Stability of asymmetrical hybrid hemoglobins. Determination by anaerobic high performance liquid chromatography

Asymmetrical hybrid hemoglobins formed in mixtures of Hb A and Hb S, Hb F and Hb S, Hb S and Hb York(beta 146 His----Pro), and Hb A and Hb York were separated by high performance liquid chromatography on cation and anion exchange columns under anaerobic conditions. The ratio of the hybrid hemoglobin to the total mixture was consistently lower than that theoretically expected and decreased with longer elution times. The hybrid tetramer appears to be unstable even under anaerobic conditions and dissociates into alpha beta dimers. The time course of dissociation of the hybrid hemoglobins was determined by varying the separation programs and thus separating the hybrid hemoglobin at different elution times. The rate of the dissociation of the hybrid hemoglobins studied follows first order kinetics. The lines representing the time course of dissociation of hybrid hemoglobins were extrapolated to time 0 to determine the fraction of the hybrid hemoglobin in the mixture prior to separation. The values obtained for equimolar mixtures of Hb A and Hb S and Hb York and Hb S or Hb A were in agreement with the expected theoretical value (50%). In contrast, the value obtained for hybrid hemoglobin FS was slightly less (about 40%). AY and SY hybrid hemoglobins dissociated into dimers at a considerably faster rate than did AS and FS hybrid hemoglobins, possibly because of the mutation at the beta 146-position in hybrid hemoglobins containing alpha beta Y dimers. This mutation hinders the formation of salt bridges that normally stabilize the "T" quaternary conformation. Since such hybrid hemoglobins have a partial "R" conformation even when deoxygenated, their rate of dissociation to dimers is expected to increase.     

Separation of asymmetrical hybrid hemoglobins by anaerobic cation-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of two structurally different hemoglobins were found to be readily separated by cation-exchange high-performance liquid chromatography under anaerobic conditions. When oxyhemoglobins A and S were mixed and deoxygenated, the resulting HPLC chromatogram showed three peaks. The distribution of the three components follow the binomial expansion a² + 2 ab + b² = 1, where a and b are the initial fractions of parent hemoglobins. The middle peak was collected in a test tube saturated with CO gas and reanalyzed under the same experimental conditions. This middle component gave two peaks of equal areas with retention times identical to those of the CO-form of the parent hemoglobins without the appearance of the hybrid hemoglobin band. No intermediate peak was observed in solutions of mixtures of liganded hemoglobins under aerobic conditions. Hybrid hemoglobins AC and SC were also formed when oxyhemoglobins A and C, S and C were mixed, respectively. The separation and the identification of hemoglobins and hybrid hemoglobin employing cation-exchange HPLC can be achieved within 30 min by gradient elution. In addition, the ability to isolate hybrid hemoglobins may be a valuable tool for the study of physical and chemical properties of hybrid hemoglobins.     

The demonstration of asymmetric hemoglobin hybrids by polyacrylamide electrophoresis.

A simpler, more economical technique than previously reported, that of conventional polyacrylamide gel electrophoresis alone, is described for the detection of asymmetric hemoglobin hybrids of the forms alphaXalphaYbeta2 and alpha2betaXbetaY when bloods from individuals with alpha and beta chain variants were examined. The presence of alpha chain variant hybrids, never before reported, is further evidence that hybrid formation is a more widespread phenomenon than has previously been thought of. Hybrids were found in artificial mixtures of hemoglobins and more importantly, are also reported here for the first time in bloods of individuals heterozygous for hemoglobin variants. These hybrid tetramers were as stable as the parent hemoglobins when examined under anaerobic conditions. The involvement of HbF in the formation of hybrids of the type alpha2betagamma is reported, and an analysis of the possible role of these as well as alpha2betaAbetaS hybrids in the sickling process is presented.     

