Cytological Mechanisms Research Articles

Amphitelic orientation of centromeres at metaphase I is an important feature for univalent-dependent meiotic nonreduction.

Univalent-dependent meiotic nonreduction, which leads to the production of unreduced gametes, is thought to be the predominant mechanism underlying allopolyploid plant formation. However, little is known about the underlying cytological mechanism. In the present study, we observed male sporogenesis in F1 amphihaploid hybrids of wheat–rye by FISH with the help of diagnostic PrCEN-1 specific for the rye centromere. Our observations indicated that at meiotic metaphase I, the chromosomes were accumulated on the equatorial plate. At this stage, the elongated centromeres were amphitelically oriented perpendicular to the equatorial plate, indicating tension from opposite poles. At late metaphase, the centromeres and sister chromatids started separating. Subsequently, the sister chromatids and centromeres split finally resulting in dyads. Our observations indicate that bipolar orientation of the sister kinetochores of univalents at the equatorial plate in metaphase I is important for the subsequent bipolar separation of sister chromatids in the first meiotic division. Allopolyploids are common in plants. Wide hybridization, the first step for the origination of allopolyploids, brings divergent genomes from different species together into an amphihaploid hybrid. Because only one set of homologous chromosomes is present, amphihaploids (analogous to haploid plants) are usually sterile due to reduced meiosis. Meiotic nonreduction (meiotic restitution), however, can lead to production of functionally unreduced gametes, and their union immediately generates an amphidiploid (allopolyploid). Meiotic nonreduction is thought to be the

Cytological Mechanisms of Leaf Rolling in Rice

ABSTRACTModerate leaf rolling, which probably results from changes of various cells that constitute leaf blades, is an important trait for rice (Oryza sativa L.) breeding. To elucidate the cytological mechanism of leaf rolling, 46 stable mutants with inward (adaxial) or outward (abaxial) rolling phenotypes were obtained from more than 100,000 transfer DNA insertion lines in rice. Histological analyses of those mutants show that changes of number, size, and pattern of bulliform cells, sclerenchyma cells, parenchyma cells, and mesophyll cells as well as vascular bundles could lead to leaf rolling. Here, eight groups have been categorized according to their cytological characteristics in these mutants: increased bulliform cell number and area, decreased bulliform cell number and area, disordered bulliform cell number and area, partial sclerenchyma cell absence, phloem expansion, increased number of parenchyma cells, changes in multiple cell types, and parenchyma‐like cells from partial mesophyll cells. Our study demonstrates that changes in each individual cell type can result in rolled‐leaf formation.

Occurrence and cytological mechanism of numerically unreduced pollen in diploid Populus euphratica

AbstractUnreduced gametes are the driving force for the polyploidizaiton of plants in nature, and are also an important tool for breeding of triploid individuals. The final heterozygosity of a 2n pollen grain depends on the cytological mechanism behind 2n pollen formation. In this study, meiotic abnormalities were analysed using fluorescent chromosome staining and indirect immunofluorescence during the microsporogenesis of 18 genotypes of diploid P. euphratica Oliv. (2n = 2x = 38). Among the 18 genotypes, 16 genotypes produce 2n pollen and two genotypes produce only normal n pollen. In all 2n pollen producers, we found that the first meiotic division was normal but that the second division was characterized by frequent abnormal spindle orientation (parallel, tripolar, and fused spindles) and premature cytokinesis. The parallel, fused spindles and premature cytokinesis were considered to be leading dyad formation, and tripolar spindles seemed to be causing triad formation at the tetrad stage. There was a higher frequency of parallel spindles than other spindle forms, but no significant correlations between parallel spindles and dyads were observed. However, a significant association (r = 0.68, P < 0.05) between the tripolar spindles and dyads was found. In some Microspore mother cells (MMCs), an indirect immunofluorescence examination of meiosis II revealed that the parallel spindles led to the gathering of one or two non-sister groups of chromosomes, causing an incorporation of RMSs from two daughter nuclei. Therefore, the incorporated RMSs established two nuclear cytoplasmic domains for the control of division plane, resulting in either triad or dyad formation.

Genetic basis for spontaneous hybrid genome doubling during allopolyploid speciation of common wheat shown by natural variation analyses of the paternal species.

