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An AFLP marker tightly linked to apomixis reveals hemizygosity in a portion of the apomixiscontrolling locus in Paspalum simplex
Paola Labombarda, Alessandra Busti, Maria Eugenia Caceres, Fulvio Pupilli, and Sergio Arcioni

Abstract: A mapping population of Paspalum simplex segregating for apomixis (asexual reproduction through seeds) was screened with AFLPs to find apomixis-linked markers. Four AFLPs linked to apomixis in coupling phase were found. Three of them did not show recombinants among the 87 individuals of the mapping population, whereas the other was more loosely linked. Integrating the AFLP data with those obtained previously with rice RFLP anchor markers, a map was drawn for the chromosome region of P. simplex encompassing apomixis. We cloned the three AFLPs tightly linked with apomixis into plasmid vectors and used them as probes to hybridize the restriction digested DNA of the mapping population. Two of them revealed RFLP bands linked to apomixis together with other alleles, whereas one was proven to belong to a hemizygous portion of the apomixis locus. The total picture resulting from AFLP and RFLP analyses was that a cluster of markers tightly linked with apomixis was detected in P. simplex together with two other markers that were more loosely linked. These two markers enclosed a relatively large chromosome segment characterized by strong repression of recombination. The block of recombination may have caused sequence divergence and, therefore, hemizygosity of some regions belonging to the apomixis-controlling chromosome segment of P. simplex. The potential of developing an apomixis-specific sequence for screening large-fragment libraries for the physical isolation of the locus encompassing apomixis is discussed. Key-words: AFLP, apomixis, hemizygosity. Rsum : Une population de cartographie de Paspalum simplex qui se reproduit par lapomixie (reproduction asexue par lintermdiaire des ses propres graines) a te tudie avec AFLPs pour trouver des marqueurs lis lapomixie. On a trouv quatre AFLPs lis lapomixie en accouplement de phase. Trois de ces marqueurs nont pas montr de recombinants entre les 87 individus de la population de cartographie, tandis que lautre est faiblement li. En compltant les rsultas obtenus avec lanalyse AFLP avec ceux-la obtenus prcdemment avec les marqueurs RFLP de riz, une carte a te dessine pour la rgion chromosomique de P. simplex relative lapomixie. Nous avons clon les trois AFLPs troitement lis lapomixie dans un plasmide vectoriel et utilis comme sondes pour hybrider le ADN de la population de cartographie digre avec un enzyme de restriction. Deux de ceux-ci ont montr quelques bandes RFLP lies avec autres allles, tandis que lautre appartient une portion hmizygote du locus de lapomixie. La carte tout entire, drivant de les deux analyses AFLP et RFLP, a dtect en P. simplex un groupe de marqueurs fortement lis lapomixie avec les deux autres marqueurs faiblement lis. Ceux derniers deux ont rempli un large segment chromosomique, caractris de une forte rpression de la recombinaison. Le bloc de la recombinaison pourrait avoir provoqu des squences divergentes et surtout hmizigotie de quelques zones appartenant au segment chromosomique qui contrle lapomixie en P. simplex. Le dveloppement de la squence spcifique pour lapomixie pourrait tre daider slectionner une librairie de grands fragments pour lisolation physique du locus de lapomixie. Mots-cls : AFLP, apomixie, hmizygotie. Labombarda et al. 519

Introduction
The most widely accepted definition of apomixis is that of Nogler (1984a), who describes this process as a form of asexual reproduction through seeds. Depending on the

differences in the origin and development of the maternal embryo, apomixis is divided into the following three types: (i) apospory, when a somatic cell of the nucellus acts as a spore; (ii) diplospory, when the megaspore mother cell circumvents part of meiosis; and (iii) adventitious embryony,

Received 29 August 2001. Accepted 18 January 2002. Published on the NRC Research Press Web site at http://genome.nrc.ca on 5 April 2002. P. Labombarda, A. Busti, M.E. Caceres, F. Pupilli,1 and S. Arcioni. Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere del CNR, via della Madonna alta 130, I-06128 Perugia, Italy.
1

Corresponding author (e-mail: f.pupilli@irmgpf.pg.cnr.it).

