Cytological characterization of the tandem repetitive sequences and their methylation status in the Antirrhinum majus genome

Tandem repetitive sequences are DNA motifs common in the genomes of eukaryotic species and are often embedded in heterochromatic regions. In most eukaryotes, ribosomal genes, as well as centromeres and telomeres or subtelomeres, are associated with abundant tandem arrays of repetitive sequences and typically represent the final barriers to completion of whole-genome sequencing. The nature of these repeats makes it difficult to estimate their actual sizes. In this study, combining the two cytological techniques DNA fiber-FISH and pachytene chromosome FISH allowed us to characterize the tandem repeats distributed genome wide in Antirrhinum majus and identify four types of tandem repeats, 45S rDNA, 5S rDNA, CentA1, and CentA2, representing the major tandem repetitive components, which were estimated to have a total length of 18.50 Mb and account for 3.59% of the A. majus genome. FISH examination revealed that all the tandem repeats correspond to heterochromatic knobs along the pachytene chromosomes. Moreover, the methylation status of the tandem repeats was investigated in both somatic cells and pollen mother cells from anther tissues using an antibody against 5-methylcytosine combined with sequential FISH analyses. Our results showed that these repeats were hypomethylated in anther tissues, especially in the pollen mother cells at pachytene stage.     

Die Aurea-Sippen von Antirrhinum majus

Ohne Zusammenfassung     

Molecular analysis of paramutant plants of Antirrhinum majus and the involvement of transposable elements

Paramutation is observed when the Antirrhinum majus lines 44 and 53 are crossed. These two lines both have insertions at the nivea locus, which encodes chalcone synthase (chs). The allele niv-53 carries the transposable element Tam1 in the promoter region of the chs gene; niv-44 carries the element Tam2 within the gene. The Tam1 element has previously been extensively characterised. Here the Tam2 element is further characterised, and the arrangement of the nivea locus in paramutant plants is analysed. The complete sequence of Tam2, and that of a partial cDNA complementary to it, have been determined. The cDNA is probably transcribed from a different copy of Tam2 from that present at the nivea locus, and does not encode a functional protein. Genomic Southerns of F1 plants from the 53/44 cross show that no major rearrangements are consistently associated with paramutation at the nivea locus of A. majus. The isolation from a paramutant plant arising from a 53/44 cross of an allele (niv-4432) resulting from the excision of Tam2 is reported. The excision of Tam2 resulted in a 32 bp deletion of chs gene sequences. Plants homozygous for the new niv-4432 allele have white flowers and are still paramutagenic, demonstrating that Tam2 need not be present at the nivea locus for paramutation to occur. Different interactions between Tam1 and Tam2 are discussed, and a possible model for paramutation is presented.     

Gonenkonkurrenz bei Antirrhinum majus L.

Zusammenfassung Die Gonenkonkurrenz (Selektion in der Haplophase) wurde bei Antirrhinum majus anhand der Spaltung in der Nachkommenschaft von Heterozygoten cae/+^cae(Farbe des Schiundflecks) untersucht. Es sollte die Frage entschieden werden, ob diese Gonenkonkurrenz eine Wirkung des Locus caeca ist oder ob ein gametophytisch wirksamer Locus in derselben Koppelungsgruppe angenommen werden muß. Es lassen sich 3 Typen von Nachkommenschaften unterscheiden: Normale Mendel-Spaltung, statistisch gesicherter Überschuß von gelb bzw. blaß. Die Hypothese Die Wirkung geht vom Locus cae aus kann auf Grund dieser Daten abgelehnt werden. Es wird die Anwesenheit eines gametophytisch wirksamen Locus ga angenommen. Aus der Sippe fim del cae stammt ga ⁺, ga^–aus Sippe 50. Den Gameten mit dem Allel ga ⁺kommt eine größere Befruchtungswahrscheinlichkeit zu als denjenigen mit ga ^–. Die Gonenkonkurrenz ist sowohl in den Pollen als auch in den Eizellennachkommenschaften festzustellen. Entweder wirkt ein Genlocus in beiden Geschlechtern gleichartig, oder es sind in der gram- Koppelungsgruppe zwei eng gekoppelte Loci vorhanden, die gleichartig auf die Gonenkonkurrenz in je einem Geschlecht einwirken. Der Locus ga ist entweder zwischen cae und del oder distal von cae zu lokalisieren.

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Competition in Antirrhinum majus L.

