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.     

Production and characterization of intertribal somatic hybrids of Raphanus sativus and Brassica rapa with dye and medicinal plant Isatis indigotica

Intertribal somatic hybrids of Raphanus sativus (2n = 18, RR) and Brassica rapa spp. chinensis (2n = 20, AA) with the dye and medicinal plant Isatis indigotica (2n = 14, I I) were firstly obtained by polyethylene glycol-induced symmetric fusions of mesophyll protoplasts. One mature hybrid with R. sativus established in field had intermediate morphology but was totally sterile. It had the expected chromosome number (2n = 32, RRI I) and parental chromosomes were distinguished by genomic in situ hybridization (GISH) analysis, and these chromosomes were paired as 16 bivalents in pollen mother cells (PMCs) at diakinesis and mainly segregated equally as 16:16 at anaphase I (A I), but the meiotic disturbance in second division was obvious. Five mature hybrids with B. rapa established in field were morphologically intermediate but showed some differences in phenotypic traits and fertility, two were partially fertile. Cytological and GISH investigations revealed that these hybrids had 2n = 48 with AAIIII complement and their PMCs showed normal pairing of 24 bivalents and mainly equal segregation 24:24, but meiotic abnormalities of lagging chromosomes and micronuclei appeared frequently during second divisions. AFLP analysis showed that all of these hybrids had mainly the DNA banding pattern from the addition of two parents plus some alterations. Some hybrids should be used for the genetic improvement of crops and the dye and medicinal plant.     

Meiosis in a triploid hybrid of <Emphasis Type="Italic">Gossypium</Emphasis>: high frequency of secondary bipolar spindles at metaphase II

Studies on meiosis in pollen mother cells (PMCs) of a triploid interspecific hybrid (3x = 39 chromosomes, AAD) between tetraploid Gossypium hirsutum (4n = 2x = 52,AADD) and diploid G. arboreum (2n = 2x = 26,AA) are reported. During meiotic metaphase I, 13 AA bivalents and 13 D univalents are expected in the hybrid. However, only 28% of the PMCs had this expected configuration. The rest of the PMCs had between 8 and 12 bivalents and between 12 and 17 univalents. Univalents lagged at anaphase I, and at metaphase II one or a group of univalents remained scattered in the cytoplasm and failed to assemble at a single metaphase plate. Primary bipolar spindles organized around the bivalents and multivalents. In addition to the primary spindle, several secondary and smaller bipolar spindles organized themselves around individual univalents and groups of univalents. Almost all (97%) of the PMCs showed secondary spindles. Each spindle functioned independently and despite their multiple numbers in a cell, meiosis I proceeded normally, with polyad formation. These observations strongly support the view that in plant meiocytes bilateral kinetochore symmetry is not required for establishing a bipolar spindle and that single unpaired chromosomes can initiate and stabilize the formation of a functional bipolar spindle.     

GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus

New Brassica napus inbred lines with different petal colors and with canola quality and increased levels of oleic (∼70%, 10% higher than that of B. napus parent) and linoleic (28%) acids have been developed in the progenies of one B. napus cv. Oro × Orychophragmus violaceus F₅ hybrid plant (2n=31). Their genetic constituents were analyzed by using the methods of genomic in situ hybridization (GISH) and amplified fragments length polymorphism (AFLP). No intact chromosomes of O. violaceus origin were detected by GISH in their somatic cells of ovaries and root tips (2n=38) and pollen mother cells (PMCs) with normal chromosome pairing (19 bivalents) and segregation (19:19), though signals of variable sizes and intensities were located mainly at terminal and centromeric parts of some mitotic chromosomes and meiotic bivalents at diakinesis or chromosomes in anaphase I groups and one large patch of chromatin was intensively labeled and separated spatially in some telophase I nuclei and metaphase II PMCs. AFLP analysis revealed that substantial genomic changes have occurred in these lines and O. violaceus–specific bands, deleted bands in ‘Oro’ and novel bands for two parents were detected. The possible mechanisms for these results were discussed.     

Cytology of higher polysomics in Coix gigantea (Poaceae): Pentasomy and hexasomy

Among a natural population of Coix gigantea a pentasomic (2n+3; 2n=23) and a hexasomic (2n+4; 2n=24) were isolated and studied cytologically for the first time. The extra homologous chromosomes in the pentasomic plant did not disturb the behaviour of the rest of the bivalents through meiosis except for the rare occurrence of a few univalents. The majority of PMCs at diakinesis showed nine bivalents and a pentavalent. The various anaphase segregations led to the formation of sub- and super-haploid male gametes with from n–1 (n=9) to n+4 (n=14) chromosomal constitutions. The four extra homologous chromosomes in the hexasomic plant, however, disturbed meiosis leading to sticky chromosomal configurations and sterility. At diakinesis, the majority of the PMCs showed nine bivalents and a hexavalent.The origin of these higher polysomics is considered to be through fusion of male and female super-haploid gametes produced by the trisomics that occurred in large numbers in the population. The occurrence and dominance of nullisomics together with other aneuploids in the diploid population of C. gigantea make it quite certain that the 2n=20 form of the species is at a tetraploid level.     