DOI: 10.1139/G02-014 2002 NRC Canada

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when the embryo develops directly from the somatic cells. The main difference between the first two types and the last is that in apospory and diplospory, commonly referred to as gametophytic apomixis (Stebbins 1950), unreduced embryo sacs are formed, whereas in adventitious embryony the formation of a female gametophyte is completely bypassed (Naumova 1993). Harnessing apomixis into outcrossing seed-propagated species would permit the full exploitation of heterosis by reseeding elite hybrids and the fixation of superior genotypes through clonal seeds. In agriculture, this would have an impact comparable with or even greater than that of the green revolution, especially in third world countries (Hanna 1995; Vielle-Calzada et al. 1996). However, attempts to directly transfer apomixis from wild relatives to crop species have failed and no apomictic genes have been isolated so far (Savidan 2000). The molecular biologists who started to study the apomictic process claimed that deepening of knowledge of the reproductive system of sexual model plants such as Arabidopsis (Chaudhury et al. 1997; Ohad et al. 1996) and Petunia (Ramulu et al. 1998) can provide new and important insights about the developmental genetics of apomixis (Grimanelli et al. 2001). Although some artificial mutants of these plants have been described as having aspects apparently indicative of parts of apomictic reproduction, no artificial apomicts arising from natural sexuals have been reported so far. In this perspective, we propose the wild apomictic grass Paspalum simplex as a model species because it presents a number of characteristics that make it useful for tagging apomixis gene(s) using a map-based approach. Some characteristics worth mentioning are the relatively small genome size (1C value = about 1.44 pg of DNA per nucleus in tetraploid apomicts) and the existence of sexually compatible apomictic and sexual lines (Caceres et al. 1999). Apospory, followed by parthenogenetic development of the embryo, is the typical female development in P. simplex (Caceres et al. 1999). However, the apomictic lines of this species segregated tetrasomically when used as male parents as a consequence of their autotetraploid genetic architecture (Pupilli et al. 1997). A mapping population segregating for apomixis was screened with rice markers, and a linkage group characterized by absence of recombination was found to control apomixis in this species (Pupilli et al. 2001). In the absence of recombination, physical isolation of the apomixis-related segment by cloning large DNA fragments using BAC or YAC vectors becomes an essential process to increase the probabilities of isolating the genes that control apomixis. The first step of these strategies is to isolate apomixis-specific sequences to be used as probes for screening the recombinant clones. Such specific fragments are unlikely to be found with heterologous markers derived from a sexual species. Therefore, the aim of the present work was the isolation of sequences specific to the region controlling apomixis of P. simplex using AFLP analysis.

