Locating a site on the maize B chromosome that controls preferential fertilization.

In maize, the B chromosome can undergo nondisjunction at the second pollen mitosis, producing sperm with two B chromosomes and sperm with zero B chromosomes. Preferential fertilization is the ability of the sperm carrying two B chromosomes to transmit more frequently to the embryo of a kernel than the sperm lacking the B chromosome. A translocation involving the B chromosome and chromosome 9, TB-9Sb, has been used to study preferential fertilization. The B-9 chromosome has the same properties of nondisjunction and preferential fertilization as the standard B chromosome. Deletion derivatives of B-9, which lack the centric heterochromatin and possibly some adjacent euchromatin, were tested for their ability to induce preferential fertilization. They were found to lack the capacity for preferential fertilization.     

Sperm Identification in Maize by Fluorescence in Situ Hybridization

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. Despite the obvious advantages of being able to recognize and then track, separate, and analyze one sperm type from the other, it has not been possible because of the lack of sufficient detectable differences between the two types of sperms. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Our results are consistent with conclusions from previous genetic studies related to B chromosome behavior during pollen formation. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random.     

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.     

B Chromosome Behavior in Maize Pollen as Determined by a Molecular Probe

The B chromosomes of maize typically undergo nondisjunction during the second microspore division (generative cell division). When the microspore nucleus contains only one B chromosome, two kinds of sperm result, one with two B chromosomes and one with no B chromosomes. The sperm with the B chromosomes preferentially fertilizes the egg cell. Previous studies of these phenomena have been limited to genetic analysis and chromosome spreads. In this study we show that a B chromosome-specific probe can be used with fluorescence in situ hybridization (FISH) analysis to detect the presence, location, and frequency of B chromosomes in intact interphase nuclei within mature pollen of maize. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe.     

Centromere function and nondisjunction are independent components of the maize B chromosome accumulation mechanism

Supernumerary or B chromosomes are selfish entities that maintain themselves in populations by accumulation mechanisms. The accumulation mechanism of the B chromosome of maize (Zea mays) involves nondisjunction at the second pollen mitosis, placing two copies of the B chromosome into one of the two sperm. The B chromosome long arm must be present in the same nucleus for the centromere to undergo nondisjunction. A centromere, containing all of the normal DNA elements, translocated from the B chromosome to the short arm of chromosome 9 was recently found to be epigenetically silenced for centromeric function. When intact B chromosomes were added to this genotype, thus supplying the long arm, the inactive centromere regained the property of nondisjunction causing the translocation chromosome 9 to be differentially distributed to the two sperm or resulted in chromosome breaks in 9S, occasionally producing new translocations. Translocation of the inactive B centromere to chromosome 7 transferred the nondisjunction property to this chromosome. The results provide insight into the molecular and evolutionary basis of this B chromosome accumulation mechanism by demonstrating that nondisjunction is caused by a process that does not depend on normal centromere function but that the region of the chromosome required for nondisjunction resides in the centromeric region.     

Dissection of rye B chromosomes, and nondisjunction properties of the dissected segments in a common wheat background

The rye B chromosome is a supernumerary chromosome that increases in number in its host by directed postmeiotic drive. Two types of rye B chromosomes that had been introduced into common wheat were dissected into separate segments by the gametocidal system to produce a number of rearranged B chromosomes, such as telosomes, terminal deletions and translocations with wheat chromosomes. A total of 13 dissected B chromosomes were isolated in common wheat, and were investigated for their nondisjunction properties. Rearranged B chromosomes, separated from their B-specific repetitive sequences on the distal part of the long arm, did not undergo nondisjunction, and neither did a translocated wheat chromosome carrying a long-arm distal segment containing the B-specific repetitive sequences. However, such rearranged B chromosomes, missing their B-specific sequences could undergo nondisjunction when they coexisted with the standard B chromosome or a wheat chromosome carrying the B-specific sequences. Deficiencies of the short arm did not completely abolish the nondisjunction properties of the B chromosome, but did reduce the frequency of nondisjunction. These results confirmed previous suggestions that the directed nondisjunction of the rye B chromosome is controlled by two elements, pericentromeric sticking sites and a trans-acting element carried at the distal region of the long arm of the B chromosome. Additionally, it is now shown that the distal region of the long arm of the B chromosome which provides this function is that which carries the B-specific repetitive sequences.     

