Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    On the evolution of azole resistance in Aspergillus fumigatus
    Zhang, J. - \ 2016
    Wageningen University. Promotor(en): Bas Zwaan; P.E. Verweij, co-promotor(en): Fons Debets; Sijmen Schoustra. - Wageningen : Wageningen University - ISBN 9789462578555 - 183
    aspergillus fumigatus - azoles - triazoles - aspergillosis - resistance - life cycle - asexual reproduction - sexual reproduction - experimental evolution - evolutionary genetics - agriculture - composting - medicine - aspergillus fumigatus - azolen - triazolen - aspergillose - weerstand - levenscyclus - ongeslachtelijke voortplanting - geslachtelijke voortplanting - experimentele evolutie - evolutionaire genetica - landbouw - compostering - geneeskunde

    During the last decade azole resistance has increasingly been reported in Aspergillus fumigatus, which is a fungal pathogen involved in the vast majority of invasive aspergillosis infections in humans, and is now a global public health concern. Antifungal azoles, especially triazoles, are the drugs of choice for medical treatment. However, this treatment is hampered by the emergence of multi-azole resistant A. fumigatus isolates, especially the highly resistant variants TR34/L98H and TR46 /Y121F/T289A. Therefore, to control this disease, it is essential to elucidate by what mechanisms resistance emerges, how resistance spreads and how resistant genotypes persist in environments without azoles. The presented thesis shows the relevance of the life cycle of A. fumigatus to the development of azole resistance and possible evolutionary routes that lead to it. The work highlights the importance of fungal biology and evolution towards understanding the development of azole resistance in fungi. We conclude that azole resistance in A. fumigatus is a consequence of selection pressure by azole in the environment on the genetic variation generated via various aspects in the A. fumigatus life cycle. This thesis also introduces an experimental evolution approach to study the dynamics and mechanisms of the evolution of azole resistance. In addition, we investigate what condition can lead an environment to be a possible hotspot for the development of resistance. Finally, we link this to the potential conditions under which resistance can emerge and spread in the lungs of humans and how this depends on the specific azole used.

    Control of Pig Reproduction IX
    Rodriguez-Martinez, H. ; Soede, N.M. ; Flowers, W.L. - \ 2013
    Leicestershire, United Kingdom : Context Products Ltd (Society of Reproduction and Fertility volume 68) - ISBN 9781899043484 - 345
    varkens - geslachtelijke voortplanting - gameten - embryo's - kunstmatige inseminatie - embryotransplantatie - zwangerschap - partus - pasgeborenen - biggen - overleving - biotechnologie - metabolomica - eiwitexpressieanalyse - kunstmatige selectie - pigs - sexual reproduction - gametes - embryos - artificial insemination - embryo transfer - pregnancy - parturition - neonates - piglets - survival - biotechnology - metabolomics - proteomics - artificial selection
    Snow shoes and sandals? : genetic aspects of heat stress sensitivity and sow reproduction
    Bloemhof, S. - \ 2013
    Wageningen University. Promotor(en): Johan van Arendonk; I. Misztal, co-promotor(en): E.F. Knol; Liesbeth van der Waaij. - S.l. : s.n. - ISBN 9789461735881 - 173
    zeugen - warmtestress - diergenetica - gevoeligheid - geslachtelijke voortplanting - voortplantingsvermogen - kritische temperatuur - hittetolerantie - selectief fokken - genetische correlatie - veredelingsprogramma's - varkensfokkerij - sows - heat stress - animal genetics - sensitivity - sexual reproduction - reproductive performance - critical temperature - heat tolerance - selective breeding - genetic correlation - breeding programmes - pig breeding

    Globally the average size of pig herds are increasing and amount of labour spent per sow / finisher pig is decreasing. These changes require sows which need less management interventions. In addition to easier manageable sows modern genotypes will also need to be more adaptable considering that global temperatures are expected to increase and pork production is partially moving to warmer climates. The end result is that commercial pigs nowadays will potentially face more heat stress challenges during their productive lives.

    In this thesis, a model was developed which was used to estimate upper critical temperatures for sows’ reproductive performance. Additionally the possibility to breed for reduced heat tolerance of sows was investigated. Therefore heritability for the random regression slope of farrowing rate against increasing temperature at day of insemination (= heat tolerance) and the genetic correlation between farrowing rate and heat tolerance was estimated.Commercial production pigs are crossbreds farmed all over the world. In contrast, selection is practiced mainly in temperate climates, in nucleus herds using purebred pigs. The success of genetic selection depends on how much genetic progress is realized in crossbred pigs. Within this thesis these genetic correlations for farrowing rate between purebreds and crossbreds were estimated.

    Sow productivity depends on a number of related traits, such as ovulation rate, the number of litters per sow per year, the number of weaned piglets per sow per year, and the length of productive live. Traditionally pig breeding programs have improved sow productivity by increasing number weaned piglets per sow per year. To improve herd-level litters per sow per year a new trait was proposed called problem free sow production by parity, which incorporates the traits interval weaning first insemination, non-return rate, farrowing rate, and selection for next parity. Heritability of problem free sow production and genetic correlations with other sow production traits were estimated.

    The main conclusion of this thesis was that it is possible to select for improved heat resistance in addition to improved commercial production levels in commercial pigs. However, genetic correlation between production in temperate and hot climates is high. This high correlation implies that, within-line, pigs with the best performance in a hot climate will be the best in temperate climate too. Most important for the success of a pig breeding program is to define appropriate breeding goals which are based on the environment(s) that market pigs are expected to perform in. The overall data collection for the genetic evaluation needs to be done in those specific environments and this will favour pigs which are able to produce over more than one specific environment.

    Reproduction in crabs: strategies, invasiveness and environmental influences thereon
    Brink, A.M. van den - \ 2013
    Wageningen University. Promotor(en): Han Lindeboom, co-promotor(en): Aad Smaal; C. McLay. - S.l. : s.n. - ISBN 9789461735232 - 164
    krabben (schaaldieren) - geslachtelijke voortplanting - geslachtsselectie - groei - voortplantingsvermogen - invasieve soorten - populatiedynamica - omgevingstemperatuur - milieufactoren - mariene ecologie - crabs - sexual reproduction - sexual selection - growth - reproductive performance - invasive species - population dynamics - environmental temperature - environmental factors - marine ecology

    This thesis provides insights into the interconnectedness of crab reproductive biology, the selective forces leading to their development, the possible links to invasiveness and the influences of environmental factors thereon. The empirical data collected and presented in this thesis can be used to compare different crab species and make predictions about the effect of climate change on their population dynamics and invasiveness.
    Two crab species in particular are examined in this thesis, Halicarcinus cookii and Hemigrapsus takanoi , which share similar size and habitat preferences, but have different reproductive strategies and statuses as endemic and invasive. The potential effects of increased water temperature due to climate change on the reproduction and eventual population changes in the species are investigated as well as the possible links reproduction and temperature have with the invasiveness of a species.
    Halicarcinus cookii is an endemic crab to the coasts of New Zealand and is not known anywhere else. The species shows determinate growth, hard shell mating, continuous brood production and ventral seminal receptacles. After the final moult the female produces numerous offspring limited only by sperm availability. With a hard shell the female also avoids mortality resulting from the regular vulnerable soft shell stage. Individuals mature over a range of sizes but do not continue growing after their pubertal moult. With such a terminal moult, brood size is limited by female size.

    Hemigrapsus takanoi is native to the north west Pacific, but has been introduced and is very successful in Europe. The species shows indeterminate growth, hard-shell mating, a defined breeding season and ventral seminal receptacles. With indeterminate growth they continue moulting and growing throughout their adult life. After their pubertal moult, these species can mate throughout the year and produce 2-3 broods between each moult. They are not limited in growth or regeneration of limbs and can safely hide from predators during the vulnerable soft-shell inter-moult period rather than mating which exposes them to predators.

    Despite their different reproductive strategies, broods of both species showed a similar reaction to increased water temperature in that the duration of development of the brood decreased as temperature increased. Extrapolating the results to a climate change scenario, it is suggested that with a temperature rise of 2°C H. cookii could produce one extra brood of over 1000 offspring per female life time, potentially leading to a 10-15% increase in fecundity and possible population growth. As H. takanoi does not show continuous brood production, predicting the effect of temperature rise is more difficult, but evidence suggests that fecundity is also likely to increase in this species with an increase in water temperature.

    Temperature increase may also lead to a change in invasiveness of a species. If areas currently below the optimum temperature for a species become warmer, it is possible that a species may spread to the new locations. Hemigrapsus takanoi may spread further north in Europe than it’s current distribution (assuming it is limited by temperature). Furthermore, if temperatures increase the rate of reproduction in a non-indigenous species, they may become more invasive in their present location.