Summary In Antirrhinum majus selection in the haplophase is investigated, using the segregation in the backcrossprogeny of heterozygotes cae/+^cae(color of petal-spot).An attempt was made to determine whether the competition depends on gene-action of the locus caeca itself or on a gametophytic gene located in the same chromosome. Three types of progenies can be distinguished: Normal mendelian segregation, or statistically significant deviations with a surplus of yellow spot or a surplus of light, nearly colorless spot respectively. The hypothesis of an action of the locus cae in the haplophase can be rejected on the basis of these data.The location of a gametophytic gene in the gram-chromosome is assumed. The allele ga ⁺ comes from the line fim del cae, the allele ga ^–from the standard line S 50. Gametes with the allele ga ⁺have a greater chance for fertilization than gametes with ga ^–. The competition is found in the progeny of pollen and of egg cells. Two explanations are possible: one gene, which acts in the haplophase irrespective of the sex, or two closely coupled loci, which act in a similar way in the haplophase of each sex respectively. The locus ga is located either between cae and del or distal from cae.

     

Transformation of Antirrhinum majus using Agrobacterium rhizogenes

Hairy roots were produced following the co-cultivation of Agrobacteriumrhizogenes cells with hypocotyls of five varieties of Antirrhinummajus. The use of a strain containing a binary plasmid withT-DNA bearing the ß-glucuronidase reporter gene resultedin the co-transformation of some root clones. Regeneration ofshoots from hairy roots occurred only with variety Golden Monarch.Regenerated plants, some of which were GUS-positive, exhibitedthe abnormal morphology common among hairy root regenerants;they were dwarfed, had an altered leaf shape, a poor root systemand were very delayed in their flowering. Attempts to allowsegregation of the two introduced T-DNAs during crossing ofprimary co-transformants with wild-type plants were not successfulsince all GUS-positive progeny possessed the abnormal morphology.However, ‘semi-dwarf’ plants with morphology muchmore similar to wild-type were produced by the vegetative propagationof selected side-shoots from the transformants. Key words: Antirrhinum, Agrobacterium rhizogenes, transformation, hairy roots     

Environmental control of flowering time in Antirrhinum majus

The effect of different environmental conditions on flowering time and the number of leaves produced before the first flower is formed has been investigated in Antirrhinum majus L. The effect of light quality has been tested by decreasing the red/far‐red ratio, generally resulting in a reduced flowering time and leaf number. Furthermore, it could be shown that photoperiod, temperature and light intensity are inversely correlated with flowering time and leaf number. However, lowering the temperature from 15 to 12°C resulted in a reduction of flowering time. This observation shows that Antirrhinum can be vernalised.
Using defined combinations of the four environmental factors we have been able to reduce flowering time to only 42 days or to delay flowering for at least 2 years. The results obtained allow an optimisation of the screening conditions for identifying flowering time mutants in Antirrhinum .     

Transmission of plastids by pollen in Antirrhinum majus

Summary Up to now Antirrhinum was classified as a typical example for a uniparentalmaternal inheritance of the plastids. However, the findings reported here prove that also the male gametophyte of Antirrhinum may occasionally transmit plastids into the egg. This conclusion is based on genetic experiments involving a form of the plastom mutant prasinizans which is described as gelbgrüne prasinizans. In contrast to all other plastid mutations known in Antirrhinum majus this mutant originated in Sippe 50 is completely viable. In plants containing plastids of this mutant type only, the mutant character is manifested during early growth stages. Cotyledons and first foliage leaves which are initially white or white yellow, slowly turn green and become indistinguishable from normal Sippe 50. Reciprocal crosses of green Sippe 50 with gelbgrüne prasinizans gave few variegated descendants; the others were exclusively plants identical with the maternal parent as far as leaf colour is concerned (Table). The variegated individuals cannot be gene mutants since selfing and crossing experiments showed non-mendelian inheritance. Furthermore it could be ruled out that in the cross Sippe 50 x gelbgrüne prasinizans the three variegated descendants represent spontaneous new plastom mutants because the pale tissue in these plants turned green in the same way as the paternal parent. Because of the typical greening of this mutant and since plastid mutations could be ruled out we have to conclude that plastids were transmitted by the pollen parent into the egg. There these plastids multiplied together with the maternal plastids giving rise to the chimeras after sorting-out of the two plastid types. This interpretation is supported by the observation of mixed cells in tissues where the leaf variegation is finely mosaiced. The results were possible only because the plastids of the pollen parent can be unequivocally recognised.     