sexual autotetraploid (2n = 4x = 40) created by treatment with colchicine (Caceres et al. 1999). This population was formed by 34 apomictic and 53 sexual individuals whose phenotypes were assigned through progeny tests carried out with the aid of molecular markers over a two-year period (Pupilli et al. 2001). DNA extraction and AFLP procedure DNA was purified from each of the BC individuals and from both parental lines according to Pupilli et al. (1997). Two bulked samples were prepared for each of the apomictic and sexual phenotypes by mixing together equal amounts of DNA from each of 10 plants. AFLP analysis was carried out as described by Vos et al. (1995) with modifications. Genomic DNA (500 ng) was restricted and ligated at 37C overnight with 1 U MseI, 5 U EcoRI, 1 Weiss U T4 DNA ligase, 5 pmol EcoRI adapters, and 30 pmol MseI adapters in a solution containing 0.05 M NaCl, 0.05 mg bovine serum albumin (BSA)/mL, and 10 T4 DNA ligase buffer in a final volume of 11 L. A preselective amplification was performed using a primer pair based on the sequences of the EcoRI and MseI adapters that had one additional selective nucleotide at the 3 end, EcoRI+G and MseI+T. The preselective amplification was performed in a final volume of 20 L with 1.5 mM MgCl2, 0.2 mM dNTPs, 10 PCR buffer, 2.5 U Taq DNA polymerase and 30 ng of preselective primers. The PCR consisted of an initial denaturation of 94C for 2 min, followed by 20 cycles of 94C for 20 s (denaturation), 56C for 30 s (annealing), 72C for 2 min (extension), and 72C for 7 min (final extension). The PCR products were diluted 10-fold in TE buffer and 5 L of the dilution was added to the selective amplification mixture that contained 60 ng of fluoresceine-labeled EcoRI+3 or +4 primer, 60 ng of MseI+3 or +4 primer, and the same reagents as the preselective amplification in a volume of 20 L. The selective amplification consisted of a touch-down PCR under the following conditions: an initial denaturation of 94C for 2 min was followed by one cycle of 94C for 20 s (denaturation), 60C for 30 s (annealing), and 72C for 2 min (extension). Over the course of the next 9 cycles, the annealing temperature was lowered gradually from 60 to 50C. The last 20 cycles had a denaturation of 94C for 20 s, an annealing of 50C for 30 s, and an extension of 72C for 2 min, followed by a final extension of 72C for 7 min. Aliquots of the selective PCRs were loaded on a 6% denaturing polyacrylamide gel and then run in a Genomyx LR (Beckman, Fullerton, Calif.) device for 2 h. After electrophoresis, the gel was scanned in the Genomyx SC (scanner Beckman) and the picture was collected as a TIF image and further analyzed with the program Adobe Photoshop version 5.0. Cloning and Southern analysis of the AFLP markers The position of the AFLP bands cosegregating with apomixis was recorded with the aid of the coordinate system of the Genomyx device. The gel sectors containing the informative bands were then cut off with a scalpel and dissolved in TE buffer at 37C for 1 h. Five microlitres of the mixture were amplified by PCR using the appropriate selective primers, ligated into the pGEM-T easy plasmid (Promega, Madison, Wis.) and transformed into Eschericia coli JM83
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Materials and methods

Plant materials The mapping population used in this study was generated by backcrossing an apomictic F1 developed from a cross between a natural tetraploid apomict (2n = 4x = 40) and a

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according to standard procedures (Maniatis et al. 1982). White colonies were screened for the presence of inserts by PCR using SP6 and T7 primers. Between 40 and 60 PCRpositive clones were blotted after amplification and probed for the first time with an insert randomly chosen from among those blotted over from the same transformation. The hybridized probe was washed out and blots were probed again with one of the other clones that had not previously hybridized. This was repeated until all of the inserts of the library were grouped according to their sequence homology. For Southern hybridization, the cloned AFLP fragments were used as probes against either amplified inserts or genomic Paspalum DNA. Probes (2550 ng) were labeled radioactively with 32P using the Ready-To-Go labelling kit (Amersham-Pharmacia, Piscataway, N.J.). Blots with genomic DNA were prepared on Hybond-N+ membranes (Amersham-Pharmacia) with 67 g of restricted Paspalum DNA according to Pupilli et al. (1997) and an identical procedure was followed for filters prepared with 5 L of amplified clones (total volume of amplification was 50 L). Probed blots containing amplified DNA were washed under high stringency conditions according to the Amersham Hybond-N+ membrane instruction manual, whereas for those blots containing the genomic DNA, the final highstringency wash was omitted. Probed membranes were exposed to X-ray films from one to five days depending on signal intensity. Data analysis For mapping purposes, only alleles segregating from the apomictic parent were taken into account. Linkage and map order were calculated using MAPMAKER/EXP3 (Lander et al. 1987; Lincoln et al. 1992). This program is able to calculate recombination frequencies between fragments in simplex allelic configuration (A000, where A is the segregating allele and 0 the corresponding null allele) and, with minor accuracy, between duplex (AA00) ones. In any case, the few AFLP fragments segregating as duplex alleles were not considered. Data were analysed using the F2 backcross option and linkage was detected with an LOD score threshold of 3. Recombination frequencies were converted to map distance (cM) using the Haldane mapping function (Haldane 1919).

