Formation of Unreduced Megaspores (Diplospory) in Apomictic Dandelions (Taraxacum officinale, s.l.) Is Controlled by a Sex-Specific Dominant Locus

Corresponding author: Peter J. van Dijk, P.O. Box 40, 6666 GA Heteren, The Netherlands., p.vandijk{at}nioo.knaw.nl (E-mail)

Communicating editor: J. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In apomictic dandelions, Taraxacum officinale, unreduced megaspores are formed via a modified meiotic division (diplospory). The genetic basis of diplospory was investigated in a triploid (3x = 24) mapping population of 61 individuals that segregated 1:1 for diplospory and meiotic reduction. This population was created by crossing a sexual diploid (2x = 16) with a tetraploid diplosporous pollen donor (4x = 32) that was derived from a triploid apomict. Six different inheritance models for diplospory were tested. The segregation ratio and the tight association with specific alleles at the microsatellite loci MSTA53 and MSTA78 strongly suggest that diplospory is controlled by a dominant allele D on a locus, which we have named DIPLOSPOROUS (DIP). Diplosporous plants have a simplex genotype, Ddd or Dddd. MSTA53 and MSTA78 were weakly linked to the 18S-25S rDNA locus. The D-linked allele of MSTA78 was absent in a hypotriploid (2n = 3x - 1) that also lacked one of the satellite chromosomes. Together these results suggest that DIP is located on the satellite chromosome. DIP is female specific, as unreduced gametes are not formed during male meiosis. Furthermore, DIP does not affect parthenogenesis, implying that several independently segregating genes control apomixis in dandelions.

SEXUAL plant reproduction involves a reduction of the somatic chromosome number by meiosis followed by a restoration of the somatic chromosome number by fertilization. Most seed plants reproduce sexually; however, 0.1% of angiosperm species reproduce asexually through seed, a process referred to as apomixis (NOGLER 1984 ; MOGIE 1992 ). In apomictic plants, embryos develop parthenogenetically (without fertilization) from unreduced somatic or gametophytic cells. Barring mutations, apomictic offspring are genetically identical to the mother plant. Apomixis is of interest for the study of the maintenance of sex, one of the great and long-standing enigmas in evolutionary biology (BARTON and CHARLESWORTH 1998 ; WEST et al. 1999 ; VAN DIJK and VAN DAMME 2000 ). Apomixis also holds great promise for agriculture, because it fixes traits regardless of their complexity in genetic control (VIELLE CALZADA et al. 1996 ; SPILLANE et al. 2001 ). Apomixis does not occur in the major crops except citrus, but it is conceivable that it can be introduced into crops in the future via genetic engineering. A solid understanding of the genetic regulation of apomixis is therefore important from both an evolutionary and an agricultural point of view.

In sexual reproduction a diploid somatic cell, the megaspore mother cell, undergoes meiosis to produce four reduced megaspores. One of these megaspores divides mitotically to form the megagametophyte or embryo sac, containing, among others, a reduced egg cell and two reduced polar nuclei. Fertilization of the egg cell by one sperm cell of the pollen grain generates a diploid embryo, while fertilization of the two polar nuclei by the second sperm cell generates the triploid endosperm. Gametophytic apomicts differ from sexually reproducing plants in that they produce unreduced embryo sacs. Circumvention of meiosis in gametophytic apomicts can be achieved either through apospory, in which meiosis is completely bypassed, or through diplospory, in which meiosis is modified (NOGLER 1984 ; ASKER and JERLING 1992 ; KOLTUNOW 1993 ). In aposporous apomicts an unreduced megagametophyte is produced ectopically from the somatic tissue of the ovule, without an intervening megasporogenesis. This aposporous megagametophyte often coexists with and outcompetes the reduced megagametophyte. In diplosporous apomicts, an unreduced gametophyte is produced from an unreduced megaspore. This unreduced megaspore results from either a mitotic-like division (mitotic diplospory) or a modified meiosis (meiotic diplospory). In both apospory and diplospory, the unreduced egg cell develops parthenogenetically into an embryo. Endosperm development in apomictic species may rely on fertilization of the reduced or unreduced polar nuclei (pseudogamy) or may proceed in the absence of fertilization (autonomous endosperm formation).

