Androgenesis and homozygous gynogenesis in muskellunge (Esox masquinongy): Evaluation using flow cytometry

The purpose of this work was to study the effects of ultraviolet (UV) irradiation on denucleation of eggs and investigate the heat-shock conditions for diploidization for induction of androgenesis in muskellunge, Esox masquinongy. Several egg incubation media, including saline, Ringer's solution, and Ringer's solution supplemented with bovine serum albumin (BSA), were found suitable to maintain the egg fertility as high as in muskellunge ovarian fluid. The optimal doses of UV radiation were 660–1320 J/m², at which 100% haploid larvae were produced at a hatching rate of 22.5 ± 2.8%. UV irradiation at low doses (165–330 J/m²) generated abnormal larvae, which were morphologically identical to haploids. Using a flow cytometry method, it was found that cellular DNA content of these larvae was close to that of diploids but significantly lower in value and had a wider distribution (expressed as coefficient of variation) than that of control fish. This suggested that a low dose of UV irradiation might cause gene mutations, alteration of chromosomal conformation and fragmentation, but did not prevent maternal DNA from participating in mitotic division. Interference of maternal DNA residues could be another reason for the poor viability of androgenetic fish. A high dose of UV radiation (1980 J/m²) caused development of severely deformed embryos, indicating that UV radiation also damaged molecules in the eggs other than the denucleation. Our results suggest that classic color and allozyme markers might not be sufficient to prove a complete androgenesis. In order to optimize time and duration of shock for induced diploidization, we investigated the heat-shock conditions for inhibiting the first mitotic cleavage through induction of homozygous gynogenesis. We found that heat-shock treatment at 31°C for 9 min starting at 1.4τ₀ (a dimensionless factor describing progress in embryo development) after fertilization produced the highest percentage of diploids at hatching. Mol. Reprod. Dev. 49:10–18, 1998. © 1998 Wiley-Liss, Inc.     

Nucleolar number variation in Hordeum species,their haploids and interspecific hybrids

Nucleolar number variation has been investigated in root tip cells by cytologically determining the number of nucleoli per cell in autoploids and alloploids of Hordeum species, their haploids and interspecific hybrids. The nucleolus organisers in autoploid types of H. vulgare or H. bulbosum did not show any alterations irrespective of the ploidy level. The nucleolar number variation in these species results from a definite pattern of fusion and the maximum number of nucleoli per nucleus corresponds to the number of secondary constrictions. Nucleolus formation in alloploids and interspecific hybrids is impaired on some of the NORs, suggesting differential amphiplasty or nucleolar dominance. A comparison of nucleolar formation in the alloploid species (brachyantherum, arizonicum, procerum and parodii), their haploids, and the interspecific hybrids revealed different degrees of variation from the expected mean and maximum numbers of nucleoli. While the deviations in hybrids between alloploids (H. arizonicum and H. brachyantherum or H. procerum and H. brachyantherum) are marginal, nucleolar dominance is more pronounced in hybrids involving H. vulgare or H. bulbosum as one of the parents and is invariably associated with the disappearance of the secondary constriction(s) from the NOR(s) contributed by one of the parents, and the number of nucleoli is appropriately reduced.     

Ribosomal RNA cistron number in Nicotiana species and derived haploids

Twelve Nicotiana species, and haploids obtained by anther culture from three of these species, were assayed for the proportion of DNA complementary to ribosomal RNA. No variation was observed in the proportion of DNA coding for ribosomal RNA between diploid and haploid plants within a given species. Wide variation was observed in this proportion between the species with values ranging between 0.068% and 0.43% of the total DNA. No relationship between the proportion of DNA complementary to rRNA and the chromosome number of the species was observed.     

Production of haploids and doubled haploids in oil palm

Background

Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.

Results

Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC₄F₂ segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 _Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 _B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 _Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 _B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 _Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.

Conclusions

Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection.     