Results and discussion

The 74 screened primer combinations yielded, on average, 110 AFLP bands per parental line with molecular weights ranging from 100 to 450 bp. Of the 7890 bands detected in total in the apomictic parent, 1045 were absent in the sexual one, but only 4 of them were specifically present in the two bulks of apomictic plants and absent in the bulks of sexual plants. All four bands were linked to apomixis, as revealed by single-plant analysis of the mapping population. The primer combinations that produced apomixis-linked bands are given in Table 1. Figure 1a shows an example of an amplification banding pattern obtained with four primer combinations on the DNA of the sexual (F) and apomictic (M) parents, and of two bulks of apomictic (BA) and sexual (BS) individuals. One of these primer combinations, EGAT MTGC, showed one band (arrowhead) to be present in the two bulks of apomictic plants and absent in the correspond-

ing bulks of sexual plants. The same primer combination was used to screen the entire BC population and, as reported in Figs. 1b and 1c, the apomixis-linked band was present in all of the 34 apomictic (A) plants and absent in all of the 53 sexual (S) plants. This indicates that this allele is linked 100% with apomixis in coupling phase. Second and third primer combinations (EGAAMTCGA and EGTA MTCGA) showed two fragments linked 100% with apomixis in coupling phase, whereas the fourth (EGCTA MTGGC) yielded a band that was more loosely linked. None of the primer combinations used to analyse the entire mapping population revealed AFLP linkage with apomixis in repulsion phase. If we assume that all of the AFLPs segregating from the apomictic parent that were screened out by bulked segregant analysis were unlinked with apomixis, we can reasonably conclude that a single locus located in a well-defined chromosome area controls the apomixis phenotype in P. simplex. Using the recombination frequencies between the apomixis locus and the AFLP markers, and supplementing the AFLP data with those obtained with rice markers as probes, a map was drawn for the chromosome region of Paspalum encompassing the apomixis locus (Fig. 2). The rice markers C901, R1759, C996A, C1069, and C454 were mapped in an area spanning 14.5 cM in the telomeric part of chromosome 12 of rice, but they were clustered together in the corresponding Paspalum area, with no recombination recorded among the 87 individuals of the mapping population; the rice marker R202, located in rice chromosome 8, was mapped at a distance of 11.4 cM from the apomixis-controlling region (Pupilli et al. 2001). The picture resulting from AFLP mapping was therefore an exact reflection of that obtained with RFLP, in that a cluster of markers tightly linked with apomixis was detected together with another marker that was more loosely linked. Both the loosely linked markers enveloped a relatively large chromosome segment characterized by a strong repression of recombination, which controls apomictic reproduction. Restriction of the recombination rate has already been observed in other apomictic systems such as Tripsacum (Grimanelli et al. 1998), in which maize heterologous markers were used, or in Pennisetum squamulatum (Ozias-Akins et al. 1998) and Cenchrus ciliaris L. (Roche et al. 1999) using the same set of homologous RAPD markers. Our data, which for the first time in the same species combine heterologous anchor markers and homologous random AFLPs for apomixis mapping, indicate that lack of recombination is not a matter of which marker is used, but is rather a characteristic of the apomictic reproduction itself. Lack of recombination could be due to the location of the apomixis locus on centromeric or other heterochromatic regions that are known to be characterized by low recombination (Wu and Tanksley 1993). However, comparative mapping data in P. simplex and in Brachiaria (Pessino et al. 1997) suggest a telomeric location of the apospory locus on the homoeologous chromosome area of rice and of maize, respectively. The presence of markers at a certain distance on both sides of the apomixis locus may indicate that the corresponding homoeologous segment, which was in a telomeric position in rice, is not so in Paspalum and could have translocated to centromeric or other heterochromatic chromosome regions during speciation. Clustered molecular
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516 Table 1. Primer combinations detecting apomixis-linked AFLP markers in Paspalum simplex Primer combination EGTAMTCGA EGAAMTCGA EGATMTGC EGCTAMTGGC EcoRI primer 5-CTCGTAGACTGCGTACCAATTCGTA-3 5-CTCGTAGACTGCGTACCAATTCGAA-3 5-CTCGTAGACTGCGTACCAATTCGAT-3 5-CTCGTAGACTGCGTACCAATTCGCTA-3 MseI primer 5-ATGAGTCCTGAGTAATCGA-3 5-ATGAGTCCTGAGTAATCGA-3 5-ATGAGTCCTGAGTAATGC-3 5-ATGAGTCCTGAGTAATGGC-3

