Heterozygous Effects of Irradiated Chromosomes on Viability in DROSOPHILA MELANOGASTER

Two large experiments were conducted in order to evaluate the heterozygous effects of irradiated chromosomes on viability. Mutations were accumulated on several hundred second chromosomes by delivering doses of 2,500r over either two or four generations for total X-ray exposures of 5,000r or 10,000r. Chromosomes treated with 5,000r were screened for lethals after the first treatment, and surviving nonlethals were used to generate families of fully treated chromosomes. The members of these families shared the effects of the first irradiation, but differed with respect to those of the second. The chromosomes treated with 10,000r were not grouped into families since mutations were accumulated independently on each chromosome in that experiment. Heterozygous effects on viability of the irradiated chromosomes were tested in both isogenic (homozygous) and nonisogenic (heterozygous) genetic backgrounds. In conjunction with these tests, homozygous viabilities were determined by the marked-inversion technique. This permitted a separation of the irradiated chromosomes into those which were drastic when made homozygous and those which were not. The results indicate that drastic chromosomes have deleterious effects in heterozygous condition, since viability was reduced by 2–4% in tests performed with the 10,000r chromosomes, and by 1% in those involving the 5,000r material. Within a series of tests, the effects were more pronounced when the genetic background was homozygous. Nondrastic irradiated chromosomes did not show detectable heterozygous effects. They also showed no homozygous effects when compared to a sample of untreated controls. In addition, there was no evidence for an induced genetic component of variance with respect to viability in these chromosomes. These results suggest that the mutants induced by high doses of X-rays are principally drastic ones which show deleterious effects on viability in heterozygous condition.     

Mutation Rate and Dominance of Genes Affecting Viability in DROSOPHILA MELANOGASTER

Spontaneous mutations were allowed to accumulate in a second chromosome that was transmitted only through heterozygous males for 40 generations. At 10-generation intervals the chromosomes were assayed for homozygous effects of the accumulated mutants. From the regression of homozygous viability on the number of generations of mutant accumulation and from the increase in genetic variance between replicate chromosomes it is possible to estimate the mutation rate and average effect of the individual mutants. Lethal mutations arose at a rate of 0.0060 per chromosome per generation. The mutants having small effects on viability are estimated to arise with a frequency at least 10 times as high as lethals, more likely 20 times as high, and possibly many more times as high if there is a large class of very nearly neutral mutations.-The dominance of such mutants was measured for chromosomes extracted from a natural population. This was determined from the regression of heterozygous viability on that of the sum of the two constituent homozygotes. The average dominance for minor viability genes in an equilibrium population was estimated to be 0.21. This is lower than the value for new mutants, as expected since those with the greatest heterozygous effect are most quickly eliminated from the population. That these mutants have a disproportionately large heterozygous effect on total fitness (as well as on the viability component thereof) is shown by the low ratio of the genetic load in equilibrium homozygotes to that of new mutant homozygotes.     

The Effects of Spontaneous Mutation on Quantitative Traits. I. Variances and Covariances of Life History Traits

We have accumulated spontaneous mutations in the absence of natural selection in Drosophila melanogaster by backcrossing 200 heterozygous replicates of a single high fitness second chromosome to a balancer stock for 44 generations. At generations 33 and 44 of accumulation, we extracted samples of chromosomes and assayed their homozygous performance for female fecundity early and late in adult life, male and female longevity, male mating ability early and late in adult life, productivity (a measure of fecundity times viability) and body weight. The variance among lines increased significantly for all traits except male mating ability and weight. The rate of increase in variance was similar to that found in previous studies of egg-to-adult viability, when calculated relative to trait means. The mutational correlations among traits were all strongly positive. Many correlations were significantly different from 0, while none was significantly different from 1. These data suggest that the mutation-accumulation hypothesis is not a sufficient explanation for the evolution of senescence in D. melanogaster. Mutation-selection balance does seem adequate to explain a substantial proportion of the additive genetic variance for fecundity and longevity.     