Studies on the inheritance of apomixis are complicated by the fact that the trait cannot be crossed into sexual plants using an apomictic female. However, most apomictic species produce viable pollen and can therefore be used as pollen donors in crosses with sexuals. The inheritance of apospory has been investigated in a considerable number of plant species and, remarkably, is always inherited as a monogenic dominant trait (GRIMANELLI et al. 2001 ; GROSSNIKLAUS et al. 2001 ). Much less is known about the inheritance of diplospory. To date the genetic control of diplospory has been extensively studied in only two species: a grass species, Tripsacum dactyloides, that reproduces by mitotic diplospory (GRIMANELLI et al. 1998 ) and a composite species, Erigeron annuus, that reproduces by meiotic diplospory (NOYES and RIESEBERG 2000 ). In these two species diplospory is also inherited as a dominant monogenic trait.

The genetic control of apomixis in another meiotic diplosporous apomict, the common dandelion, Taraxacum officinale, is disputed. Both diploid sexuals (2x = 16) and polyploid autonomous apomicts (mostly triploid, 3x = 24) occur within this species (RICHARDS 1970 , RICHARDS 1973 ). RICHARDS 1970 , RICHARDS 1973 proposed a model for the control of apomixis in Taraxacum with two unlinked dominant genes, one for diplospory and one for parthenogenesis. This model was based on the observations of SØRENSEN and GUDJÓNSSON (1946) and SORENSEN 1958 that loss of a chromosome in disomic offspring (2n = 3x - 1) from triploid apomicts results in the loss of either diplospory or parthenogenesis, depending on which chromosome is lost. On the basis of the chromosome terminology used by SØRENSEN and GUDJÓNSSON (1946), RICHARDS 1970 suggested that the genotypic constitutions for diplospory and parthenogenesis would be Hhh and Ddd, respectively.

MOGIE 1988 , MOGIE 1992 challenged the dominant model because of the poor quality of the previous observations: the small numbers of disomics studied, the unreliable karyology, and the ambiguous interpretation of the phenotypes. Moreover, MOGIE 1992 argued that all known meiotic mutants in sexual species are recessive rather than dominant (BAKER et al. 1976 ; KAUL and MURTHY 1985 ). Therefore, Mogie suggested a single locus control of apomixis, in which diplospory is recessive and parthenogenesis is a pleiotropic effect of diplospory. MOGIE 1992 regarded parthenogenesis as "an innate capacity which will find expression if fertilization can be avoided through mechanisms such as precocity." Precocious megagametophyte development could be a direct consequence of meiotic diplospory, which, according to MOGIE 1992 , is an "abbreviated version of normal meiosis." In Mogie's model, the dominance relationships between the sexual wild-type allele and the mutant apomixis allele are dosage dependent. Triploid apomicts contain one dominant wild-type allele A and excess copies of the apomixis allele a: Aaa, whereas sexual triploids are AAA or AAa. The triplex genotype aaa is weak or inviable due to deleterious mitotic effects of the apomixis mutation.