The complex process of allopolyploid speciation includes various mechanisms ranging from species crosses and hybrid genome doubling to genome alterations and the establishment of new allopolyploids as persisting natural entities. Currently, little is known about the genetic mechanisms that underlie hybrid genome doubling, despite the fact that natural allopolyploid formation is highly dependent on this phenomenon. We examined the genetic basis for the spontaneous genome doubling of triploid F1 hybrids between the direct ancestors of allohexaploid common wheat (Triticum aestivum L., AABBDD genome), namely Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). An Ae. tauschii intraspecific lineage that is closely related to the D genome of common wheat was identified by population-based analysis. Two representative accessions, one that produces a high-genome-doubling-frequency hybrid when crossed with a T . turgidum cultivar and the other that produces a low-genome-doubling-frequency hybrid with the same cultivar, were chosen from that lineage for further analyses. A series of investigations including fertility analysis, immunostaining, and quantitative trait locus (QTL) analysis showed that (1) production of functional unreduced gametes through nonreductional meiosis is an early step key to successful hybrid genome doubling, (2) first division restitution is one of the cytological mechanisms that cause meiotic nonreduction during the production of functional male unreduced gametes, and (3) six QTLs in the Ae . tauschii genome, most of which likely regulate nonreductional meiosis and its subsequent gamete production processes, are involved in hybrid genome doubling. Interlineage comparisons of Ae . tauschii ’s ability to cause hybrid genome doubling suggested an evolutionary model for the natural variation pattern of the trait in which non-deleterious mutations in six QTLs may have important roles. The findings of this study demonstrated that the genetic mechanisms for hybrid genome doubling could be studied based on the intrinsic natural variation that exists in the parental species.

Isolation of gastric cancer stem cells based on colony morphology and detection of their sensitivity to 5-fluorouracil

To investigate the sensitivity of gastric cancer stem cells (GCSCs) to 5-fluorouracil (5-FU), and to explore the cytological mechanism of drug-resistance of gastric cancer. Immunohistochemical staining was employed to examine expression of stem cell marker CD44 and drug-resistance protein thymidylate synthase (TS) in 71 cases with gastric cancer. Based on morphology of cell colonies derived from AGS gastric cell line, colonies consisting of GCSCs were isolated, and expression of CD44 and TS, as well as self-renewal capacity of GCSCs were detected. Sensitivity of GCSCs to 5-FU was examined through CCK-8 assay. The positive rates of CD44 and TS were 59.2% (42/71) and 56.3% (40/71) in gastric cancer, and expression of CD44 was associated with that of TS (χ(2)=12.76, P<0.01; Kappa=0.41). Serial sections indicated that CD44+ cancer cells simultaneously expressed TS. AGS developed morphologically diverse colonies, and the GCSCs colonies exhibited a tight and regular shape, which were called holoclone. Holoclones expressed CD44 and TS strongly, possessing capacity of robust self-renewal and forming a lot of second passage clones after incubation. Subclones expressed CD44 and TS weakly, forming less second passage clones. Paraclones almost did not express CD44 and TS, forming no second passage clone after incubation. Affected by 5-Fu, three holoclones showed less growth inhibition compared with another colony type and wild-type AGS cells. Furthermore, IC50 of 5-FU for three holoclones was (113.43±5.81), (272.68±25.75) and (118.14±17.75) μmol/L respectively, which were significantly higher than that of one subcolony type [(16.97±1.01) μmol/L] and AGS cells [(27.52±0.59) μmol/L] (all P<0.05). GCSCs possess lower sensitivity to 5-FU, and may play a critical role in drug-resistance of gastric cancer.

Histochemical comparison of the nonhost tomato with resistant wheat against Blumeria graminis f. sp. tritici

The nonhost interaction of tomato-Blumeria graminis f. sp. tritici (Bgt) and resistant host interaction of wheat-Bgt were compared histochemically. The percentage of appressorium formation had no significant difference on tomato and wheat leaves. Papilla formation occurred earlier and more on host wheat than on nonhost tomato leaves, while the incidence of hypersensitive cell death was much higher in the nonhost interaction. Whole-cell H2O2 accumulation and hypersensitive cell death usually appeared in haustorium-invaded wheat epidermal cells. In contrast, the vast majority of non-haustorium epidermal cells were associated with H2O2 accumulation and hypersensitive cell death on tomato. Localized H2O2 accumulation and hypersensitive response were detected in effective papillae in both interactions. The peak percentage of haustorium formation was less than 7% in the nonhost interaction while reached 43% in the incompatible host interaction. These results indicate that hypersensitive cell death and papillae are likely to play an important role in preventing Bgt penetration and development on tomato and wheat leaves, both defense responses involving H2O2 accumulation. This study further implies that the nonhost and incompatible interactions share similar cytological mechanisms.