Experimental gynogenesis in the newt species Pleurodeles waltlii and P. poireti

Eggs of diploid females of Pleurodeles waltlii, inseminated by genetically inactivated sperm of Salamandra salamandra, have been treated by heatshock or increased hydrostatic pressure during the first hour of their development. The resulting viable gynogenetic individuals show different degrees of ploidy although they mostly are diploid. The use of females with a pericentric inversion as a marker chromosome allow the chromosomal constitution to be clarified. Eggs of females heterozygous for a recessive semilethal mutation (ascite caudale, ac) subjected to the same experimental treatment gave 50% ac/ac embryos. Experiments with Pleurodeles poireti gave the same results as those with P. waltlii. These observations prove that gynogenetic Urodeles can be produced in large numbers. In their offspring the detection of inherent or spontaneous recessive mutations is greatly facilitated.

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Sommaire Des oeufs de femelles diploïdes de Pleurodeles wahlii sont inséminés par du sperme de Salamandra salamandra inactivé du point de vue génétique. Ils sont soumis pendant la première heure de développement soit à une élévation de température (37°1 pendant 5 minutes), soit à une compression (450 bars pendant 6 minutes). On obtient des individus gynogénétiques viables de ploïdie variable mais en majorité diploïdes. L'utilisation de femelles diploïdes de P. waltlii présentant un chromosome marqueur permet de démontrer l'origine gynogénétique des descendants et de préciser la manière dont s'est constitué leur équipement chromosomique. Des oeufs de femelles diploïdes de P. waltlii hétérozygotes pour la mutation récessive semi-léthale ascite caudale, inséminés et choqués selon le même protocole conduisent à 50% d'embryons gynogénétiques homozygotes pour cette mutation. La même technique appliquée à des oeufs de femelles diploïdes de Pleurodeles poireti permet d'obtenir également des individus gynogénétiques, de ploïdie variable. Ces résultats confirment la possibilité d'obtenir en grand nombre des individus gynogénétiques viables chez les Urodèles. Chez les individus hétérozygotes pour un gène récessif, la détection et l'analyse des mutations spontanées ou induites peuvent être grandement facilitées par ce mode de reproduction.

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The authors would like to dedicate this article to the memory of the late Jean Rostand (1894–1977) pioneer of experimental diploid gynogenesis in amphibia     

Negative evidence of parthenogenesis induction by Wolbachia in a gallwasp species, Dryocosmus kuriphilus

The alpha‐proteobacteria of the genus Wolbachia is a widespread group of maternally inherited endosymbionts of arthropod and nematode hosts. Wolbachia infection induces a range of host phenotypes, including cytoplasmic incompatibility, male killing, feminization, and induction of thelytokous parthenogenesis. Heterogony (cyclical parthenogenesis) is a remarkable characteristic of oak gallwasps, Cynipini, the largest tribe of the Cynipidae. A few species of Cynipini are exceptional in that they are univoltine and exhibit thelytokous parthenogenesis, probably because they lost the arrhenotokous generation of their heterogonic ancestor species due to Wolbachia infection. In this study, the presence of Wolbachia was detected using polymerase chain reaction primers for the wsp genes in a thelytokous parthenogenetic species [Dryocosmus kuriphilus (Yasumatsu)] (Hymenoptera: Cynipidae: Cynipini). Approximately 29.8 and 87.1% of adults of the Zhuzhou and Fuzhou strains, respectively, were infected with Wolbachia while all females of the remaining four strains collected from other localities in China were Wolbachia free. The length of the wsp fragment of Zhuzhou and Fuzhou strains was found to be 573 and 561 bp, respectively. The nucleotide sequence of the bacterial wsp fragment indicated that the endosymbiotic bacteria of the Zhuzhou and Fuzhou strains are members of supergroup A, but belong to different clades; they probably originated from two independent infection events. In conclusion, thelytokous parthenogenesis of D. kuriphilus is not caused by Wolbachia infection and the deletion of the arrhenotokous generation is thus not associated with such an infection.     

Production of haploids and doubled haploids from unfertilized ovule culture of various wild species of gentians (Gentiana spp.)