Segregation studies of apomixis in Taraxacum that test these competing models have not been published. Crosses between diploid sexuals and triploid apomictic dandelions generate low numbers of hybrids because of the low frequencies of haploid and diploid pollen grains produced by the irregular pollen meiosis of triploids. Moreover, the high loads of inviable or weak aneuploid pollen produced by triploids induce high selfing rates in normally self-incompatible sexual diploids (MORITA et al. 1990 ; TAS and VAN DIJK 1999 ). The few hybrids obtained in these diploid sexual-triploid apomict crosses could be interpreted as phenotypic recombinants lacking different elements of apomixis (VAN DIJK et al. 1999 , VAN DIJK et al. 2003 ). Although this finding suggests that apomixis in Taraxacum is controlled by several independent genes, the numbers of hybrids were too low for segregation ratio analysis. We now have circumvented this problem for diplospory by the use of a tetraploid nonparthenogenetic diplosporous pollen donor (4x = 32), which produces higher quality pollen. A cross between a sexual diploid and this tetraploid pollen donor generated a large triploid population that segregated for diplospory. Here we show that diplospory in Taraxacum is inherited as a monogenic dominant trait, consistent with Richards' model. Moreover, both genetic mapping and cytogenetic analysis indicate that the diplospory locus is located on one of the satellite chromosomes, as was originally suggested by SØRENSEN's (1958) study.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant materials:
A triploid population segregating for diplospory was made by crossing a diploid sexual seed parent (S2-125) with a tetraploid diplosporous pollen parent (PAX). The origin of these two plants is described in detail in FALQUE et al. 1998 and VAN DIJK et al. 1999 . The reciprocal cross in which tetraploid PAX was used as the seed parent produced only pentaploid (5x = 40) offspring, indicating that PAX produced unreduced egg cells that were fertilized (P. J. VAN DIJK, unpublished data). PAX eggs cells are not parthenogenetic, as PAX requires fertilization for seed set. PAX may have lost this element of apomixis by recombination during pollen meiosis in its apomictic grandfather (VAN DIJK et al. 1999 ).

The plants of the triploid segregating population were phenotypically classified as diplosporous or nondiplosporous. This was done using testcrosses in which each plant was crossed with a diploid pollen donor. The ploidy levels of the progeny were then evaluated. From each 3x x 2x testcross a random sample of 25 well-developed, brown seeds was germinated. The nuclear DNA amounts of 10 randomly selected seedlings were determined by flow cytometry. If <10 seedlings were available, then all available seedlings were analyzed. The maternal parent was classified as diplosporous if a 3x x 2x testcross produced only near-tetraploid offspring and as nondiplosporous if a testcross produced offspring with nuclear DNA amounts mainly in between diploid and triploids. Occasionally, incomplete nuclear restitution in a diplosporous triploid mother plant may produce offspring with a few chromosomes less than the full tetraploid complement (VAN DIJK et al. 1999 ). Conversely, a nondiplosporous triploid mother plant may occasionally produce offspring with more chromosomes than the triploid complement owing to the fact that a highly disturbed triploid meiosis can induce partial nuclear restitution (RAMSEY and SCHEMSKE 1999 ). Therefore the whole progeny sample was taken into consideration for the diplosporous vs. nondiplosporous classification of the mother plant.

DNA flow cytometry and cytogenetics:
Ploidy levels were determined with a flow cytometer (Ploidy analyzer, Partec, Münster, Germany) using 4',6-diamidino-2-phenylindole as fluorescent stain (TAS and VAN DIJK 1999 ). A diploid reference plant was used as an internal standard. DNA fluorescence was calibrated with chromosome number counts in an aneuploid series (VAN DIJK et al. 2003 ), which allowed us to estimate the chromosome number. Mitotic root tip preparations were made as described in VAN BAARLEN et al. 2000 .

Molecular markers:
The inheritance of three codominant microsatellite markers (FALQUE et al. 1998 ) was studied. MSTA72 and -78 were selected for their high levels of polymorphism in the cross. MSTA78 was a lucky choice, as one of the alleles turned out to be associated with diplospory. A third microsatellite locus, MSTA53, was also analyzed because this locus was known to be linked to MSTA78 (FALQUE et al. 1998 ).

DNA was extracted from young leaves according to the protocol of ROGSTAD 1992 . PCR conditions were identical to those described in FALQUE et al. 1998 , with the exception that one of the primers was fluorescently labeled with Cy5 (Amersham Pharmacia Biotech). The PCR products were analyzed on an ALF express II automatic sequencer (Amersham Pharmacia Biotech). Microsatellite genotypes were scored in the Curve view mode of the ALFwin sequence analyzer 2.00 software.

