Cytogenetical studies in the bridge cross Allium cepa× (A. fistulosum×A. roylei)

  Allium fistulosum harbours a number of desirable agronomical traits for the breeding of onions. However exploitation of A. fistulosum for onion breeding via direct sexual hybridization is problematic. Therefore, we examined if a bridge cross, using A. roylei as a bridging species, might provide an alternative. By means of genomic in situ hybridization (GISH) we showed that each of the three parental genomes can be distinguished from the others in interspecific hybrids, suggesting that these genomes contain sufficiently different repetitive DNA families. We succeeded in carrying out multi-colour GISH to metaphase spreads of a first-generation bridge-cross individual [A. cepa× (A. fistulosum×A. roylei], which is composed of three parental genomes. Recombination between the genomes of A. fistulosum and A. roylei took place to a large extent: 7 recombined chromosomes were observed, and it could be shown that the proximal regions of the recombined A. fistulosum/A. roylei chromosomes belonged to the former, whereas the distal parts belonged to the latter. The high percentage of bound bivalent arms in metaphase I of pollen mother cells of a fertile bridge-cross individual suggests the introgression of A. fistulosum genes, mediated by A. roylei, into the genome of A. cepa. However, the presence of univalents reflects decreased pairing and recombination between the three genomes. Pollen fertility and pollen-tube growth of the first-generation bridge-cross individual seem to be sufficient to produce a second generation bridge-cross (A. cepa×first-generation bridge cross) progeny. Received: 27 May 1997 / Accepted: 30 June 1997     

Bulb-type onion introgressants posessing Allium fistulosum L. genes recovered from interspecific hybrid backcrosses between A. cepa L. and A. fistulosum L.

Allium fistulosum possesses a number of traits which would be desirable in A. cepa. Thus far, no commercial A. cepa cultivars have been released which harbor Allium fistulosum traits. F₁BC₃ populations were generated for this study by backcrossing A. cepa to A. cepa×A. fistulosum hybrids. The F₁BC₃ plants were evaluated for plant morphology, floral characters, male- sterile cytoplasm, soluble solids and pungency, and isozymes. Overall growth habit and floral characters of the F₁BC₃ plants were much like A. cepa. We report here the recovery of recombinant, bulbing, and fertile A. cepa-type onions that exhibit A. fistulosum isozyme alleles and morphological markers. Recombination between A. cepa and A. fistulosum genomes was achieved using the introgression strategy of backcrossing A. fistulosum into A. cepa, thereby ameliorating the nuclear cytoplasmic barriers that occurred in previous less successful introgression attempts when plants were not in A. cepa cytoplasm. We believe this report to be the first demonstration of onion introgressants that are like A. cepa in appearance, are male- and female-fertile, and possess A. fistulosum genes. Received: 29 May 1999 / Accepted 22 June 1999     

Recombinant chromosomes of advanced backcross plants between Allium cepa L. and A. fistulosum L. revealed by in situ hybridization

Cytological analysis of (Allium cepa L.×Allium fistulosum L.)×A. cepa L. F₁BC₃ plants revealed most plants were diploid with 16 chromosomes. Karyotypes of these plants showed recombinant chromosomes. Fluorescence and genomic in situ hybridization patterns of interspecific F₁ hybrid and F₁BC₃ plants revealed A. fistulosum chromosomes or chromosomal segments. A highly repetitive 376-bp DNA sequence and genomic DNA of A. fistulosum revealed similar telomeric hybridization sites when hybridized onto A. fistulosum chromosomes. Cytogenetic evidence showed that A. fistulosum DNA has recombined into the A. cepa genome. Received: 20 October 1999 / Accepted: 11 November 1999     

Chromosomal location of rDNA in Allium: in situ hybridization using biotin- and fluorescein-labelled probe

Summary A biotin- and fluorescein-labelled probe of Helianthus argophyllus has been used to map specific repeated rDNA sequences by in situ hybridization on mitotic chromosomes of Alliwn cepa, Allium fistulosum, a diploid interspecific (Allium fistulosum x Allium cepa) F₁ hybrid, and a triploid interspecific (2 x = A. cepa, 1 x = A. fistulosum) shallot. Hybridization sites were restricted to satellited and smallest pairs of chromosomes in both A. cepa and A. fistulosum. The number, size, and position of the hybridization sites distinguish homologous chromosomes and identify the individual chromosomes carrying the nucleolus organizing region (NOR) at the secondary constriction, as well as the individual chromosomes carrying an additional NOR. This in situ hybridization technique is the first reported in a plant species and offers new cytogenetic markers in Allium.     