The production of haploids and doubled haploids (DHs) on unfertilized ovule culture was examined in 19 wild species of gentians (Gentiana ssp.) classified into eight sections. Of the 19 species including 35 strains, embryo-like structures (ELSs) were obtained from unfertilized ovule culture in 15 species, and regenerated plants were produced in 11 species. ELS production has varied greatly among the15 species, i.e., 0.5–79.2% frequency of responding flower buds and 0.01–1.99 ELSs per flower bud. Of the ELS-producing species, almost all were classified into the sects. Pneumonanthe or Cruciata. Species in sect. Pneumonanthe showed higher responses than those in sect. Cruciata. In examining the effect of flower bud stage on ELS production, more than twice as many ELSs were observed at the anther-dehiscent stage than that at the anther-indehiscent stage. Ploidy level was determined in 117 randomly selected regenerated plantlets, which suggests that most were diploid (46.2%) and haploid (32.5%). When 12 diploid plants were examined using simple sequence repeat (SSR) markers, 8 (66.7%) were DH. This study revealed that unfertilized ovule culture can be applied not only on cultivated gentian species but also on a number of wild species. The production of haploids and DHs in wild gentians provides novel prospects for ornamental and/or medicinal gentian breeding.

     

Adaptation of cucurbit species to changes in substrate temperature: Root growth,antioxidants, and peroxidation

To investigate their response to changes in substrate temperatures, the roots from six species of cucurbit plants were exposed to 14°C, 24°C, or 34°C, while their aerial portions were maintained at natural ambient temperatures (23°C to 33°C). These species could be classified into three groups based on their stress response: Group A,Cucurbita ficifolia and C.maxima, heatsensitive but cold-tolerant; Group B,Cucumis sativus and C.melo, heat- and cold-sensitive; and Group C,Luffa cylindrica andBenincasa hispida, heat-tolerant but cold-sensitive. The highest growth rates and lowest contents of malondialdehyde (MDA) for plants in Groups A, B, and C were achieved at temperatures of 14°C, 24°C, and 24°C to 34°C, respectively. Superoxide dismutase (SOD) activity was lowest in the roots exposed to optimal growth temperatures while activities of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (G-POD) operated coordinately in a complicated manner to prevent the accumulation of reactive oxygen species (ROS) in the root cells. Moreover, all plants, regardless of species, responded to unfavorable temperatures by increasing their synthesis of ascorbate and glutathione as well as by reducing the redox ratio of those two important antioxidants.     

高等植物的雄核发育

雄核发育的过程可以人为划分为3个阶段：小孢子胚性潜能的获得、细胞分裂的起始和胚胎的分化形成。文章介绍每一阶段所发生的细胞和分子水平的变化,其中侧重点在基因表达的变化,包括转录因子基因、与胁迫有关的蛋白质基因、淀粉合成及蛋白质分解相关基因以及近年来发现的与胚胎发生有关的调节基因的研究进展。     

Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species

The induction of triploidy and gynogenesis by chromosome set manipulation has traditionally been studied more intensively in freshwater than in marine fish. In the last years, however, several studies have applied these manipulations in about a dozen marine species, including mainly sparids, moronids and flatfishes. This paper focuses on the methodologies used to induce, verify, and assess performance of both triploids and gynogenetics of these marine species. Since many of them are batch spawners and have small and fragile eggs and larvae, peculiarities relating to broodstock management, gamete quality and mortality assessment during early larval stages are also taken into account. However, data show that if handling is correct and the treatments are optimized, triploid and gynogenetic rates of 100% can be easily achieved. Survival of triploids with respect to the controls is about 70–80%, whereas in gynogenetics it is generally low and more variable, depending on the species considered. In the marine fish investigated so far, triploidy has not resulted in significantly higher growth rates. On the other hand, the induction of gynogenesis has resulted in the production of both all-female and mix-sex stocks. Throughout the paper, special reference is made to the European sea bass (Dicentrarchus labrax L.), a species of both basic and applied interest, for which a comprehensive study has been carried out on the induction, verification and performance of triploids and gynogenetics.Author for correspondence     

The Leptynia hispanica species complex (Insecta Phasmida): polyploidy, parthenogenesis, hybridization and more