On the mechanism of chromatin loss induced by the B chromosome of maize

Knobbed regions of the regular maize complement frequently are eliminated at the second microspore division in spores which have two or more B chromosomes. Evidence is presented that no or little loss occurs in spores with one B and that the rate is not increased in spores with more than two B's.—The B chromosomes from an unrelated strain proved as effective in inducing loss as did the B's of the original high loss stock.—Chromatin loss induced by B's is restricted to knobbed A chromosomes and occurs only at the second microspore division. Knobbed chromosomes 3, 5, and 9 have been tested and all interact with B's to give loss. Chromosomes with large knobs are more frequently broken than are those with smaller knobs and knobless chromosomes show negligible loss.—Although knobs and B's are essential for chromatin elimination, modifying genes can markedly affect the rate of loss.——Two knobbed heterologous chromosomes undergo simultaneous loss more frequently than expected from independent events. The data indicate that joint loss occurs in competent cells and that preferential assortment of the two deficient chromosomes to specific poles is unlikely.—B chromosomes and deficient chromosomes assort independently at the second microspore anaphase.—Genetic data from crosses with marker genes in both arms of chromosome 3 show that breakage of the postulated dicentric bridge does not occur solely at the centric region since a variety of deficient chromosomes were recovered.—Nondisjunction of B chromosomes and elimination of knobbed chromatin take place during the second microspore mitosis. The argument is advanced that the two phenomena result from faulty replication of heterochromatic segments. The position of the nonreplicating segment in the two kinds of chromosomes determines whether nondisjunction or breakage takes place.—Finally, it is suggested that all of the reported effects of the B chromosome can be accounted for if the B is a parasitic entity having no genetic function other than controlling the replication of its proximal heterochromatic knob and increasing the ability of B-containing sperm cells to compete successfully for fertilization of the egg.     

Sporophytic nondisjunction of the maize B chromosome at high copy numbers

It has been known for decades that the maize B chromosome undergoes nondisjunction at the second pollen mitosis.Fluorescence in-situ hybridization (FISH) was used to undertake a quantitative study of maize plants with differing numbers of B chromosomes to observe if instability increases by increasing B dosage in root tip tissue.B chromosome nondisjunction was basically absent at low copy number,but increased at higher B numbers.Thus,B nondisjunction rates are dependent on the dosage of B's in the sporophyt...     

B chromosomes of maize (Zea mays L.) are positioned nonrandomly within sperm nuclei

We used fluorescence in situ hybridization to identify and map the position of B chromosomes (supernumerary chromosomes) within maize sperm cells. Observations on over 1,000 sperm cells from several genotypes show that, on average, the B chromosomes are positioned in the tip one-fourth of the sperm nucleus two-thirds of the time. In contrast, the centromeres and knobs of the A chromosomes (the normal set) are not restricted to the tip portion of the nucleus. To our knowledge, this is the first example of specific chromosome positioning within a plant gamete. Studies on nuclear architecture of somatic cells in both plants and animals suggest that chromosome behavior and gene expression may correlate with chromosome position within the nucleus. The functional significance of nonrandom positioning of the B chromosomes within maize sperm is as yet unclear. Received: 10 May 2000 / Revision accepted: 6 September 2000     

