for growth habit. In this study, 153 restriction fragment length polymorphisms (RFLP) and one morphological marker (Dt1) were used to identify QTLs associated with plant height, lodging, and maturity in 111 F₂-derived lines from a cross of PI 97100 and Coker 237. The F₂-derived lines and two parents were grown at Athens, Ga., and Blackville, S.C., in 1994 and evaluated for phenotypic traits. The genetic linkage map of these 143 loci covered about 1600 cM and converged into 23 linkage groups. Eleven markers remained unlinked. Using interval-mapping analysis for linked markers and single-factor analysis of variance (ANOVA), loci were tested for association with phenotypic data taken at each location as well as mean values over the two locations. In the combined analysis over locations, the major locus associated with plant height was identified as Dt1 on linkage group (LG) L. The Dt1 locus was also associated with lodging. This locus explained 67.7% of the total variation for plant height, and 56.4% for lodging. In addition, two QTLs for plant height (K007 on LG H and A516b on LG N) and one QTL for lodging (cr517 on LG J) were identified. For maturity, two independent QTLs were identified in intervals between R051 and N100, and between B032 and CpTI, on LG K. These QTLs explained 31.2% and 26.2% of the total variation for maturity, respectively. The same QTLs were identified for all traits at each location. This consistency of QTLs may be related to a few QTLs with large effects conditioning plant height, lodging, and maturity in this population.     

Relationship of cyst nematode gene frequencies to soybean resistance

Summary Soybean (S, Glycine max (L.) Merr.) lines with relatively few cysts of soybean cyst nematode (CN, Heterodera glycines Ichinohe) populations are usually called CN-resistant. The phenotype of number of cysts per plant is of the CN-S (Cyst Nematode-Soybean) association and determined by the interactions of genes for avirulence-resistance. The acronym alins was proposed for these alleles for incompatibility, with xalin representing the interaction X of one microsymbiont malin with its host h-alin. These alins are dominant in the gene-for-gene model but may be mostly recessive with CN-S. Definitive genetic studies have been hindered by the heterogeneity of sexually reproducing CN populations and lack of the appropriate genetic models. Loegering's abstract interorganismal genetic model was modified so that one model represented all four possible interactions of dominant-recessive alins for an incompatible phenotype. This involved redefining the Boolean algebra symbol 1 to represent both the alins AND their frequencies. The model was used to derive the relationship: {ie893-01} where the expectation E of cysts (of any CN-S combination, as proportion of number of cysts on a check cultivar) is proportional to the product of CN genotypic frequencies expressed as functions of m-alin frequencies. Each m-alin is at a different locus, i.e., {ie893-02}. The number of terms multiplied for each CN-S is equal to the number of alins in the S line (or F₂ plant). There are too many unknowns in the equation to solve for any of them. The relationship does explain the continuous distributions of phenotypes that were nearly always observed. Basic genetic principles were used to concurrently derive the models and to obtain discontinuous distributions of numbers of cyst phenotypes in segregating generations due to one recessive alin in a CN-susceptible soybean line.Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 9739     

Restriction fragment length polymorphism diversity in soybean

Summary Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.     

Evaluation of soybean RFLP marker diversity in adapted germ plasm

Summary Soybean RFLP markers have been primarily developed and genetically mapped using wide crosses between exotic and adapted genotypes. We have screened 38 soybean lines at 128 RFLP marker loci primarily to characterize germ plasm structure but also to evaluate the utility of RFLP markers identified in unadapted populations. Of these DNA probes 70% detected RFLPs in this set of soybean lines with an average polymorphism index of 0.30. This means that only 1 out of 5 marker loci was informative between any particular pair of adapted soybean lines. The variance associated with the estimation of RFLP genetic distance (GD_R) was determined, and the value obtained suggested that the use of more than 65–90 marker loci for germ plasm surveys will add little precision. Cluster analysis and principal coordinate analysis of the GD_R matrix revealed the relative lack of diversity in adapted germ plasm. Within the cultivated lines, several lines adapted to Southern US maturity zones also appeared as a separate group. GD_R data was compared to the genetic distance estimates obtained from pedigree analysis (GD_P). These two measures were correlated with r = 0.54 for all 38 lines, but the correlation increased to r = 0.73 when only adapted lines were analyzed.     