Sexual polyploidization in plants – cytological mechanisms and molecular regulation

In the plant kingdom, events of whole genome duplication or polyploidization are generally believed to occur via alterations of the sexual reproduction process. Thereby, diploid pollen and eggs are formed that contain the somatic number of chromosomes rather than the gametophytic number. By participating in fertilization, these so-called 2n gametes generate polyploid offspring and therefore constitute the basis for the establishment of polyploidy in plants. In addition, diplogamete formation, through meiotic restitution, is an essential component of apomixis and also serves as an important mechanism for the restoration of F1 hybrid fertility. Characterization of the cytological mechanisms and molecular factors underlying 2n gamete formation is therefore not only relevant for basic plant biology and evolution, but may also provide valuable cues for agricultural and biotechnological applications (e.g. reverse breeding, clonal seeds). Recent data have provided novel insights into the process of 2n pollen and egg formation and have revealed multiple means to the same end. Here, we summarize the cytological mechanisms and molecular regulatory networks underlying 2n gamete formation, and outline important mitotic and meiotic processes involved in the ectopic induction of sexual polyploidization.

Cytological investigation of anther development in DGMS line Shaan-GMS in Brassica napus L.

The cytological mechanism of male sterility of Shaan-GMS, a natural mutant dominant genic male sterile (DGMS) line in Brassica napus L., is not well studied. Cytological observation was made on different-size buds of DGMS line 0A30A derived from Shaan-GMS line. The pollen mother cells (PMCs) of DGMS line 0A30A were degenerating at the beginning of meiosis and could not pass the anaphase I stage, with no dyads or tetrads formed, suggesting that the DNA damage checkpoint and spindle assembly checkpoint were activated in sterile anthers. During the meiosis process of sterile anthers in the sterile plants, several kinds of abnormal meiotic cells could be observed: nuclei condensed PMCs, cells with micronuclei, collapsed cells, plasmolysis cells, cells connected with nucleoplasmic bridge, and microspore analogue developed from PMCs without meiosis but enclosed by the exine wall. The results suggested Shaan-GMS to be a new type of DGMS line in B. napus.

Thelytokous Parthenogenesis in Eusocial Hymenoptera

Female parthenogenesis, or thelytoky, is particularly common in solitary Hymenoptera. Only more recently has it become clear that many eusocial species also regularly reproduce thelytokously, and here we provide a comprehensive overview. Especially in ants, thelytoky underlies a variety of idiosyncratic life histories with unique evolutionary and ecological consequences. In all eusocial species studied, thelytoky probably has a nuclear genetic basis and the underlying cytological mechanism retains high levels of heterozygosity. This is in striking contrast to many solitary wasps, in which thelytoky is often induced by cytoplasmic bacteria and results in an immediate loss of heterozygosity. These differences are likely related to differences in haplodiploid sex determination mechanisms, which in eusocial species usually require heterozygosity for female development. At the same time, haplodiploidy might account for important preadaptations that can help explain the apparent ease with which Hymenoptera transition between sexual and asexual reproduction.

Cytological Mechanisms of Induced Gynogenesis in Nigorobuna Crucian Carp Eggs Exposed to Heat or Cold Shock for Retention of Second Polar Body

Cytological Mechanisms of Induced Gynogenesis in Nigorobuna Crucian Carp Eggs Exposed to Heat or Cold Shock for Retention of Second Polar Body

Asexual reproduction in introduced and native populations of the ant Cerapachys biroi

Asexual reproduction is particularly common among introduced species, probably because it helps to overcome the negative effects associated with low population densities during colonization. The ant Cerapachys biroi has been introduced to tropical and subtropical islands around the world since the beginning of the last century. In this species, workers can reproduce via thelytokous parthenogenesis. Here, we use genetic markers to reconstruct the history of anthropogenic introductions of C.biroi, and to address the prevalence of female parthenogenesis in introduced and native populations. We show that at least four genetically distinct lineages have been introduced from continental Asia and have led to the species' circumtropical establishment. Our analyses demonstrate that asexual reproduction dominates in the introduced range and is also common in the native range. Given that C.biroi is the only dorylomorph ant that has successfully become established outside of its native range, this unusual mode of reproduction probably facilitated the species' worldwide spread. On the other hand, the rare occurrence of haploid males and at least one clear case of sexual recombination in the introduced range show that C.biroi has not lost the potential for sex. Finally, we show that thelytoky in C.biroi probably has a genetic rather than an infectious origin, and that automixis with central fusion is the most likely underlying cytological mechanism. This is in accordance with what is known for other thelytokous eusocial Hymenoptera.