Segregation of the amphitelically attached univalent X chromosome in the spittlebug <Emphasis Type="Italic">Philaenus spumarius</Emphasis>

In meiosis I, homologous chromosomes combine to form bivalents, which align on the metaphase plate. Homologous chromosomes then separate in anaphase I. Univalent sex chromosomes, on the other hand, are unable to segregate in the same way as homologous chromosomes of bivalents due to their lack of a homologous pairing partner in meiosis I. Here, we studied univalent segregation in a Hemipteran insect: the spittlebug Philaenus spumarius. We determined the chromosome number and sex determination mechanism in our population of P. spumarius and showed that, in male meiosis I, there is a univalent X chromosome. We discovered that the univalent X chromosome in primary spermatocytes forms an amphitelic attachment to the spindle and aligns on the metaphase plate with the autosomes. Interestingly, the X chromosome remains at spindle midzone long after the autosomes have separated. In late anaphase I, the X chromosome initiates movement towards one spindle pole. This movement appears to be correlated with a loss of microtubule connections between the kinetochore of one chromatid and its associated spindle pole.     

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.     

Co-segregation of sex chromosomes in the male black widow spider <Emphasis Type="Italic">Latrodectus mactans</Emphasis> (Araneae,Theridiidae)

During meiosis I, homologous chromosomes join together to form bivalents. Through trial and error, bivalents achieve stable bipolar orientations (attachments) on the spindle that eventually allow the segregation of homologous chromosomes to opposite poles. Bipolar orientations are stable through tension generated by poleward forces to opposite poles. Unipolar orientations lack tension and are stereotypically not stable. The behavior of sex chromosomes during meiosis I in the male black widow spider Latrodectus mactans (Araneae, Theridiidae) challenges the principles governing such a scenario. We found that male L. mactans has two distinct X chromosomes, X₁ and X₂. The X chromosomes join together to form a connection that is present in prometaphase I but is lost during metaphase I, before the autosomes disjoin at anaphase I. We found that both X chromosomes form stable unipolar orientations to the same pole that assure their co-segregation at anaphase I. Using micromanipulation, immunofluorescence microscopy, and electron microscopy, we studied this unusual chromosome behavior to explain how it may fit the current dogma of chromosome distribution during cell division.     

Meiosis in Drosophila melanogaster

Individual bivalents or chromosomes have been identified in Drosophila melanogaster spermatocytes at metaphase I, anaphase I, metaphase II and anaphase II in electron micrographs of serial sections. Identification was based on a combination of chromosome volume analysis, bivalent topology, and kinetochore position. — Kinetochore microtubule numbers have been obtained for the identified chromosomes at all four meiotic stages. Average numbers in D. melanogaster are relatively low compared to reported numbers of other higher eukaryotes. There are no differences in kinetochore microtubule numbers within a stage despite a large (approximately tenfold) difference in chromosome volume between the largest and the smallest chromosome. A comparison between the two meiotic metaphases (metaphase I and metaphase II) reveals that metaphase I kinetochores possess twice as many microtubules as metaphase II kinetochores. — Other microtubules in addition to those that end on or penetrate the kinetochore are found in the vicinity of the kinetochore. These microtubules penetrate the chromosome rather than the kinetochore proper and are more numerous at metaphase I than at the other division stages.     

东乡野生稻与栽培稻正反交种间杂种F1的雄配子发生与发育

该文以东乡野生稻与栽培稻(美国光壳稻P1003)的正反交种间杂种F₁(正交为光壳稻P1003×东乡野生稻; 反交为东乡野生稻×光壳稻P1003)为试材, 研究其各个减数分裂时期的染色体行为特征、染色体交换重组及雄配子发育特点。结果表明: 正反交杂种F₁花粉母细胞细胞核减数分裂的同步性较高, 细胞质为连续型胞质分裂类型。在细胞核分裂的过程中, 核仁在前期I到中期I逐渐消失。染色体在前期I到中期I逐渐收缩, 变得更加清晰可见。在终变期和中期I, 90.54%以上的花粉母细胞能形成12个二价体(含少数棒状二价体和8字型二价体), 部分花粉母细胞(正交9.46%, 反交7.65%)出现少量的单价体、三价体和四价体。后期I观察到1.27%–1.35%的花粉母细胞含有1至数条落后染色体。最终有92.6%–94.8%的小孢子能发育成形态正常、染色能力强的成熟花粉粒。另外, 在正交杂种F₁的粗线期中存在2个核仁, 而反交杂种F₁及其双亲的粗线期只有1个核仁。这些研究结果可为作物品种改良、种质资源创新以及种间亲缘关系研究提供细胞学证据。     