    The colonisation of a new habitat will involve new interactions, such as predation and competition, with species not previously encountered. The interactions of the two invasive crab species H. takanoi and Hemigrapsus sanguineus with the native Carcinus maenas in the delta waters of SW Netherlands was also investigated in this thesis. Whereas C. maenas was the most common shore crab in these waters, its numbers have declined on the soft sediment substrates during the last 20 years. As the two invasive crab species were first recorded in the Dutch delta in 1999, they could not have initiated the decline of the native C. maenas. However, within a few years H. takanoi completely dominated the intertidal hard substrate environments; the same environments on which juvenile C. maenas depend. On soft sediment substrate the native and invasive crab species are presently more or less equally abundant. Nowadays H. takanoi appears to be a fierce interference competitor or predator for small C. maenas specimens by expelling them from their shelters. However, due to the habitat generalist nature of C. maenas, it is unlikely that the Hemigrapsus species will cause it’s local extinction. More likely is that they will learn to live together.

    The objective to provide new information about a rarely studied species (Halicarcinus cookii) was fulfilled in this thesis and the information can be used as bases for comparison for future research.

    The hypothesis that temperature has no effect on the reproductive rate of crabs was rejected as both study species showed similar increases in brood development rate with increased temperatures. This suggests that global temperature rises may increase the reproductive rate of wider crab populations.

    The hypothesis that the arrival, presence and effect of Hemigrapsus takanoi in the Dutch delta waters has had no effect on the native green crab Carcinus maenas was complicated by the fluctuations and the decrease in C. maenas numbers prior to the arrival of H. takanoi. It was concluded that while H. takanoi did not cause the initial decrease in the C. maenas population, it did take advantage of it and now dominates niches previously occupied by juvenile C. maenas where size dependent competition and/or predation on juvenile C. maenas occurs.

    Sexual selection in Fungi
    Nieuwenhuis, B.P.S. - \ 2012
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Duur Aanen. - S.l. : s.n. - ISBN 9789461733580 - 156
    schimmels - geslachtsselectie - genetica - geslachtelijke voortplanting - evolutionaire genetica - evolutie - fungi - sexual selection - genetics - sexual reproduction - evolutionary genetics - evolution

    Sexual selection is an important factor that drives evolution, in which fitness is increased, not by increasing survival or viability, but by acquiring more or better mates. Sexual selection favours traits that increase the ability of an individual to obtain more matings than other individuals that it is in competition with. For many sexually reproducing organisms, obtaining mates is an essential part of the lifecycle, sexual selection can therefore be very strong. A trait that leads to more matings can be selected, even if it strongly reduces other components of fitness, for instance predator escape. Often sexual selection leads to sex specific traits, which can become very extravagant. In animals and plants, it has been well established that this form of selection is an important evolutionary force, but it has not been considered for fungi. This thesis revolves around the idea that in this aspect, fungi are not fundamentally different from animals and plants and that also for species from this kingdom sexual selection influences evolution. Many fungi reproduce sexually and need to find a partner before reproduction can proceed. Furthermore, it is likely that not all individuals that benefit from mating can perform mating, hence a struggle for mate acquisition will occur.
    In my research I have investigated how likely it is that in fungi such struggles occur and which mechanisms might act during competitions. For these studies I used the mushroom forming basidiomycete fungus Schizophyllum commune as a model organism. I studied the potential for mate competition in natural populations, performed laboratory mating essays to test competition and preference, and experimentally tested if sexual selection can increase competitive ability.
    Sexual reproduction in fungi is highly regulated. Many molecular mechanisms are known that modulate each step, from meiosis to gamete production and from mate finding to gamete fusion. In most fungi these characteristics are regulated by genes located on the mating type locus or loci. These genes do not only regulate mating, but also define compatibility between the gametes: gametes with the same alleles at a mating type locus cannot fuse. Because of this double function, fungal mating types are potentially very important for sexual selection. Besides that mating types are a target for sexual selection because they affect traits that might increase competitive ability, since the mating types determine compatibility, they also define who competes with whom. Sexual selection describes how within one sex mating occurs for individuals of the other sex and is therefore always intra-sexual. Fungi do not have different sexes, but do have sex roles. Sexual selection will therefore act if there is competition for mating in the male or female role. Compatibility between sex roles is different from compatibility between mating types. The first is defined by the size of the mycelium, and the second by a genetic recognition mechanism. This difference is of importance to understand how sexual selection can act in fungi and is explained in Chapter 2.
    Mating in mushroom fungi occurs by reciprocal exchange of nuclei. In the female role nuclei from a compatible mate are incorporated into the haploid mycelium. These nuclei migrate though the mycelium until in each cell of the mycelium two haploid nuclei are present; it becomes a so called dikaryon. Only a dikaryon can produce mushrooms that produce spores. In the male role, a mycelium can donate nuclei to a haploid mycelium. A mycelium can thus be considered hermaphroditic. After fertilize, the dikaryon can still act as a nucleus donor, but not incorporate more nuclei – this type of mating is known as the Buller phenomenon. Also spores can act as male, but as they have no mycelium, not as a female. Due to the presence of more individuals that can mate in the male role than there are female mycelia (monokaryons), competition over fertilizations is expected.
    Competition can only occur when there are multiple individuals. Fungi are sessile organisms that can only meet other individuals when they are in the same locality. To test whether there is potential for sexual selection in nature, the number of individuals that meet each other needs to be defined. Not much knowledge on numbers of individuals is known, because mushroom fungi generally grow by mycelium expansion inside a substratum and each part of the mycelium can produce mushrooms. Therefore, all mushrooms on a tree can be one genetic individual, but it is also possible that each mushroom is a separate individual. We sampled 24, 12 and 24 mushrooms from the same substrate of three natural populations to analyse how mating occurred (Chapter 3). We determined the identity of the two different nuclei in each mushroom, as well as the mitochondria. Because mitochondria do not migrate during mating, they are specific for each female mycelium. We found that multiple genetic individuals (3, 3 and 8) are present in a small area, and that many matings must have occurred. Even though it is generally assumed that matings occur between two monokaryons, none such matings were found. The data suggest that mating in nature occurs between a monokaryon and a spore, or a monokaryon and a dikaryon.
    During a dikaryon-monokaryon (di-mon) mating only one of the two nucleus types from the dikaryon is successful in fertilizing the monokaryon. The nucleus type that is successful will likely increase its fitness considerably, as the entire female mycelium becomes colonized. Sexual selection is expected to select for nuclei that are better in performing this fertilization. Furthermore, during mating the receiving monokaryon meets two different nuclei, and might be able to choose between them. We performed crosses between 15 dikaryons and six different monokaryons to test if selection occurs, and whether selection occurs by male-male competition, or by female choice (Chapter 4). When confronting the same dikaryon with different monokaryon, in some of the cases the female mycelium decided which of the two nuclei won. In most cases however, the same nucleus always fertilized the monokaryon, irrespective of which monokaryon. This suggests that nuclei are able to either manipulate the monokaryon in incorporating them into the mycelium and not the other type, or that the nuclei of one type can directly suppress mating by the other nuclei in the dikaryon.
    Nuclei in a dikaryon have a strict way of cell division in which the different types divide in synchrony. Probably the two nuclei keep each other in check to assure this synchrony. Experiments in which the two nuclei in a dikaryon are separated into monokaryons suggest that the two nuclei suppress each other’s mitotic division, and that one of the two nuclei is better in suppression than the other. Consequently, after de-dikaryotisation more monokaryons of one type are recovered than of the other. We tested if this mechanism of suppression might be responsible for the dominant nuclei in the di-mon matings (Chapter 5). Separating the two nuclei confirmed earlier findings that always one of the two nuclei is dominant and that a hierarchy in dominance exists. This pecking order did not correspond with the results from the winner in the di-mon matings, which suggests that the mechanism of suppressed mitotic division is not responsible for dominance in di-mon matings. Nevertheless, we argue that the hypothesis that a link between the two mechanisms exists should not be completely written off. Because the interactions that take place during di-mon matings are very complex, the functioning of this mechanism might be obscured during mating.
    The observed variance in mating success described above might lead to sexual selection, however, it does not show that sexual selection actually led to traits that improve increased mate acquisition. To show that traits can evolve that increase fitness by higher mating success, we performed an evolution experiment (Chapter 6). An evolving population of nuclei was continuously mated with a non-evolving monokaryon. This setup selected for novel traits that increase competitive ability over matings. After 20 transfers, four out of twelve evolved lines had increased in competitive fitness and one line had decreased. Different fitness components were measured to investigate which traits had resulted in changed fitness. Fertilization success was mainly determined at the moment of fusion with and in initial migration into the receiving monokaryon. Two strains showed increased spores production, but this did not add to the increased fitness caused by fusion and initial migration. Little fitness change occurred during migration or in the dikaryon phase. We observed no clear trade-offs between the competitive ability of fertilizing in the male role, and female characteristics. This experiment showed that sexual selection can act in mushroom fungi.
    Sexual selection can also play a role in other groups of fungi than the mushroom forming fungi. So far this has not been considered, and little research has been done to show how mate competition might influence evolution. We reinterpreted the current knowledge on mating in fungi and assessed whether and when sexual selection might play a role (Chapter 7). Sexual selection is most likely to occur when sex roles can be observed during mating, as this can lead to skewed sex ratios. Also when there is large difference in quality between potential mates sexual selection might lead to evolution of choice. Directions are given where sexual selection is expected to function in fungal mating. Examples are given of how sexual selection might have led to for instance the evolution of micro-conidia in ascomycetes and pheromone redundancy in basidiomycetes. Furthermore, the existence of different sex roles in fungi, can lead to sexual conflict between the genomes derived from the paternal and the maternal gametes of which examples are given. The realization that sexual selection can also act in fungi gives great opportunity to test how universal general theories of sexual selection are in another important group of organisms. Additionally, because fungi are easy to manipulate, predictions on sexual selection can be tested experimentally using fungi.
    Mushroom forming fungi have a life history which differs from animals and plants. Sexual selection will therefore affect mushroom fungi in a different manner than it would animals and plants. In the general discussion of this thesis (Chapter 8) I will assess how mating influences fungal fitness, teasing apart the benefits and costs of mating in the male and female roles. I give directions for future research and discuss a setup to directly measure the effect of pheromones on female choice in mushroom fungi.
    There are still many unanswered fundamental questions about sexual selection. Adding knowledge from a third important kingdom can help increase the understanding of the principles that drive evolution by sexual selection. Furthermore, applying sexual selection theory to fungi might elucidate the functioning of the sometimes very complex mechanisms that have evolved for fungal mating.

