Centromere orientation,reorientation, and segregation in an interchange quadrivalent during anther development in pearl millet

Prometaphase I orientation, reorientation and anaphase I segregational behaviour of a chain-forming interchange quadrivalent involving one of the long chromosomes and the long arm of the seventh (nucleolar) chromosome was studied during anther development in pearl millet. The data obtained from 34 anthers showed that by early prometaphase I about 90% of the bivalents have attained stable bipolar orientation but about 48% of the quadrivalents are mal-oriented. There seems to be an interaction between bivalents and quadrivalents during mal-orientation and reorientation. The mal-oriented bivalents reoriented before the quadrivalents. For quadrivalent mal-orientation four types, 4/0, 3/1, 2/1/1/1 and 2/2 (adjacent 1), were distinguished in addition to the regular types, adjacent 2 and alternate. Based on their potential to reorient, the order of the mal-oriented quadrivalent types was 4/0 > 3/1 > 2/1/1; 2/2 led to anaphase I disjunction as for an adjacent 1 segregation. The data from 36 anthers at anaphase I showed alternate segregation of chromosomes in nearly 50% of pollen mother cells (PMCs) up to a developmental index of about 65. In late anthers about 35% PMCs showed alternate segregation. This suggests that the PMCs that reached metaphase I later had more adjacent 2 orientations since mal-oriented configurations delay meiotic development, and implies preferential reorientation behaviour of the maloriented quadrivalent types.     

Spontaneous structural rearrangements in Solanum phureja Juz. et Buk. 2. Meiotic behaviour and identification of interchange chromosomes using primary trisomics.

Meiosis was studied in two diploid (2n = 2x = 24) siblings of Solanum phureja Juz. et Buk. and in 11 disomic and 2 trisomic descendants. The diploid siblings carry the same heterozygous interchange and either one or two inversions. The frequency of quadrivalents at diakinesis/metaphase I in these clones was 0.56 and 0.62 per pollen mother cell. In two plants from the first inbred generation (I1) this frequency was about the same but in some other I1 plants and a full sib the frequency was substantially lower, varying from 0.00 to 0.16. Most quadrivalents, 78-83%, were rings. A variety of quadrivalent configurations at diakinesis and metaphase I was observed, giving rise to balanced and unbalanced gametes. The absence of ring quadrivalents in trisomic descendants of one of the siblings implied that tertiary trisomics or primaries being homozygous for the interchange were present in the I1 generation. Regular chromosome distribution (12-12) at anaphase I occurred in 46.5 and 73.2% of the pollen mother cells studied in the two original clones. Irregularities, such as 11-13 distribution, lagging chromosomes, and a bridge and fragment, were detected on average in 2.7, 3.3, and 32.5%, respectively, of the anaphase I cells analysed. In hybrids from crosses between 6 primary trisomics as females with the interchange heterozygote, the involvement in the interchange of chromosomes 3 and 12 was clearly demonstrated.     

A quadrivalent studied in living and fixed grasshopper spermatocytes

A single specimen of the grasshopper Melanoplus differentialis was discovered which was heterozygous for an interchange involving two of the largest chromosomes of the complement. The resulting quadrivalent occurred as a ring-of-four, chain-of-four, and, rarely, as two heteromorphic bivalents. Metaphase orientation and anaphase disjunction of some of these quadrivalents were recorded in living cells. The chain-of-four was stable in the configuration in which two pairs of kinetochores were oriented to each pole. The configuration in which three pairs of kinetochores were oriented to one pole and one pair to the other pole was also stable, but was shifted off the equator toward the pole to which three pairs of kinetochores were oriented. Given the nature of this quadrivalent the unusual stability of the 31 configuration is expected from current hypotheses of chromosome orientation and reorientation.     