Chromosomal Behavior during Meiosis in the Progeny of Triticum timopheevii × Hexaploid Wild Oat

The meiotic behavior of pollen mother cells (PMCs) of the F₂ and F₃ progeny from Triticum timopheevii × hexaploid wild oat was investigated by cytological analysis and sequential C-banding-genomic in situ hybridization (GISH) in the present study. A cytological analysis showed that the chromosome numbers of the F₂ and F₃ progeny ranged from 28 to 41. A large number of univalents, lagging chromosomes, chromosome bridges and micronuclei were found at the metaphase I, anaphase I, anaphase II and tetrad stages in the F₂ and F₃ progeny. The averages of univalents were 3.50 and 2.73 per cell, and those of lagging chromosomes were 3.37 and 1.87 in the F₂ and F₃ progeny, respectively. The PMC meiotic indices of the F₂ and F₃ progeny were 12.22 and 20.34, respectively, indicating considerable genetic instability. A sequential C-banding-GISH analysis revealed that some chromosomes and fragments from the hexaploid wild oat were detected at metaphase I and anaphase I in the progeny, showing that the progeny were of true intergeneric hybrid origin. The alien chromosomes 6A, 7A, 3C and 2D were lost during transmission from F₂ to F₃. In addition, partial T. timopheevii chromosomes appeared in the form of univalents or lagging chromosomes, which might result from large genome differences between the parents, and the wild oat chromosome introgression interfered with the wheat homologues’ normally pairing.     

An original meiotic mutation in Paspalum regnellii

 Cytogenetic studies carried out over a period of 2 consecutive years on a native Brazilian accession of Paspalum regnellii (2n=40) revealed a meiotic mutation that has not been previously reported for any other species. Among 13 inflorescences investigated during the first collection year, three presented anomalous meiotic behavior starting from metaphase I. At the beginning of this phase, the chromosomes occupied the entire equatorial plate in a membrane-to-membrane arrangement, and the spindle fibers, which were clearly visible, did not converge towards the poles. Degeneration of spindle fibers occurred at the end of metaphase I. Chromosome segregation did not occur and the bivalents were left scattered at random in the cytoplasm. Remnants of chromosome fibers could be seen close to the centromere during this stage. The bivalents gave origin to micronuclei in telophase I, with extremely wide variations in number and size among cells. With the absence of spindle formation during meiosis II, metaphase and anaphase II were not observed. Second cytokinesis occurred in prophase II cells after the occurrence of first cytokinesis. The final product of meiosis was completely abnormal, with a predominance of polyads with microspores of different sizes that resulted in abortive pollen grains. In the affected inflorescences, all microsporocytes presented this anomaly, which caused total sterility. Received: 27 March 1997 / Revision accepted: 7 July 1997     

Morphological, yield, cytological and molecular characterization of a bread wheat X tritordeum F1 hybrid

The morphological, yield, cytological and molecular characteristics of bread wheat X tritordeum F₁ hybrids (2n =6x = 42; AABBDH^ch) and their parents were analysed. Morphologically, these hybrids resembled the wheat parent. They were slightly bigger than both parents, had more spikelets per spike, and tillered more profusely. The hybrids are self-fertile but a reduction of average values of yield parameters was observed. For the cytological approach we used a double-target fluorescencein situ hybridization performed with total genomic DNA fromHordeum chilense L. and the ribosomal sequence pTa71. This technique allowed us to confirm the hybrid nature and to analyse chromosome pairing in this material. Our results showed that the expected complete homologous pairing (14 bivalents plus 14 univalents) was only observed in 9.59% of the pollen mother cells (PMCs) analysed. Some PMCs presented autosyndetic pairing of Hch and A, B or D chromosomes. The average number of univalents was higher in the wheat genome (6.8) than in the Hch genome (5.4). The maximum number of univalents per PMC was 20. We only observed wheat multivalents (one per PMC) but the frequency of trivalents (0.08) was higher than that of quadrivalents (0.058). We amplified 50 RAPD bands polymorphic between the F₁ hybrid and one of its parents, and 31 ISSR polymorphic bands. Both sets of markers proved to be reliable for DNA fingerprinting. The complementary use of morphological and yield analysis, molecular cytogenetic techniques and molecular markers allowed a more accurate evaluation and characterization of the hybrids analysed here.     