Sporophytic nondisjunction of the maize B chromosome at high copy numbers

It has been known for decades that the maize B chromosome undergoes nondisjunction at the second pollen mitosis.Fluorescence in-situ hybridization(FISH)was used to undertake a quantitative study of maize plants with differing numbers of B chromosomes to observe if instability increases by increasing B dosage in root tip tissue.B chromosome nondisjunction was basically absent at low copy number,but increased at higher B numbers.Thus,B nondisjunction rates are dependent on the dosage of B's in the sporophyte.Differences in nondisjunction were also documented between odd and even doses of the B.In plants that have inherited odd humbered doses of the B chromosome,B loss is nearly twice as likely as B gain in a somatic division.When comparing plants with even doses of B's to plants with odd doses of B's,plants with even numbers had a significantly higher chance to increase in number.Therefore,the B's nondisjunctive capacity,previously thought to be primarily restricted to the gametophyte,is present in sporophytic cells.     

Chromosome nondisjunction and instabilities in tapetal cells are affected by B chromosomes in maize

Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B's), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus formation, but the B's form more micronuclei than do knobbed chromosomes and knobbed chromosomes form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm, but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A's) in tapetal cells. Our results support the conclusion that nondisjunction and micronucleus formation are regular events in the process of the tapetal cell death program, but B's strongly increase A chromosome instability.     

Accumulation of multiple copies of maize minichromosomes

Multiple copies of B chromosomes in maize (Zea mays) can accumulate in the genome using the B chromosome's accumulation mechanism, specifically nondisjunction at the second pollen mitosis and preferential fertilization of the egg. Using this mechanism, we accumulated 4 different-sized minichromosomes derived from the B chromosome to test the chromosome limits of the cell. The accumulation of normal B chromosomes is associated with multiple phenotypes including white stripes and asymmetric leaf blades, but when minichromosomes are accumulated these symptoms are absent. We also found that multiple B chromosome-derived minichromosomes can coexist with A chromosome-derived minichromosomes. During the years that these experiments were conducted, we found many B chromosome rearrangements and fragments, 2 recoverable A chromosome fragments, and observed a minichromosome breakage-fusion-bridge cycle in roots.     

A stably transmitted pair of translocated supernumerary chromosomes in maize.

A pair of stably transmitted supernumerary chromosomes of unknown source has been found in a maize stock carrying a desynaptic mutant. The presence of the supernumerary chromosome appears to be unrelated to the meiotic mutant, but is believed to have been derived from a translocated B chromosome contaminant. The supernumerary chromosomes carry a segment of a A chromosome in this stock where there appear to be two normal copies of each of the 10 A chromosomes. Thus, this A chromosome segment is present in quadruplicate. Surprisingly, a quadrivalent configuration is formed in most microsporocytes, which involves not only synapsis but also chiasma formation in the A chromosome segments involved in the quadrivalent. This represents a strong preferential pairing of supernumeraries with the normal A chromosome segments. Such nonrandom association and crossing over might provide information on the nature of early homologue alignment at meiosis.     

Meiosis in Male DROSOPHILA MELANOGASTER I. Isolation and Characterization of Meiotic Mutants Affecting Second Chromosome Disjunction

Two second chromosome, EMS-induced, meiotic mutants which cause an increase in second chromosome nondisjunction are described. The first mutant is recessive and causes an increase in second chromosome nondisjunction in both males and females. It causes no increase in nondisjunction of the sex chromosomes in either sex, nor of the third chromosome in females. No haplo-4-progeny were recovered from either sex. Thus, it appears that this mutant, which is localized to the second chromosome, affects only second chromosome disjunction and acts in both sexes.-The other mutant affects chromosome disjunction in males and has no effect in females. Nondisjunction occurs at the first meiotic division. Sex chromosome disjunction in the presence of this mutant is similar to that of sc(4)sc(8), with an excess of X and nullo-XY sperm relative to Y and XY sperm. In some lines, there is an excess of nullo-2 sperm relative to diplo-2 sperm, which appears to be regulated, in part, by the Y chromosome. A normal Y chromosome causes an increase in nullo-2 sperm, where B(s)Y does not. There is also a high correlation between second and sex chromosome nondisjunction. Nearly half of the second chromosome exceptions are also nondisjunctional for the sex chromosomes. Among the double exceptions, there is an excess of XY nullo-2 and nullo-XY diplo-2 gametes. Meiotic drive, chromosome loss and nonhomologous pairing are considered as possible explanations for the double exceptions.     