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Approximate size of the marker (bp) 180 120 250 120

Fig. 1. AFLP pattern of bulked segregant and single-plant analyses of apomictic and sexual P. simplex. (a) Amplified banding pattern of the female (F) and male (M) parents, two bulked sets of 10 plants each of sexual (BS) and apomictic (BA) backcross progenies obtained with four primer combinations; arrowhead indicates an apomixis-linked band revealed by the primer combination EGATMTGC. (b and c) AFLP pattern of the entire mapping population obtained with the primer combination EGATMTGC; arrowhead indicates the same band as in a that is present in all 34 apomictic (A) and absent in all 53 sexual (S) backcross individuals.

markers tightly linked to apomixis may indicate the presence of a complex locus. Such loci are quite common among living organisms and all are characterized by strong repression or absence of recombination among critical genes. Welldocumented examples include the following: (i) the mating type loci of some algae (Ferris and Goodenough 1994), yeasts (Astell et al. 1981; Kelly et al. 1988), and fungi (Arnaise et al. 1993; Debuchy and Coppin 1992); (ii) the incompatibility locus in Brassica (Casselman et al. 2000); (iii) the DM3 cluster of disease resistance genes in lettuce (Meyers et al. 1998); and (iv) the t-haplotype system in the mouse (Silver 1985). The genetic structure of most of these systems is characterized by highly heterozygous rearranged areas in the forms of inversions, deletions, and (or) translocations that are reported to be related to recombination suppression in both Drosophila (Lyttle 1993) and mouse (Hammer et al. 1991). However, recombination between two critical traits related to apomictic reproduction (i.e., apospory or diplospory and pathenogenesis) has been observed in Taraxacum (van Dijk et al. 1999), Erigeron (Noyes and Rieseberg 2000), Hypericum, and Poa (Matzk et al. 2000), whereas in other systems, such as Panicum (Savidan 1980), Ranunculus (Nogler 1984b), and Paspalum simplex (Caceres et al. 2001), the same traits are inherited together.

The absence of recombination must have induced intensive sequence divergence and, therefore, hemizygosity (i.e., uniqueness of sequences) is expected in at least portions of the apomixis-controlling chromosome region in P. simplex. To isolate some of these unique sequences, the three AFLP bands tightly linked to apomixis were reamplified, inserted into plasmid vectors, and used as probes against libraries of cloned AFLPs and blots of genomic DNA of both parental lines and BC progenies. If the reamplified bands were used directly as probes on genomic DNA, the resulting hybridization pattern was a dark smear that indicated the probe used was a pool of different sequences of similar molecular weight. Therefore, to isolate the apomixis-related sequences, we cloned the reamplified fragments and screened the resulting libraries for homology of sequence. Figure 3 displays the hybridizing banding pattern of a blot containing the amplified DNA of 19 colonies resulting from the cloning of the fragment EGTAMTCGA and probed with the inserts of three colonies. All of these probes showed a major hybridizing band (arrowheads) whose molecular weight of ~200 bp was consistent with that of the cloned fragment (Table 1). The insert of colony 1 revealed the most abundant fragment of the library, being present in 11 of 19 colonies (colonies 1, 3, 4, 5, 7, 8, 11, 12, 13, 18, and 19; Fig. 3a), the insert of colony 16 showed homology with five other clones (colonies
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Labombarda et al. Fig. 2. A map of the chromosome segment encompassing apomixis in P. simplex. C901, R1759, C996A, C1069, and C454 are markers of rice chromosome 12, and R202 of rice chromosome 8. Distances are expressed in centimorgans.