    Aspects of sexual reproduction in Mycosphaerella species on wheat and barley : genetic studies on specificity, mapping, and fungicide resistance
    Ware, S.B. - \ 2006
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema; M.A. de Waard. - [S.l.] : S.n. - ISBN 9789085045274 - 190
    triticum aestivum - tarwe - hordeum vulgare - gerst - mycosphaerella graminicola - geslachtelijke voortplanting - plantenziekteverwekkende schimmels - septoria - gastheerspecificiteit - genetische kartering - resistentie tegen pesticiden - virulentie - pathogeniteit - ziekteresistentie - overleving - triticum aestivum - wheat - hordeum vulgare - barley - mycosphaerella graminicola - septoria - sexual reproduction - survival - plant pathogenic fungi - host specificity - virulence - pathogenicity - genetic mapping - pesticide resistance - disease resistance

    Mycosphaerella species are haploid ascomycetes that cause major economic losses in crops that include cereals, citrus fruits, and bananas, among others. Two organisms in this genus are Mycosphaerella graminicola (Fuckel) .I. Schröt (anamorph Sepioria tritici) and Septoriapasserinii. M graminicola is the causal agent of septoria tritici blotch of both bread wheat and durum wheat species, and S. passerinii causes septoria speck!ed 1eaf blotch of barley. M. graminicola is a heterothaliic fungus with a very active sexual cycle, while no sexual cycle has been reported for S. passerinii.

    This thesis inciudes studies on mating and genetics of both M. graminicola and S. passerinii. Chapter 1 gives an introduction to these pathogens and an overview of the research topics. In Chapter 2. we studied the possibility of in planta generation of sexual progeny of the fungal wheat pathogen M graminicola when one of the parents was avirulent on a resistant host. We found that avirulent isolates are able to survive and even increase in biomass after inoculation onto resistant wheat cultivars and can complete sexual cycles on resistant cultivars to yield viable ascospores as long as the other parent is virulent. To our knowledge, this is the first time such a phenomenon has been described, and the possibility to generate such crosses opened the door for studies in Chapters 3 and 4.

    Chapter 3 describes the construction of two high-density genetic linkage maps of M graminicola using Diversity Arrays Technology (DArT) and the integration of these into a core map with common markers due to a common parental isolate. One of the maps was constructed based on segregations of progeny of two bread wheat-derived isolates, IPO323 and IPO94269, and the other was constructed from segregations of progeny of IPO323 and the durum wheat-derived isolate IPO95052. In total, 1,144 markers made up the integrated core map. Analyses from this study revealed that progeny had translocations, diploid and partial diploid linkage groups, and loss of entire linkage groups.

    Although M. graminicola causes disease on both bread wheat and durum wheat, isolates within the population show clear distinctions in either virulence on bread wheat or on durum wheat (host specificity)- In Chapter 4, we studied the genetic basis of host specificity in M. graminicola using 163 progeny from crosses between the Dutch bread wheat-derived 1PO323 and the Algerian durum wheat-derived TPO95052. Phenotyping of progeny was performed on a set of seven differential cultivars, and progeny crossed on either bread wheat or durum wheat could infect cultivars of bread wheat, durum wheat, both, or neither. These results were used to map nine quantitative trait loci (QTLs) on seven linkage groups in the high-densiry genetic linkage map from Chapter 3. One of these loci was previously mapped for cultivar specificity of IPO323in bread wheat, and the same locus was now mapped for host specificity of IPO323 to durum wheat. Our results show that the reported host specificity is probably the result of combinations of a number of independently inherited avirulence factors.

    In addition to avirulence genes, fungi can inherit other traits for survival. One such heritable trait is a point mutation in the mitochondrial genome that conveys resistance to strobilurin fungicides. Chapter 5 describes a study on the inheritance of strobilurin resistance. Resistant and sensitive isolates of M. graminicola were crossed on wheat seedlings that were both untreated and preventively treated with various concentrations of azoxystrobin (Amistar™), and progeny were analyzed to determine the rate of inheritance of the aforementioned mutation. Preventive rates from 3.125-200% Amistar™ resulted in completely resistant progeny populations despite the fact that the segregation of nuclear genes confirmed regular meiotic behavior. We conclude that sensitive isolates overcome the disruption of mitochondrial respiration and participate in sexual reproduction even under high fungicide pressure and that fungicide stress induces or results in preferential mating in M. graminicola.

    The barley pathogen S. passerinii clusters closely to M. graminicola in phylogenetic studies based on ITS sequences, and a high degree of genetic variation among isolates is found in nature. However, no teleomorph has been reported for this S. passerinii, and hence, it was considered to be asexual. Nevertheless, mating type idiomorphs were recently detected and isolated. In Chapter 6, we studied the possibility of a Mycosphaerella teleomorph associated with S. passerinii. Isolates with opposite mating types were co-inoculated onto barley cultivars, and leaves were monitored for the discharge of ascospores. Characterization of a segregating population by both molecular and phenotypic analyses confirmed that we successfully generated the hitherto unknown Mycosphaerella teleomorph of S. passerinii.

    Finally, the results of this thesis are discussed in a broader perspective in Chapter 7 in relation to epidemiology, co-evolution, and durability of resistance in the wheat-M. graminicola pathosystem. The proven ability of avirulent isolates of M. graminicola to generate sexual progeny on resistant cultivars represents a new dynamic in population genetics that has not'previously been considered in epidemiology. Resu!ts from this thesis emphasize the complex ways in which the sexual cycle contributes to the overall success of M. graminicola on wheat.

    Controlled reproduction of penaeid shrimp: a contribution to its improvement
    Alfaro Montoya, J. - \ 2001
    Wageningen University. Promotor(en): E.A. Huisman; J. Komen. - S.l. : S.n. - ISBN 9789058084002 - 149
    aquacultuur - dierfysiologie - garnalen - penaeus - geslachtelijke voortplanting - gametogenese - schaal- en schelpdierenteelt - aquaculture - animal physiology - shrimps - penaeus - sexual reproduction - gametogenesis - shellfish culture

    This dissertation deals with controlled reproduction of penaeid shrimp. New knowledge about natural reproductive activity of Penaeus occidentalis in Gulf of Nicoya, Costa Rica, is presented. Since in vitro fertilization of open thelycum shrimp proved unsuccessful, a hypothesis is given to explain experimental results. In P. setiferus , the Male Reproductive Blackening Disease was studied, and bacterial infection was found to be associated with the male's condition. Production of spermatophores in captivity was explored in two species, P. stylirostris and P. vannamei . Adequate husbandry as well as successive ejaculation improved spermatophore quality. Deterioration of spermatophores was observed as part of a normal process for renewal in P. vannamei , without pathological implications. In order to further improve spermatophore quality, the injection of 17-alpha-methyltestosterone and 17-alpha-hydroxyprogesterone at 0.01 and 0.1 µg g -1 body weight was evaluated. 17-alpha-methyltestosterone significantly improved the quality of spermatophores, whereas 17-alpha-hydroxyprogesterone did not. Serotonin injection was evaluated as an alternative to female's eyestalk ablation for induction of ovarian maturation and spawning in P. vannamei . This neurotransmitter induced lower maturation and spawning with 3 doses of 50 µg g -1 body weight, than eyestalk ablation. In other to lay a basis for cryopreservation, penaeid embryos were evaluated in terms of their tolerance to cooling, cryoprotectants, and hypersaline solutions. T. byrdi morulae and advanced embryos (10 h) were tolerant to cooling at 10 °C, but were very sensitive to 0 °C. Embryos showed high tolerance to methanol and intermediate tolerance to dimethyl sulfoxide. Morulae were more resistant to hypersaline treatment at 55 ppt than advanced embryos.

