Tetraploid Males Research Articles

Impact of seasonality on the reproductive physiology of diploid and tetraploid eastern oyster, Crassostrea virginica

Aquaculture production of triploid Eastern oyster, Crassostrea virginica, in the Gulf of Mexico (GoM) is reliant on naturally conditioned diploid females and tetraploid males. However, limited knowledge is available on their reproductive biology, including the gametogenic cycle. The aim was to quantify monthly changes in gametogenesis and gamete quality for triploid production in Eastern oyster in the GoM from June 2022 to July 2023. Monthly changes in temperature and salinity were recorded. Shell morphometrics and sex were determined. Sperm density, sperm activity, Spermatogenic Maturity Index (SMI), oocyte diameter, Oogenesis Maturity Index (OMI), and gonad coverage area were quantified. Mean monthly temperature ranged from 15.1 to 31.2 °C and salinity ranged from 12.5 to 27.6 PSU. Shell morphometrics increased over the annual cycle. When gametes were detectable, 48–80% of tetraploids and 45–70% of diploids were males. Hermaphrodites were identified in both ploidies in June; an additional tetraploid hermaphrodite was identified in March. Sperm density exhibited monthly fluctuations with high densities reported in June–July, September, and April–May. Sperm were motile when present, with high motility in June–July. High sperm velocity was also recorded in June–July and March–May. Tetraploids exhibited high SMI from June–September and April–May with increased variability in December–January. Mature sperm cells were predominant in tetraploids in June–July. Gonadal coverage was high from June–September and March–May. OMI was highest from June–September and March–May, while variability was high from August–March. Large oocyte diameters and gonad coverage areas were observed in June–July, September, and April–May. Together, these findings provide novel insights into reproductive dynamics for triploid production of Eastern oyster in GoM.

Successive selection responses of tetraploid Crassostrea angulata: Comparison of growth, survival, sex ratio and ploidy composition between selected and unselected groups

Triploid Crassostrea angulata has the advantages of fast growth, poor fertility and high quality. However, due to poor fertility, direct genetic improvement is difficult. Mating between tetraploid males and diploid females has been shown to be the best method for commercial triploid production, so genetic improvement of tetraploids will directly affect the phenotypic traits of triploid offspring. However, to date, few studies have investigated the genetic improvement of tetraploid oysters. Based on the induced tetraploid C. angulata population, this study carried out three successive generations of growth selective breeding research, and systematically compared the growth, survival, ploidy composition and sex ratio of the selected group and the control group. The results showed that there was no significant difference in the survival rate from planktonic larvae to adult between the two groups. However, the growth of the selected group was significantly higher than that of the control group, that was, the shell height and whole weight of S3 were significantly higher than that of C3, and the selection advantage of shell height increased from 7.80% of S1 to 34.68% of S3. The ploidy composition of the two tetraploid groups was relatively stable before the 90th days, but on the 270th day, some tetraploids underwent chromosome loss and transformed into diploids or triploids, which might be related to gametogenesis of tetraploids. Moreover, with the increase of generations, the number of individuals with chromosome loss in the selected group gradually decreased, and their ploidy became more stable than that in the control group. In addition, both groups were fertile, with a sex ratio of more males than females, and a proportion of hermaphrodite individuals. In conclusion, this study successfully selected a fast growth line of tetraploid C. angulata, which can be used for commercial production of fast-growing triploid C. angulata, and provides a basis and reference for the genetic improvement of tetraploid oysters.

Deviations in the Z:A ratio disrupt sexual development in the eri silkmoth, Samia cynthia ricini.

Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety, and 2 models, W-dominance and Z-counting, have been proposed to determine sex. The W-dominant mechanism is well known in Bombyx mori. However, little is known about the Z-counting mechanism in Z0/ZZ species. We investigated whether ploidy changes affect sexual development and gene expression in the eri silkmoth, Samia cynthia ricini (2n = 27♀/28♂, Z0♀/ZZ♂). Tetraploid males (4n = 56, ZZZZ) and females (4n = 54, ZZ) were induced by heat and cold shock, and then, triploid embryos were produced by crosses between diploids and tetraploids. Two karyotypes (3n = 42, ZZZ and 3n = 41, ZZ) were identified in triploid embryos. Triploid embryos with 3 Z chromosomes showed male-specific splicing of the S. cynthia doublesex (Scdsx) gene, whereas 2-Z triploid embryos showed both male- and female-specific splicing. From larva to adult, 3-Z triploids showed a normal male phenotype, except for defects in spermatogenesis. However, abnormal gonads were observed in 2-Z triploids, which showed both male- and female-specific Scdsx transcripts not only in the gonads but also in somatic tissues. Two-Z triploids were thus obviously intersexes, suggesting that sexual development in S. c. ricini depends on the Z:A ratio and not only on the Z number. Moreover, mRNA-seq analyses in embryos showed that relative levels of gene expression are similar between samples with different doses of Z chromosomes and autosome sets. Our results provide the first evidence that ploidy changes disrupt sexual development but have no effect on the general mode of dosage compensation in Lepidoptera.

Comparative analysis of growth, survival and sex proportion among tetraploid-based autotriploid (Crassostrea gigas and C. angulata) and their allotriploid oysters

Triploid is widely cultivated in China and many other oyster-producing countries for the advantages of fast growth, favorable infertility and better meat quality. However, triploid Crassostrea gigas is found to have disadvantages of slow growth in later grow-out stage and high summer mortality, while the triploid C. angulata has the disadvantages of fragile shells and a relatively high ratio of developed gonads in population, and therefore, both of them have space for further improvement. Due to the limitations of poor fertility and low survival of offspring, triploid genetic improvement cannot be carried out directly. Hybridization between species is a useful method for indirect genetic improvement of triploids, and natural crosses between tetraploid males and diploid females have proven to be the best method for commercial promotion of triploids. The growth, survival and gonad development of autotriploids (DGTG-diploid C. gigas ♀ × tetraploid C. gigas ♂, DATA-diploid C. angulata ♀ × tetraploid C. angulata ♂) and allotriploids (DATG-diploid C. angulata ♀ × tetraploid C. gigas ♂, DGTA-diploid C. gigas ♀ × tetraploid C. angulata ♂) were investigated in this study. It is showed that the fertilization rates of autotriploids were significantly higher than that of allotriploids, and DATG larvae grew significantly faster than the other three groups (P < 0.05). DGTA had significant shell height, whole weight and survival advantages at Laizhou site (P < 0.05), while DATG exhibited significantly fastest growth and highest survival at Zhangpu site (P < 0.05), but both DATG and DATA showed significantly faster growth and higher survival at Beihai site (P < 0.05), with no significant difference between them (P > 0.05). Sites, genotypes and their interaction always presented significant effects on shell height and whole weight from the 90th to 360th day, but not on survival with the 180th day as an exception. Gonad examination confirmed that the proportions of fertile individuals in the groups with maternal C. gigas (DGTG and DGTA) were lower than that in the groups with maternal C. angulata (DATA and DATG), and DATA always had the highest rate of developed gonad at three sites. These results indicate that DGTA has advantages in growth, survival and infertility in northern China while DATG performs best in southern China, which exhibits that there is a significant maternal effect of hybridization in allotriploids, and promise a guidance for their commercial production.

Successful creation of seedless (sterile) leucaena germplasm developed from interspecific hybridisation for use as forage