Metaphase I orientation of chain-forming interchange quadrivalents: a theoretical consideration

The orientation behavior of chain forming interchange quadrivalents at metaphase I was studied in three interchange heterozygotes of pearl millet [Pennisetum americanum (L.) Leeke] which involve chromosomes 1, 3, 6 and 7 in various combinations. Of these, two combinations predominantly produced rings and the third was a chain-forming type. The chain quadrivalents derived from the two ring-forming interchanges, as well as the chain quadrivalent generated by the third interchange, all showed one adjacent orientation at metaphase I (adjacent-1 or -2, depending upon the formation or failure of chiasmata and their positions in the different segments of the pachytene cross). Homologous centromere co-orientation leading to adjacent-1 and alternate-1 occurs following chiasma failure in the noncentric arms of the pachytene cross, and nonhomologous centromere co-orientation leading to adjacent-2 and alternate-2 occurs following chiasma failure in the centric arms of the pachytene cross. Thus, it has been proposed that, unlike in ring quadrivalents, a specific chain quadrivalent will have only homologous or nonhomologous centromere co-orientations at metaphase I.     

Interchange quadrivalents and chromosome order at meiotic metaphase I in Briza L. (Gramineae)

In interchange heterozygotes of Briza humilis and B. media the interchange quadrivalent is shown to be preferentially positioned in flattened, lateral spreads at metaphase I. The positioning of the interchange quadrivalents is different in the two species but in both the frequency of alternate or adjacent orientation is different for different positions on the metaphase plate. B chromosomes in B. humilis are found to alter the positioning of the quadrivalent and the B chromosomes themselves are also found to show a nonrandom distribution on the metaphase plate.     

Position and orientation in the metaphase equator of an interchange quadrivalent of hybrid rye

The position and orientation of an interchange quadrivalent in flattened lateral views of metaphase I were studied in pollen mother cells of hybrid rye. Five quadrivalent types showed three positional distributions in the equator, these distributions having elements both similar to and very different from those found previously for an interchange quadrivalent of Allium triquetrum.     

Centromeric dots in crane-fly spermatocytes: meiotic maturation and malorientation

During the first meiotic division in crane-fly spermatocytes, the two homologs of a metaphase bivalent each bear two sister kinetochores oriented toward the same pole. We have previously reported treatments that increase the percentage of metaphase bivalents in which one or both homologs have bipolar malorientations: kinetochore microtubules] extending from a homolog toward both poles. The maloriented homologs lag at anaphase. Treatments that induce this behavior include: (a) recoverey from exposure to low temperatures or Colcemid or Nocodazole concentrations that prevent spindle formation but allow nuclear membrane breakdown, and (b) exposure to 6° C, a temperature that permits spindle assembly but slows progression through meiosis. Giemsa staining methods reveal two 0.5 m diameter dots at the centromeric region of each metaphase homolog; these often are more separated in maloriented homologs. This investigation was undertaken to assess whether this separation precedes the establishment of bipolar malorientation, and hence may be a cause of it, or is only a consequence of forces resulting from bipolar malorientation. Analysis showed that, in untreated cells, the average center-to-center distance between sister centromeric dots increases during the course of meiosis I. After the above-mentioned treatments, center-to-center distances similar to those normally seen in untreated half-bivalents at anaphase I were seen in bivalents, both after and before nuclear membrane breakdown. Longer exposure to temperatures that arrested meiosis increased the degree of dot separation. Based on our data, we conclude that normal orientation during the first meiotic division is aided by the close apposition of centromeric dots, and that a time-dependent maturation occurs causing centromeric dots to separate for the second meiotic division and facilitating orientation of sister kinetochores to opposite poles. If centromeric maturation occurs either prior to or during early stages of the first meiotic division, then it may contribute to persisting bipolar malorientation.     

