Division Restitution Research Articles

Cold-Induced Male Meiotic Restitution in Arabidopsis thaliana Is Not Mediated by GA-DELLA Signaling.

Short periods of cold stress induce male meiotic restitution and diploid pollen formation in Arabidopsis thaliana by specifically interfering with male meiotic cytokinesis. Similar alterations in male meiotic cell division and gametophytic ploidy stability occur when gibberellic acid (GA) signaling is perturbed in developing anthers. In this study, we found that exogenous application of GA primarily induces second division restitution (SDR)-type pollen in Arabidopsis, similar to what cold does. Driven by the close similarity in cellular defects, we tested the hypothesis that cold-induced meiotic restitution is mediated by GA-DELLA signaling. Using a combination of chemical, genetic and cytological approaches, however, we found that both exogenously and endogenously altered GA signaling do not affect the cold sensitivity of male meiotic cytokinesis. Moreover, in vivo localization study using a GFP-tagged version of RGA protein revealed that cold does not affect the expression pattern and abundance of DELLA in Arabidopsis anthers at tetrad stage. Expression study found that transcript of RGA appears enhanced in cold-stressed young flower buds. Since our previous work demonstrated that loss of function of DELLA causes irregular male meiotic cytokinesis, we here conclude that cold-induced meiotic restitution is not mediated by DELLA-dependent GA signaling.

Abnormal meiosis in an intersectional allotriploid of Populus L. and segregation of ploidy levels in 2x × 3x progeny.

Triploid plants are usually highly aborted owing to unbalanced meiotic chromosome segregation, but limited viable gametes can participate in the transition to different ploidy levels. In this study, numerous meiotic abnormalities were found with high frequency in an intersectional allotriploid poplar (Populus alba × P. berolinensis ‘Yinzhong’), including univalents, precocious chromosome migration, lagging chromosomes, chromosome bridges, micronuclei, and precocious cytokinesis, indicating high genetic imbalance in this allotriploid. Some micronuclei trigger mini-spindle formation in metaphase II and participate in cytokinesis to form polyads with microcytes. Unbalanced chromosome segregation and chromosome elimination resulted in the formation of microspores with aneuploid chromosome sets. Fusion of sister nuclei occurs in microsporocytes with precocious cytokinesis, which could form second meiotic division restitution (SDR)-type gametes. However, SDR-type gametes likely contain incomplete chromosome sets due to unbalanced segregation of homologous chromosomes during the first meiotic division in triploids. Misorientation of spindles during the second meiotic division, such as fused and tripolar spindles with low frequency, could result in the formation of first meiotic division restitution (FDR)-type unreduced gametes, which most likely contain three complete chromosome sets. Although ‘Yinzhong’ yields 88.7% stainable pollen grains with wide diameter variation from 23.9 to 61.3 μm, the pollen viability is poor (2.78% ± 0.38). A cross of ‘Yinzhong’ pollen with a diploid female clone produced progeny with extensive segregation of ploidy levels, including 29 diploids, 18 triploids, 4 tetraploids, and 48 aneuploids, suggesting the formation of viable aneuploidy and unreduced pollen in ‘Yinzhong’. Individuals with different chromosome compositions are potential to analyze chromosomal function and to integrate the chromosomal dosage variation into breeding programs of Populus.

Male Parent Identification of Triploid Rubber Trees (Hevea brasiliensis) and the Mechanism of 2n Gametes Formation

Eight triploids were screened among offspring of the rubber tree clone GT1 × different clones by flow cytometry and chromosome counting. Twenty-five simple sequence repeat (SSR) markers were screened to identify the origin of 2n gametes, to determine the male parents of these triploids, and to evaluate the mechanism of 2n gamete formation using band configurations and microsatellite DNA allele counting peak ratios (MAC-PR). The results showed that 2n gametes originated from the maternal rubber tree clone GT1, contributing the extra genome copy present in the triploids. It was confirmed that GT1 is able to produce a 2n megagametophyte spontaneously. Many male parents were shown to provide pollen for formation of triploid rubber trees, including clones RRIC 103, Yunyan 277-5, and three other clones. The second division restitution (SDR) was likely the main mechanism involved in formation of megagametophytes in GT1, as the rate of maternal heterozygosity restitution (HR) of all eight triploids varied from 27.78% to 75.00%, with a mean of 51.46%, and all 25 markers varied from 0% to 100%, with a mean of 51.69%. Elucidation of the origin and formation of 2n gametes will help optimize further sexual hybridization of polyploid rubber trees.