Increased rate of nondisjunction in sex cells derived from low-quality semen

The relationship between chromosomal nondisjunction and semen quality was studied in two groups of males who differ highly in their semen quality: 12 individuals with low-quality semen caused by varicocele, and 8 subjects with high-quality semen, selected from sperm donors for in vitro fertilization. Chromosomal nondisjunction was inferred from the rate of disomy found in mature sperm cells. To determine the rate of disomy, we applied fluorescence in situ hybridization using satellite-specific probes for chromosomes 1, 15, 18, X and Y. In sperm cells of males with low-quality semen, the mean rate of disomy for each of the autosomes and of hetero-disomy for the sex chromosomes (XY) was significantly higher than that observed in the high-quality semen samples: more than 15-fold higher for chromosomes 1 and 15, and 7-fold higher for chromosomes 18 and XY. Yet, the homo-disomy rate for each of the sex chromosomes (XX and YY) was almost the same in both types of semen. The large discrepancy between the low- and high-quality semen in the rate of sex chromosome hetero-disomy versus the similar rate of homo-disomy strongly suggests that the abnormal chromosomal segregation in meiocytes of males with low-quality semen resulted from chromosomal nondisjunction at the first meiotic division. The results indicate that men showing poor semen quality are at an increased risk for meiotic nondisjunction, similar to women at the end of their reproductive years. Received: 30 June 1997 / Accepted: 17 September 1997     

A comparative study of retrotransposons in the centromeric regions of A and B chromosomes of maize

Bacterial Artificial Chromosomes (BACs) derived from the B chromosome, based on homology with the B specific sequence, were subcloned and sequenced. Analysis of DNA sequence data indicated the presence of 23 common retroelements, as well as novel sequences of B chromosome origin. Generally, where the same retrotransposon type was observed in both A and B chromosomes, there were more copies per unit of sequence in the B centromeric region (the major site of B repeat) than in the A centromere, except for Huck-1. Based on previous estimates of the age of the major burst of transposition into the maize genome, the oldest retrotransposons (Ji-6 and Tekay, approximately 5.0 and 5.2 million years ago, respectively) were found in the B centromere region only, while the next two oldest (Huck-1 and Opie-1) were found in both the A and B sequences. Phylogenetic analysis of Opie retroelements from both A and B centromeres indicated that some of the B Opie centromeric sequences share a more recent common ancestor with A Opie retroelements than they do with other B Opie centromeric sequences. These results imply that the supernumerary maize B chromosome has coexisted with the A chromosomes during that period of transposition. They also support the hypothesis that the B chromosome had its origins from A chromosome elements, or that alternative origins, such as being donated to the maize genome in a wide species cross, preceded six million years ago, because the spectrum of retrotransposons in the two chromosomes is quite similar.     

Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction.

While it is known that all chromosomes are susceptible to meiotic nondisjunction, it is not clear whether all chromosomes display the same frequency of nondisjunction. By use of multicolor FISH and chromosome-specific probes, the frequency of disomy in human sperm was determined for chromosomes 1, 2, 4, 9, 12, 15, 16, 18, 20, and 21, and the sex chromosomes. A minimum of 10,000 sperm nuclei were scored from each of five healthy, chromosomally normal donors for every chromosome studied, giving a total of 418,931 sperm nuclei. The mean frequencies of disomy obtained were 0.09% for chromosome 1; 0.08% for chromosome 2; 0.11% for chromosome 4; 0.14% for chromosome 9; 0.16% for chromosome 12; 0.11% for chromosomes 15, 16, and 18; 0.12% for chromosome 20; 0.29% for chromosome 21; and 0.43% for the sex chromosomes. Data for chromosomes 1, 12, 15, and 18, and the sex chromosomes have been published elsewhere. When the mean frequencies of disomy were compared, the sex chromosomes and chromosome 21 had significantly higher frequencies of disomy than that of any other autosome studied. These results corroborate the pooled data obtained from human sperm karyotypes and suggest that the sex chromosome bivalent and the chromosome 21 bivalent are more susceptible to nondisjunction during spermatogenesis. From these findings, theories proposed to explain the variable incidence of nondisjunction can be supported or discarded as improbable.     

