Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Why are triploid zebrafish all male?

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal.     

Effect of ploidy variation on amino acid composition ofDatura innoxia (Solanaceae)

In haploid, diploid, triploid, tetraploid and hexaploid cytotypes of the scopalamine-yieldingDatura innoxia, the amino acid profiles were studied. The results suggest that the amino acid composition is influenced quantitatively by increasing ploidy levels, but in an irregular way.     

Durum wheat as a candidate for the unknown female progenitor of bread wheat: an empirical study with a highly fertile F<Subscript>1</Subscript> hybrid with <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss.

Hexaploid bread wheat was derived from a hybrid cross between a cultivated form of tetraploid Triticum wheat (female progenitor) and a wild diploid species, Aegilops tauschii Coss. (male progenitor). This cross produced a fertile triploid F₁ hybrid that set hexaploid seeds. The identity of the female progenitor is unknown, but various cultivated tetraploid Triticum wheats exist today. Genetic and archaeological evidence suggests that durum wheat (T. turgidum ssp. durum) may be the female progenitor. In previous studies, however, F₁ hybrids of durum wheat crossed with Ae. tauschii consistently had low levels of fertility. To establish an empirical basis for the theory of durum wheat being the female progenitor of bread wheat, we crossed a durum wheat cultivar that carries a gene for meiotic restitution with a line of Ae. tauschii. F₁ hybrids were produced without using embryo rescue techniques. These triploid F₁ hybrids were highly fertile and spontaneously set hexaploid F₂ seeds at the average selfed seedset rate of 51.5%. To the best of our knowledge, this is the first example of the production of highly fertile F₁ hybrids between durum wheat and Ae. tauschii. The F₁ and F₂ hybrids are both similar morphologically to bread wheat and have vigorous growth habits. Cytological analyses of F₁ male gametogenesis showed that meiotic restitution is responsible for the high fertility of the triploid F₁ hybrids. The implications of these findings for the origin of bread wheat are discussed.     

Synthetic polyploid production of Miscanthus sacchariflorus,Miscanthus sinensis,and Miscanthus x giganteus

Plants from the genus Miscanthus are potential renewable sources of lignocellulosic biomass for energy production. A potential strategy for Miscanthus crop improvement involves interspecific manipulation of ploidy levels to generate superior germplasm and to circumvent reproductive barriers for the introduction of new genetic variation into core germplasm. Synthetic autotetraploid lines of Miscanthus sacchariflorus and Miscanthus sinensis, and autoallohexaploid Miscanthus x giganteus were produced in tissue culture from oryzalin treatments to seed‐ and immature inflorescence‐derived callus lines. This is the first report of the genome doubling of diploid M. sacchariflorus. Genome doubling of diploid M. sinensis, M. sacchariflorus, and triploid M. x giganteus to generate tetraploid and hexaploid lines was confirmed by stomata size, nuclear DNA content, and chromosome counts. A putative pentaploid line was also identified among the M. x giganteus synthetic polyploid lines by nuclear DNA content and chromosome counts. Comparisons of phenotypic performance of synthetic polyploid lines with their diploid and triploid progenitors in the greenhouse found species‐specific differences in plant tiller number, height, and flowering time among the doubled lines. Stem diameter tended to increase after polyploidization but there were no significant improvements in biomass traits. Under field conditions, M. x giganteus synthetic hexaploid lines showed greater phenotypic variation, in terms of plant height, stem diameter, and tiller number, than their progenitor lines. Production of synthetic autopolyploid lines displaying significant phenotypic variation suggests that ploidy manipulation can introduce useful genetic diversity in the limited Miscanthus germplasm currently available in the United States. The role of polyploidization in the evolution and breeding of the genus Miscanthus is discussed.     