Holocentric chromosome behaviour inCuscuta (Cuscutaceae)

The present study demonstrates thatCuscuta babylonica Choisy has holocentric chromosomes. Evidence for this phenomenon comes from three different observations. (1) Mitosis: During metaphase and anaphase the sister-chromatids are situated parallel to the equatorial plane with no sign of localized kinetochore activity. (2) Inverted meiosis in microsporocytes. (3) X-rayed microsporocytes, in which the numerous chromosome fragments do not show any lagging or formation of micronuclei. We assume that only one out of the three subgenera inCuscuta, namely subg.Cuscuta, has holocentric chromosomes, while the two other subgenera have monocentric chromosomes.     

Cytogenetic Studies on Five Intermediate Forms of Progeny of Triticum aestivum Agropyron glaucum

A stuty has been made of the chromosome number of root-tip cells and the chro- mosome configuration of PMCs in five intermediate forms of progeny of Triticum aestivum × Agropyron glaucum. The somatic cells in each of these intermediate forms have 28 pairs of chromosomes (2n=56) and the PMCs at diakinesis or metaphase Ⅰ of meiosis have 28 bivalents. The process of meiosis in most PMCs is regular. Lagging chromosomes and fragments at anaphase Ⅰ or telophase Ⅰ were observed only in few PWCs. According to our results, all the five intermediate forms of progeny of T. aestivum × Ag. glaucum are allooctoploids. The application of these allooctoploids is also briefly discussed.     

Cytogenetic studies on microsporogenesis and male gametophyte development in autotriploid cucumber (Cucumis sativus L.): implication for fertility and production of trisomics

To understand the correlation between chromosomes behavior and fertility in autotriploid cucumber (Cucumis sativus L.), microsporogenesis in pollen mother cells (PMCs) and male gametophyte development were studied using improved staining and chromosome preparation techniques. Meanwhile, for more efficient selection of trisomics from the progeny of autotriploid-diploid crosses, fertilization rates of ovules from reciprocal crosses were counted to observe the transfer rate of gametes in the autotriploid cucumber. Variable chromosome configurations, e.g. multivalents, quadrivalents, trivalents, bivalents and univalents were observed in the most PMCs of the autotriploids at metaphase I. Chromosome lagging and bridges at anaphase in both meiotic divisions resulted from irregular chromosome separation and asynchronization was frequently observed as well, which led to formation of micronuclei and inviable gametes. The frequency of normal PMCs in autotriploids at the stage of tetrad was only 40.6%. Among those normal microspores, most of them (91.2%) could develop into normal gametophytes with 2 cells and 3 germ pores. Stainability and germination rate of pollen grains were only 18.8 and 13.5%, respectively. However, chromosomes separated to form gametes with 8 chromosomes at anaphase I, suggesting a possible method for the production of primary trisomics from the progeny of autotriploid-diploid crosses. Fruit set of 3n × 2n and 2n × 3n were 80 and 70%, respectively. It obtained an average of 6.2 plump seeds per fruit in 3n × 2n, while 4.9 in 2n × 3n crosses. Transfer rates of gametes through the gastrula or the pollen in autotriploids were 13.4 and 10.4%, respectively. Some aneuploid gametes (n + 1 = 8, n + 2 = 9) also have capability of setting seed and sexual reproduction besides normal gametes containing whole chromosome sets (n = 7, 2n = 14). Further, some primary trisomic plants were selected from the progeny of autotriploid-diploid crosses. Based on the results obtained we suggest that abnormal meiosis in PMCs was the cytogenetic reason for low fertility of autotriploid cucumber pollen. 3n × 2n cross was more efficient for selecting primary trisomic plants in cucumber.     

THE QUESTION OF POST-REDUCTION IN SPHAGNUM

The 19 spatially distinct chromosomal units at first meiotic metaphase in sporophytically diploid species of Sphagnum have usually been considered to be bivalents, but one investigator (Sorsa, 1956) has interpreted them as chromosomes from dissociated bivalents and meiosis as post-reductional. The present studies on diploid S. squarrosum (Pers.) Crome establish the chromosome number on the basis of the following evidence: there are in addition to m-chromosomes, 19 pairs of chromosomes in early prophase, 19 bivalents at diakinesis, 19 chromosomes in each of the two sets at second metaphase, 19 daughter chromosomes in each of the four sets at late second anaphase, and 19 chromosomes in gametophytic mitoses. The 19 bodies at first meiotic metaphase in diploid species are true bivalents in loose secondary association, which has led to their erroneous interpretation as chromosomes of dissociated bivalents. The gametic chromosome number in sporophytically diploid Sphagnum is therefore, without doubt, n = 19, and this evidence negates the claim for post-reduction in Sphagnum.     