Ultrastructural and cytochemical studies on oogenesis of the fern Pteridium aquilinum

Egg development in Pteridium aquilinum var. latiusculum was studied using ultrastructural and cytochemical methods to examine structural features influencing fertilization in leptosporangiate ferns. Ultrastructural observations indicate a separation cavity is first formed above the egg during oogenesis with a pore region persistently connecting the egg and the ventral canal cell. The egg envelope is formed by deposition of amorphous materials in the separation cavity on the outer surface of plasmalemma. The egg envelope was not formed across the pore region; instead, a fertilization pore was formed. During oogenesis, the egg nucleus produced extensive evaginations containing osmiophilic bodies. Cytochemical experiments revealed that the egg envelope displays strong periodic acid-Schiff reaction indicative of polysaccharides, with negligible Sudan black B staining for lipids, suggesting that the egg envelope is composed principally of polysaccharides, and not lipids. The present manuscript provides new insights into egg structure and development of Pteridium, including discovery and characterization of the fertilization pore and observations on the chemical nature of the egg envelope, thus contributing to the understanding of the cytological mechanism of the sexual reproduction of ferns.

Androgenesis: a review through the study of the selfish shellfish Corbicula spp.

Among the asexual reproductive modes, androgenesis is probably one of the most astonishing and least studied mechanisms. In this 'paternal monopolization', the maternal nuclear genome fails to participate in zygote development and offspring are paternal nuclear clones. Obligate androgenesis is known in only a few organisms, including multiple species of clam in the genus Corbicula. Corbicula is a good system to review the evolutionary consequences of this 'all-male asexuality' because the cytological mechanisms of androgenetic reproduction have been described. In Corbicula, sperm are unreduced and, after fertilization, the maternal nuclear chromosomes are extruded as two polar bodies. Hermaphroditic lineages of Corbicula have a worldwide distribution and seem to reproduce through androgenesis, whereas their sexual relatives have restricted ranges. The invasive success of these androgenetic Corbicula lineages may be linked to their asexual mode of reproduction. We review the phenomenon of androgenesis, focusing on evolutionary perspectives, using the genus Corbicula as an exemplar system.

A Novel Lactone-Forming Carboxylesterase: Molecular Identification of a Tuliposide A-Converting Enzyme in Tulip

Tuliposides, the glucose esters of 4-hydroxy-2-methylenebutanoate and 3,4-dihydroxy-2-methylenebutanoate, are major secondary metabolites in tulip (Tulipa gesneriana). Their lactonized aglycons, tulipalins, function as defensive chemicals due to their biological activities. We recently found that tuliposide-converting enzyme (TCE) purified from tulip bulbs catalyzed the conversion of tuliposides to tulipalins, but the possibility of the presence of several TCE isozymes was raised: TCE in tissues other than bulbs is different from bulb TCE. Here, to prove this hypothesis, TCE was purified from petals, which have the second highest TCE activity after bulbs. The purified enzyme, like the bulb enzyme, preferentially accepted tuliposides as substrates, with 6-tuliposide A the best substrate, which allowed naming the enzyme tuliposide A-converting enzyme (TCEA), but specific activity and molecular mass differed between the petal and bulb enzymes. After peptide sequencing, a novel cDNA (TgTCEA) encoding petal TCEA was isolated, and the functional characterization of the recombinant enzyme verified that TgTCEA catalyzes the conversion of 6-tuliposide A to tulipalin A. TgTCEA was transcribed in all tulip tissues but not in bulbs, indicating the presence of a bulb-specific TgTCEA, as suggested by the distinct enzymatic characters between the petal and bulb enzymes. Plastidial localization of TgTCEA enzyme was revealed, which allowed proposing a cytological mechanism of TgTCE-mediated tulipalin formation in the tulip defensive strategy. Site-directed mutagenesis of TgTCEA suggested that the oxyanion hole and catalytic triad characteristic of typical carboxylesterases are essential for the catalytic process of TgTCEA enzyme. To our knowledge, TgTCEA is the first identified member of the lactone-forming carboxylesterases, specifically catalyzing intramolecular transesterification.