517 Fig. 3. Hybridizing banding pattern of a blot containing 19 amplified inserts arising from cloning the AFLP apomixis-linked fragment revealed by the primer combination EGTAMTCGA. The hybridizing pattern of inserts present in colonies 1, 16, and 17 is reported in a, b, and c, respectively. Arrowheads indicate hybridizing fragment of expected molecular weight.

2,6,9,14, and 16; Fig. 3b) that were different from the previous ones, and the insert of colony 17 (Fig. 3c) was shown to be a unique sequence even if a weak signal arising from cross hybridization with some of the positive clones seen in Fig. 3a is evident. Colonies 10 and 15 were false positives in that although they were white colonies, no insert of the expected molecular weight arose after PCR amplification. The inserts carried by colonies 1, 3, and 4 were then used as probes on blots containing the genomic DNA of the parental lines digested with HindIII, EcoRI, BamHI, EcoRV, and BglII to detect informative polymorphisms. The inserts isolated from colonies 3 and 4 were not polymorphic with any of the enzymes used, whereas clone No. 1 (hereafter referred to as EM180) proved to be an apomixis-specific sequence in that it revealed hybridizing bands in the apomictic parent only. An identical cross-hybridization strategy was used for the other two AFLP fragments: apomixis-related RFLPs were revealed in libraries constructed with the AFLPs EGAAMTCGA and EGATMTGC by one insert out of six and one out of five, respectively, but in no cases were apomixis-specific sequences found. Figure 4 reports the hybridizing banding pattern of a blot containing the restricted DNA of apomictic (M) and sexual (F) parents together with

17 apomictic (A) and an equal number of sexual (S) BC progenies probed with the polymorphic probe revealed by the primer combination EGAAMTCGA (Fig. 4a) and EM180 (Fig. 4b). The latter probe hybridized to two fragments in the apomictic parent, both of which were always present in the apomictic BC progenies; no other fragments were present in the sexual genotypes of the same progeny nor in a randomly collected sample of natural sexual genotypes of P. simplex (data not shown), indicating that this sequence belongs to a hemizygous area of the apomixiscontrolling locus in this species. Three bands were revealed when the same blot was probed again with the polymorphic probe EGAAMTCGA. Of the two of these that segregated from the apomictic parent, one (arrowhead) was linked to apomixis and the other was not. In conclusion, the fragment EM180 is a suitable probe for screening large-fragment libraries for a physical isolation of the apomixis-related region in P. simplex. The apomixis locus in P. simplex presents a number of similarities with the R domain of the mating-type locus of the green alga C. reinardtii, chiefly among them the repression of recombination and the presence of hemizygous sequences that are phenotype-specific. Such sequences are regarded as prime candidates for the location of genes that function in only one mating type in C. reinardtii (Ferris and Goodenough 1994). The application of the modern tools of molecular biology to advantageous model systems such as Arabidopsis has led to the identification of mutants that seem to have some typical aspects of apomictic development. In the mutant fie, the endosperm forms without fertilization (Ohad et al. 1996) and in a similar mutant, fis, seeds develop in the absence of fertilization (Chaudhury et al. 1997). It is reasonable to suppose that both the fie and the fis genes control processes that are downstream from the start of both the aposporic and the diplosporic development (Savidan 2000). Therefore, we believe that the factors that are able to trigger the apomictic
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Fig. 4. Hybridizing banding patterns of one blot containing the DNA digests (EcoRI) of sexual (F) and apomictic (M) parental lines, and of 34 apomictic (A) or sexual (S) BC individuals, probed with the apomixis-related clone EGAAMTCGA (a) and the apomixisspecifc clone EM180 (b). Arrowhead indicates an apomixis-linked fragment.

process may be found in apomixis-specific chromosome segments such as those present in wild apomicts and revealed by the probe EM180. Further studies using PCR-based strategies are underway to explore the apomixis-related area beyond both the 5 and 3 ends of the E180 fragments to define the limits of its specificity.