    Non-homologous chromosome synapsis during mouse meiosis : consequences for male fertility and survival of progeny
    Peters, A.H.F.M. - \ 1997
    Agricultural University. Promotor(en): C. Heyting; P. de Boer. - S.l. : Peters - ISBN 9789054857761 - 182
    muridae - muizen - meiose - geslachtelijke voortplanting - parthenogenese - polyembryologie - vruchtbaarheid - overleving - levensvatbaarheid - interacties - milieu - uitsterven - stofverplaatsing - chromosoomtranslocatie - chromosomen - cytologie - histologie - muridae - mice - meiosis - sexual reproduction - parthenogenesis - polyembryony - fertility - survival - viability - interactions - environment - extinction - translocation - chromosome translocation - chromosomes - cytology - histology
    In the mouse, heterozygosity for several reciprocal and Robertsonian translocations is associated with impairment of chromosome synapsis and suppression of crossover formation in segments near the points of exchange during prophase of meiosis. This thesis describes the analysis of the consequences of the occurrence of non-homologous synapsis and/or suppression of meiotic crossover formation over many successive generations for male fertility and viability of the progeny.

    For studying chromosome synapsis, we modified a drying down technique which results in high yields of nuclei of all first meiotic prophase stages in both male and female from only small amounts of tissue (chapter 2). Preparations are suitable for synaptonemal complex (SC) analysis by normal light and electron microscopy (chapters 2, 3 and 7), for fluorescence immunocytochemistry and in situ hybridization (chapters 2, 8).

    In the study presented in chapter 3, we analysed the variation in male fertility of mice double heterozygous for two near identical reciprocal translocations T(1;13)70H and T(1;13)1Wa in relation to the synaptic behaviour of two differently sized heteromorphic bivalents during meiotic prophase. Male fertility rises when non-homologous synapsis in the small 1 13heteromorphic bivalent, leading to a "symmetrical" SC, is more frequent at the initial prophase stages. Based on the data presented, we favour the "unsaturated pairing site" model as the primary cause for male sterility.

    In T70H/T1Wa females not all heterologous synapsis within the small heteromorphic bivalent is effectuated during the early stages of meiosis; some is achieved lateron by the mechanism of "synaptic adjustment" (chapter 3). Each heteromorphic bivalent contains a copy of the chromosome 1 region between the T70H and T1Wa breakpoints which is about 10 cM in size (Δ1 segment). Although axial elements representing these Δ1 segments are seen to approach each other during early meiotic prophase stages, they never successfully constitute a synaptonemal complex in either sex (chapter 3). This agrees with the fact that in earlier cytogenetic studies quadrivalents were never seen at both male and female diakinesismetaphase 1.

    In chapter 7, we demonstrate that male fertility of the T70H/T1Wa mice is not only determined by the chromosomal constitution of the carrier but is additionally influenced by the pairing or synaptic history in previous meioses of especially the T70H and T1Wa short translocation chromosomes. Fertility of T70H/T1Wa males is more impaired after one or more successive transmissions of the T1Wa translocation chromosomes through a heteromorphic bivalent configuration, irrespective of the sex of the transmitting parent.

    Furthermore, we show that the introduction of the Robertsonian translocation Rb(l1.13)4Bnr into the T70H/T1Wa karyotype restores fertility of double heterozygous males by stimulating non-homologous synapsis of the small heteromorphic bivalent. We speculate that this Rb4Bnr effect is mediated by a prolongation of the early stages of meiotic prophase I.

    Successive female transmissions of the T1Wa translocation chromosomes in the presence of Rb4Bnr inititially resulted in an increase of the capacity for early meiotic nonhomologous synapsis within the small heteromorphic bivalent, leading to a restoration of fertility for the majority of carriers. Subsequently, a decrease of the capacity of the small heteromorphic bivalent to fully synapse was noticed, although a higher than original (F1) background level of male fertility remained.

    These variations in male fertility are most likely based on epigenetic variance, reflected as the capacity to engage into non-homologous synapsis early in male meiosis leading to a "symmetrical" SC, despite the different amounts of chromatin to accommodate.

    In chapter 4, the localization of several microsatellite markers and single copy genes relative to the T70H and T1Wa breakpoints, using quantitative PCR, quantitative Southern blotting and in situ hybridization, is described.

    In chapter 5, we investigated the level of suppression of meiotic recombination and impairment of chromosome synapsis in T70H heterozygotes in relation to the viability of the progeny. For T70H/+ females, the introgression of the D1Mit4, D1Mit20 and D1Mit122 microsatellite marker alleles positioned distal of the T70H breakpoint on the normal chromosome 1 into the 13 1T70H long translocation chromosome was suppressed in a distance dependent manner. This effect was more pronounced in T70H/+ females, additionally homozygous for Rb4Bnr. The delay in introgression was paralleled by a reduction of the frequency and extent of non-homologous synapsis in segments near the T70H breakpoints of the pachytene translocation multivalents in T70H/+ and Rb4BnT70H/Rb4Bnr+ males. The extend of non-homologous synapsis around the centre of the synaptic cross configuration in these males correlated with fluctuations in prenatal viability of segregating translocation homozygotes in crosses between (Rb4Bnr)T70H homozygous males and heterozygous females when meiotic drive at the female second meiotic division is excluded. The reduction in viability is explained by the gain of mutations resulting from incorrect processing of recombination intermediates which is due to non-homologous synapsis around the translocation breakpoints.

    In chapter 6, we analysed the consequences of the absence of crossing over for regions between the T70H and T1Wa breakpoints (Δ1 and Δ13 segments) of the Rb4BnrT1Wa translocation chromosomes, which have been transmitted for over 20 generations via heteromorphic bivalents in Rb4BnrT70H/Rb4BnrT1Wa females. Survival of heterozygous and homozygous carriers for these segments was taken as the phenotypic endpoint. The viability of progeny of crosses between Rb4BnrT70H homozygous males and Rb4BnrT70H/Rb4BnrT1Wa females, of which the latter principally produce 4 types of gametes, was estimated using a haplotype analysis of microsatellites in the Δ1 segment for genotyping (see chapter 4). We observed no differences in the pre- and postnatal survival rates of the double heterozygous and 13 1H, 13 1H, 1 13Wa 1 13H "duplication" progeny in which the Δ1 and Δ13 segments of the T1Wa translocation chromosomes had either no, an onegeneration or a multi-generation history of non-homologous synapsis in heteromorphic bivalents during previous female meioses. In addition, intercrossing of Rb4BnrT70H/Rb4BnrT1Wa double heterozygotes after genetic isolation of these Δ1 and Δ13 segments for 20 to 22 generations, showed that the viability of the Rb4BnrT1Wa homozygotes was not different from the Rb4BnrT70H homozygous and Rb4BnrT70H/Rb4BnrT1Wa karyotypes generated by this cross. Thus, exclusion of the Δ1 and Δ13 segments from meiotic crossing over within non-homologous synapsed heteromorphic bivalents during 20 to 25 successive generations does not result in an accumulation of recessive lethal mutations or an increased susceptibility for gaining dominant lethal mutations.

    For the D1Mit122 microsatellite used in offspring haplotyping a higher mutation frequency was observed after transmission through a double heterozygous than after transmission through a T70H homozygous karyotype (chapter 6). On the basis of the identity of the mutations, the ectopic pairing of the St2 gene copies (containing D1Mit122) during meiosis of T70H/T1Wa males (chapter 8) and the observation of ectopic homologous contacts of the Δ1 segments during the zygotene stage without SC formation (chapter 3), we speculate that these mutations are the result of ectopic homologous gene conversion events most likely occurring in the absence of a synaptonemal complex.

    The crossover suppressive influence of the Rb translocation on the Δ1 segment (chapter 5) enabled us to analyze the effects of introgression of genetic material from the Swiss +/+ stock into the translocation karyotypes. Introgression of "new" genetic material correlated with an increase in littersize of Rb4BnrT70H homozygotes (chapter 5), an improvement of the life expectancy of Δ1 duplication offspring from double heterozygous mothers (chapter 6) and a clear improvement of male fertility in double heterozygous and T70H homozygous males also carrying Rb4Bnr (chapter 7). These pleiotrophic findings are discussed in chapter 8 in terms of genetic versus epigenetic mechanisms of inheritance.

    Finally, when T1Wa was backcrossed for many generations to the Rb4BnrT70H/Rb4BnrT70H karyotype, essentially precluding genetic recombination in the Δ1 and Δ13 segments, or when T1Wa was combined with Rb4Bnr after many successive transmissions via alternating T1Wa heterozygotes and homozygotes, stable Rb4BnrT1Wa homozygous lines could not be bred (chapter 8). Especially female reproductive performance decreases after repeated male and female homologous meiosis. As non-homologous synapsis in the centre of the synaptic cross configuration in T1Wa/+ males is common too (unpublished results), more work into the genetic stability of chromosome segments, that have a history of hindered homologous interaction, is indicated (chapter 8).

    SCP1, a major protein component of synaptonemal complexes of the rat
    Meuwissen, R.L.J. - \ 1997
    Agricultural University. Promotor(en): C. Heyting. - S.l. : Meuwissen - ISBN 9789054856498 - 129
    meiose - eiwitten - polypeptiden - aminozuren - aminozuursequenties - ratten - chromosomen - cellen - geslachtelijke voortplanting - meiosis - proteins - polypeptides - amino acids - amino acid sequences - rats - chromosomes - cells - sexual reproduction

    Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes during meiotic prophase. SCs consist of two proteinaceous axes, one along each homologue, that are connected along their length by numerous transverse filaments (TFs). The assembly and disassembly of SCs closely correlates with the successive events at the chromosomal level: the condensation, pairing, recombination and segregation of homologous chromosomes, In Chapter 1, recent advances in the analysis of the structure and composition of SCs are described, and possible roles of SCs in meiotic chromosome pairing and recombination are considered. The regulation of meiotic recombination and the formation and maintenance of stable chiasmata are now considered as possibly the principal functions of SCs.