The Leptynia hispanica stick insect species complex includes bisexuals, triploid and tetraploid parthenogenetic populations, suggesting that polyploidy has played a central role in the evolution of this complex. An analysis of karyotype, mitochondrial DNA (cox2) and nuclear DNA (ef1-alpha) markers was carried out to clarify phylogenetic relationships and microevolutionary/phylogeographical patterns of the L. hispanica complex. Our analyses suggested a subdivision of bisexual populations into four groups, tentatively proposed as incipient species. Moreover, triploids and tetraploids showed two independent origins, the latter being more ancient than the former. From ef1-alpha analysis, triploids showed hybrid constitution, while the hybrid constitution of tetraploids is likely, but more data are needed. We suggest that L. hispanica is a case of 'geographical parthenogenesis' with parthenogenetic strains colonizing large peripheral ranges, and bisexuals confined to glacial refuge areas. Moreover, the age, wide distribution and competitive advantage of polyploids over diploids, demonstrate their significance in the evolution of the L. hispanica species complex.     

Hybridization, glaciation and geographical parthenogenesis

Parthenogenetic organisms are all female and reproduce clonally. The transition from sex to parthenogenesis is frequently associated with a major change in geographical distribution, often biasing parthenogenetic lineages towards environments that were severely affected by the glacial cycles of the Late Pleistocene. It is difficult to interpret these patterns as arising simply as a result of selection for the demographic effects of parthenogenesis because many parthenogenetic organisms are also hybrids. Here, I argue that many cases of geographical parthenogenesis might be best seen as part of a broader pattern of hybrid advantage in new and open environments. Parthenogenesis in these cases could have a more secondary role of stabilizing strongly selected hybrid genotypes. In this context, geographical parthenogenesis might tell us more about the role of hybridization in evolution than about the role of sex.     

Geographical parthenogenesis, genome size variation and pollen production in the arctic-alpine species Hieracium alpinum

Hieracium alpinum L. (Asteraceae) is an arctic-alpine species distributed throughout Europe with both diploid and triploid cytotypes. We determined the ploidy levels of plants from 23 populations from Austria, Bosnia and Herzegovina, Finland, Italy, Norway, Romania, Slovakia, Switzerland and Ukraine. Data showed a non-overlapping pattern of cytotype distribution: sexually reproducing diploids (2n = 2x = 18) occur solely in the Eastern and Southern Carpathians, while apomictic triploids (2n = 3x = 27) cover the rest of the range. Such clear-cut allopatry is rather rare in vascular plants with geographical parthenogenesis. Comparison of absolute genome size indicates genome downsizing (by on average 3.7%) of haploid DNA amount in triploids relative to diploids. Genome size further correlated with longitude and latitude in the Alps, with decreasing absolute DNA content from west to east, and from south to north. While previously published data indicated complete male sterility of triploid plants, we found that plants from the Alps and Bosnia and Herzegovina commonly produced some pollen, whereas populations from the Western Carpathians and Scandinavia seemed to be almost completely pollen sterile. Scenarios about the evolution of geographical parthenogenesis in H. alpinum are discussed.

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Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