A current perspective on apomixis in ferns

AbstractThis review provides a synopsis of apogamous reproduction in ferns and highlights important progress made in recent studies of fern apomixis. First, a summary of the apomictic fern life cycle is provided, distinguishing between two pathways to diploid spore production that have been documented in apomictic ferns (premeiotic endomitosis and meiotic first division restitution) and briefly discussing the evolutionary implications of each. Next, recent trends in fern apomixis research are discussed, exposing a shift in focus from the observation and characterization of apomixis in ferns to more integrated studies of the evolutionary and ecological implications of this reproductive mode. Peer‐reviewed contributions from the past decade are then summarized, spanning the identification of new apomictic lineages through to the developmental, phylogenetic, and population genetic insights that have been made in studies of fern apomixis during that time. Gaps in our understanding are also discussed, including the extent and implications of recombinant apomixis in ferns, the possible reversibility of reproductive mode (from apomictic to sexual) in ferns, and the genomic causes and consequences of apomixis in seed free vascular plants. To conclude, future directions for fern apomixis research are proposed in the context of modern technological advances and recent insights from studies of apomixis in other groups.

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis.

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate.

Abnormal meiotic chromosome behavior and gametic variation induced by intersectional hybridization in Populus L.

Abnormal meiotic chromosome behavior and pollen variation in two intersectional hybrids between Populus simonii Carr. (section Tacamahaca Spach) and Populus euphratica Oliv. (section Turanga Bge.) were investigated by the squashed technique and indirect immunofluorescence. Both hybrids produced abnormal flower buds characterized by a primary bud with an accessary bud at frequencies of 39.0 and 17.8 %, respectively. Chromosome pairing analysis at late diakinesis showed average configurations 20.31 I + 7.04 II + 1.42 III + 0.04 IV in hybrid “Xhu-DK” and 19.18 I + 7.16 II + 1.45 III + 0.04 IV in hybrid “Xhu-TL”, indicating high divergence between their parent species. Meiotic abnormalities, including precocious chromosome migration, lagging chromosomes, chromosome bridges, and micronuclei, were observed at high frequency, resulting in imbalanced chromosome segregation and chromosome elimination and caused high gamete sterility. Some micronuclei triggered formation of minispindles in Metaphase II, which were responsible for the formation of microcytes. Fused and tripolar spindles in the second meiotic division developed into dyads and triads, respectively, leading to viable first meiotic division restitution (FDR) type unreduced pollen formation. However, Xhu-TL was completely sterile because the high frequency of precocious cytokinesis during the second division prevented unreduced pollen formation though spindle fusion. Xhu-DK produced 29.6 % viable pollen grains. The diameter of viable pollen ranged from 19.6 to 64.9 μm, suggesting that Xhu-DK might be capable of producing unreduced pollen or pollen with a higher ploidy level. The strategy of using the distant hybrids for chromosome introgression and polyploidy breeding in Populus L. was considered.

Unreduced gamete formation in wheat: Aegilops triuncialis interspecific hybrids leads to spontaneous complete and partial amphiploids

Production of unreduced or 2n gametes during meiosis is a common consequence of interspecific hybridization in plants. In the present work, five different bread wheat cultivars were crossed with an accession of Ae. triuncialis and interspecific F1 hybrids (MVT, NAT, OMT, PIT and ZAT) were produced. Meiosis in pollen mother cells (PMCs) of F1 hybrids, pollen viabilities, and seed set ratio were analyzed. Restitution meiosis and consequently unreduced viable pollen were observed. Immunolabeling against phosphorylated histone H3 at serine-10 (phosphoH3S10) suggested that unreduced male gametes were produced by first division restitution (FDR). FDR was more frequent in some hybrids, resulting in higher rates of unreduced male gamete formation and F2 seed set. The F2 embryos were highly variable in chromosome number ranging from 35 to 70 suggesting that unreduced gamete formation led to spontaneous partial and complete amphiploids.