Comparison of triploid and diploid cucumber in long-term liquid cultures

Triploid suspensions generally grew more vigorously in modified MS medium with 2,4-D than those of diploids. The embryogenic potential of 26-month-old auxin-dependent suspension cultures depended on the line. Neither triploid nor diploid BOR (Borszczagowski line) were able to produce somatic embryos. Similarly, 12–20-month-old cytokinin-dependent suspensions from the same triploid line were not capable of regeneration. Only aggregates from 26-month-old auxin-dependent suspension of triploid line 603 differentiated into somatic embryos. In contrast, 18-month-old diploid and triploid liquid cultures of meristematic clumps (LMC) of BOR retained their regeneration potential. The ploidy level of triploid and diploid auxin-dependent suspension cultures was stable during the first 8 months. However, the ploidy level of triploids remained stable over 26 months of culture, whereas 66.7% of diploid cultures underwent chromosome doubling. No ploidy changes were observed among plants regenerated from 18-month-old LMC. Our data suggest that loss of embryogenic potential in suspension culture was independent of ploidy level.     

Polyploidy and aneuploidy inOphrys,Orchis, andAnacamptis (Orchidaceae)

Studies on chromosome numbers and karyotypes in Orchid taxa from Apulia (Italy) revealed triploid complements inOphrys tenthredinifera andOrchis italica. InO. tenthredinifera there is no significant difference between the diploid and the triploid karyotypes. The tetraploid cytotype ofAnacamptis pyramidalis forms 36 bivalents during metaphase I in embryo sac mother cells. Aneuploidy was noticed inOphrys bertolonii ×O. tarentina with chromosome numbers n = 19 and 2n = 38. There were diploid (2n = 2x = 36), tetraploid (2n = 4x = 72), hexaploid (2n = 6x = 108) and octoploid (2n = 8x = 144) cells in the ovary wall of the diploid hybridOphrys apulica ×O. bombyliflora. Evolutionary trends inOphrys andOrchis chromosomes are discussed.     

Contrasting reproductive strategies of triploid hybrid males in vertebrate mating systems

The scarcity of parthenogenetic vertebrates is often attributed to their ‘inferior’ mode of clonal reproduction, which restricts them to self‐reproduce their own genotype lineage and leaves little evolutionary potential with regard to speciation and evolution of sexual reproduction. Here, we show that for some taxa, such uniformity does not hold. Using hybridogenetic water frogs (Pelophylax esculentus) as a model system, we demonstrate that triploid hybrid males from two geographic regions exhibit very different reproductive modes. With an integrative data set combining field studies, crossing experiments, flow cytometry and microsatellite analyses, we found that triploid hybrids from Central Europe are rare, occur in male sex only and form diploid gametes of a single clonal lineage. In contrast, triploid hybrids from north‐western Europe are widespread, occur in both sexes and produce recombined haploid gametes. These differences translate into contrasting reproductive roles between regions. In Central Europe, triploid hybrid males sexually parasitize diploid hybrids and just perpetuate their own genotype – which is the usual pattern in parthenogens. In north‐western Europe, on the other hand, the triploid males are gamete donors for diploid hybrids, thereby stabilizing the mixed 2n‐3n hybrid populations. By demonstrating these contrasting roles in male reproduction, we draw attention to a new significant evolutionary potential for animals with nonsexual reproduction, namely reproductive plasticity.     

Diploid Ancestors of Triploid Export Banana Cultivars: Molecular Identification of 2<Emphasis Type="Italic">n</Emphasis> Restitution Gamete Donors and n Gamete Donors

The origin of triploid export banana cultivars was investigated. They all belong to Cavendish and Gros Michel subgroups of triploid clones and have a monospecific Musa acuminata origin. The appearance of these cultivars is thought to be result of hybridization between partially sterile diploid cultivars producing non reduced gametes and fertile diploids producing normal haploid gametes. To trace these diploid ancestors we compared the RFLP patterns, revealed by 36 probe/enzyme combinations, of 176 diploid clones representing the worldwide available variability with that of clones from the Cavendish and Gros Michel subgroups. This lead us to the identification of the common putative diploid ancestor of cultivars from Cavendish and Gros Michel subgroups which contributed to triploid cultivar formation through the production of 2n restitution gametes. For cultivars of Gros Michel subgroup we also propose a normal gamete donor that may have complemented the triploid allele set.     