Evidence for nuclear-cytoplasmic incompatibility between Allium fistulosum and A. cepa

An F₂ population (Allium fistulosum x A. cepa) of 20plants, 10 BC₁,[(A. fistulosum x A. cepa) x A. cepa], and 50 BC₂ plants, [(A. fistulosum x A. cepa) x A. cepa] x A. cepa were studied cytogenetically and characterized for four isozyme alleles plus various morphological characteristics. All of the progenies were in A. fistulosum (the bunching onion) cytoplasm. In the F₂ population we observed non-random chromosomal and allelic segregation, suppression of bulb onion allelic expression, and abnormalities in mitosis and meiosis. Most BC₂ plants resembled A. cepa (the bulbing onion) morphologically, but anthers, filaments, pistils, and petals were abnormal. Only 3 plants, and these were most nearly like the F₁ hybrid morphologically, produced any seeds.The data and observations support the hypothesis of nuclear-cytoplasmic incompatibility interactions between the bunching and bulb onion species.Use of trade names does not imply endorsement of the products named nor criticism of similar ones not named. This research was supported by the New Mexico Agricultural Experiment Station.     

GISH study of advanced generation of the interspecific hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew

Genomic in situ hybridization (GISH) was used for a chromosomal composition study of the later generations of interspecific hybrids between A. cepa L. and A. fistulosum L., which are relatively resistant to downy mildew (peronosporosis). GISH revealed that F₂ hybrids, which did not produce seeds, were triploids (2n = 3x = 24) with 24 chromosomes and possessed in their complements 16 chromosomes of A. fistulosum L. and eight chromosomes of A. cepa L. or eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L. The advanced F₅ hybrid, which produced few seeds, was amphidiploid with 32 chromosomes. BC₁F₅ hybrid was triploid with eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L., which did not produce seeds. BC₂ (BC₁F₅) plant was amphidiploid that possessed 4 recombinant chromosomes and produced few seeds. GISH results point to 2n-gametes formation in macro- and microsporogenesis of the hybrids. The mechanism of 2n-gametes formation and the possibility of apomixes events in the backcrossing progeny are discussed.     

Introgression of Allium fistulosum L. into Allium cepa L.: cytogenetic evidence

Summary A diploid Allium cepa plant was recovered from the backcross of an interspecific triploid (2 x A. cepa + 1 x A. fistulosum) to an A. cepa diploid which exhibited both A. cepa and A. fistulosum Adh-1 alleles. Cytogenetic analyses revealed a recombinant sub-telocentric chromosome. The ADH-1 locus is believed to be on the long arm of the sub-telocentric A. fistulosum chromosome 5. Meiosis of the triploid progenitor gives strong evidence that recombination occurred. A. fistulosum chromosome 8 has been substituted for A. cepa chromosome 1.Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-275     

The chromosomal locations of enzyme-coding genes Adh-1 and Pgm-1 in Allium fistulosum L.

Summary The chromosomal locations of two enzymecoding genes were investigated using allele dosage effects in two Allium cepa alien addition lines possessing different A. fistulosum chromosomes as the trisomes. Adh-1 has been assigned to the A. fistulosum sub-telocentric chromosome 5, while Pgm-1 is on the A. fistulosum submetacentric chromosome 4. Karyotype data of a shortday cultivar of A. cepa, Temprana, A. fistulosum Ishikura Long White, and the relevant chromosomes of the two trisomies are presented.Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-224     

Chromosome characteristics and behavior differences in Allium fistulosum L., A. cepa L,their F1 hybrid,and selected backcross progeny

Mitotic and meiotic studies were performed on Allium fistulosum, A. cepa, their F₁ hybrid, and ten selected backcross (BC)₁ plants [(A. fistulosum x A. cepa) x (A. cepa)]. Each BC₁ plant had at least one A. cepa isozyme allele (Pgi, Idh, or Adh). Chromosome morphology and behavior differed among plants. Meiocytes were observed with one, two, or three bridges and/ or fragments, indicating at least three paracentric inversions between A. fistulosum and A. cepa. Unusual crossing over and multivalent associations suggest that the 5F subtelocentric chromosome of A. fistulosum is involved in at least one translocation. The number of bridges and fragments and multivalent associations varied between the F₁ hybrid and BC₁ progenies. The F₁ hybrid and all BC₁ progenies were either sterile or had very little seed set. Fertility was not restored in any of the selected BC₁ plants.The use of trade names does not imply endorsement of the products named nor criticism of similar ones not named. This research was supported by the New Mexico Agricultural Experiment Station     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.     