Effect of high temperature on the production of 2n pollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization

AbstractTo integrate the gene pool of a wild species (primarily diploid) into a cultivated pool (primarily tetraploid), a crossing between a dihaploid cultivated rose and a hybrid of Rosa wichurana allowed to obtain interspecific diploid hybrids that produced 2n pollen grains. A return to a tetraploid level sought by breeders can then be considered using sexual polyploidization, obtained by crossing a tetraploid cultivated rose with these hybrids. Application of a high‐temperature regime led to a small but significant increase in the percentage of 2n pollen grains in these hybrids of up to 4.6%. This result was obtained by applying high temperatures close to 32°C during the day to plants cultivated in a glasshouse during recurrent cycles of bloom. Crosses were made between an unreleased tetraploid hybrid tea rose, as a female, and the diploid hybrid that produces the most 2n pollen grains. Tetraploid (42.1%) and triploid (57.9%) offspring were obtained. The use of these 2n pollen grains of the first division restitution type should facilitate the introgression of complex traits of interest.

LOCATION OF A CHROMOSOME REGION LINKED TO ALTERNARIA BROWN SPOT RESISTANCE FROM THE EVALUATION OF TRIPLOID MANDARIN POPULATIONS

Alternaria brown spot (ABS) in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternate, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. In the present work, we have taken advantage of the particular genetic structures of unreduced megagametophytes resulting from second division restitution (SDR) (leading to triploid hybrid recovery) to map a genome region linked to ABS resistance in citrus. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a region linked to ABS resistance near the centromere of chromosome III. SSR and SNP markers were developed for efficient early marker-assisted selection (MAS) of ABS resistant hybrids. (Resume d'auteur)

Gene expression differences associated with growth vigor in Populus full-sib allotriploid progeny following manipulated first division restitution of the diploid maternal parent

Polyploidy has played an important role in Populus breeding because allopolyploid trees generally display characteristics of increased height growth over diploids, and tree growth is determined by cell division and expansion in the apical region. However, the transcriptome characteristics of the shoot apical meristem involved in stem segment elongation and plant height growth remain unclear. In this study, apical buds were sampled from high and low growth vigor plants from a single full-sib cross between the parental clones, and their gene expression profiles were compared using high-throughput sequencing. In total, 4,870 genes were identified with differential expression between the high growth vigor allotriploid and its parents, and 821 genes were nonadditively expressed in the high growth vigor allotriploid. The differences in gene expression between the allotriploid and the male parent were more significant than those between it and its female parent. Many of the differentially expressed genes related to cell proliferation, meristem initiation, transcription factors, DNA and RNA methylation. Our results indicated that Populus allotriploids can generate extensive transcriptomic diversity compared with their parents. In addition, we found that the DEGs between high and low growth vigor Populus allotriploids are involved in important biological processes. Major changes in gene expression probably provide a driving force for phenotypic variation and adaptation in allopolyploids. The DEGs selected in this study provide important insight to obtain a better understanding of height growth heterosis in Populus allotriploids.

FDR and SDR processes in meiosis and diploid gamete formation in poplars (Populus L.) detected by centromere-associated microsatellite markers

Sexual polyploidisation is one of the appropriate approaches in poplar breeding. Controlled pollinations were carried out with spontaneously formed, as well as induced, 2n gametes. Among the offspring individuals, 36 triploid plants and 1 tetraploid individual were detected by flow cytometry. The parental clones and all polyploid offspring individuals were genotyped by 18 nuclear microsatellite markers. The allelic configurations, especially tri-allelic patterns, and dosage effects were used to recognise diploid contributions of the male or female gamete. Three out of 18 markers localised near the centromeres of linkage groups I, X and XV. They are assumed to be unaffected by crossing over events and, therefore, able to ascertain the mechanism of first division restitution (FDR) or second division restitution (SDR) to generate diploid gametes. The applied three unlinked centromere-associated microsatellite markers allow a very effective determination of FDR resp. SDR processes. Altogether, 21 diploid pollen (10 FDR and 11 SDR) and 13 diploid ovules (1 FDR and 12 SDR) as well as 2 cases of postmeiotic reconstitution were determined with no inconsistency for the three markers. A female hybrid aspen clone (Populus tremula × Populus tremuloides) was assured to be able to frequently spontaneously form diploid ovules by the SDR mechanism. The transferred average heterozygosity in FDR gametes was assessed to be remarkably higher than that in SDR gametes. However, a selective inducement to favour FDR gametes seems not to be feasible with the current thermo-treatment techniques.

QTug.sau-3B is a major quantitative trait locus for wheat hexaploidization.