A natural triploid in Trichomycterus davisi (Siluriformes,Trichomycteridae): mitotic and meiotic characterization by chromosome banding and synaptonemal complex analyses

Within a total of 50 analyzed specimens a male individual of Trichomycterus davisi has been recorded with 81 chromosomes including 60 metacentric, 18 submetacentric and three subtelocentric chromosomes. When compared with diploid individuals (2n = 54) and the morphological standard of chromosomes, this male is a triploid with 3n = 81 chromosomes. Since staining with silver nitrate indicates three active nucleolar organizer regions (NORs), the three NOR-bearing chromosomes in this individual are genetically active. Analysis of the synaptonemal complex (SC) by electronic microscopy shows that there is an incomplete pairing of the third set of chromosomes in the triploid individual.     

三倍体丹参杂交种的花粉形态研究

三倍体丹参是以二倍体丹参为父本、人工染色体加倍的四倍体白花丹参为母本杂交选育的杂交种。为深入了解三倍体丹参花粉的特性,以及为三倍体种质利用提供孢粉学依据,该文以二倍体丹参为对照,研究了三倍体丹参杂交种花粉的形态变异规律。利用光学显微镜和扫描电镜对二倍体和三倍体丹参的花粉萌发沟、外壁纹饰、花粉粒形状等特征进行了显微和超微形态观察,综合进行了花粉形态差异比较,并对花粉大小和形状数据进行了差异显著性分析和正态检验。结果表明:(1)二倍体丹参为6沟花粉,三倍体花粉萌发沟有6沟和8沟两种类型,沟内疣状颗粒分布不匀,出现畸形萌发沟。(2)二倍体和三倍体花粉外壁均为网状雕纹。二倍体花粉网眼内具多个多边形穿孔,穿孔大; 6沟和8沟两种类型的三倍体花粉网眼无穿孔或仅有几个小穿孔,6沟和8沟花粉的外壁雕纹相同。(3)三倍体花粉的极轴长(P)和赤道宽(E)均值显著小于二倍体花粉,花粉大小呈偏正态分布,P*E的差异系数大于二倍体花粉,且有极值存在。三倍体和二倍体丹参的萌发沟和雕纹存在差异,而花粉形状差异不显著。综上结果表明三倍体丹参花粉在倍性效应和杂合性的双重影响下发生了形态变异,且有多种形态变化。     

Triploid females and diploid males: underreported phenomena in <Emphasis Type="Italic">Polistes</Emphasis> wasps?

Summary In hymenopteran species, males are usually haploid and females diploid. However, in species that have complementary sex determination (CSD), diploid males arise when a female produces offspring that are homozygous at the sex-determining locus. Although diploid males are often sterile, in some species they have been shown to produce diploid sperm, thus producing triploid daughters if they mate successfully. Diploid males have been observed in very few species of social wasps, and we know of no published reports of triploid females. In this paper, we review the existing literature on diploid males and triploid females in the Hymenoptera, and report the observation of triploid females in three species of Polistes paper wasps. Although polyploid offspring may be produced parthenogenetically, the more likely scenario is that Polistes wasps have CSD and produce diploid males via homozygosity at the sex-determining locus. Therefore, female triploidy indicates that diploid males do exist in Polistes species where they are presumed to be absent, and are likely to be even more frequent among species that have experienced a genetic bottleneck. We conclude by cautioning against the assumption of a selective advantage to the production of early males, and by discussing the implications of male diploidy and female triploidy for measurement of sex ratio investment and assumptions of reproductive skew theory.Received 5 December 2003; revised 20 March 2004; accepted 19 April 2004.     