Taxonomic implications of genome size for all species of the genus Gasteria Duval (Aloaceae)

Nuclear DNA content (2C) is used as a new criterion to investigate all species of the genus Gasteria Duval including the three recently described species Gasteria polita van Jaarsv., G. pendulifolia van Jaarsv. and G. glauca van Jaarsv.. The 122 accessions investigated have the same chromosome number (2n=2x=14), with exception of three tetraploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with Propidium Iodide, is demonstrated to range from 32.8–43.2 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. Based on DNA content the species could be divided in five groups: G. rawlinsonii Oberm. with 32.8 pg, 13 mostly inland species with 34.3–36.0 pg, five coastal species with 36.5–39.0 pg and Gasteria batesiana Rowley with 43.2 pg. The thirteen species with 34.3–36.0 pg could be divided further, in a group of eight species occupying mainly very restricted areas with 34.3–35.1 pg and a second group of five species with 35.2–36.0 pg mainly occupying large areas. These five groups did not coincide very well with the two sections and four series of Gasteria based on a cladistic analysis by van Jaarsveld et al. (1994). Based on its long leafy branches, location in the centre of Gasteria species distribution and its by far lowest DNA content, G. rawlinsonii might be the most primitive member of the genus. Nuclear DNA content as measured by flow cytometry is shown to be relevant to provide additional information on the relationships between Gasteria species.     

Fertile progeny of a hybridization between soybean [Glycine max (L.) Merr.] and G. tomentella Hayata

Summary A colchicine-doubled F₁ hybrid (2n=118) of a cross between PI 360841 (Glycine max) (2n=40) x PI 378708 (G. tomentella) (2n=78), propagated by shoot cuttings since January 1984, produced approximately 100 F₂ seed during October 1988. One-fourth of the F₂ plants or their F₃ progeny have been analyzed for chromosome number, pollen viability, pubescence tip morphology, seed coat color, and isoenzyme variation. Without exception, all plants evaluated possessed the chromosome number of the G. max parent (2n=40). Most F₂ plants demonstrated a high level of fertility, although 2 of 24 plants had low pollen viability and had large numbers of fleshy pods. One F₂ plant possessed sharp pubescence tip morphology, whereas all others were blunt-tipped. All evaluated F₂ and F₃ plants expressed the malate dehydrogenase and diaphorase isoenzyme patterns of the G. max parent and the endopeptidase isoenzyme pattern of the G. tomentella parent. Mobility variants were observed among progeny for the isoenzymes phosphoglucomutase, aconitase, and phosphoglucoisomerase. This study suggests that the G. Tomentella chromosome complement has been eliminated after genetic exchange and/or modification has taken place between the genomes.Journal Paper No. J-13776 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, USA, Project 2763     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Symbiotic interactions between soybean and competing strains ofBradyrhizobium japonicum

Selected symbiotic characteristics of fiveBradyrhizobium japonicum strains were assessed in association with ‘Ransom’ soybean plants (Glycine max [L.] Merr.). In the first of two greenhouse experiments, relative nodulation competitiveness of the strains was examined. Strains were grouped into pairs, and corresponding cells were applied to surface-disinfected seeds so as to provide seven ratios of cell numbers between the two strains. Tap root nodules were harvested 28 days after sowing and serotyped by means of an enzyme-linked immunosorbent assay. Strains differed considerably in nodulation competitiveness, and these differences were successfully quantified using relationships previously proposed in the literature. A second experiment involved assessment of the reproducibility of this technique and characterization of the symbiotic response to single- and double-strain inocula. Differences in relative nodulating abilities of strains were apparent between experiments and were possibly related to observed variations in greenhouse temperatures. Plant shoot weight and total N content were not significantly correlated with nodule number or weight when evaluated across inoculation treatments, but these correlations were often significant within inoculation treatments. Certain double-strain inocula produced either positive or negative effects on shoot weight, N content, and nodulation, when compared with values predicted from corresponding controls receiving single-strain inocula. Paper No. 11741 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Trehalase activity in mycorrhizal and nonmycorrhizal roots of leek and soybean