Meiotic nonreduction resulting in unreduced gametes is thought to be the predominant mechanism underlying allopolyploid formation in plants. Until now, however, its genetic base was largely unknown. The allohexaploid crop common wheat (Triticum aestivum L.), which originated from hybrids of T. turgidum L. with Aegilops tauschii Cosson, provides a model to address this issue. Our observations of meiosis in pollen mother cells from T. turgidum×Ae. tauschii hybrids indicated that first division restitution, which exhibited prolonged cell division during meiosis I, was responsible for unreduced gamete formation. A major quantitative trait locus (QTL) for this trait, named QTug.sau-3B, was detected on chromosome 3B in two T. turgidum×Ae. tauschii haploid populations. This QTL is situated between markers Xgwm285 and Xcfp1012 and covered a genetic distance of 1 cM in one population. QTug.sau-3B is a haploid-dependent QTL because it was not detected in doubled haploid populations. Comparative genome analysis indicated that this QTL was close to Ttam-3B, a collinear homolog of tam in wheat. Although the relationship between QTug.sau-3B and Ttam requires further study, high frequencies of unreduced gametes may be related to reduced expression of Ttam in wheat.

2n megagametophyte formed via SDR contributes to tetraploidization in polyembryonic 'Nadorcott' tangor crossed by citrus allotetraploids.

2 n megagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement. Tetraploid plants are frequently observed in the seedlings of diploid polyembryonic citrus genotypes. However, the mechanisms underlying the formation of tetraploids are still indistinct when apomictic citrus genotypes are used as female parent to cross with tetraploids. Herein, 54 tetraploid progenies, which were unexpectedly obtained previously from four 2x × 4x crosses using polyembryonic 'Nadorcott' tangor as seed parent, were analyzed by 22 simple sequence repeat (SSR) markers, aiming to reveal their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent; while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confirmed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2n megagametophytes. Based on the genotypes of 2n megagametophytes, the analysis of maternal heterozygosity restitution (HR) for each marker showed that it varied from 0.00 to 87.80 % with a mean value of 40.89 %. In addition, it was observed that 13 markers displayed a lower rate than 50 %. On the basis of the above results, it can be speculated that the second division restitution (SDR) is the mechanism underlying the 2n megagametophyte formation in 'Nadorcott' tangor. The elucidation of the mechanism of 2n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus.

Breeding of polyploid heather (Calluna vulgaris)

Calluna vulgaris is an important landscaping plant in Northern Europe. As C. vulgaris is the only species within the genus Calluna, the available gene-pool for breeding is rather narrow and phenotypic variation is limited. Hence, a breeding program for polyploids was set up in order to broaden phenotypic variation in this important ornamental crop. Therefor basic genetic characteristics of polyploid C. vulgaris were analyzed using the progeny of a spontaneously occurring tetraploid genotype. With the help of morphological and molecular marker analysis, it was determined that the autotetraploid plant probably arose from unreduced gametes resulting from first division restitution. Tetraploids displayed tetrasomic inheritance with free combination of homologous and homeologous chromosomes. Triploids were semi-fertile with a high rate of aneuploids in their progeny. As the so-called bud-blooming flower type is a trait of outstanding interest in breeding of C. vulgaris, a strategy for efficient breeding of triploid bud-bloomers was deduced.

Pollen viability and meiotic behaviour in intraspecific hybrids of Hydrangea aspera subsp. aspera Kawakami group x subsp. sargentiana

AbstractPollen viability and male meiosis in intraspecific hybrids of Hydrangea aspera subsp. aspera Kawakami group (2n = 2x = 36) and subsp. sargentiana (2n = 2x = 34) were investigated. Although it had been assumed that they were sterile, pollen viability was observed; it varied from 2.5% to 12.1%. The production of gametes with different chromosome numbers was implied by the analysis of the dispersion of the diameter distribution of pollen grains. Analysis of male meiosis made it possible to identify the origins with two major categories of meiotic aberrations: abnormal chromosome distribution (early chromosome migration at metaphase, lagging chromosomes at anaphase, micronuclei at telophase), leading to the formation of unbalanced tetrads and/or ones with supernumerary microspores; and abnormal spindle orientation in metaphase II (tripolar, fused and parallel spindles), leading to the formation of dyads or triads. The mode of 2n pollen formation is of the First Division Restitution type. The high level of parental heterozygosity that is normally associated with them should facilitate the transfer of a polygenic trait in breeding programme.