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.     

Meiosis in four trisomic and one double trisomic males of the newt Pleurodeles waltlii

In the newt Pleurodeles waltlii, meiosis was studied in four trisomic and one double trisomic males. Study of first prophase shows that trivalent frequencies and trivalent configurations are correlated with chromosome length; moreover, trivalent configurations indicate that long chromosomes have multiple points of initiation of synapsis whereas two points might be adequate to secure synapsis of short chromosomes. From the study of metaphase II it appears that the extra chromosomes segregate in half of the spermatocytes II. Some abnormal spermatocytes, resulting from nondisjunction of chromosomes at mitosis or at first division of meiosis, or from precocious division of chromosomes at first division of meiosis were observed. In the male double trisomic meiosis fails at anaphase of second division; this accounts for the sterility of the individual.     

Phylogenetic conclusions from Giemsa banding and NOR staining in top onions (Liliaceae)

The chromosomes of different strains of the top or tree onion, ofAllium cepa andA. fistulosum, as well as of cloned progenies from reciprocal crosses between these two taxa have been studied by application of Feulgen- or aceto carmine-, Giemsa- and silver staining. It was possible to differentiate between the satellite chromosomes and 2–4 other chromosome pairs ofA. cepa andA. fistulosum. The phylogenetic origin of the top onions [A. ×proliferum (Moench)Schrad.] from hybridization ofA. cepa andA. fistulosum is substantiated, taking into consideration the variability in size and position of satellites and of active NORs.     

Electrophoretic analysis of Allium alien addition lines

Summary Meiotic pairing in an interspecific triploid of Allium cepa and A. fistulosum, Delta Giant, exhibits preferential pairing between the two A. cepa genomes, leaving the A. fistulosum genome as univalents. Multivalent pairing involving A. fistulosum chromosomes occurs at a low level, allowing for recombination between the genomes. Ten trisomies were recovered from the backcross of Delta Giant x A. cepa cv., Temprana, representing a minimum of four of the eight possible alien addition lines. The alien addition lines possessed different A. fistulosum enzyme markers. Those markers, Adh-1, Idh-1 and Pgm-1 reside on different A. fistulosum chromosomes, whereas Pgi-1 and Idh-1 may be linked. Diploid, trisomic and hyperploid progeny were recovered that exhibited putative pink root resistance. The use of interspecific plants as a means to introgress A. fistulosum genes into A. cepa appears to be successful at both the trisomic and the diploid levels. If introgression can be accomplished using an interspecific triploid such as Delta Giant to generate fertile alien addition lines and subsequent fertile diploids, or if introgression can be accomplished directly at the diploid level, this will have accomplished gene flow that has not been possible at the interspecific diploid level.Journal article No. 1140, Agr. Expt. Stn. N.M. State Univ., Las Cruces, NM, USA     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

AFLP linkage group assignment to the chromosomes of Allium cepa L. via monosomic addition lines

Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei. Received: 16 April 1999 / Accepted: 13 August 1999     

RABL DISTRIBUTION OF INTERPHASE AND PROPHASE TELOMERES IN ALLIUM CEPA NOT ALTERED BY COLCHICINE AND/OR ULTRACENTRIFUGATION

Neither colchicine nor ultracentrifugation, singly or in sequence, significantly alters the normal Rabl distribution of interphase or prophase telomeres in root tip cells of Allium cepa L. The position of telomeres was determined by C-banding, which stains A. cepa chromosomes only at the telomeres. Centrifugation displaces mitotic figures toward one side of the cell, but otherwise their mitotic configurations are little changed. These light microscope results are interpreted to show that a) interphase and prophase telomeres are attached strongly to some component of the nuclear envelope; b) a colchicine-sensitive component apparently does not attach interphase and prophase telomeres to the nuclear envelope; and c) chromosomes at all stages of the cell cycle are attached to some structure, nuclear envelope, and/or spindle fibers.     

Comparison of the Giemsa C-Band karyotypes and the relationships of Allium cepa,A. fistulosum and A. galanthum

Giemsa C-band staining of somatic chromosomes in Allium cepa, A. cepa cv. Shallot, A. fistulosum and A. galanthum shows that these four taxa are characterised by all chromosomes having one band at each telomere, except chromosome arms with satellites, in which case the band is proximal to the nucleolar constriction. This similarity is correlated with common morphological characteristics and an ability of the three species to hybridize with one another, indicating that they have a close genetic relationship. The results indicate that Giemsa C-bands are evolutionarily conservative in this group and provide a useful criterion for determining relationships.