Study of a translocation in tetraploid rye

Colchicine induced tetraploids (4x=28) from diploidSecale cereale heterozygous for a translocation showed a strong tendency of non-preferential pairing for the interchanged chromosomes. The normal chromosomes associated in configurations up to quadrivalents, and the translocation complex formed multivalents up to octavalents. Most of the interchanged chromosome associations were characterized by their heteromorphic nature. The percentage of the chromosomes in the interchange complex forming multivalent associations was far higher than that of the remaining twenty chromosomes. Abnormalities were observed at anaphase I and II in the pollen mother cells. The tetraploids appeared to be completely sterile. It is suggested that the high frequency of multivalent formation may be explained on the basis that the interchange might have involved a region of localized chiasmata. The absence of polyploidy in the genusSecale as against its widespread occurrence in the related grass genera may be accounted for, in part, on the basis of non-preferential pairing.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

In autotetraploids, chromosome pairing may be in the form of quadrivalents or bivalent pairs. Whether or not the quadrivalents are maintained until first meiotic metaphase depends on the formation of chiasmata. The relative frequencies of M I configurations thus contain information both on pairing and on chiasma formation. With distal chiasma localisation six configurations can be recognised and their relative frequencies determined: ring quadrivalents, chain quadrivalents, trivalents (with univalent), ring bivalents, open (rod) bivalents, univalent pairs. These represent five degrees of freedom permitting five parameters to be estimated: the frequency (f) of quadrivalent pairing; the frequencies of chiasmate association of the two ends (arms in metacentrics), a′, b′, after quadrivalent pairing, and a, b after bivalent pairing. — The appropriate formulae have been derived and applied to observations on Tradescantia virginiana (4n=24) which has pronounced distal chiasma localisation. Slight modifications make the model applicable to autotetraploids with interstitial in addition to distal chiasmata. In T. virginiana, chromosome pairing appeared to be random between homologues (65.8% quadrivalent pairing; 55.4% observed at M I). After quadrivalent pairing chiasmate association is frequent in the “average long” arm (95.0%) and much less so in the other arm (60.5%). This is attributed to partner exchange. After bivalent pairing chiasma frequencies are still different for the two arms (93.8% and 83.5% association respectively) but much less pronounced. Various complications are discussed.     

Chromosomal interchange inEleutherine plicata Herb

Chromosome behaviour at metaphase I and anaphase I of meiosis inEleutherine plicata Herb. (2n=14) is studied. Cells with chromosome associations comprising an association of four long chromosomes, in addition to five bivalents were observed more frequently than those with seven bivalents. it is concluded that the ring of four is due to a segmental interchange between the two long non-homologous chromosome pairs. The ring of four at anaphase I showed delayed disjunction, bridge formation and irregular separation of chromosomes in a number of cells while the behaviour of the other bivalents was normal.     

The spatial arrangement ofAllium triquetrum chain quadrivalents at metaphase I as viewed by confocal microscopy

We examined the three-dimensional arrangement of bivalents and, in particular, a chain of four chromosomes (chain quadrivalent) in the metaphase I spindle of pollen mother cells ofAllium triquetrum by confocal microscopy. Firstly, we show by optical sectioning and three-dimensional image reconstruction that the cooriented pairs of centromeres of all seven bivalents lie virtually parallel to each other in the metaphase I spindle, parallel to the long axis of the spindle. Secondly, we like-wise show that the four centromeres of the chain quadrivalent are aligned in the metaphase I spindle in, essentially, atwo-dimensional array, not in a three-dimensional array, as proposed by some other authors. This two-dimensionality has its basis, we argue, in the principle that poleward directed spindle forces minimise centromere-to-pole distances and therefore align pairs of centromeres connected to opposite poles most axially (vertically) in the spindle. These distances are minimised for the quadrivalent as a whole only when it lies in two dimensions, i.e. in aplane parallel to the spindle axis.     