Cytologische Untersuchungen an Antirrhinum majus mut. cancroidea

Zusammenfassung Die canc-Keimlinge sind diploid. Durch früh einsetzende, regellose Teilungsstörungen, Unterbleiben der Zellwandbildung, unvollständige oder fehlende Entwicklung der Kernwände entstehen mixoploide Gewebe wechselnder Polyploidstufen. Zellen und Kerne wachsen zu oft riesenhaften Gebilden heran. Auch in der älteren Pflanze finden diese Störungen ihren Fortgang.Die Gewebsentartung und Polyploidisierung sind also entwickelt, bevor irgendwelche Form- oder Größenänderungen der Chromosomen auftreten.Ein später einsetzender und bis zur Blütenbildung sich verstärkender Gestaltswechsel der Chromosomen wurde im wesentlichen unterhalb des Vegetationskegels in dem postembryonalen Sproßteil gefunden. Alle Veränderungen können in Kernen verschiedenster Valenz vor sich gehen, ihre Entwicklung ist in den Metaphasen am besten zu verfolgen.Die Metaphasechromosomen zeigen eine zunehmende Verkürzung und Verbreiterung und bilden späterhin einen Längsspalt aus. Die Aufteilung der Spalthälften erfolgt erst mit der Mitose, die Chromosomenzahl bleibt die gleiche. In der weiter herangewachsenen Pflanze können die Metaphasen das Aussehen der 1936 (b) beschriebenen Doppel chromosomen des prämeiotischen Archespors haben. Die Wachstumsphasen zeigen, daß diese Zweiergruppen endomitotische Abkömmlinge eines Chromosoms sind. Die innere Teilung wird aber hier unterbrochen, die Paare trennen sich erst in der Mitose, und es liegt weder eine Reduktion noch eine Verdoppelung der Chromosomenzahl vor. Eine auffallende Volumenzunahme der Chromosomen erfolgt in der Bildung von Vierergruppen. Ihre Wachstumsphasen können ebenfalls beobachtet werden. Wenn ihre Aufteilung in Zweiergruppen erst durch die mitotische Spindel erfolgt, so bleibt auch hier die Endomitose unvollständig und die Chromosomenzahl unverändert.Die Vierergruppen können aber in der Metaphase bereits in ihre Zweiergruppen aufgeteilt sein, und solche Chromosomen haben durch Vollendung der inneren Teilung ihre Zahl verdoppelt.Die Polyploidisierung auf dem Wege der inneren Teilung kann auch in anderer Form erfolgen: Metaphasechromosomen ganz normaler Gestalt liegen häufig in strenger oder bereits mehr oder weniger gelockerter Paarung. Die Endomitose ist auch hier zum Abschluß gelangt.Die Vorgänge sind wechselnd und sehr labil. Sie verlaufen häufig nicht einmal einheitlich innerhalb des gleichen Kerns.Die in Endomitose befindlichen Chromosomen sind in der frühen Prophase längsgespalten. Die Spalthälften sind nur zuerst umeinandergeschlungen. Manche Längshälften können bereits in dieser Phase ohne Bindung gepaart nebeneinander liegen. In anderen Fällen löst sich die Umschlingung erst in der späteren Prophase.Im prämeiotischen Archespor sind die endomitotischen Vorstufen in diesem Jahr nicht aufgetreten, ebensowenig waren sie im Cyclus der Meiosis zu beobachten. Daß sie aber auch hier vorkommen können, wurde aus früherem Material erwiesen.Es muß angenommen werden, daß das zur Endomitose führende Chromosomenwachstum bei den canc-Pflanzen weitgehend von Außenbedingungen (Licht und Wärme) abhängig ist.Die gesamten Erscheinungen sind der Ausdruck einer durch Radiunibestrahlung erzeugten, rezessiv mendelnden Mutation.     

Multiplication of Antirrhinum majus L. by shoot-tip culture

Nine varieties of Antirrhinum majus L. have been used in a study of in vitro multiplication of plants using shoot-tip culture. Acceptable multiplication rates were obtained in several media with only variety Victory showing significantly lower rates of shoot production. Wounded shoots of this variety produced callus in the absence of added auxin and some of this callus produced prolific roots.     

Regeneration of plants from Antirrhinum majus L. callus

Somaclone production in Antirrhinum majus plants by regeneration of plants from callus cultures has been achieved using three types of explant tissue. Regeneration from mature stem internode-derived callus was extremely poor. Callus derived from seedling shoot tips could be induced to form new shoots in six of seven cultivars tested. Regeneration was achieved in all seven cultivars when callus was produced from segments of hypocotyl and was most effective using agar-solidified medium containing 0.25 mgl^-1 naphthoxyacetic acid + 10% coconut milk. In this case, five of the cultivars produced shoots directly, one produced leaves from the petioles of which new shoots emerged, and one regenerated plants chiefly through the production of embryoids.