First evidence of sex chromosome pre-reduction in male meiosis in the Miridae bugs (Heteroptera)

The karyotype and male meiosis of Macrolophus costalis Fieber (Insecta, Heteroptera, Miridae) were studied using C-banding, AgNOR-banding and DNA sequence specific fluorochrome staining. The chromosome formula of the species is 2n = 28(24+X1X2X3Y). Male meiotic prophase is characterized by a prominent condensation stage. At this stage, two sex chromosomes, "X" and Y are positively heteropycnotic and always appeared together, while in autosomal bivalents homologous chromosomes were aligned side by side along their entire length, that is, meiosis is achiasmatic. At metaphase I, "X" and Y form a pseudobivalent and orient to the opposite poles. At early anaphase I, the "X" chromosome disintegrates into three separate small chromosomes, X1, X2, and X3. Hence both the autosomes and sex chromosomes segregate reductionally in the first anaphase, and separate equationally in the second anaphase. This is the first evidence of sex chromosome pre-reduction in the family Miridae. Data on C-heterochromatin distribution and its composition in the chromosomes of this species are discussed.     

Somatic association of telocentric chromosomes carrying homologous centromeres in common wheat

Summary Measurements of distances between telocentric chromosomes, either homologous or representing the opposite arms of a metacentric chromosome (complementary telocentrics), were made at metaphase in root tip cells of common wheat carrying two homologous pairs of complementary telocentrics of chromosome 1 B or 6 B (double ditelosomic 1 B or 6 B). The aim was to elucidate the relative locations of the telocentric chromosomes within the cell. The data obtained strongly suggest that all four telocentrics of chromosome 1 B or 6 B are spacially and simultaneously co-associated. In plants carrying two complementary (6 B ^S and 6 B ^L) and a non-related (5 B ^L) telocentric, only the complementary chromosomes were found to be somatically associated. It is thought, therefore, that the somatic association of chromosomes may involve more than two chromosomes in the same association and, since complementary telocentrics are as much associated as homologous, that the homology between centromeres (probably the only homologous region that exists between complementary telocentrics) is a very important condition for somatic association of chromosomes. The spacial arrangement of chromosomes was studied at anaphase and prophase and the polar orientation of chromosomes at prophase was found to resemble anaphase orientation. This was taken as good evidence for the maintenance of the chromosome arrangement — the Rabl orientation — and of the peripheral location of the centromere and its association with the nuclear membrane. Within this general arrangement homologous telocentric chromosomes were frequently seen to have their centromeres associated or directed towards each other. The role of the centromere in somatic association as a spindle fibre attachment and chromosome binder is discussed. It is suggested that for non-homologous chromosomes to become associated in root tips, the only requirement needed should be the homology of centromeres such as exists between complementary telocentrics, or, as a possible alternative, common repeated sequences of DNA molecules around the centromere region.Dedicated to Professor Dr. Marcus M. Rhoades on his 70th birthday.     

Asynchronous meiotic rhythm as the cause of selective chromosome elimination in an interspecific <Emphasis Type="Italic">Brachiaria</Emphasis> hybrid

This paper reports the occurrence of chromosome elimination during microsporogenesis in an interspecific hybrid between a sexual diploid accession (SEX) of Brachiaria ruziziensis (2n=2x=18) and an apomictic tetraploid accession (APO) of B. brizantha (2n=4x=36). Meiosis was very abnormal in the triploid hybrid (2n=3x=27); we observed a distinct asynchrony from metaphase I to the end of meiosis. The APO and the SEX genomes did not show the same meiotic rhythm. When the former, with nine bivalents, was in metaphase I, the nine SEX univalents were not yet aligned; when the latter reached the plate, the APO genome was already in anaphase. In subsequent stages, the APO genome had reached the poles while the SEX was undergoing sister-chromatid segregation. As the SEX genome always remained temporally behind, it gave rise to one extra-nucleus in each pole. In the second division, the behavior was the same but anaphase II did not occur for the SEX genome, and only one extra-nucleus was observed in each cell in telophase II. Chromosome elimination for the SEX genome ranged from partial to total. The importance of these findings with respect to Brachiaria breeding programmes is discussed.