Effect of FS (alpha 2 gamma beta s) hybrid hemoglobin on Hb S nucleation and aggregation

Asymmetrical cross-linked FS (alpha 2 gamma beta s) hybrid hemoglobin (Hb FS-fumarate) was prepared by reacting mixtures of hemoglobins F and S with double-headed aspirin, bis(3,5-dibromosalicyl) fumarate. When the molar ratio of hemoglobin to the cross-linking agent was 1 to 2 in a 1:1 FS mixture, the relative ratio of the products, cross-linked hemoglobins F (Hb F-fumarate), FS (HB FS-fumarate), and S (Hb S-fumarate), was 1.0:2.6:2.0, in contrast to a 1:2:1 ratio of cross-linked hemoglobins A, AS, and S in a 1:1 AS mixture. These results suggest that the fumaryl group reacts differently with Hb F, Hb FS and Hb S, and that the difference could be attributed to the difference in the structure in the vicinity of the EF6 Lys of non alpha-chains. The oxygen-binding properties of Hb F-fumarate, Hb FS-fumarate, and Hb S-fumarate were similar, except that the n-value of Hb F-fumarate was slightly lower than n-values of Hb S-fumarate and Hb FS-fumarate. Kinetic studies on aggregation showed that the addition of Hb FS-fumarate to unmodified Hb S did not affect the delay time prior to aggregation, but did increase the total turbidity. Electrophoretic and densitometric scanning analysis of the aggregate phase of this mixture showed the fraction of Hb FS-fumarate to be 19%. Hb F-fumarate's effect on the delay time is concentration-dependent; the greater the concentration of Hb F-fumarate, the longer the delay time. The turbidity after aggregation of the mixture of Hb S and Hb F-fumarate was much less than that of Hb S and Hb FS-fumarate. However, the fraction of Hb F-fumarate in the aggregate phase was 19%, which is similar to that of Hb FS-fumarate. These data suggest that Hb F and FS hybrid hemoglobin cannot participate in nuclei formation, but can participate in aggregation after sufficient amounts of nuclei are formed from Hb S, and that increased levels of Hb F do not have an inhibitory effect on the formation of nuclei but on the growth of aggregates.     

The dissociation of human deoxyhemoglobin and mixed state hemoglobin into monomer.

The experimental hybridizations between fully deoxygenated human and canine hemoglobins and between half-ligated human hemoglobin and canine cyanomethemoglobin show that new two hybrids in addition to the parent hemoglobins were clearly formed in the mixtures at the high concentration of KI. Thus, human deoxyhemoglobin under the present conditions is in an equilibrium with three species, tetramer in equilibrium dimer in equilibrium monomer. This means that the deoxyhemoglobin is in R-T equilibrium, and shifts considerably toward the R state under the present conditions. On the other hand, the half-ligated hemoglobin in 1.5 M KI becomes much more dissociable than the deoxy T state and appears to be completely transformed into the R state. Nevertheless, the co-operativity, n, is still high (n = 2.0).     

Hemoglobin synthesis in cell hybrids formed between teratocarcinoma and friend erythroleukemia cells

Viable hybrid cells have been isolated following fusion of Friend erythroleukemia cells and undifferentiated teratocarcinoma cells. The hybrids formed between near-diploid parental cells resembled Friend cells in their ability to grow in suspension and to synthesize hemoglobin in the presence of the chemical inducers dimethyl sulfoxide (DMSO) and ouabain. Erythropoietin (EPO) was effective in inducing hemoglobin synthesis in some of the hybrid cell lines. The hemoglobins synthesized by the hybrids were of the adult forms, but were quantitatively different from those hemoglobins synthesized by the parental Friend cells, suggesting that the fusion event modulated the expression of the hemoglobin chain genes.     

Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

Reconstitution of human fetal hemoglobin from isolated alpha and gamma chains

The reconstitution of hemoglobin F from isolated alpha and gamma chains was studied. An equimolar amounts of the alpha and gamma chains were mixed and incubated in 10 mM potassium phosphate buffer, pH 7.0, at 25 degrees C. Formation of hemoglobin F in the mixture was measured by separating hemoglobins on a cation exchange HPLC. Time courses of the formation of Hb F were independent of the protein concentration and could be analyzed on an exponential process with a first-order rate constant of (2.0 +/- 0.4) x 10(-3) h-1. Under the experimental conditions the isolated gamma chain existed as tetramer dominantly. These results suggest that the overall reaction of the reconstitution of hemoglobin F is limited by the dissociation step of the self-associated gamma chain.     