The association between diplospory and the 18S-25 S rDNA locus was also studied. Natural populations had previously revealed that rDNA-intergenic spacer (IGS) HinfI digests were hyperpolymorphic in the 1- to 1.6-kb region due to variation in the number of 21- and 30-bp repeats (P. J. VAN DIJK and L. M. KING, unpublished observations). The whole 3.2-kb IGS was amplified using two conserved primers (SpaF, 5'-GACGACTTAAATACGCGACGG, and SpaR, 5'-GACTACTGGCAGGATCAACC; POLANCO and DE LA VEGA 1994 ). Amplifications were performed on a Hybaid OmniGene thermal cycler, with a 2-min initial denaturation step at 94°; followed by 30 cycles of 1-min denaturation at 94°, 1-min annealing at 55°, and 4-min extension at 72°; and a final extension of 7 min at 72°. The reaction mixture contained 1.0 mM MgCl₂, 200 µM of each dNTP, 0.2 µM primers, 2.6 units Expand polymerase (Boehringer Mannheim, Mannheim, Germany) and 25 ng template DNA. The total volume was 50 µl. The amplified products were digested with HinfI restriction enzyme. DNA fragments were separated on a 2% agarose gel and stained with ethidium bromide.

Linkage analysis:
Linkage between the marker loci and the putative diplospory locus was estimated as follows. Each triploid offspring microsatellite genotype was split into a haploid egg cell genotype originating from the diploid mother S2-125 and a diploid pollen grain genotype, originating from the tetraploid father PAX. The recombination frequencies in the egg cells were calculated using the DH module of Joinmap 3.0 (VAN OOIJEN and VOORRIPS 2001 ) using the Kosambi mapping function. The estimation of linkage between single-dose markers in polyploids is essentially equivalent to that of a diploid backcross (WU et al. 1992 ). For the diploid pollen grains, the codominant microsatellite genotypes were rewritten as single-dose genotypes for each of the four paternal alleles. The four alleles of a locus were thus treated as four loci. For example, for the "locus" 78a, the genotypes ab, ac, and ae were all coded as 10 heterozygotes and the genotypes bc, be, and ce as 00 homozygotes. Similarly, for the locus 78b, the genotypes ab, bc, and be were all coded as 10 heterozygotes and the genotypes ac, ae, and ce as 00 homozygotes. The same coding was applied to loci 78c and 78e. Linkage between all loci was calculated using the BC1 module of Joinmap 3.0. In tetraploid PAX, diplospory was mapped assuming a single dominant gene (DIP) controlling diplospory. In diploid S2-125, the linkages between a segregating rDNA-IGS fragment and the three microsatellite loci were also calculated. Significance of linkage was evaluated by the LOD score given by Joinmap and by Mather's equation, ²[1] = (a - b - c + d)²/(a + b + c + d), where a, b, c, and d are the observed number of plants in the four classes in the progeny (MATHER 1951 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Segregation of diplospory in the mapping population:
The cross S2-125 x PAX generated 86 F₁ offspring plants, all of which were triploids (Fig 1). No diploid selfed progeny were found, indicating that PAX pollen did not induce breakdown of the self-incompatibility system. Sixty-one triploid F₁ plants were crossed with diploid pollen donors for testcrosses. Five plants died before testcrossing and the remaining 20 plants could not be tested due to the lack of flowering synchrony between the cross partners.

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. The segregation of diplospory in the cross S2-125 x PAX. Seed parents in a cross are to the left, pollen parents to the right. The sizes of the rectangles are proportional to the ploidy level. The numbers above a cross fork indicate the number of crosses for which the ploidy levels of the progeny were analyzed. The numbers along the progeny line indicate the total number of offspring analyzed, as summed over the crosses. Diplosporous plants are indicated in gray.

The triploid F₁ plants fell in two highly distinct classes with respect to the ploidy levels of their progeny in the 3x x 2x testcrosses: 27 plants produced almost exclusively tetraploid offspring, and 34 plants produced offspring with aneuploid chromosome numbers in between diploid and triploid. This first group of 27 F₁ plants was therefore classified as meiotic restitutional (diplosporous), and the second group of 34 F₁ plants was classified as meiotic reductional (nondiplosporous). Fig 2 shows the overall distribution of the offspring chromosome numbers obtained in 3x x 2x testcrosses. The penetrance of diplospory was high. A total of 98.5% of the offspring carried the full tetraploid chromosome set and none of the diplosporous offspring had <29 chromosomes. There was little overlap in chromosome numbers between the diplosporous and the nondiplosporous progeny: only 2.1% of the nondiplosporous progeny had 29 or more chromosomes. Of the nondiplosporous progeny plants, 1.45% were full tetraploids, indicating that triploidy per se can also induce nuclear division restitution, albeit at a rate much lower than that of the diplospory factor.