Heterozygous Effects on Fitness of Ems-Treated Chromosomes in DROSOPHILA MELANOGASTER

The heterozygous effects on fitness of second chromosomes carrying mutants induced with different doses of EMS were ascertained by monitoring changes in chromosome frequencies over time. These changes were observed in populations in which the treated chromosomes, as well as untreated competitors, remained heterozygous in males generation after generation. This situation was achieved by using a translocation which links the second chromosome to the X chromosome; however, only untranslocated second chromosomes were mutagenized. Chromosomes were classified according to their effects on viability in homozygous condition. A preliminary homozygosis identified completely lethal chromosomes; secondary tests distinguished between drastic (viability index < 0.1) and nondrastic chromosomes. Chromosomes that were nondrastic after treatment were found to reduce the fitness of their heterozygous carriers by 3-5%. The data show that flies homozygous for these chromosomes were about 2.7% less viable per treatment with 1 mm EMS than flies homozygous for untreated chromosomes. By comparing the fitness-depressing effects of nondrastic EMS-induced mutants in heterozygous condition with the corresponding viability-depressing effects measured by Temin, it is apparent that the total fitness effects are several times larger than the viability effects alone. Completely lethal chromosomes derived from the most heavily treated material reduced fitness by 11% in heterozygous condition; approximately half of this reduction was due to the lethal mutations themselves.     

Effects of P Element Insertions on Quantitative Traits in Drosophila Melanogaster

P element mutagenesis was used to construct 94 third chromosome lines of Drosophila melanogaster which contained on average 3.1 stable P element inserts, in an inbred host strain background previously free of P elements. The homozygous and heterozygous effects of the inserts on viability and abdominal and sternopleural bristle number were ascertained by comparing the chromosome lines with inserts to insert-free control lines of the inbred host strain. P elements reduced average homozygous viability by 12.2% per insert and average heterozygous viability by 5.5% per insert, and induced recessive lethal mutations at a rate of 3.8% per insert. Mutational variation for the bristle traits averaged over both sexes was 0.03Ve per homozygous P insert and 0.003Ve per heterozygous P insert, where Ve is the environmental variance. Mutational variation was greater for the sexes considered separately because inserts had large pleiotropic effects on sex dimorphism of bristle characters. The distributions of homozygous effects of inserts on the bristle traits were asymmetrical, with the largest effects in the direction of reducing bristle number; and highly leptokurtic, with most of the increase in variance contributed by a few lines with large effects. The inserts had partially recessive effects on the bristle traits. Insert lines with extreme bristle effects had on average greatly reduced viability.     

Viabilities of Third Chromosomes of DROSOPHILA PSEUDOOBSCURA Differing in Relative Competitive Fitness

Arrowhead (AR) third chromosome arrangements of Drosophila pseudoobscura, whose competitive fitnesses had been determined in population cages, were tested for their genetic loads in homozygous, heterozygous (homokaryotypic), and heterokaryotypic (AR/CH) combinations. The results showed that their competitive population cage performances were correlated to their viabilities as homozygotes but were not correlated to their viabilities as heterozygotes or as heterokaryotypes. However, the results do not fit in too simply with the mutational model of population structure, since the improvement of homozygous viability with increased competitive fitness was not accompanied by a significant degree of dominance as measured by the regression of viabilities of heterozygotes on homozygotes. Only the AR chromosomes derived from the population with poorest competitive fitness showed marked partial dominance (h=.35). The viabilities of heterokaryotypes were markedly uniform for all chromosomes tested and produced significantly greater numbers of flies per culture than the homokaryotypes. In general, the results show that the ranking of relative competitive fitnesses of these chromosomes is not a simple extrapolation of their viabilities, although marked changes in the populations tested have occurred. It is proposed that the differences in competitive fitness, homozygous viability, and degree of dominance observed among these chromosomes, arise from differences in genetic variability which enable different linkage relationships to be established for genes affecting these attributes.     