Acknowledgements
This research has been carried out in the frame of the European Union (EU) project entitled Introducing and controlling asexual reproduction through seeds in apomictic systems and sexual crop plants, contract No. FAIR5-CT973730. Financial support was obtained from the EC project entitled Natural apomixis as a novel tool in plant breeding, contract No. QLG2-CT-2000-00603 and from CNR (Consiglio Nazionale delle Ricerche), Progetto Strategico Caratterizzazione e valorizzazione delle risorse genetiche vegetali, animali e microbiche.

References
Arnaise, S., Zickler, D., and Glass, N. 1993. Heterologous expression of mating-type genes in filamentous fungi. Proc. Natl. Acad. Sci. U.S.A. 90: 66166620. Astell, C.R., Alstrom-Jonasson, L., Smith, M., Tachell, K., Nasmyth, K.A., and Hall, D.B. 1981. The sequence of the DNAs coding for the mating-type loci of Saccaromyces cerevisiae. Cell, 17: 1523. Caceres, M.E., Pupilli, F., Quarn, C.L., and Arcioni, S. 1999. Feulgen densitometry of embryo sacs permits discrimination between sexual and apomictic plants in Paspalum simplex. Euphytica, 110: 161167. Caceres, M.E., Matzk, F., Busti, A., Pupilli, F., and Arcioni, S. 2001. Apomixis and sexuality in Paspalum simplex: characterization of the mode of reproduction in segregating progenies by different methods. Sex. Plant Reprod. 14: 201206 Casselman, A.L., Vrebalov, J., Conner, J.A., Singhal, A., Giovannoni, J., Nasrallah, M.E., and Nasrallah, J.B. 2000. Determining the physical limits of the Brassica S locus by recombinational analysis. Plant Cell, 12: 2333. Chaudhury, A.M.L., Ming, L., Miller, C., Craig, E., Dennis, E.S., and Peacock, W.J. 1997. Fertilization- independent seed devel-

opment in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 94: 42234228. Debuchy, R., and Coppin, E. 1992. The mating types of Podospora anserina, functional analysis and sequence of the fertilization domains. Mol. Gen. Genet. 233: 113121. Ferris, P.J., and Goodenough, U.W. 1994. The mating type locus in Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell, 76: 11351145. Grimanelli, D., Leblanc, O., Espinosa, E., Perotti, E., Gonzales de Leon, D., and Savidan, Y. 1998. Mapping diplosporus apomixis in tetraploid Tripsacum: one gene or several genes? Heredity, 80: 3339. Grimanelli, D., Leblanc, E., Perotti, E., and Grossniklaus U. 2001. Developmental genetics of gametophytic apomixis. Trends Genet. 17: 597604. Haldane, J.B.S. 1919. The combination of linkage values and the calculation of diestances betwen the loci of linked factors. J. Genet. 8: 299309. Hammer, M.F., Bliss, S., and Silver, L.M. 1991. Genetic exchange across a paracentric inversion of the mouse t complex. Genetics, 128: 799812. Hanna, W.W. 1995. Use of apomixis in cultivar development. Adv. Agron. 54: 333350. Kelly, M., Burke, J., Smith, M., Klar, A., and Beach, D. 1988. Four mating-types genes control sexual differentiation in the fission yeast. EMBO J. 7: 15371547. Lander, E., Green, P., Abrahmson, J., Barlow, A., Daly, M., Lincoln, S., and Newburg, L. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps in experimental and natural populations. Genomics, 1: 174181. Lincoln, S., Daly, M., and Lander, E. 1992. Constructing genetic maps with MAPMAKER/EXP 3.0. Whitehead Institute Technical Report. 3rd edition. Whitehead Institute, Cambridge, Mass. Lyttle, T.W. 1993. Cheaters sometimes prosper: distortions of Mendelian segregation by meiotic drive. Trends Genet. 9: 205210. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Matzk, F., Meister, A., and Schubert, I. 2000. An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Plant J. 21: 97108. Meyers, B.C., Chin, D.B., Shen, K.A., Sivaramakrishnan, S., Lavelle, D.O., Zhang, Z., and Michelmore, R.W. 1998. The ma 2002 NRC Canada