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. The overall distribution of offspring chromosome numbers obtained in testcrosses with sexual diploid pollen donors for triploid F1 plants that were classified as diplosporous (solid bars; N = 263) or meiotic (hatched bars; N = 172).

In nondiplosporous progeny, high numbers of aneuploid individuals with low viability are expected due to an unbalanced triploid meiosis. The germination rates in testcrosses with nondiplosporous F₁ progeny were significantly lower than those of diplosporous F₁ progeny (means ± SE: 0.40 ± 0.03 and 0.75 ± 0.06, respectively; Kruskal-Wallis, one-way ANOVA, P < 0.001). Moreover, many nondiplosporous seedlings died at an early stage, before they could be analyzed by flow cytometry. Therefore the average number of analyzed seedlings for nondiplosporous progeny was only 5.1 (range 3–10) vs. 9.7 for diplosporous progeny (range 9–10).

Associations between diplospory and microsatellites:
Six and five different alleles, respectively, were present in the cross between S2-125 and PAX for MSTA72 and -78. Table 1 gives the observed segregation ratios of the marker loci for the parents. No significant deviations from the expected Mendelian segregation ratios were observed during female meiosis in diploid S2-125 or during male meiosis in tetraploid PAX.

The alleles at MSTA72 were evenly distributed over the diplosporous and the nondiplosporous F₁ progeny, whereas at MSTA78, the a-allele was strongly associated with diplospory. The MSTA78a frequency among diplosporous plants was 0.46 but only 0.03 among nondiplosporous plants (chi-square test for heterogeneity, 30.00; d.f. = 1; P < 0.001). Since this association could be due to chromosomal linkage, we also screened the mapping population for MSTA53, which is linked to MSTA78 (at 10 cM distance; FALQUE et al. 1998 ). The segregation ratios for MSTA53 did not differ significantly from the expected Mendelian ratios (Table 1). The MSTA53b allele showed the same strong positive association with diplospory as the MSTA78a allele, supporting the hypothesis of chromosomal linkage of the MSTA78a allele with a diplospory factor.

Linkages between the three microsatellite loci were estimated using all F₁ plants, including the nonflowering plants. Table 2 lists the significant recombination frequencies between pairs of loci and the LOD scores. MSTA78 and -53 were closely linked and both were unlinked to MSTA72, consistent with the findings of FALQUE et al. 1998 . In PAX the allelic phases could be determined for the four homologous chromosomal segments: MSTA78a was linked with MSTA53b, -78b with -53e, -78c with -53null, and -78e with -53c. Recombination was detected on three homologous chromosomal segments, but was not detected between the diplospory-associated alleles MSTA78a and -53b (81 x 4 = 324 chromosomes sampled; on average 0.009 crossovers per chromosome). Four crossovers were detected between MSTA53 and -78 during female meiosis in S2-125 (80 x 2 = 160 chromosomes; on average 0.025 crossovers per chromosome).

The associations between MSTA78a, MSTA53b, and diplospory strongly suggest chromosomal linkage; for the estimation of linkage, however, it was necessary to first determine the diplospory genotype of tetraploid PAX and its mode of inheritance.

A genetic model for the inheritance of diplospory:
The many different alleles present at the three microsatellite loci allow one to deduce the two-allele genotypes of the diploid PAX pollen grains from the F₁ genotypes. Table 3 shows that all of the six possible two-allele pollen genotypes at the three microsatellite loci were formed, implying the absence of strict disomic inheritance. The observed numbers did not differ significantly from the numbers expected with random tetrasomic chromosome segregation. No homozygous diploid pollen genotypes were observed, indicating that chromatid segregation or double reduction did not take place.