The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer.

A new turbidimetric method for the direct measurement of the solubility of oxy- and deoxyhemoglobins (Hb) in concentrated phosphate buffer has been established. The principle of the method is the formation of a homogeneous emulsion when hemoglobin is introduced in concentrated phosphate buffer. The solubility of the oxy and deoxy forms of Hb A, Hb S, Hb C, Hb F, and Hb CHarlem (beta 6Glu leads to Val, beta 73Asp leads to Asn) has been studied. The solubility of deoxy-Hb S was the lowest and the solubility curve was broader than those of the other hemoglobins indicating that the aggregates of deoxy Hb S require more water to be dissolved. The solubility of oxy- and deoxyhemoglobins depends on temperature and pH. The solubility of hemoglobins is increased as the temperature is lowered and the pH is raised. The pH dependency of the solubility of deoxy-Hb S in high phosphate buffer was opposite to that of the minimum gelling concentration of deoxy-Hb S. The order of the solubility of Hb CHarlem, Hb FS, Hb AS, Hb CS, and Hb S in concentrated phosphate buffer corresponds to the order of minimum gelling concentration of these hemoglobins or hemoglobin mixtures. Solubility studies of a 1:1 mixture of deoxy-Hb A and deoxy-Hb S show that deoxy-Hb A aggregates in 2.42 M phosphate buffer in which pure deoxy-Hb A is totally soluble. This result indicates that deoxy-Hb S interacts with deoxy-Hb A and decreases its solubility.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

Hydroxylamine reduction to ammonium by plant and cyanobacterial hemoglobins

Plants often face hypoxic stress as a result of flooding and waterlogged soils. During these periods, they must continue ATP production and nitrogen metabolism if they are to survive. The normal pathway of reductive nitrogen assimilation in non-legumes, nitrate, and nitrite reductase can be inhibited during low oxygen conditions that are associated with the buildup of toxic metabolites such as nitrite and nitric oxide, so the plant must also have a means of detoxifying these molecules. Compared to animal hemoglobins, plant and cyanobacterial hemoglobins are adept at reducing nitrite to nitric oxide under anaerobic conditions. Here we test their abilities to reduce hydroxylamine, a proposed intermediate of nitrite reductase, under anaerobic conditions. We find that class 1 rice nonsymbiotic hemoglobin (rice nsHb1) and the hemoglobin from the cyanobacterium Synechocystis (SynHb) catalyze the reduction of hydroxylamine to ammonium at rates 100-2500 times faster than animal hemoglobins including myoglobin, neuroglobin, cytoglobin, and blood cell hemoglobin. These results support the hypothesis that plant and cyanobacterial hemoglobins contribute to anaerobic nitrogen metabolism in support of anaerobic respiration and survival during hypoxia.     

Geminate combination of oxygen with iron-cobalt hybrid hemoglobins

Photodissociation of oxygen from the ferrous subunits of hybrid hemoglobins in which the heme of either the alpha or the beta chain has been replaced by cobalt protoporphyrin IX shows large differences between the subunits. With a 25-ns light pulse, the apparent quantum yield at the end of the flash is greater for the beta-iron hybrid than for the alpha-iron hybrid. With the beta-iron hybrid, the yield is greater when solution conditions favor the T-state. After the flash, a part of the oxygen which has been dissociated recombines with a half-time of the order of tens of nanoseconds. The proportion is greatest in the R-state at low temperature and least in the T-state. With the alpha-iron hybrid, oxygen is much less readily removed, and the rapid recombination is slight or absent. It is seen most clearly at low temperatures in conditions which favor the T-state. The long term (greater than 100 ns) effect is that oxygen is much more readily removed from the beta-iron hybrid in the T-state than under any other condition. Analogous flash experiments performed with human hemoglobin A may be closely simulated by superposition of the results obtained with the two hybrid hemoglobins under the same conditions. Isolated human alpha and beta--SH chains show differences similar to, but less marked than, those of the iron-cobalt hybrids.     