Rates of movement of transposable elements on the second chromosome of Drosophila melanogaster

The rates of movement of 11 families of transposable elements of Drosophila melanogaster were studied by means of in situ hybridization of probes to polytene chromosomes of larvae from a long-term mutation accumulation experiment. Replicate mutation-accumulation lines carrying second chromosomes derived from a single common ancestral chromosome were maintained by backcrosses of single males heterozygous for a balancer chromosome and a wild-type chromosome, and were scored after 116 generations. Twenty-seven transpositions and 1 excision were detected using homozygous viable and fertile second chromosomes, for a total of 235,056 potential sources of transposition events and a potential 252,880 excision events. The overall transposition rate per element per generation was 1.15 x 10(-4) and the excision rate was 3.95 x 10(-6). The single excision (of a roo element) was due to recombination between the element's long terminal repeats. A survey of the five most active elements among nine homozygous lethal lines revealed no significant difference in the estimates of transposition and excision rates from those from viable lines. The excess of transposition over excision events is in agreement with the results of other in situ hybridization experiments, and supports the conclusion that replicative increase in transposable element copy number is opposed by selection. These conclusions are compared with those from other studies, and with the conclusions from population surveys of element frequencies.     

Partial Dominance of Ems-Induced Mutations Affecting Viability in DROSOPHILA MELANOGASTER

More than 700 EMS-treated second chromosomes marked with either cn (cinnabar) or bw (brown), and derived from long-inbred stocks, were measured for their heterozygous effects on viability in both isogenic (homozygous) and nonisogenic (heterozygous) backgrounds. Each test was replicated five times. When the background was homozygous, flies heterozygous for a treated chromosome were an average of 2.1% less viable, per 0.005 m EMS, than flies heterozygous for an untreated chromosome. Classified according to their homogous effect in an accompanying series of crosses, the lethal-bearing chromosomes (L), which carry genes of less drastic effects as well, reduced the viability of their heterozygous carriers by 3.3%, severe detrimentals (D(s)) by 2.2%, and mild detrimentals (D(m)) by 1.2% at this dose. In the heterozygous background, the mean heterozygous disadvantage for the entire group was 1%, or about half as large.--When computed separately for each count from a single mating, the heterozygous disadvantage was consistently greatest for the earliest counts (4.8%), next highest for the middle count (0.8%), and lowest in the latest count (0.5%), in the homozygous background, indicating that mutant heterozygotes were delayed in time of emergence. The figures in the heterozygous background were, again, reduced, but in the same direction.-The relative viability disadvantage of the cn marker was about 2(1/2) times greater in the homozygous than in the heterozygous background, further supporting the conclusion that the homozygous background can accentuate differences. The enhancement of treatment and marker effects could be a direct result of the level of background heterozygosity per se or attributable to the reduced vigor of the inbred strain.-Dominance, a measure of the heterozygous effect of a mutant relative to its homozygous effect, is greater for genes with small homozygous disadvantage than for more drastic genes. In the homozygous background the average dominance for lethals was 0.019 in contrast to 0.183 for mild detrimentals, supporting other published results suggesting that genes with milder effect, because they occur more frequently, have a greater impact on a population.-The homozygous D:L ratio of EMS mutations was 0.266 and the D(m): L ratio, 0.092, which are lower than comparable load ratios for spontaneous mutations, but greater than for X-ray induced mutations.     