Both Richards' dominant and Mogie's recessive models were tested for their fit to the observed diplospory segregation ratio. Only tetrasomic inheritance with random chromosome pairing was considered, as the linked markers MSTA53 and -78 indicated the absence of disomic inheritance or chromatid segregation. In the case of the recessive model, two sexual diploid genotypes are possible: AA and Aa. Both Mogie's and Richards' models were tested for a single and a double dose of the diplospory allele. Table 4 gives the six possible cross combinations with their goodness of fit to the observed segregation ratio. Three models were rejected, but three other genotype combinations fit the observed data with the possible genotypes for PAX being Aaaa, AAaa, or Hhhh.

The three possible genotype combinations lead to different predictions for the frequencies of linked microsatellite markers among diplosporous and nondiplosporous progeny (Table 5). As shown in Table 5 the dominant Hhhh model (3) has a good fit with the observed allele frequencies for MSTA78, whereas both recessive diplospory models (1 and 2) are rejected. Assuming a simplex Hhhh genotype for PAX, the recombination fractions between MSTA53, -78, and the diplospory locus can be estimated (Table 2). Four plants were recombinant for both microsatellite loci and for the diplospory locus. The recombination frequency between MSTA78a and -53b and DIP is 7 cM. Both markers are located on the same side of the diplospory locus. No linkages between the diplospory locus and the other alleles at MSTA53 and -78 were found, supporting the hypothesis that the dominant diplospory factor is present in a single dose in tetraploid PAX.

The association between MSTA78 and diplospory in the pedigree:
PAX was produced from a cross between a triploid diplosporous seed parent (H6-3) with a diploid sexual pollen donor. In turn, H6-3 was produced from a cross between a diploid sexual seed parent with a triploid full apomict SE3x-6. This pedigree is shown in Fig 3. The genotypes of the apomictic founder SE3x-6 for MSTA53 and -78 are, respectively, b c null and a c e and that of H6-3 is b c e and a b e. Thus, as expected, PAX inherited the diplospory-linked chromosome segment b-a from its apomictic grandfather SE3x-6. The nondiplospory segment c-e was also inherited from SE3x-6. The other two chromosome segments in PAX, e-b and null-c, originated from diploid sexual parents that were no longer alive.

View larger version (22K): In this window In a new window Download PPT slide   Figure 3. The full pedigree of the apomictic founder SE3x-6 with the genotypes for MSTA78. The siblings of H6-3 are listed in Table 6. Diplosporous plants are indicated in gray. Dots represent unknown alleles. G, generation.

The MSTA78 genotypes of other surviving plants of the second generation (G₂) were also determined (Table 6). The strict association between the MSTA78a allele and diplospory in the G₂ was fully consistent with the linkage that was found in the mapping population (G₄). All nine diplosporous G₂ plants carried the diplospory-linked MSTA78a allele whereas none of the three nondiplosporous triploid G₂ plants carried this allele. The MSTA78a allele was also absent in the four diploid, nondiplosporous G₂ plants.

The diplospory locus is located on the satellite chromosome:
SORENSEN 1958 suggested that a diplospory factor was located on a satellite chromosome. Therefore we investigated the association between the diplospory and the 18S-26S rDNA locus. Digestion of the rDNA-IGS with HinfI produced complex polymorphic banding patterns. Only the presence or absence of a 1350-bp S2-125 fragment could be reliably scored in the F₁ mapping population. The segregation ratio of the presence or absence of this fragment did not differ significantly from the expected 1:1 Mendelian ratio (Table 1). According to MATHER's (1951) chi-square test, MSTA53 and -78 are both significantly linked to the rDNA-IGS locus, at 0.31 and 0.36 cM, respectively (Table 2). MSTA53 and -78 are linked to the diplospory locus in PAX and are also linked to rDNA-IGS in S2-125, providing indirect evidence that the diplospory locus is located on the satellite chromosome.