Radioimmunochemical characterization of hemoglobins Lepore and Kenya: unique antigenic determinants located on hybrid hemoglobins.

Antisera were produced in rabbits to the three known types of Lepore hemoglobins, which contain hybrid delta-beta non-alpha-chains, and to hemoglobin Kenya, which has a hybrid gamma-beta non-alpha-chain. By using a sensitive radioimmunoassay technique, the absorbed antisera were shown to contain an antibody population that was specific for the hybrid hemoglobin and did not cross-react with normal hemoglobins. However, with the absorbed Lepore-specific antisera, the three known types of Lepore hemoglobins were antigenically indistinguishable from each other, suggesting that antibodies are not produced to the primary structural differences which define the three non-alpha-chains of the Lepore hemoglobins. These studies demonstrate that the non-alpha-subunits of hemoglobins Lepore and Kenya possess unique antigenic determinant sites, evidently resulting from an altered polypeptide conformation.     

Hybrid bridges to gene flow: a case study in milkweeds (Asclepias)

Natural hybridization occurs throughout areas of sympatry for the North American milkweeds Asclepias exaltata and A. syriaca (Asclepiadaceae), even though the formation of F1 hybrid seed is a rare event. For introgressive hybridization to proceed, F1 and advanced hybrids must be released from reproductive barriers and successfully mate with one or both parental species. I investigated the mating system of natural hybrids between A. exaltata and A. syriaca in three populations in Shenandoah National Park, Virginia. Allozyme data and a maximum-likelihood procedure were used to estimate the frequency of six genotypic classes (parentals, F1, F2, and backcrosses) of the hybridizing populations, the pollinia received by hybrid plants, and the paternal parents of seeds produced by hybrids. F1 hybrids, backcross A. syriaca, and parental A. syriaca individuals were common in three hybrid populations. Even though self-pollinations and interhybrid pollinations were common, F2 seed production and the occurrence of F2 individuals were rare in hybrid populations. Hybrid plants received more pollen from A. syriaca than A. exaltata, which resulted in the production of more backcross-A. syriaca seed than backcross-A. exaltata seed. Asclepias exaltata was rare in the hybrid populations, but A. exaltata pollinia were received by hybrids and this species sired between 15% and 36% of the seeds produced on hybrids. The potential for introgression with A. exaltata populations is lower because this species is unsuccessful as the maternal parent in interspecific and backcross hand-pollinations. The asymetry of hybridization with A. syriaca as the maternal parent is further supported by the incorporation of maternally inherited chloroplast DNA markers in hybrids. Hybrid milkweeds frequently backcross with both parental species and may be released from the reproductive barriers that limit the formation of F1 hybrids in natural populations. The direction of interspecific gene flow and introgression in milkweeds is influenced by the reproductive biology of hybrids, the constituency of the surrounding population, and failure of some crosses to produce seeds. Finally, introgressive hybridization remains an important evolutionary force even when the initial formation of F1 hybrids in natural populations is rare.     

Genetic hemoglobin abnormalities in 2363 Cuban newborns

Summary A total of 2363 Cuban newborns were screened for genetic hemoglobin abnormalities; 2187 (92.56%) had a normal electrophoretic pattern. Of the 176 samples with abnormal electrophoretic patterns, 102 (4.32%) had hemoglobins A, F plus Bart's; 54 (2.29%) had hemoglobins A, F and S; 3 (0.13%) had hemoglobins A, F, S plus Bart's; 14(0.59%) had hemoglobins A, F and C; 1 (0.04%) had hemoglobins A, F, C and Bart's. The frequency of Hb Bart's was 4.46% in AA phenotype, 5.25% in AS, and 6.67% in AC. Two newborns were found to have rare variants. A close correlation was found between the observed and expected phenotypes, which indicates the accuracy of the diagnostic methods used. The results of all hemoglobin abnormalities were entered on the infants' hospital records. In addition, these families received genetic counseling.     