    The experimental work described in this thesis is focused on the structure and function of SCP 1, a major protein component of rat SCs (Chapters 2-5).

    Chapter 2 describes how cDNA clones encoding SCP1 of the rat were isolated by screening a cDNA library with monoclonal antibodies that recognize a 125,000 M r SC component. A polyclonal antiserum raised against the translation product of one of the isolated cDNA clones recognizes a single protein on Western blots of proteins of isolated SCs with identical electrophoretic mobility as the protein recognized by the monoclonal antibody that was used for screening. The protein encoded by the cDNAs (called SCP 1: lynaptonemal -complex protein 1) has a predicted molecular weight of 117 kDa. 'Me central part of SCP1 is capable of forming an amphipathic (alfa-helix, whereas the C-terminal domain carries motifs that are characteristic of DNA-binding proteins (Ser[]Fhr-Pro motifs). In immunogold labeling experiments, the monoclonal antibody that was used for screening and the polyclonal antiserurn that was elicited against the translation product of the cDNA both labeled the central region of the SC. We conclude that SCP I is most probably a major component of the transverse filaments of SCs, and speculate that SCP I has evolved by specialization of a nuclear matrix protein.

    In order to identify conserved features of SCPI, we isolated and characterized cDNAs encoding human SCP 1 (Chapter 3). Human SCP 1 and rat SCP 1 have 75% amino acid identity. Most of the prominent structural features of rat SCP1 are conserved in human SCP1. The human SCP1 gene was localized on human chromosome 1p12-p13. We conclude that human SCP 1 is most probably the functional homologue of rat SCP 1, and that SCP 1 is not a very conserved protein.

    Because SCP1 contains amino acid sequence motifs which are characteristic of DNAbinding proteins, we analysed the DNA binding capacity of SCP1, by means of a quantitative south-western blot assay (Chapter 4). The DNA binding capacity was confined to the C-terminal domain. This domain has about the same affinity for total rat genomic DNA as for a Drosophila SAR DNA probe, but its affinity for E. coli DNA is significantly lower. Similar results were obtained for full-length SCP 1.

    Analysis of the kinetics of DNA binding revealed that there is probably one type of DNA binding site on SCP I. Distamycin A could completely inhibit binding of DNA by the C- terminal domain. We therefore concluded that the C-terminal domain, and thus also full- length SCP1, binds to DNA through interaction with the minor groove, The binding of SCP I to DNA in vivo was shown by paraformaldehyde crosslinking of living spermatocytes. The DNA-binding capacity of the C-terminal domain of SCP1 is in agreement with the localization of this domain in the LEs of SCs, where most of the DNA is situated. We speculate about the DNA sequences to which SCP1 binds in vivo and about the implications of this for the role of SCP I in the assembly and function(s) of SCs.

    Chapter 5 describes the analysis of the organization of SCP1 molecules within SCs. Using polyclonal antibodies elicited against non-overlapping fragments of SCP1, we performed immunogold of SCs. Two types of SC preparations were used for this purpose, namely surface-spread spermatocytes and ultrathin sections of Lowicryl-embedded testicular tissue of the rat. The distribution of immunogold label on surfacespread spermatocytes differed significantly from the distribution of label on sections. This difference is probably due to masking of SCP1 epitopes within the SCs in surfacespread preparations and/or the surface morphology of the surface spreads. The results obtained on sections were therefore used for the localization of subdomains of SCP1 within the SC structure. We present the following model for the organization of SCP1 molecules within SCs: the C- terminus of SCP1 lies in the inner half of the lateral element (LE), and the molecules protrude from the LE through the central region into the central element (CE), so that N-termini of SCP1 molecules from opposite LEs overlap. The implications of this model for the assembly of SCs and for the possible functions of SCP 1 are discussed.

    In Chapter 6, the cytological and biochemical features of SCP 1 are compared with data about TF components in other organisms. Especially the possible function(s) of a putative TF component of yeast, Zip1p, are discussed, and 1 consider the relevance of the Zip1p data for the possible function(s) of SCP1. Finally, 1 present a model for the function(s) of SCP 1 during meiotic prophase.

    Natuurlijke verjonging van grove den in de boswachterij Ommen
    Oosterbaan, A. ; Tempel, J. - \ 1995
    Bosbouwvoorlichting 34 (1995)6. - ISSN 0166-8986 - p. 63 - 64.
    bosbouw - natuurlijke verjonging - zaadkieming - groei van zaailingen - geslachtelijke voortplanting - bomen - pinus sylvestris - forestry - natural regeneration - seed germination - seedling growth - sexual reproduction - trees
    Onderzoek naar verjonging door natuurlijk bezaaiing. In de tabellen gegevens over het gemiddelde aantal zaden per vierkante meter en het gemiddelde gewicht, aantal, kieming en kiemingspercentage van het opgevangen grove dennenzaad
    Research for the development of sago palm (Metroxylon sagu Rottb.) cultivation in Sarawak, Malaysia
    Jong, F.S. - \ 1995
    Agricultural University. Promotor(en): M. Flach. - S.l. : Jong - 139
    metroxylon sagu - sago - plantenvermeerdering - beplanten - formatie - distributie - voedingsstoffenreserves - geslachtelijke voortplanting - sarawak - metroxylon sagu - sago - propagation - planting - formation - distribution - nutrient reserves - sexual reproduction - sarawak
    General introduction (Chapter 1)

    This chapter contains an overview of knowledge with respect to the cultivation of the true sago palm (Metroxylon sagu). The palm flowers once and forms suckers or tillers. Seedlings grow into a rosette stage of leaves and trunks are only formed after 4-6 years. The trunk may reach a length of 6-14 m and possess a trunk of 7-24 feathered leaves. An enormous inflorescence heralds the end of the life cycle. Formation of the inflorescence, for which the starch in the trunk is being used, begins 4-14 years after the start of trunk formation.

    Continuous suckering multiplies the palm vegetatively, forming a cluster around the leader palm. Suckers are commonly used for vegetative propagation by man. Since time immemorial, man uses the sago palm trunk as a source of food starch in South East Asia. It has been a commercial crop of smallholder farmers on peat soils in Sarawak for a long time. At present, Sarawak exports nearly 50,000 tonnes air-dried sago palm starch.

    Since 1982, the Sarawak Government tries to improve and increase its cultivation, as it is one of the few crops that can be grown with reasonable success on the rather wet deep peat soils. For this purpose, a research station has been established at Sungai Talau Station and a laboratory in Mukah. The semi-govern mental agency for land development, Land Custody and Development Authority, is planting sago on some ten thousands of hectares, in order to develop part of the 1.5 million ha of poorly drained peat soils.

    The chapter ends with an overview of the main limiting factors for sago palm cultivation. This lead to the research presented in this thesis.

    Factors affecting the subsequent survival rate of sago palm suckers in the nursery (Chapter 2).

    Suckers are the most popularly used planting material for establishing sago palms in smallholder gardens and plantations in Sarawak. In nurseries, the mortality rate of suckers is around 20-40%. In the dry season, higher mortality rates are common.

    Factors suspected to affect the subsequent survival rate of sago palm suckers in nurseries were investigated. The survival of suckers was significantly enhanced if they were planted promptly, best if it was within three days after removal from the parent palm. Suckers stored for more than two weeks before planting generally showed a marked decrease in their subsequent survival in the nursery. When the cutends and the whole or part of the rhizome were completely buried in the soil, an increased rate of survival was also obtained. Rhizomes planted 8 cm below the soil surface or just placed on top of the soil surface had lower survival rates. Trimming of roots to as short as 1 em did not affect the subsequent survival of the suckers. Trimming of the rhizomes to a length close to the growing point of the sucker was deleterious. Shading of suckers during the dry season appeared to contribute positively to their successful establishment.

    When planting of suckers was delayed, treatment with a wide-spectrum fungicide while storing the suckers in cool and moist places was shown to reduce their mortality rate.

    Effects of sucker size on the establishment of sago palms (Chapter 3).

    The effects of sucker size on their subsequent establishment were investigated. Suckers of all sizes from 5 to 25 em in base diameter were established successfully without mortality. In general, larger suckers of 15-25 em in base diameter are faster in their establishment. However, these suckers are heavy, bulky, less abundant, and expensive to handle. In large- scale cultivation of sago palms, the use of large suckers has to be weighed in financial terms and availability of the material. It appears that larger suckers may be suitable for smallholder cultivation. In plantations, smaller suckers of 7-10 em in base diameter are recommended.

    Effects of plant spacing on the growth and development of sago palms on undrained deep peat (Chapter 4).

    The growth of sago palms was compared at 4.5 m, 7.5 m, 10.5 m and 13.5 m square planting. Palms spaced at 4.5 m had the lowest frond emergence rate, smallest trunk circumference at the base and at 1 m above ground level (a.g.l.), shortest prostrate (ground)trunk, longest fronds with thinnest rachides and the smallest crown size. They produced the least numbers of trunks and suckers, and their canopy was closed before trunk formation commenced in the third to fourth year after field planting.