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Résumé   Hieracium alpinum L. (au sens strict) est une espèce arctique-alpine d’aire de répartition très large, comprenant les régions nordiques (le Groenland, l’Islande, l’Ecosse, la Scandinavie et le nord de la Russie) et les montagnes de l’Europe continentale (les Alpes, les Carpates, les Sudètes, les Vosges et le plateau de Vranica). Dans cette étude, nous avons compté le nombre chromosomique et estimé la plo?die par cytométrie de flux de plantes provenant de 23 populations échantillonnées en Autriche, Bosnie et Herzégovine, Finlande, Italie, Norvège, Roumanie, Slovaquie, Suisse et Ukraine. Ces données et celles de la littérature montrent une nette séparation spatiale entre deux cytotypes différents: Les populations diplo?des sexuées sont réparties uniquement dans les Carpates orientales et occidentales (Roumanie et Ukraine), tandis que les populations triplo?des apomictiques occupent l’aire de répartition restante. Ce type d’allopatrie stricte est rare chez les plantes avec parthénogenèse géographique. En comparant la taille du génome haplo?de (1Cx) des plantes triplo?des avec celui des plantes diplo?des, nous avons identifié une sensible réduction de taille du génome polyplo?de (la divergence moyenne est 3.7%). Parmi les plantes triplo?des, les individus du plateau de Vranica (Bosnie et Herzégovine) ont significativement moins d’ADN que les triplo?des provenant des Alpes ou des Carpates occidentales (2C = 10.28 pg d’ADN contre 11.02 et 10.93 pg, respectivement). Une corrélation significative entre la taille du génome et la longitude et la latitude a été révélée dans les Alpes, avec des valeurs décroissantes d’ouest en est, et du sud vers le nord. Tandis que les données publiées indiquaient une stérilité male complète chez les triplo?des, nous avons trouvé des plantes triplo?des provenant des Alpes et du plateau de Vranica produisant du pollen, bien qu’en faible quantité et de taille hétérogène. Divers scénarios sur l’évolution de la parthénogénèse géographique chez H. alpinum sont discutés à la lumière de ces nouveaux résultats.

     

Androgenesis in recalcitrant solanaceous crops

Tomato, eggplant, and pepper are three solanaceous crops of outstanding importance worldwide. For hybrid seed production in these species, a fast and cheap method to obtain pure (homozygous) lines is a priority. Traditionally, pure lines are produced by classical inbreeding and selection techniques, which are time consuming (several years) and costly. Alternatively, it has become possible to accelerate the production of homozygous lines through a biotechnological approach: the induction of androgenesis to generate doubled haploid (homozygous) plants. This biotechnological in vitro tool reduces the process to only one generation, which implies important time and costs savings. These facts make androgenic doubled haploids the choice in a number of important crops where the methodology is well set up. Unfortunately, recalcitrant solanaceous crops such as tomato, eggplant, and pepper are still far from an efficient and reliable technology to be applied on a routine basis to different genotypes in breeding programs. In eggplant and pepper, only anther cultures are known to work relatively well. Unfortunately, a more efficient and promising technique, the culture of isolated microspores, is not sufficiently developed yet. In tomato, none of these methods is available nowadays. However, recent advances in the knowledge of embryo development are filling the gaps and opening new ways to achieve the final goal of an efficient protocol in these three recalcitrant species. In this review, we outline the state of the art on androgenic induction in tomato, eggplant, and pepper, and postulate new experimental ways in order to overcome current limitations.     

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.     

Multiple origins of parthenogenesis,and a revised species phylogeny for the Southeast Asian butterfly lizards,Leiolepis

Hybridization often occurs in areas of secondary contact between closely related species. In some cases these hybridization events can create hybrid offspring that are reproductively viable as new parthenogenetic species. The genus Leiolepis contains nine species that collectively range throughout continental Southeast Asia. Of these, four are unisexual (some diploid and some triploid). We analyzed a multi‐locus dataset within a multi‐lineage coalescent framework to infer the origins of these parthenogenetic hybrid species. Our results provide evidence that repeated hybridization events between L. reevesii and L. guttata have led to the formation of all four distinct parthenogenetic species. Our data further suggest there have been low levels of mitochondrial introgression between L. belliana and L. reevesii at their contact zone in southern Cambodia. This work addresses contentious species boundaries and provides the first taxon‐complete hypothesis of relationships for the butterfly lizards. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 1080–1093.     

Karyotypes, supernumerary chromosomes, rudimentary parthenogenesis and polyploidy in 2 species of the genus Phronima (Crustacea, Amphipoda)]

The diploid number of Phronima sedentaria and P. atlantica is 30, all the chromosomes are metacentric or submetacentric; the caryotypes of these two species are compared with those of other Amphipods. Some individuals of each species have a supernumerary chromosome. This element, found in both sexes, remains as an univalent at meiosis and goes earlier to one pole. In some cases nonfertilized ova undergo segmentation; this rudimentary parthenogenesis stops quickly and is partly related to polyploidization.