Cytological evidence also supports this chromosomal location of the diplospory locus. One of the triploid G₂ plants, H6-4, a full-sib of H6-3, is apomictic as shown by fixed heterozygosity for the four studied microsatellite loci (MSTA61, -64, -72, and -78; P. J. VAN DIJK, unpublished results) in the offspring. However, one of its apomictically produced offspring plants (H6-4-4) lacked the maternal MSTA78a allele. Moreover, flow cytometry indicated that H6-4-4 is a hypotriploid (3x - 1). Chromosome spread preparations showed that H6-4-4 contains only two instead of the normal three satellite chromosomes (Fig 4). The simultaneous loss of one of the three MSTA78 alleles and one of the three satellite chromosomes provides cytological evidence that MSTA78 and, therefore, also the linked diplospory locus are located on a satellite chromosome.

View larger version (92K): In this window In a new window Download PPT slide   Figure 4. Root tip metaphase in the disomic plant H6-4-4 (3x - 1 = 23). This is an offspring plant from the apomictic mother H6-4. H6-4-4 is missing one of the three maternal MSTA78 alleles (see Curve view print; small arrows) and also one of the three satellite chromosomes (large arrows).

H6-4-4 was a slender plant that produced only a few inflorescences. H6-4-4 flower heads were smaller than those of H6-4 and had an irregular appearance because of the curled floret ligules, as was also described by SØRENSEN and GUDJÓNSSON (1946) for the tenuis disomic aberrant. Attempts to determine whether H6-4-4 had lost diplospory by testcrossing it with a diploid pollen donor were unsuccessful. Four flower heads were crossed, but were completely sterile.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A model for the inheritance of diplospory in Taraxacum:
The inheritance of diplospory and the association with two linked microsatellite loci strongly suggests that diplospory in Taraxacum is controlled by a dominant genetic factor. This diplospory gene, DIP, is present in a single dose in the triploid apomictic founder of the pedigrees. The diplospory locus may be heterozygous or hemizygous. Hemizygosity was demonstrated for the apospory locus in the grass species Pennisetum squamulatum (OZIAS-AKINS et al. 1998 ) and Paspalum simplex (PUPILLI et al. 2001 ; LABOMBARDA et al. 2002 ).

Diplospory affects only female meiosis, resulting in unreduced megaspores. Male meiosis in diplosporous plants is reductional and allows for genetic mapping of the trait. The weak, but significant, linkage between the rDNA locus and the diplospory-linked markers MSTA53 and -78 in the sexual plant S2-125 is consistent with a satellite chromosome location of the diplospory gene. The physical location of the DIP locus was cytogenetically confirmed in a disomic plant (3x - 1 = 23) H6-4-4 that lacked both the diplospory-linked MSTA78a allele and a satellite chromosome. Unfortunately, this plant was sterile and it could not be directly determined whether this plant also lacked diplospory. Nevertheless both the linkage and the cytogenetic evidence presented in this article support the suggestion by SORENSEN 1958 that the DIP gene is located on the satellite chromosome. Given the weak linkage between MSTA53 and -78 and the rDNA locus, it is likely that the diplospory locus is located on the short arm, opposite the nucleolar organizer region.

Our results corroborate Richards' two dominant loci model for apomixis in Taraxacum. Mogie's alternative model of a single recessive gene for diplospory with a pleiotropic effect on parthenogenesis is incompatible with our results and therefore has to be rejected.

In Richards' model the putative diplospory gene is named H and the putative parthenogenesis gene is named D, referring to SØRENSEN and GUDJÓNSSON's (1946) chromosome terminology. However, this lettering is counterintuitive and therefore confusing. Now that the diplospory locus has been mapped, we suggest the name DIPLOSPOROUS, abbreviated to DIP, in which the dominant D allele controls diplospory and the recessive d allele controls normal meiotic reduction.

The apomictic founder SE3x-6 expressed three elements of apomixis: diplospory, parthenogenesis, and autonomous endosperm development. The G₁ plant H6-3 was diplosporous and exhibited autonomous endosperm development, but lacked parthenogenesis (VAN DIJK et al. 1999 ). Parthenogenesis did not reappear in later generations (PAX and its F₁ progeny) and was thus permanently lost. Some siblings of H6-3 were diplosporous and parthenogenetic, but lacked autonomous endosperm development (VAN DIJK et al. 2003 ). This suggests that the three elements of apomixis in Taraxacum can be inherited independently and can be separated by recombination during male meiosis. Again this is inconsistent with a single-locus pleiotropic model for apomixis in Taraxacum.