    The vegetative growth was intermediate in the 7.5 m spacing treatment. Compared to 4.5 m spacing, these palms had significantly higher frond emergence rate, larger crown size and trunk circumference, better stem formation and suckering ability as well as shorter fronds with thicker rachides. Their canopy was closed by the fifth to sixth year after planting. However, when compared to the 10.5 m and 13.5 m spacing treatments, the 7.5 m treatment palms had significantly smaller crown size, lower frond production rate, longer fronds and thicker rachides, smaller trunks at 1 m a.g.l. and slower trunk formation. There were no differences among these treatments in the suckering ability and the size of the basal circumference of the trunk.

    Significant differences between spacing treatments of 10.5 m and 13.5 m were only found in the frond length, sucker number and frond production rate. However, the canopy of palms spaced at 10.5 m was about to close in the eighth year whereas those spaced at 13.5 m remained open. No difference was found in the average trunk height between any two spacing treatments.

    The formation and growth of sago palm trunks were suppressed at 4.5 and 7.5 m spacing whereas at a spacing of 13.5 m, the field was under-utilized. Among the spacing treatments, the maximum trunk production per unit area was from palms spaced at 10.5 m. This suggests that sago palm on peat should be cultivated at a spacing of about 10 m.

    Distribution and variation in the starch and moisture contents of sago palms at different growth stages (Chapter 5).

    Sago palms of similar growth stages established on shallow peat vary in length, circumference and weight of their trunks. From the time of planting, a sago palm remains in the rosette growth stage for about 5.5 years before trunks are formed. Flower initiation occurs at 12.5 years and the fruit drop is completed in 14.5 years.

    The average content and density of dry starch increases with increasing maturity of the sago palm until flowering. Maximum starch content of 18-20% is found between the full trunk growth stage (just before the emergence of inflorescence structure) and flowering stage. Thereafter, the starch content decreases sharply and remains finally at about 4 - 6%.

    The moisture content is high and remains rather constant along the trunk of young sago palms. As the palm matures, moisture content decreases, especially in the lower portion of the trunk.

    The lowest mean moisture content is found in palms from the full trunk growth stage to flowering stage, corresponding to the highest starch content in the trunk. In young and over- mature palms, the mean moisture content is higher. A high negative correlation (r2 = -0.85) is found between moisture and starch contents, showing the mutual replacement of starch and moisture in the trunk.

    Within each growth stage, the density of the fresh trunk is constant along the entire trunk length. However, among different growth stages, the mean density of the sago palm trunk increases gradually from the early trunk formation stage. It reaches a maximum between the full trunk growth and flowering stage before decreasing in the subsequent over-mature stages.

    This study provides an understanding of the pattern of starch accumulation and the relationship between the fresh density of the trunk and the starch content in it. This enables the harvesting of palms at the correct growth stage for maximum starch yield per unit time, and facilitates the grading of sago logs for starch yield based on their buoyancy.

    Flowering biology of the sago palm (Chapter 6).

    Scaffolds were constructed below the gigantic inflorescence of sago palms to investigate the flowering biology. In the early stage of development, flower buds occur in pairs in a bracteole. One is a staminate (male) and the other a hermaphrodite (perfect) flower. During development, abortion of either the staminate or the hermaphrodite flower buds occurs. By anthesis, mainly single flower buds are left in each bracteole. The sago palm is andromonoecious as indicated by the presence of staminate and hermaphrodite flowers in the same inflorescence. In three of the seven palms investigated, abnormal hermaphrodite flowers which opened prematurely were encountered.

    The duration of flower opening in the entire inflorescence is about 30 days for the staminate flowers and 50 days for the hermaphrodite flowers. For the abnormal hermaphrodite flowers, opening may stretch over a period of three months and the premature stamens die during the opening. The peak of daily flower opening is between 1100-1400 hours. To a large extent, the sago palm is protandrous but overlaps in the opening of staminate and hermaphrodite flowers do occur. Seedless fruits are formed in all these palms. Visiting insects ( predominantly Trigona itama, Trigona apicalis and Apis dorsata) are found in great numbers during anthesis. However, only seedless fruits develop in most palms. This suggests that the pollen and pistil of sago palms may be self - incompatible. Bagging experiments to exclude visiting insects suggest that cross- pollination is obligatory in sago palms.

    .In the current investigation, each sago palm produced between 276,000-864,000 mature flower buds and 2174-6675 mature fruits. The duration of fruit growth from anthesis to last fruit drop is between 19-23 months.

    Germination of sago palm seeds (Chapter 7 ).

    Some practical methods to increase the germination rate of sago palm seeds were investigated. The capability of seeds to germinate was found to increase as seed maturity advanced. Germination tests conducted on mature sago palm seeds using wet sand tray showed that removal of the husk and husk plus fleshy tissue (sarcotesta) enhanced germination. Loosening of the operculum and treatment with 10' M gibberellin also increased the number and speed of germination. Brief treatment with concentrated sulphuric acid was found fatal to the seeds. Germination of the entire sago palm fruit required an environment with high humidity. An easy and effective way of achieving this is to put mature sago palm fruits in partially permeable Hessian sacks placed in a damp atmosphere.

    General conclusions (Chapter 8)

    In this chapter, an assessment of the research results for practical application in sago cultivation is given.

    Application of the findings from experiments on the survival of suckers may lead to an increase of survival between 10 and 40%. At the current cost of about RM 3.50 per sucker, this will decrease cost of planting, at the present planting distance, between RM 72 and RM 290 per hectare. Selecting suckers with a base diameter between 7 and 10 cm and a weight between 2 and 5 kg can reduce transportation and handling costs. This may also prevent price increase due to temporary sucker shortage when larger planting materials are preferred . Planting sago palms in a 10 m square pattern, with possibly a temporary plant in the middle of the square for oneharvest trunk , appears optimal for ultimate production. Findings in the distribution of starch in the trunk at different growth stages leads to harvesting just before flowering. Based on the ralation found between starch content, moisture content and trunk density, it may lead to the development of a practical method to estimate the starch content of a trunk based on its buoyancy. The results obtained in flowering and seed germination appear to be important and may be used for future breeding and research programmes. Sago palm seeds may also be used for new planting if suckers are either too expensive or in short supply.

    The most pressing problems that needs attension in sago research is fertilizer application on the notoriously poor and badly drained peat soils. It is also important to start research on shortening the unproductive phase of the cultivation.

    Visie vermeerdering boomkwekerijgewassen : ontwikkeling, knelpunten, prioriteiten en mogelijkheden voor onderzoek
    Kunneman, B.P.A.M. - \ 1990
    Boskoop : Proefstation voor de Boomkwekerij (Rapport / Boomteeltpraktijkonderzoek nr. 5) - 35
    ontwerp - bosbouw - houtachtige planten als sierplanten - plantenvermeerdering - geslachtelijke voortplanting - theorie - vegetatieve vermeerdering - design - forestry - ornamental woody plants - propagation - sexual reproduction - theory - vegetative propagation
    Clones of common carp, Cyprinus carpio = New perspectives in fish research
    Komen, J. - \ 1990
    Agricultural University. Promotor(en): E.A. Huisman; W.B. van Muiswinkel, co-promotor(en): C.J.J. Richter. - S.l. : S.n. - 169
    cyprinidae - karper - geslachtelijke voortplanting - parthenogenese - polyembryologie - genotypen - genetische variatie - cyprinidae - carp - sexual reproduction - parthenogenesis - polyembryony - genotypes - genetic variation

    The absence of well defined inbred lines is an important problem associated with scientific research on fish. Inbred lines can be produced by conventional full-sib mating, but at least 10-15 generations are needed to produce homozygous inbred lines. Using common carp, which reach maturity at 1.5 years, this would last some 15-30 years. Nowadays experimental fishes are usually obtained from commercial fish farms, or bred in the laboratory using a limited number of broodstock fish. In both cases the genetic background and the degree of inbreeding of the experimental animal is unknown.

    In consequence the results from various laboratories are difficult to compare. Bioassays often show a large variation in the experimental results and a relative low reproducability. Moreover, large numbers of fish are needed to obtain statistically significant results. In order to solve these problems this research project was started with the aim to develop homozygous inbred lines of fish by gynogenetic breeding. Furthermore, in our university there was a high need for inbred lines with specific (mutant) genotypes, which could be used in the ongoing research on the immune system and sex determination of common carp.

    In gynogenesis, eggs are fertilized with genetically inactivated sperm. The resulting haploid embryo can be made diploid by inhibition of the second meiotic division (retention of the second polar body or 2PB method), or by inhibition of the first mitotic division (endomitosis or EM method). In the first case the gynogenetic offspring will be partly heterozygous due to recombination during the preceeding meiotic prophase. In the second case the haploid genome of the embryo is duplicated while the first cell division is prevented. The resulting diploid offspring will be fully homozygous.