Homozygous and Hemizygous Viability Variation on the X Chromosome of DROSOPHILA MELANOGASTER

We report here a study of viability inbreeding depression associated with the X chromosome of Drosophila melanogaster. Fifty wild chromosomes from Mt. Sinai, New York, and 90 wild chromosomes from Death Valley, California, were extracted using the marked FM6 balancer chromosome and viabilities measured for homozygous and heterozygous females, and for hemizygous males, relative to FM6 males as a standard genotype. No statistically significant female genetic load was observed for either chromosome set, although a 95% confidence limit estimated the total load <0.046 for the samples pooled. About 10% of the Death Valley chromosomes appear to be "supervital" as homozygotes. There is little evidence for a pervasive sex-limited detrimental load on the X chromosome; the evidence indicates nearly identical viability effects in males and homozygous females excluding the supervital chromosomes. The average degree of dominance for viability polygenes is estimated between 0.23 to 0.36, which is consistent with autosomal variation and implies near additivity. We conclude that there is little genetic load associated with viability variation on the X chromosome and that the substantial reduction in total fitness observed for chromosome homozygosity in an earlier study may be due largely to sex-limited fertility in females.     

Nonhomologous Synapsis and Reduced Crossing over in a Heterozygous Paracentric Inversion in Saccharomyces Cerevisiae

Homologous chromosome synapsis (``homosynapsis') and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination. We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to ~55% of that in the homozygotes, and that positive interference still modulates crossing over. Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains. When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores. We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level.     

Fitness and Density-Dependent Population Growth in DROSOPHILA MELANOGASTER

The density-dependent rates of population growth were determined for 26 populations of Drosophila melanogaster maintained in the serial transfer system. Twenty-five populations were homozygous for an entire chromosome 2 sampled from nature; the other was a random heterozygous population. Rates of population growth around the carrying capacity cannot explain the large fitness depression of these lines. However, the homozygous lines show large differences in rates of population growth at low densities relative to the random heterozygous standard. The average relative fitness of the homozygous lines, as determined from the growth rates at the lowest density, is 0.51.     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

     

Inbreeding and fitness in captive populations: Lessons from Drosophila

The avoidance of inbreeding is a primary goal of endangered species population management. In order to fully understand the effects of inbreeding on the fitness of natural and captive populations, it is necessary to consider fitness components which span the entire life cycle of the organism. Using Drosophila melanogaster as a model organism for conservation genetics studies, we constructed 18 experimental lines derived from wild-type stocks which were homozygous for chromosome 2 (this chromosome constitutes 38% of the genome or is equivalent to F = 0.38). For six of these lines which exhibited a reduced homozygous fitness, we estimated the relative values of fitness components operating at both the juvenile stage (pre-adult viability) and adult stage (female fecundity and male-mating ability) of the life cycle. Males in these lines showed a markedly reduced mating ability, while viability and female fecundity were much less affected. Equilibrium values of the wild-type chromosomes in these lines were accurately predicted using a model that incorporated into it these independently estimated fitness components. These results emphasize the importance of studying all fitness components directly to determine overall fitness. A reduced mating ability among inbred males of a captive population can have serious consequences for its future sustainability, and can further jeopardize reintroduction efforts; consequently, a program to carefully monitor the reproductive success of individual males, as well as other fitness components, is recommended. © 1993 Wiley-Liss, Inc.     

Genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Crustacea: Copepoda). II. Crossbreeding experiments, cytogenetics, and a model of chromosomal evolution

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.     

Genetical variation for enzyme activity in a population of Drosophila melanogaster. IV. Analysis of alcohol dehydrogenase activity in chromosome substitution lines.

Chromosome substitution lines derived from two inbred strains of Drosophila melanogaster homozygous for the AdhS allele of alcohol dehydrogenase but differing significantly in ADH activity have been analysed. Variation in activity can be attributed to all three major chromosomes. The effect of the second chromosome, where the ADH structural gene is located, can be modified significantly by the genotype of both the first and the third chromosomes. The most substantial single effect results from homozygous differences between the third chromosomes. In contrast, differences between the X chromosomes are revealed only when the second or second and third chromosomes are heterozygous.     