Cytogerontological studies of biological activity of oregano essential oil

In order to clarify possible cytological mechanisms that underlie the beneficial effects of carvacrol-bearing essential oils on health and mental abilities, we studied one of them (oregano essential oil) in experiments on transformed cultured Chinese hamster cells. Possible cytotoxic or mitogenic effects of the preparation at various concentrations were preliminarily estimated by analyzing the cell culture density after 4 days of cultivation. The preparation concentration in the growth medium (on carvacrol basis) varied from 1 × 10−15 up to 5 × 10−4 M (on carvacrol basis). As a result, two concentrations were selected for further experiments, including 2.5 × 10−5 M as the maximal absolutely non-toxic concentration and 2.5 × 10−4 M as the concentration at which the oregano essential oil decreased approximately 2-fold the final cell density of the grown culture. It was found that the preparation at 2.5 × 10−5 M had no effect on either the colony-forming ability of the cells or the saturation density of the culture (which is a marker of its “biological age”) or kinetics of its “stationary phase aging” (degradation of cultured cells in the stationary phase of growth, similar to age-related changes of the cells in aging organism). On the contrary, the oregano essential oil at 2.5 × 10−4 M abruptly diminished colony-forming ability of the cells and influenced as a “pro-aging” factor on the saturation density of the cell culture and kinetics of the cell death induced by “stationary phase aging.” Based on our own concept of aging and the data obtained, we assumed that detected increase in the life span of mice under the influence of the oregano essential oil could be determined by certain functional changes at the organismal level only, but is not associated with any geroprotective (anti-aging) activity of the preparation, which is manifested at the cellular level and improves the cell viability.

Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon.

Cytological mechanism of 2n pollen formation in Chinese jujube (Ziziphus jujuba Mill. ‘Linglingzao’)

In order to elucidate the cytological mechanism of 2n pollen formation in Chinese jujube, a cultivar named ‘Linglingzao’ (2n = 2x = 24) which produces relative more 2n pollens naturally was employed for microsporogenesis analysis. Chromosomes paired in 12 bivalents at diakinesis and the first meiotic division was normal, whereas, the second division was characterized by frequent abnormal spindle orientation (parallel and tripolar spindles). Perpendicular, tripolar, and parallel spindles at metaphase II accounted for 72.93, 17.22, and 9.85% respectively. Perpendicular and tripolar spindles led to tetrads and triads formation respectively. Two types of parallel spindles were observed and a significant association correlation (r = 0.84, P < 0.05) between parallel spindle II (fused spindles) and dyads was found which meant only fused spindles can form dyads, while parallel spindle I with a long distance between two sets of chromosomes seemed to lead tetrad formation. At tetrad stage, the observed frequencies of tetrads, triads and dyads were 80.99, 16.40, and 2.61% respectively. By the rule that each tetrad can form four n pollens, each triad can form one 2n pollen and two n pollens, each dyad can form two 2n pollens, the frequency of 2n pollen estimated was 5.71%. Based on the pollen diameter difference between n and 2n pollens, frequency of 2n pollen observed was 6.15%. No significant difference was observed between frequency of 2n pollen estimated and that of observed. The results indicated that, tripolar spindles and fused spindles at metaphase II followed by more triads and few dyads formation at tetrad stage were responsible for the production of 2n pollen in Chinese jujube. 2n gametes observed correspond to first division restitution. Research on the cytological mechanism of unreduced pollen will provide a platform for unreduced pollen induction and polyploidy breeding in Chinese jujube.

The cytological instability of neoallopolyploids suggesting a potent way for DNA introgression: The case of synthetic hexaploid wheat × Aegilops peregrina

SHW-L1 is a newly synthetic hexaploid wheat, which has a genomic combination analogous to that of natural common wheat. In the present study, intergeneric F(1) hybrids of SHW-L1 - Aegilops peregrina AS24 were produced without embryo rescue. Among the 31 F(1) hybrid plants observed, 20 had the expected chromosome number 2n = 35 and were euploids (genome ABDUS(I)). The remainder 11 plants were aneuploids with 33, 34, 36 and 37 chromosomes, indicating that the neoallohexaploid SHW-L1 was cytologically instable and produced aneuhaploid gametes with 19, 20, 22 and 23 chromosomes, respectively. The hybrid plant lacking chromosome 5B carrying Ph1 showed a very high level of homoeologous chromosome pairing. The absence of Ph gene provides a potent cytological mechanism for DNA introgression among homoeologous chromosomes between newly synthetic wheat and wild species.