    In a first series of experiments (chapter 3) the optimal conditions for irradiation and dilution of milt, and for administration of a temperature shock to inhibit the second meiotic division, were investigated. Milt was irradiated with U.V. light (235.7 nm). Dilution (in physiological saline) and irradiation duration were important parameters for the survival of spermatozoa. Sperm, diluted 1:3, could be irradiated for 60 minutes (2200 J/m2,min) without loss of fertilization capacity. This fertilization capacity was considerably reduced when higher dilutions were used, while a shorter irradiation period failed to inactivate all spermatozoa.

    The effectiveness of genetic inactivation was checked by using sperm from scaled males (a dominant trait) and eggs from scattered females (recessive trait). Gynogenetic offspring turned out to be all scattered. Inhibition of the second meiotic division was achieved by administering eggs, fertilized with genetically inactivated sperm, a temperature shock at various moments after fertilization. Consistent yields of 25-50 % viable fry were obtained when eggs were cold shocked (0°C) for 45 minutes, 1-2 or 7-9 minutes after fertilization (at 24 °C). This bimodal response was typical for common carp, but essentially different from other investigations on common carp gynogenesis, where lower incubation temperatures and degumming of egg was practised.

    In a second series of experiments (chapter 4) the optimal conditions for inhibition of the first mitotic division were investigated. The occurrence of metaphase of the first mitotic division was histologically determined. Consistent yields of 5 - 15 % viable fry were obtained when eggs were heat shocked at 40 °C). for 2 minutes, 28-30 minutes after fertilization (i.e. at metaphase). Accurate timing of the heat shock, as well as the heat shock temperature and duration, were critical in obtaining an optimal yield of diploid fry. The homozygous nature of the gynogenetic fry was demonstrated by the Mendelian segregation patterns of two recessive colour mutations (chapter 4).

    An important aspect of the described gynogenetic breeding techniques is the effect of the expected homozygosity in a first generation of gynogenetic offspring. In order to investigate this effect, we compared homozygous carps (EM method) with heterozyous gynogenetic carps (2PB method) and a group obtained by full-sib mating (chapter 5). The three groups were all obtained from the same mother, and allowed a comparison of the effects of increasing levels of homozygosity. Skin grafts were exchanged between animals of the same group and between animals of different groups. Skin allografts exchanged among heterozygous gynogenetic carp exhibited prolonged survival. Furthermore a strong histocompatibility (H) locus was seen to segregate in this group. In contrast skin allografts exchanged among homozygous gynogenetic siblings or among normal full-sibs were all rejected in an acute manner, with homozygous fish showing the most vigorous allograft reactions. These findings were explained by assuming that acute allograft reactions were the result of a single strong H-locus disparity, or of a multiple minor H-loci barrier which mimics a strong H-locus effect (chapter 5).

    In a follow-up experiment (chapter 6) the effects of increasing levels of homozygosity on sex, gonad development and fertility of carps from these three groups were compared. Surprisingly nearly 50 % males and fishes with intersex gonads were found in the EM group while males were absent in the 2PB group. This excluded a possible contamination with non-irradiated (non-inactivated) sperm. Inbreeding significantly increased the mean gonad weight as well as the variation in gonad weights. Full sib (FS) and heterozygous gynogenetic offspring (2PB) were normal in gonad development, but gonads from homozygous gynogenetic (EM) carp were often retarded in vitellogenesis. The ovulation response was significantly reduced with increasing levels of inbreeding. Eggs from ovulated females of the FS, 2PB and EM groups were fertilized with milt from males of the FS and EM groups. Yields of normal fry were reduced in crosses involving FS and 2PB eggs when compared to crosses with EM eggs or milt. This indicated that homozygous fish were essentially free of recessive lethal genes affecting embryo survival (chapter 6).

    New inbred lines were produced using a combination of both gynogenetic techniques. Homozygous inbred strains were produced by gynogenetic reproduction (2PB method) of homozygous gynogenctic (EM) females. F 1 hybrid strains were produced by crossing homozygous females with homozygous gynogenetic male siblings. The clonal nature of these strains was unequivocally demonstrated by reciprocally exchanged skin allografts. All grafts exchanged among members of the same strain were permanently accepted. Likewise grafts from homozygous strain members were accepted by fish from the related half-sib F 1 hybrid strains, while the reverse grafts were rejected. These results provided evidence for the idea that in carp, as in other vertebrates studied so far, histocompatibility genes exist as major and minor loci which are codominantly expressed (chapter 5).

    The inbred strains and F 1 hybrids were comparable in body weight and gonad development (chapter 6), but the F 1 hybrids showed a much lower variation in body weight and gonad development. In contrast the phenotypic variation was considerably enlarged in the homozygous inbred strains. This phenomenon is well known in inbred strains of mice and rats, and are generally attributed to developmental instability. The F 1 hybrids are therefore more suited for use in bioassay's, especially since they might possess an increased viability.

    One of the advantages of the described gynogenetic inbreeding system is that selection of the most interesting and viable genotypes is required only in the first generation. The selected females can be propagated to produce inbred strains are identical to their parents in overall performance. However, in order to obtain males within a gynogenetic inbred line, some females should be sex-inversed by hormonal treatment. Therefore juvenile, non-inbred carps were treated with various doses of orally administrated 17αmethyltestosterone during different periods after hatching. The treatment periods were 3-8 weeks, 6-11 weeks and 10-15 weeks after hatching. The tested hormone concentrations in the food were 50 and 100 ppm, while a dose of 150 ppm was also applied during 6-11 weeks after hatching. The gonads were inspected at 6 months after hatching. Administration of 50 ppm 17α-MT in the food between 6 and 11 weeks after hatching resulted in 92,7% males. Earlier treatments with 17α-MT in concentrations of 50 and 100 ppm of hormone in the food resulted in high percentages of sterile fish while later treatments produced a high percentage of intersex gonads (chapter 7). Surprisingly a similar experiment using 178 estradiol failed to induce female gonads in any of the periods tested and irrespective of the concentrations of hormone used.

    The optimal treatment with methyltestosterone was used to induce sex-inversion in the produced homozygous inbred strains and F1 hybrids (chapter 8). The untreated groups contained females and a single fish with intersex gonads. In the treated groups however, mainly intersex gonads were observed. Only one F 1 hybrid group contained significantly more males (60 %) than animals with intersex gonads. These results can only be explained by assuming that the success of hormone induced sex inversion is genetically determined.

    Maleness in common carp is thought to be determined by dominant sex determining genes, since heterozygous gynogenetic offspring were all female. However, in some homozygous gynogenetic offspring nearly 50 % males and intersexes were found. It was therefore suggested that maleness in these groups might he caused by recessive mutations in sex determining genes. The mother of one offspring group, probably heterozygous for a putative mutation, was crossed with an unrelated gynogenetic male from another experimental group. The offspring of this cross was exclusively female, but crosses of these females with gynogenetic males contained again 50 % males and intersexes. It was concluded that these males and intersexes were homozygous for a recessive mutant sex determining gene termed mas-1. To our knowledge such mutations have not been described in fish before (chapter 8).

    In conclusion, it can be stated that gynogenesis is a very successful and rapid method for the production of homozygous inbred lines of the common carp, Cyprinus carpio. Such inbred lines have until now only been produced in two small aquarium fish species, zebrafish ( Brachydanio rerio ), and medaka ( Oryzias latipes ). Our new inbred lines of common carp will be very important for future scientific research. The use of F 1 hybrids in endocrinological and immunological bioassays will result in an increased standardisation and thus in a reduction of the number of experimental animals needed. Perhaps the inbred lines can also provide an alternative for the use of other experimental vertebrate animals. The present study also demonstrated the possibilities of gynogenetic breeding in unravelling complex biological processes as graft rejection and sex determination. Moreover, the rapid isolation of specific mutants with an abnormal development may offer important possibilities for future research.

    Oospore formation by Phytophthora infestans in host tissue after inoculation with isolates of opposite mating type found in The Netherlands.
    Frinking, H.D. ; Davidse, L.C. ; Limburg, H. - \ 1987
    Netherlands Journal of Plant Pathology 93 (1987). - ISSN 0028-2944 - p. 147 - 149.
    phytophthora - plantenziekteverwekkende schimmels - aardappelen - geslachtelijke voortplanting - solanum tuberosum - sporen - phytophthora - plant pathogenic fungi - potatoes - sexual reproduction - solanum tuberosum - spores
    Sexual reproduction in seed plants, ferns and mosses
    Willemse, M.T.M. ; Went, J.L. van - \ 1985
    Wageningen : Pudoc - ISBN 9789022008799 - 206
    bryologie - bryophyta - mossen - geslachtelijke voortplanting - vaatplanten - bryology - bryophyta - mosses - sexual reproduction - vascular plants
    Behandeling van de biologie met betrekking tot de geslachtelijke voortplanting van deze plantesoort en onderzoek- en vermeerderingstechnieken op dit gebied. Aandacht voor de ontwikkeling van sporen en microsporen; mannelijke steriliteit; vorming van de stuifmeelbuis en invloeden op diverse ontwikkelingsstadia; enkele nieuwe methoden met betrekking tot het gebruik van "fluorochromen", immunologische technieken en kwantitatieve metingen aan pollen; oppervlakte van de stigma; incompatibiliteit; kieming van stuifmeel; megasporogenese; zaadcellen; structuur van de embryozak; bevruchting; apomixis en embryogenese
    Reproduction in Spinacia Oleracea L. : ultrastructural aspects of pistil development, pollination and fertilization
    Wilms, H.J. - \ 1981
    Landbouwhogeschool Wageningen. Promotor(en): J.L. van Went, co-promotor(en): M.T.M. Willemse. - Wageningen : Wilms - 129
    chenopodiaceae - geslachtelijke voortplanting - spinazie - spinacia oleracea - chenopodiaceae - sexual reproduction - spinach - spinacia oleracea
    Ontwikkeling en samenstelling van de zaadknop

    De ontwikkeling en differentiatie van de integumenten, nucellus en de megagametofyt resulteert in spinazie in een ortho-amphitrope zaadknop. In het nucellus worden vier verschllende weefsels onderscheiden: het geleidend weefsel, het oorspronkelijke chalazale weefsel, het uiteindelijk chalazaal "proliferating" weefsel en het laterale weefsel. De cellen van de onderscheiden weefsels hebben overeenkomstige als ook sterk verschillende eigenschappen, die gerelateerd zijn aan de positie binnen het geheel en aan hun functie.