Root extracts of leek (Allium porrum L.) and soybean (Glycine max L. Merr.) showed trehalase activity which was inhibited by phloridzin and was several times higher than the activity of general -glucosidase. The activity had an acidic optimum. Trehalase activity in extracts of sporocarps and extraradical mycelium of the arbuscular mycorrhizal fungus Glomus mosseae Nicol. & Gerd. (Trappe & Gerd.) was higher than in root extracts and had an optimum at pH 7. Following inoculation with G. mosseae, trehalase activity increased in mycorrhizal roots above the levels observed in nonmycorrhizal roots. Irrespective of fungal colonization, root trehalase activity increased in the presence of Mg²⁺, decreased in the presence of Mn²⁺ and Zn²⁺, and was unaffected by Na₂EDTA.     

Anther culture and cold treatment of floral buds increased symmetrical and extra nuclei frequencies in soybean pollen grains

Androgenic response is characterized by a multinucleate or multicellular stage of pollen development. Histological sections stained with toluidine blue and squashes in propionic-carmine and in 4-6-diamidino-2-phenylindole (DAPI) were used for serial observations (0, 14 and 28 days) in soybean pollen grains from cultured anthers and floral buds submitted to cold treatment at 4 °C. In a total of 62,536 pollen grains, it were observed general averages of 2.06% of pollen grains with two symmetrical nuclei and of 1.41% pollen grains with typical extra nuclei (i.e. additional nuclei with typical morphology). Symmetrical and extra nuclei frequencies increased in both treatments but only the number of pollen grains with typical extra nuclei increased significantly with time of exposure to treatments. In addition, 8.59% of multinucleate pollen grains were recorded with atypical nuclei, smaller than vegetative or generative-types and with a fragmented shape. The frequency of these grains increased significantly with time of exposure to treatments. Thus, soybean multinucleate grains occurrence was not an exclusive response to culture. These preliminary results point to the need of further studies to clarify the relationship between typical and fragmented extra nuclei with both androgenesis and programmed cell death.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

5S ribosomal gene variation in the soybean and its progenitor

Summary The soybean, Glycine max and its wild progenitor, Glycine soja, have been surveyed for repeat length variation for the nuclearly encoded 5S ribosomal RNA genes. There is little variation among the 33 accessions assayed, with a common repeat length of 345 bases being typical of both taxa. A 334 base size variant was encountered in individuals from two populations of G. soja from China. The low level of variability is in marked contrast to the variation observed within and between the species of the perennial subgenus Glycine.     

Optimization of somatic embryogenesis and embryo germination in soybean

Summary Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container. This research was funded by Lubrizol Genetics, Inc., Madison, WI.     

Relationships between nuclear DNA content and seed and leaf size in soybean

 A correlation between genome size and agronomically important traits has been observed in many plant species. The goal of the present research was to determine the relationship between genome size, seed size, and leaf width and length in soybean [Glycine max (L.) Merr.] Twelve soybean strains, representing three distinct seed size groups, were analyzed. Flow cytometry was used to estimate their 2C nuclear DNA contents. Data on seed size and leaf size of the 12 strains were obtained from 1994 and 1995 field experiments. Variation of 2C nuclear DNA among the 12 soybean strains was 4.6%, ranging from 2.37 pg for a small-seed strain to 2.48 pg for a large-seed strain. Strain seed size was positively associated with leaf width (r=0.92) and leaf length (r=0.93). Genome size was highly correlated with seed size (r=0.97), leaf width (r=0.90) , and leaf length (r=0.93). The results of our study indicate that there is a significant correlation between genome size and leaf and seed size in soybean. It is possible that selection for greater seed size either leads to, or results from, greater genome size. If so, this relationship might be worth exploring at a more fundamental level. Received: 5 April 1997 / Accepted: 9 January 1998     

Photosynthesis at ambient and elevated humidity over a growing season in soybean

Daytime rates of net photosynthesis of upper canopy leaflets of soybeans were compared on 17 days for leaflets exposed to air at the ambient humidity and at a higher humidity. Leaflets at the higher humidity had higher rates of net photosynthesis on 16 of the 17 days. The daily total of net photosynthesis of leaflets at the higher humidity was on average 1.32 times that for leaflets at ambient humidity. A strong limitation of net photosynthesis by ambient humidity was found throughout the growing season.