Effect of oilseed rape genotype on the spontaneous hybridization rate with a weedy species:an assessment of transgene dispersal

Spontaneous outcrossing of different malesterile rapeseed lines and transgenic hybrids with a population of a weedy species, Raphanus raphanistrum L., has led to the harvest of numerous seeds showing a size dimorphism. Flow cytometry analysis correlated with chromosome counts showed that all of the large seeds belonged to rapeseed, whereas the small seeds were a mixture of mostly interspecific triploid hybrids, with some trigenomic amphidiploids, diploid and haploid rapeseed plants. Significant differences were revealed between the rapeseed lines and transgenic hybrids in their ability to form interspecific hybrids with Raphanus raphanistrum under natural conditions. Resistance to the herbicide Basta was properly expressed in the triploid and amphidiploid hybrids. Low male fertility of the interspecific triploid hybrids was not correlated with seed set in the subsequent generation.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos

Between 1 and 4% of human oocytes fertilized in vitro are tripronuclear. It has been reported that these tripronuclear oocytes can develop to grossly normal-appearing morulae and that chromosomally, these embryos could be triploid, diploid, or severely depleted. The etiology and proportion of apparently diploid and aneuploid embryos deriving from tripronuclear human oocytes is unknown. This study provides evidence for the first time that most (18 of 29) tripronuclear human oocytes cleave directly to 3-cells at the first cleavage division. These embryos have a severely abnormal (but not triploid) chromosomal complement. Furthermore, some (4 of 29) tripronuclear human oocytes cleave to 2-cells plus an extrusion, and these embryos are diploids, whereas some (7 of 29) cleave to 2-cells, and these embryos are triploid after the first cleavage division. These findings demonstrate that most tripronuclear human oocytes have an altered cleavage pattern at the first cleavage division, that most tripronuclear human oocytes (76% in this study) do not develop into triploid embryos, and that a correlation exists between the pattern of the first cleavage division and the subsequent karyotype of these embryos. Insight into the mechanisms by which these oocytes fail to develop into triploid embryos is also provided.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

DNA markers for sex: Molecular evidence for gender dimorphism in dioecious Mercurialis annua L.

Male specific Random Amplified Polymorphic DNA (RAPD) markers, OPB01-1562 and OPC07-303, were identified and sequenced in dioecious Mercurialis annua. Sequence Characterized Amplified Region (SCAR) primers were designed. Several internal segments of OPB01-1562 were amplified as male specific SCAR markers. These markers were PCR amplified from strong, intermediate and weak male subtypes selected according to their resistance to feminization by cytokinin. Nucleotide sequence of OPB01-1562 isolated from three male subtypes were near identical. The OPB01-1562 and derived SCAR markers were absent in females as well as hexaploid Mercurialis male and monoecious individuals. The gender relationship of the markers was maintained in all ecotypes tested. There were 2 internal fragments of OPB01-1562, which were PCR amplified from all genotypes of diploid and hexaploid Mercurialis. It is argued that identification of gender specific DNA suggests a dimorphic differentiation of the genome of dioecious Mercurialis annua.     

Synthesis and evaluation of Triticum durum — T. monococcum amphiploids

Summary Nine Triticum durum — T. monococcum amphiploids (AABBA^mA^m) were synthesized by chromosome doubling of sterile triploid F₁ hybrids involving nine T. durum (AABB) cultivars and a T. monococcum (A^mA^m) line. The triploid F₁ hybrids had a range of 4–7 bivalents and 7–13 univalents per PMC. The synthetic amphiploids, however, showed a high degree of preferential pairing of chromosomes of the A genomes of diploid and tetraploid wheats. The amphiploids were meiotically stable and fully fertile. Superiority of four amphiploids for tiller number per plant, 100-grain weight, protein content and resistance to Karnal bunt demonstrated that these could either be commercially exploited as such after overcoming certain inherent defects or used to introgress desirable genes into durum and bread wheat cultivars. Methods for improvement of these amphiploids are discussed.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.