    De integumenten vertonen verschillen in hun ontwikkeling. Het buiten integument omvat 3-5 cellagen met daartussen intercellulaire holten van variabele grootte. Het binnen integument wordt gevormd door twee zich verschillend ontwikkelende cellagen. Het plasmodesmaal contact tussen deze twee cellagen vermindert en is verdwenen bij volgroeidheid.

    Demegagametofyt heeft vIak na de coenocytische periode plasmodesmata in de chalazale celwand tussen de chalazale antipode en de aangrenzende nucelluscellen.

    De localisering van reservemateriaal, zoals zetmeel, andere polysacchariden, proteinen en lipiden, is bestudeerd in de zich ontwikkelende zaadknop om de voedingsbaan naar de embryozak en het embryo vast te stellen. In de jonge zaadknop gaat de aanvoer van voedingsstoffen via het oorspronkelijk chalazale weefsel naar de zich ontwikkelende vrouwelijke gametofyt en het geleidend weefsel , waarbij opslag vooral in dit laatste plaatsvindt.

    Tijdens de groei van de zaadknop ontwikkelt het uiteindel ijk chalazaal "proliferating"weefsel zich waarbij dit het oorspronkelijk chalazaleweefsel wegdrukt en de transportfunctie ervan overneemt. Hiermee gaan veranderingen gepaard in de waargenomen hoeveelheden reservematerialen. De opslag van zetmeel neemt sterk toe in het buiten integumenten in de cellen, die de baan van oorspronkelijke chalazale cellen - embryozak omgeven. Na de bevruchting wordt dit zetmeel geleidelijk afgebroken.

    Proteinen worden vooral aangetroffen in de weefsels die betrokken zijn bij de groei en penetratie van de pol
    lenbuis naar de embryozak.

    Ontwikkeling van de embryozak

    Vanaf de vorming van de coenocyt toont de ontwikkel ing van de embryozak twee fasen: de eerste start met de vorming van de cellen en gaat tot het bereiken van de uiteindelijke grootte van de cellen. Nu begint de tweede fase, die loopt tot het stadium waarop de embryozak bevruchtingsrijp is. Gedurende de eerste fase zijn de dimensies, oppervlakten en volumen van de verschillende cellen en celonderdelen gemeten en met elkaar vergeleken. De celvergroting is in hoofdzaak het gevolg van vacuolisatie. Het protoplasma van de antipoden blijkt nauwelijks toe te nemen, terwijl dat van de synergide verzesvoudigd en dat van de eicel en de centrale cel ongeveer vertienvoudiqd. Gedurende de tweede fase ontwikkelen de cellen tot hun uiteindelijke ulstrastructuur.

    Bij de vorming van de cellen biijkt kwalitatief de samenstelling van iedere cel ongeveer gelijk: een kern met een onregelmatige vorm, veel ER en ribosomen, de nodige mitochondriën en dictyosomen, weinig plastiden en geen lipid. De ontwikkeling en differentiatie van de onderscheiden cel typen geschledt verschillend voor zowel de wanden als hun plasmatische onderdelen.

    De antipoden bereiken hun functionele structuur snel, leven korstondig en degenereren volgens een vast patroon.

    De eicel groeit eerst zeer snel, vervolgens langzaam en ondergaat in het volgroeide stadium niet lang voor de bevruchting een hernieuwde vermeerderingsactiviteit van mitochondrien.

    Deontwikkelling van synergiden en centrale cel is geleidelijk. Voor de bevruchting degenereert een van de synergiden, terwijl in de centrale cel de poolkernen talrijke kernuitlopers vormen, die gedeeltelijk fuseren.

    De structurele veranderingen in ieder cel type zijn gerelateerd aan hun mogelijke functies. Opslag en afbraak van reservemateriaal wordt verklaard. In de bijna rijpe embryozak is het aanbod groter dan nodig; dit leidt tot opsIag in de vorm van zetmeel . Dit geschiedt achtereenvolgend in de eicel, centrale cel en synergiden en tenslotte ook nog iets in de antipoden. Wanneer de zaadknop volgroeid is stopt de aanvoer wegens het niet meer functioneren van de aanvoerweg. Het zetmeel wordt geleidelijk afgebroken, beginnende in de meest chalazale cel. Ten tijde van de bevruchting is het vooral de eicel die nog zetmeel bezit.

    Stempel en stijI

    Het receptieve gedeelte van de stamper bestaat uit 4 large stempels. Het bovenste gedeelte van een stempel bevat uitsluitend papilcellen met een breed centraal deel en een smal spiraliserend staartgedeelte. Het onderste gedeelte bezit bovendien nog cylindrische parenchymcellen. In iedere stempel wordt een centrale zone gevormd, allereerst door de staartgedeelten, later samen met de smalle parenchymatische cellen. In de stijl komen deze centrale zones bijeen en fuseren tot één zone van geleidend weefsel. Aan de basis buigt dit geleidend weefsel af in de richting van de micropyle. De intercellulairen tussen de verschillende cellen van de stempel en van de stijl verschillen sterk in grootte en in electronen-dichtheid van hun matrix.

    Pollenbuisgroei in stempel en stijl.

    De kieming geschiedt binnen 10-20 minuten na bestuiving, terwijl de Pollenkitt reeds binnen 7-10 minuten met de pellicula van de stempelpapil fuseert. De pollenbuls dringt door de pellicula en de aarigetaste cuticula en groeit via de buitenste wandlaag naar de basis van de papil en vervolgt zijn weg via intercellulairen. De pollenbuisgroei door stempel en stijl veroorzaakt geen structurele veranderingen in de aangrenzende cellen. In de stijl kan de pollenbuisgroei ook via celwanden en/of tussen celwand en plasmamembraan door. In alle gevallen bereikt de pollenbuis de ruimte tussen buiten integument en vruchtwand. Soms groeit een pollenbuis eerst nog een periode via de cellen van de vruchtwand. Een afsluitende cuticula ontbreekt op deze plaats aan de binnenzijde van de vruchtwand. Ongeveer 6 uur na bestuiving bereiken de eerste pollenbuizen de micropyle.

    Penetratie in de zaadknop

    Indien de zaadbeginsels nog niet volgroeid zijn, kunnen pollenbuizen dit nucellus niet penetreren en klusteren dien tengevolge in en om de micropyle tesamen. Wanneer de zaadknop volgroeid is, blijkt bestuiving uitscheiding van "substanties" vanuit de synergiden te stimuleren. Deze uitscheiding leidt tot aantasting van de longitudinale middenlamellen van het nucellus dat tussen de embryozak en de micropyle gelegen is. Uiteindelijk wordt ook de nucellaire cuticula nabij de micropyle aangetast. Vanaf dit tijdstip kunnen pollenbuizen het nucel lusweefsel penetreren. De pollenbuisgroei geschiedt aanvankelijk uitsluitend intercellulair en vervolgens via wegen, overeenkomstig aan die in de stijl. Meerdere pollenbuizen kunnen de embryozak bereiken, naar slechts één pollenbuis penetreert de degenererende synergide via het faden-apparaat. Spermacellen, vegetatieve kern en pollenbuis cytoplasma met vele karakteristieke amyloplasten worden via een terminale opening geloosd in de degenererende synergide.


    De fusie van de gameten vinct 7-9 uur na de bestuiving plaats,waarbij blijkbaar geen of zeer weinig organellen van de spermacellen zijn betrokken. Celfusie vindt plaats tussen spermacel en eicel, respectievelijk centrale cel, waarbij de desbetreffende membranen fuseren. De fusie tot zygotekern geschiedt snel , de fusie tot endospermkern is veel geleidelijker. De eerste mitotische deling is in de endospermkern 16-17 uurna de feitelijke bevruchting voltooid, terwijI dan de zygotekern nog in de aanloopfase verkeerd.

    Vernieuwing en verscheidenheid : geslachtelijke voortplanting als ontwikkelingsproces
    Willemse, M.T.M. - \ 1976
    Wageningen : L.H. - 67
    geslachtelijke voortplanting - parthenogenese - polyembryologie - sexual reproduction - parthenogenesis - polyembryony
    Rede Wageningen (no. 346)
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