Bivalent orientation and behavior in crane-fly spermatocytes recovering from cold exposure

At metaphase in crane-fly primary spermatocytes, the two sister kinetochores at the centromere of each homologue in a bivalent normally are adjacent and face the same pole; one homologue has all its kinetochore microtubules (kMTs) extending toward one pole and its partner has all its kMTs extending toward the opposite pole. In contrast, during recovery from exposure to 2 degrees C, one or both homologues in many metaphase bivalents had bipolar malorientations: all kMTs of one kinetochore extended toward one pole and some or all those of its sister extended toward the other. Metaphase sister kinetochores that had most of their kMTs extending toward the same pole were adjacent, and those with most extending toward opposite poles were separated from each other. Distances between homologous centromeres were similar to those in properly oriented bivalents. Maloriented bivalents were tilted relative to the spindle axis, and analysis of living cells showed that tilted configurations were rare during prometaphase in untreated cells but frequently arose in cold-recovering cells as initial configurations, then persisted through metaphase. This was in contrast to unipolar configurations of bivalents (configurations suggesting orientation of both homologous centromeres toward the same pole), which always reoriented shortly after the configuration arose. We conclude that in cold-recovering cells, bipolar malorientations are more stable than unipolar malorientations, and the orientation process is affected such that bipolar malorientations arise in bivalents upon initial interaction with the spindle and persist through metaphase.     

Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes

Exposing crane fly larvae to 6 degrees C or returning them to 22 degrees C after exposure to 6, 2, or 0.2 degrees C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J. R. LaFountain, 1982, Chromosoma, 85:619-631). We report here documentation that lagging autosomes in cold-treated and cold- recovering cells are maloriented. During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles: one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and non-lagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold- recovering cells was similar to that observed in untreated anaphase cells. Giemsa-stained centromeric dots of sister chromatids were contiguous in non-laggards and separated in laggards at anaphase. We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.     

Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other

To test the "traction fiber" model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are "smart," meaning they embody a position-sensitive mechanism that controls their activity.     

CYTOLOGICAL ANALYSIS OF ANTHURIUM ANDRAEANUM (ARACEAE), ITS RELATED TAXA AND THEIR HYBRIDS

Karyotypes and meiotic configurations of Anthurium andraeanum and closely related taxa were analyzed. The karyotypes of A. andraeanum, A. caperatum, A. formosum, A. kamemotoanum, A. lindenianum, A. roseospadix, A. cf. sanctifidense, A. subsignatum, A. garagaranum, and an unidentified Anthurium sp. commonly consisted of four large metacentric or submetacentric chromosomes, two fairly large acrocentric chromosomes, two satellite chromosomes, and 22 smaller chromosomes. Variation in the karyotypes of A. nymphaeifolium and A. ochranthum suggested chromosomal rearrangement in the genus. All taxa showed 15 pairs of chromosomes at prometaphase I of meiosis in pollen mother cells. Four large chromosomes appeared as ring bivalents, and the rest of the chromosomes appeared as either ring or rod bivalents. Regular bivalent formation at prometaphase I of meiosis in pollen mother cells of species hybrids suggested close genomic relationships among parental taxa. On the other hand, reduction of pollen fertility estimated by pollen stainability in those hybrids suggested genetic divergence of species.     

Cytological studies on meiosis and male gametophyte development in autotetraploid cucumber

With improved staining and chromosome preparation techniques, meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetraploid cucumber (Cucumis sativus L.) was studied to understand the correlation between chromosomes behaviour and fertility. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalents were observed in most PMCs at metaphase I. Lagging chromosomes were frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyads, triads, tetrads with micronuclei and polyads were observed at tetrad stage, and the frequencies of normal tetrad with four microcytes were only 55.4 %. The frequency of abnormal behaviour in each stage of meiosis was counted, and the average value was 37.2 %. The normal meiotic process could be accomplished to form the microspore tetrads via simultaneous cytokinesis. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages: single-nucleus early stage, single-nucleus late stage and 2-celled stage. The frequency of abnormalities was low during the process of male gametophyte development. The germination rate of pollen grains was 46.9 %. These results suggested that abnormal meiosis in PMCs was the reason for low pollen fertility in the autotetraploid cucumber.     