Genetic Load and Viability Distribution in Central and Marginal Populations of DROSOPHILA SUBOBSCURA

Relative viabilities of individuals homozygous or randomly heterozygous for wild O chromosomes derived from a marginal (Norwegian) and a central (Greek) population of D. subobscura were obtained by means of a newly prepared marker strain. In the central and marginal populations 20.8 and 28.8 percent of all chromosomes proved lethal or semilethal in homozygous condition. Mean viability was higher for +/+ random heterozygotes than for +/+ homozygotes. This remained the case for the marginal, but not for the central populations, after exclusion of the detrimental chromosomes from the calculations. The variances of viabilities were higher for homozygotes than for heterozygotes, but the test crosses with chromosomes from the marginal population had generally higher variances than those with chromosomes from the central population. No correlation was found in either populations between the action of a chromosome in homozygous condition with its action in heterozygous condition. This is interpreted as complete recessiveness of genetic load. The results are discussed in terms of the observed reduction of the inversion polymorphism which is not paralleled by a reduction in enzyme and, as shown here, by reduction in viability variation. It is thought that the heterotic effect of inversions is due to their homeostatic action, which depends less on structural genes than on higher orders of organization due to gene interaction or regulation. Whatever the causes, it is very likely that marginal populations differ from central populations with respect to their genetic system.     

Fitness Effects of Ems-Induced Mutations on the X Chromosome of DROSOPHILA MELANOGASTER. I. Viability Effects and Heterozygous Fitness Effects

Drosophila melanogaster X chromosomes were mutagenized by feeding males sucrose solutions containing ethyl methanesulfonate (EMS); the concentrations of EMS in the food were 2.5 mM, 5.0 mM, and 10.0 mM. Chromosomes were exposed to the mutagen up to three times by treating males in succeeding generations. After treatment, the effective exposures were 2.5, 5.0, 7.5, 10.0, 15.0, and 30.0 mM EMS. X chromosomes treated in this manner were tested for effects on fitness in both hemizygous and heterozygous conditions, and for effects on viability in hemizygous and homozygous conditions. In addition, untreated X chromosomes were available for study. The viability and heterozygous fitness effects are presented in this paper, and the hemizygous fitness effects are discussed in the accompanying one (MITCHELL and SIMMONS 1977). Hemizygous and homozygous viability effects were measured by segregation tests in vial cultures. For hemizygous males, viability was reduced 0.5 percent per mM EMS treatment; for homozygous females, it was reduced 0.7% per mM treatment. The decline in viability appeared to be a linear function of EMS dose. The viabilities of males and females were strongly correlated. Heterozygous fitness effects were measured by monitoring changes in the frequencies of treated and untreated X chromosomes in discrete generation populations which, through the use of an X-Y translocation, maintained them only in heterozygous condition. Flies that were heterozygous for a treated chromosome were found to be 0.4% less fit per mM EMS than flies heterozygous for an untreated one.     

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.     

Genetic variation for total fitness in Drosophila melanogaster: complex yet replicable patterns

The extent of genetic variation in fitness is a crucial issue in evolutionary biology and yet remains largely unresolved. In Drosophila melanogaster, we have devised a method that allows the net effects on fitness of heterozygous wild-type chromosomes to be measured, by competing them against two different "balancer" chromosomes. We have applied the method to a large sample of 40 wild-type third chromosomes and have measured fitnesses of nonlethal chromosomes as well as chromosomes bearing recessive lethals. The measurements were made in the environment to which the population was adapted and did not involve inbreeding. The results show an extraordinary similarity in the behavior of replicates of the same chromosome, indicating consistent genetic effects on total fitness. Some invading chromosomes increased rapidly and some slowly, and some rose to appreciable frequency after several months, but then declined again: in every case, the same pattern was seen in each replicate. We estimated relative fitnesses, rates of change of fitness, and relative viabilities, for each chromosome. There were significant fluctuations around the fitted model, which were also highly replicable. Wild-type chromosomes varied substantially in their effects on heterozygous fitness, and these effects vary through time, most likely as a result of genotype x environment interactions.     

Reverse breeding: a novel breeding approach based on engineered meiosis

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity . Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.