Magnesium(II) and zinc(II)-protoporphyrin IX's stabilize the lowest oxygen affinity state of human hemoglobin even more strongly than deoxyheme.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha2(Fe)beta2(M) and alpha2(M)beta2(Fe); M=Mg(II), Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha2(Fe)beta2(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effectors, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha2(Fe)beta2(Mg) and alpha2(Fe-CO)beta2(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effectors is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution.We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins.     

Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes

Concerns exist that transgenic crop x weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa x transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations.     

Maintenance of hemoglobin inducibility in somatic cell hybrids of tetraploid (2S) mouse erythroleukemia cells with mouse or human fibroblasts.

It has previously been reported that Friend mouse erythroleukemia (MEL) cells synthesize hemoglobin when exposed to 2% dimethylsulfoxide, and that hybrids between MEL cells and fibroblasts (or other nonerythroid cells) do not synthesize hemoglobin. We have been successful in obtaining hybrids (3/15) between MEL cells and mouse L-cell fibroblasts that maintain hemoglobin inducibility by preserving nonadherent cells after fusion. The proportion of hemoglobin inducible hybrids can be increased (8/11) by using a stable 2S (pseudotetraploid) MEL parent in addition to preserving nonadherent cells after fusion. All hybrids which were nonadherent were hemoglobin inducible, and all hybrids which were adherent were not. Five nonadherent hybrid clones were analyzed from fusions between a stable 2S MEL parent and a human fibroblast (WI-38, VA-2). All these clones were inducible for hemoglobin. It is concluded that gene dosage is effective in increasing the proportion of hemoglobin inducible hybrids, but hybrid morphology is the phenotype characteristic that correlates most closely with expression of hemoglobin inducibility.     

Oxygen equilibria of hemoglobin A and hemoglobin S valency hybrids.

Oxygen equilibrium determinations with “unsymmetrical” MetHb/Hb hybrids derived from human hemoglobins A and S are reported. All four of the possible hybrids have higher oxygen affinity than the parent hemoglobins. The α₂^Metβ₂^S hybrid has a lower oxygen affinity than that of α₂^Metβ₂^S. However, both the β^Met hybrids have similar oxygen affinity. The Bohr value of α₂^Metβ₂^S is more negative than that of α₂^Metβ₂^A while the β^Met hybrids appear to have almost identical Bohr values. These findings favor the view that α and β chains in hemoglobin A have different conformations and indicate that hemoglobin S has a β-chain conformation different from that of β-chain of hemoglobin A. This difference is probably carried into the oxygenation properties of the α-chain in such a way as to be reflected only when the β chain is oxidized.     

No effect of transgene and strong wild parent effects on seed dormancy in crop–wild hybrids of rice: implications for transgene persistence in wild populations

Soil seed banks act as a gene pool for local plant species and, as such, can buffer local populations, especially those experiencing challenging environmental conditions. Seed dormancy has important implications to dynamics of soil seed banks. Therefore, estimating the seed dormancy of transgenic crop–wild hybrids could shed light on the persistence of transgenes in wild‐plant soil seed banks. Individuals from eight populations of wild rice Oryza rufipogon were crossed with those of three insect‐resistant transgenic rice lines. Selfed (F2–F4) and backcrossed populations (BC1, BC1F2 and BC1F3) were then made from the hybrids. Seed germination was tested under three treatments: (a) normal; (b) overwintering in soil; and (c) one‐week heat‐shocking. The effects of transgene, wild parent and hybrid generation on hybrid seed germination were examined. No significant effect of insect‐resistant transgenes (Bt and CpTI) was detected on the seed dormancy of crop–wild hybrids, while a significant wild parent effect was found. The seeds of advanced generation hybrids have higher germination percentages and lower dormancy than do those of F1 and BC1 generations. The study showed that the dormancy of hybrid seeds was determined mainly by their genetic backgrounds. All hybrid seeds have higher germination percentages and lower dormancy (and, consequently, a poorer overwintering ability), compared with wild seeds, and reduce dormancy would contribute to a fitness disadvantage, compared with wild types. Therefore, such seeds might form part of naturally occurring soil seed banks, through which crop genes would persist in wild populations.