Labombarda et al. jor resistence gene cluster in lettuce is highly duplicated and spans several megabases. Plant Cell, 10: 18171832. Naumova, T.N. 1993. Apomixis in angiosperms nucellar and integumentary embryony. CRC Press, Boca Raton, Fla. Nogler, G.A. 1984a. Gametophytic apomixis. In Embryology of angiosperms. Edited by B.M. Johri. Springer Verlag, Berlin. pp. 475518. Nogler, G.A. 1984b. Genetics of apospory in apomictic Ranunculus auricomus V. Conclusions. Bot. Helv. 94: 411422. Noyes, R.D., and Rieseberg, L.H. 2000. Two independent loci control agamospermy (apomixis) in the triploid flowering plant Erigeron annuus. Genetics, 155: 379390. Ohad, N., Margossian, L., Hsu, Y.C., Williams, C., Repetti, P., and Fischer, R.L. 1996. A mutation that allows endosperm development without fertilization. Proc. Natl. Acad. Sci. U.S.A. 93: 53195324. Ozias-Akins, P., Roche, D., and Hanna, W.W. 1998. Tight clustering and hemizygosity of apomixis-linked markers in Pennisetum squamulatum implies genetic control of apospory by a divergent locus that may have no allelic form in sexual genotypes. Proc. Natl. Acad. Sci. U.S.A. 95: 51275132. Pessino, S.C., Ortiz, J.P.A., Leblanc, O., do Valle, C.B., Evans, C., and Hayward, M.D. 1997. Identification of a maize linkage group related to apomixis in Brachiaria. Theor. Appl. Genet. 94: 439444. Pupilli, F., Caceres, M.E., Quarn, C.L., and Arcioni, S. 1997. Segregation analysis of RFLP markers reveals a tetrasomic inheritance in apomictic Paspalum simplex. Genome, 40: 822828. Pupilli, F., Labombarda, P., Caceres, M.E., Quarn, C.L., and Arcioni, S. 2001. The chromosome segment related to apomixis in Paspalum simplex is homoeologous to the telomeric region of the long arm of rice chromosome 12. Mol. Breed. 8: 5361. Ramulu, K.S., Dijkhuis, P., Pereira, A., Angenent, G.C., Van Lookeren Campagne, M.M., and Dons, J.J.M. 1998. EMS and transposon mutagenesis for the isolation of apomictic mutants in

519 plants. In Somaclonal variation and induced mutations in crop improvement. Edited by S.M. Jain, D.S. Brar, and B.S. Ahloowalia. Kluwer Academic Publishers, Dordrecht, The Netherlands. pp. 379400. Roche, D., Cong, P., Chen, Z., Hanna, W.W., Gustine, D.L., Sherwood, R.T., and Ozias-Akins, P. 1999. An apospory-specific genomic region is conserved between buffelgrass (Cenchrus ciliaris L.) and Pennisetum squamulatum Frensen. Plant J. 19: 203208. Savidan, Y. 1980. Chromosomal and embryological analysis in sexual apomictic hybrids of Panicum maximum Jacq. Theor. Appl. Genet. 57: 153156. Savidan, Y. 2000. Apomixis: genetics and breeding. Plant Breed. Rev. 18: 1386. Stebbins, G.L. 1950. Variation and evolution in plants. Columbia University Press, New York, N.Y. Silver, L.M. 1985. Mouse t haplotypes. Annu. Rev. Genet. 19: 179208. van Dijk, P., Tas, I.C.Q., Falque, M., and Akx-Schotman, T. 1999. Crosses between sexual and apomictic dandelions (Taraxacum): II. The breakdown of apomixis. Heredity, 83: 7157212. Vielle-Calzada, J.P., Crane, C.F., and Stelly, D.M. 1996. Apomixisthe asexual revolution. Science (Washington, D.C.), 274: 13221323. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van Dalee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23: 44074414. Wu, K.S., and Tanksley, S.D. 1993. PFGE analysis of the rice genome: estimation of fragment sizes, organization of repetitive sequences and relationships between genetic and physical distances. Plant Mol. Biol. 23: 243254.

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