Meiosis in some species and cultivars ofCrocus (Iridaceae)

Meiosis is described in thirty-seven taxa ranging from low to high chromosome numbers. Most were regular and bivalent forming.C. flavus subsp.flavus was observed at first anaphase only and was highly irregular.C. speciosus had one or two possible interchange quadrivalents and theC. vernus aneuploid cultivars exhibited multivalents, bivalents and univalents together with some anaphase bridges and fragments. Chiasma frequency is determined here for the first time.     

CYTOLOGY AND REPRODUCTIVE CHARACTERISTICS IN PENNISETUM SETACEUM

A green ecotype of fountaingrass, Pennisetum setaceum (Forsk.) Chiov., was found to be triploid (3x = 27) with 9 II + 9 I at metaphase I. Meiotic behavior of the bivalents was normal, but the univalents lagged and divided precociously at anaphase I. The univalent halves lagged at anaphase II and were distributed at random. Micronuclei were common, and stainable pollen was approximately 40%. The green type produced four progeny with 54 chromosomes. A purple strain of P. setaceum was found to be hexaploid (6x = 54) with varying chromosomal associations. Meiotic behavior was highly irregular and stainable pollen was almost zero. Megasporogenesis was initiated in both types, but the megaspores degenerated and nucellar and integument cells developed into four-nucleate, aposporous embryo sacs. Following self-pollination, the green type set approximately 10% seed and the purple type set 0.05% seed. Purple fountaingrass set as high as 18% seed following the application of pollen from Pennisetum ciliare (L.) Link (4x = 36), exemplifying a rare form of pseudogamy between two species. Improvement of P. setaceum through a standard breeding program appears unlikely.     

Studies on male and female meiosis in Indian Allium

Male meiosis in autotetraploid Allium tuberosum (4×=32) is fairly regular, keeping in view its cytological status, with 81 percent of the chromosomes associated in quadrivalents and trivalents. About 5% of the cells have 32 univalents. Anaphase segregation is slightly irregular. While 48% of the pollen mitoses show 16 chromosomes, 87% of the mature pollen is viable as indicated by carmine or iodine staining. — Megaspore mother cells have 64 chromosomes associated in 32 bivalents at metaphase I. Anaphase segregation is normal. In three out of 56 cells studied multivalents, bivalents and univalents are observed as in male meiosis. — It is concluded that the species reproduces by pseudogamous parthenogenesis made possible by meiotic modification. This modification is almost perfect and almost completely specific for female meiosis. Slight effects are observed in male meiosis.     

The distribution of male meiotic pairing sites on chromosome 2 of Drosophila melanogaster: meiotic pairing and segregation of 2-Y transpositions

The distribution of meiotic pairing sites on a Drosophila melanogaster autosome was studied by characterizing patterns of prophase pairing and anaphase segregation in males heterozygous for a number of 2-Y transpositions, collectively coveringall of chromosome arm 2R and one-fourth of chromosome arm 2L. It was found that all transpositions involving euchromatin from chromosome 2, even short stretches, increased the frequency of prophase I quadrivalents involving the sex and second chromosome bivalents above background levels. Quadrivalent frequencies were the same whether the males carried both elements of the transposition or just the Dp (2;Y) element along with two normal chromosome 2s, indicating that pairing is non-competitive. The frequency of quadrivalents was proportional to the size of the transposed region, suggesting that pairing sites are widely distributed on chromosome 2. Moreover, all but the smallest transpositions caused a detectable bias in the segregation ratio, in favor of alternate segregations, indicating that the prophase associations were effective in orienting centromeres to opposite poles. One transposition involving only heterochromatin of chromosome 2 had no effect on quadrivalent frequency, consistent with previous evidence that autosomal heterochromatin lacks meiotic pairing ability in males. One region at the base of chromosome arm 2L proved to be especially effective in stimulating quadrivalent formation and anaphase segregation, indicating the presence of a strong pairing site in this region. It is concluded that autosomal pairing in D. melanogaster males is based on general homology, despite the lack of homologous recombination.by A.C. Spradling