Comparisons with other apomictic species:
Dominant monogenic control of apomeiosis has been reported in all apomictic species studied so far (see GRIMANELLI et al. 2001 ; GROSSNIKLAUS et al. 2001 , for reviews). Most of these species are aposporous. T. officinale, after E. annuus (NOYES and RIESEBERG 2000 ), is now the second species in which meiotic diplospory is shown to be inherited as a monogenic dominant trait.

Taraxacum and Erigeron are both members of the Asteraceae (Compositae) family, but belong to different subfamilies (BREMER 1994 ). Diplospory in the two species is cytogenetically very similar (BERGMAN 1950 ). Diplospory in triploid Erigeron is inherited disomically (NOYES and RIESEBERG 2000 ), suggesting that the diplospory chromosome does not pair with the two other homologous chromosomes. In contrast, diplospory in Taraxacum is inherited tetrasomically, without obvious preferential chromosome pairing.

In most species apomixis is inherited as a single dominant trait (GROSSNIKLAUS et al. 2001 ), whereas in both Erigeron and Taraxacum diplospory and parthenogenesis are inherited independently. Likewise, apospory and parthenogenesis are regulated independently in Poa pratensis (ALBERTINI et al. 2001 ). It is therefore conceivable, in species where apomixis is inherited as a monogenic trait, that apomixis is in fact controlled by a complex of closely linked genes with different functions.

What is the function of the DIP gene?
Many meiotic mutants have been described in sexual species like maize and Arabidopsis. These mutations affect meiotic processes such as chromosome pairing, recombination, and chromatid cohesion (KAUL and MURTHY 1985 ; BHATT et al. 2001 ; MERCIER et al. 2001 ).

Two cytological mechanisms of diplospory have been described in Taraxacum: (i) first division restitution (FDR) and (ii) pseudohomeotypic division. In both cases the univalents move to the equatorial plane to form a metaphase plate. In the case of FDR, the univalent group undergoes strong contraction to form a so-called contraction nucleus (GUSTAFSSON 1934 ; FAGERLIND 1947 ; BERGMAN 1950 ), which is followed by a normal meiosis II. In contrast, in the pseudohomeotypic division, the univalents at the metaphase plate of meiosis I divide, and the chromatids move to opposite poles and form two nuclei (GUSTAFSSON 1934 ). Meiosis II is omitted in this type of diplospory.

Diplospory in Taraxacum is characterized by the (near) absence of bivalents. FAGERLIND 1947 did not observe chromosome pairing during early prophase, which suggests asynapsis rather than desynapsis. Both FDR and pseudohomeotypic division produce a dyad of two megaspores. A number of mutants have been described in which dyads rather than tetrads are formed during meiosis. The dyad mutation in Arabidopsis affects only female meiosis, as with diplospory in Taraxacum; however, the dyads result from the omission of meiosis II (SIDDIQI et al. 2000 ). The Spo13 mutation in Saccharomyces cerevisiae also produces dyads, affecting meiosis I, but no Arabidopsis Spo13 ortholog has been found so far (MERCIER et al. 2001 ). At present we are not aware of any meiotic mutant in sexual plants with a phenotype similar to that of DIP. Moreover, all known meiotic mutants are recessive and generally highly sterile whereas DIP in Taraxacum is dominant and diplosporous plants are highly fertile. This makes the DIP gene especially intriguing and interesting for further characterization.

	ACKNOWLEDGMENTS

We acknowledge Hans de Jong and Henny Verhaar for the cytogenetic analyses performed in this study and for providing Fig 4. We thank Kim Boutilier, Hans de Jong, Peter van Baarlen, Kitty Vijverberg, and the reviewers of Genetics for their comments, which helped to improve the manuscript considerably. Part of this work was funded by the European Union as part of the project "Natural apomixis as a novel tool in plant breeding (ApoTool)," contract number QLG2-2000-00603 of the Quality of Life and Management of Living Resources section. This is publication 3226 of the Netherlands Institute of Ecology (NIOO-KNAW).

Manuscript received May 9, 2003; Accepted for publication October 1, 2003.

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