The origin of unusual chromosome constitutions among newly formed allopolyploids

Wide hybridization followed by spontaneous chromosome doubling of the resulting hybrids plays an important role in plant speciation. Such chromosome doubling is usually accomplished via unreduced gametes produced by altered meiosis, the so-called 'meiotic restitution'. Unreduced gametes are expected to carry somatic chromosome numbers and constitutions. However, it has been shown recently that new allopolyploids often carry unusual chromosome constitutions which include compensating and noncompensating nulli-tetrasomies and monotrisomies, and translocations of homoeologues. We have reanalyzed meiotic divisions in a wheat-rye hybrid by in situ probing with labeled DNA focusing on deviations from the standard pattern of meiotic restitution. In a typical first division restitution in a wide hybrid, there is no chromosome pairing, univalents separate sister chromatids in anaphase I, and there is no meiosis II. Here we illustrate that occasional pairing of homoeologous chromosomes in metaphase I, combined with separation of sister chromatids of univalents, generates diads with compensating nulli-disomies and associated translocations of homoeologues. Similarly, precocious metaphase I migration to the poles of some undivided univalents generates a wide range of noncompensating simple and complex nulli-disomies in the gametes. Both alterations to the standard pattern of meiotic restitution tend to maintain the somatic chromosome numbers in the gametes; chromosome constitutions are variable but mostly genetically balanced. This source of variation among progeny may be an important factor contributing to greater success of natural allopolyploids.

Occurrence of a Spontaneous Triploid Progeny from Crosses between Diploid Asparagus (Asparagus officinalis L.) Plants and Its Origin Determined by SSR Markers

The origin of a spontaneous triploid asparagus plant from crosses of 2x × 2x was investigated by SSR and flow cytometric analyses. One hundred and twenty-four progeny were obtained from crosses between a diploid female ‘Gold Schatz’ and a diploid male ‘Hokkai 100’. SSR analysis proved that two and one genes were transmitted from the maternal and paternal parents, respectively, at each SSR locus of one progeny, 07M-61, whereas one gene each was from the female and male parents in the other diploid progeny. Triploidy of 07M-61 was confirmed by flow cytometric analysis. It was suggested that the triploid plant was derived from fertilization between an unreduced egg and reduced sperm nuclei, given its SSR genotypes. It was also suggested that the unreduced maternal gamete was derived from first division restitution (FDR) or second division restitution (SDR) with chiasma occurrence during meiosis. There were no noticeable morphological differences between the triploid and diploid progeny.

Complete asynapsis resulting in 2n pollen formation in Paspalum jesuiticum Parodi (Poaceae)

Cytological investigation revealed complete asynapsis during microsporogenesis in 2 wild accessions of Paspalum jesuiticum collected in distinct Brazilian regions. Both accessions were hexaploid (2n = 6x = 60) and 60 univalents could be counted at diakinesis. In this phase, the majority of meiocytes exhibited univalents with both chromatids. After alignment at the metaphase plate, the chromatids segregated to the poles. Only 1 meiotic division (equational) occurred, and after cytokinesis, a dyad with 2n microspores was formed. The genetic constitution of the 2n gametes was equivalent to that of first division restitution (FDR). Since recombination did not occur, 100% transmission of parental heterozygosity could be expected from the FDR 2n gametes. The meiotic behavior of both accessions suggested that they resulted from a recent natural hybridization event. The potential use of the 2n gametes in Paspalum breeding programs has been discussed.

Genetically based location from triploid populations and gene ontology of a 3.3-mb genome region linked to Alternaria brown spot resistance in citrus reveal clusters of resistance genes.

Genetic analysis of phenotypical traits and marker-trait association in polyploid species is generally considered as a challenge. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from second division restitution (SDR) to map a genome region linked to Alternaria brown spot (ABS) resistance in triploid citrus progeny. ABS in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata. This pathogen produces ACT-toxin, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a 3.3 Mb genomic region linked to ABS resistance near the centromere of chromosome III. Clusters of resistance genes were identified by gene ontology analysis of this genomic region. Some of these genes are good candidates to control the dominant susceptibility to the ACT-toxin. SSR and SNP markers were developed for efficient early marker-assisted selection of ABS resistant hybrids.

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.)

We induced a fdr1 mutation in maize which makes haploid plants malefertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified. Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0mM sodium azide solution for 2h. The 0.5mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43×B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3% [13/85]) was recorded with the 2.5mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48% and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.