Analysis of Nondisjunction Induced by the R-X1 Deficiency during Microsporogenesis in Zea Mays L

The r-X1 deficiency in maize induces nondisjunction at the second mitotic division during embryo sac formation. However, it was not known if this deficiency also induces nondisjunction during the microspore divisions. Microsporogenesis in plants lacking or containing this deficiency was compared using two approaches. First, chromosome numbers were determined in generative nuclei. Many (8.3%) of the generative nuclei in r-X1-containing plants were aneuploid; however, those from control plants were all haploid. Thus, this deficiency induces nondisjunction during the first microspore division. Second, nucleoli were analyzed in microspores. The only nucleolar organizing region in maize is on chromosome 6. If chromosome 6 underwent nondisjunction during the first microspore division, one nucleus in binucleate microspores would contain no nucleolus and the other would contain two nucleoli (or one nucleolus if the nucleoli fused). Only one (0.03%) microspore of this type was observed in control plants while 1.12% were found in r-X1-containing plants. Thus, the r-X1 deficiency induces nondisjunction of chromosome 6 during the first microspore division. However, both of the sperm nuclei in trinucleate microspores contained one nucleolus in r-X1-containing and control plants; thus, this deficiency does not induce nondisjunction of chromosome 6 (and presumably other chromosomes) during the second microspore division.     

A procedure for localizing genetic factors controlling mitotic nondisjunction in the B chromosome of maize

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis at rates as high as 98% (Roman, 1948; Carlson, 1969a). Nondis-junction is controlled by at least two separable regions on the B chromosome (Roman, 1949; Longley, 1956; Carlson, 1969b; Ward, 1972). A procedure for identifying and localizing the chromosomal sites required for nondisjunction is reported here. A translocation between the B and chromosome 9 (TB-9b) was utilized. Plants carrying TB-9b were screened for mutants of nondisjunction, i.e. translocations in which nondisjunction does not occur. Two such translocations were identified in a small screening. While the mutant translocations have not been analyzed in pachytene, they are most likely deletions or rearrangements of regions on the B chromosome vital to nondisjunction. Diminutive and rearranged B chromosomes are known to arise spontaneously in small populations (Randolph, 1941; Longley, 1956). — Also reported here are the nondisjunctional properties of the B⁹ isochromosome (Carlson, 1970) and several telocentric (or subtelocentric) derivatives of this chromosome. Some derivatives of the isochromosome are virtually incapable of nondisjunction, and should provide information on the role of the centromere in nondisjunction.     

Molecular evidence for transcription of genes on a B chromosome in Crepis capillaris

Dispensable, supernumerary (B) chromosomes are found in diverse eukaryotic species. The origin and genetic consequences of B chromosomes have been the subjects of speculation for more than a century. Until now, there has been no molecular evidence that B chromosome DNA is transcribed and there is no unequivocal evidence as to their origin. B chromosomes are considered to be genetically inert although they appear to cause a variety of phenotypic effects. We report that members of one of two ribosomal RNA gene families that are confined to the B chromosomes of a plant, Crepis capillaris, are transcribed--thus providing the first molecular evidence of gene activity on B chromosomes. Sequence analysis of part of the A and B chromosome rRNA genes, together with comparisons with related species, indicates that the B chromosome rRNA genes originate from the A chromosome.