Meiotic restitution in amphihaploids in the tribe Triticeae

In haploid and diploid organisms of the plant kingdom, meiotic division of diploid cells proceeds in two consecutive stages, with DNA replicating only once. In amphihaploids (interspecific or iontergeneric hybrids), where homologs are absent, the reduction of the chromosome number does not occur, meiosis is abnormal, and the plants are sterile. Gamete viability in F1 hybrids is ensured by a single division when chromosomes are separated into sister chromatids in either the first or the second division. Such gametes ensure partial fertility of amphihaploids, thereby facilitating their survival and stabilization of the polygenome. The frequency of the formation of viable gametes varies from a few cases to 98.8% in different anthers of the hybrids. Here, studies on the cytological mechanisms and genetic control of chromosome unreduction or restitution in different amphihaploids of the tribe Triticeae are reviewed. The current notions on the control of formation of restitution nuclei based on the principles of a prolonged metaphase I and different types of meiocytes. The main terms used for systematization of restitution mechanisms are first-division restitution (FDR), single-division meiosis (SDM), and unreductional meiotic cell division (UMCD). It has been assumed that archesporial cells of remote hybrids may have two cell division programs, the meiotic and the mitoyic ones. The possible approaches to the analysis of the genetic control of chromosome restitution in amphihaploids are discussed.

Automictic parthenogenesis and rate of transition to homozygosity

The production of diploid daughters from unfertilized eggs can be achieved through various cytological mechanisms. Under apomictic parthenogenesis, meiosis is suppressed, and the absence of recombination or re-assortment of alleles leads to the production of a ‘true clone' of the mother. By contrast, under automictic parthenogenesis, the first stages of meiosis are conserved and diploidy is restored by fusion of two nuclei originating from the same primary oocyte (terminal, central or random fusion) or by gamete duplication (Suomalainen et al., 1987). Automictic parthenogenesis increases homozygosity at each generation, but the rate of increase depends on the cytological mechanism restoring diploidy. In a previous paper in Heredity, we determined the mode of thelytokous parthenogenesis in the ant Cataglyphis cursor, by comparing the observed generational increase in homozygosity at several informative loci with theoretical expectations (Table 1 in Pearcy et al., 2006). We argued that the expected rate of transition from heterozygosity to homozygosity at each generation was 0.33 (rather than 0.50) for loci with high recombination under terminal and central fusion. Here we clarify the reasoning behind the calculations. During gametogenesis, the duplicated homologous chromosomes of the 4n primary oocyte pair at the prophase of meiosis I and crossovers can occur between chromatids (Figure 1a). The probability of crossover in the region between the centromere and a given locus increases with the distance between the two. However, when multiple crossovers occur, the original location of the alleles can, by chance, be restored (locus 2 in Figure 1b). Also, if alleles are swapped for both chromatid pairs, the effects of the two recombination events cancel out each other (locus 2 in Figure 1c). In short, the probability that the alleles are effectively swapped between homologous chromosomes is not equal to the probability of observing at least one crossover. Figure 1 Effects of recombination during oogenesis. Chromosomal crossovers (black triangles) result in alleles being swapped between homologous chromatids (a), leading to alternate allele segregation in the egg nucleus and the polar bodies. However, multiple crossovers ... In theory, when the distance between a given locus and the centromere is maximum (that is, infinite), crossovers are so frequent that one may consider that alleles segregate independently. In this case, each offspring of a [ab] heterozygous mother producing [a1a2b1b2] 4n primary oocytes has a 1/3 probability of being homozygous [a1a2] or [b1b2]. For example, if the chromatid in the egg nucleus bears allele a1, there is 1 chance out of 3 that fusion will occur with a polar body possessing the allele a2 rather than b1 or b2. Of course, the same reasoning holds for the other alleles. Interestingly, this probability of transition to homozygosity is independent of the mode of fusion (terminal, central or random), as alleles segregate randomly from the primary oocyte to the polar bodies. Thus, effective recombination probability for any given locus is lower than the rate of crossover and, in theory, should never reach 1. Under automictic parthenogenesis with terminal, central or random fusion, extreme rate of crossover should result in alleles segregating independently. For these loci, the rate of transition to homozygosity is equal to the statistical probability of picking two identical items among two pairs without replacement, that is 1/3.