Context The legume shrub/tree leucaena (Leucaena leucocephala (Lam.) de Wit.) is highly regarded as a cattle fodder, although it is considered an environmental weed in many parts of Australia. Aims We investigated the feasibility of developing a forage variety of triploid leucaena through interspecific hybridisation that is sterile (without seeds) as a strategy to mitigate its weed risk. Methods A conventional breeding strategy was used to create triploid hybrids from hand-crosses between tetraploid and diploid species of leucaena. Molecular markers were developed to confirm successful crosses and flow cytometry was used to confirm the ploidy level. The plants are being evaluated for flowering behaviour, seed and herbage production across two environmentally diverse sites at Carnarvon and Kununurra in northern Western Australia. Key results During 2018/19 and 2019/20, 2260 crosses between 45 different combinations of diploid females by tetraploid males and their reciprocals have created over 3000 putative triploid hybrid plants. This is the first time that triploids have successfully been created in Australia and 10 new parental combinations were created as a world-first. Conclusions A cohort of sterile triploid plants has been identified and selections will be made based on their edible biomass productivity, insect tolerance, capacity for regrowth after cutting, nutritive value and plant chemistry (including anti-methanogenic responses). A sterile leucaena variety will have application not only in Australian grazing systems but also in other tropical regions of the world. Implications A cost-effective technology for mass vegetative propagation of leucaena will be required for commercialisation of a sterile variety.

Tetraploid induction of Crassostrea hongkongensis and C. sikamea by inhibiting the polar body 1 release in diploid fertilized eggs.

The production of an all-triploid population by mating tetraploid males with diploid females is the best and most fundamental method for the large-scale production of triploid oysters. Obtaining a stable tetraploid population is essential for guaranteed production in industrialized triploid cultivation. C. hongkongensis and C. sikamea are important oyster breeding species in southern China, and have great economic value. However, there are not any published data on inducing tetraploid C. hongkongensis or C. sikamea. Therefore, we investigated tetraploid induction in these two oyster species by inhibiting the PB1 release in diploid fertilized eggs using Cytochalasin B (CB) under 31°C, 15 ‰ salinity. The results confirmed that the optimal tetraploid induction conditions for C. hongkongensis were a CB concentration of 0.50mg/L with induction starting at 9.0min after fertilization, and stopping at 21.0min after fertilization; the induction efficiency index reached 0.123 under these conditions. The optimal tetraploid induction conditions for C. sikamea were a CB concentration of 0.50mg/L, with induction starting at 7.5min after fertilization and stopping at 18min after fertilization; the induction efficiency index could be as high as 0.281 under these conditions. However, we confirmed that the tetraploid rate decreased with larval growth, and no tetraploids were detected in the juvenile period of either C. hongkongensis or C. sikamea. This may be attributed to the very low survival of the tetraploid larvae induced by this method, especially as most tetraploid larvae died during the first three days. In summary, it is simple to directly induce tetraploid C. hongkongensis and C. sikamea larvae by inhibiting the PB1 release of diploid zygotes, but the low survival rate makes it challenging to obtain viable juvenile tetraploids.

Diploid and aneuploid sperm in tetraploid ginbuna, Carassius auratus langsdorfii

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratusauratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types.

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish.

Polyploids in vertebrates are generally associated with unisexual reproduction, but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown. Here, we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus. The synthetic octoploids were revealed to have more than 200 chromosomes, in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C. auratus into the unisexual gynogenetic hexaploid C. gibelio. Significantly, a few synthetic octoploid males were found to be fertile, and one octoploid male was confirmed to regain sexual reproduction ability, which exhibits characteristics that are the same to sexual reproduction tetraploid males, such as 1:1 sex ratio occurrence, meiosis completion and euploid sperm formation in spermatogenesis, as well as normal embryo development and gene expression pattern during embryogenesis. Therefore, the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.

High percentages of larval tetraploids in the yellowtail tetra Astyanax altiparanae induced by heat-shock: The first case in Neotropical characins

Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra Astyanax altiparanae, triploids present sterility and increased carcass yield, suggesting the generation of tetraploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40° for 2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 °C were compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping, and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 ± 9.6%), 24 (16.0 ± 9.1%), and 26 mpf (10.3 ± 6.2%) in comparison with the control group (62.2 ± 7.7%). The flow cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf presented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat shock treatment at 26 mpf (40 °C for 2 min) is optimum for tetraploid induction in the A. altiparanae. Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in basic and applied sciences.

Pollen donor effects on the expression of red flesh colour in kiwifruit

The choice of pollen donor may sometimes affect fruit characteristics of kiwifruit. In this study, flowers of a red-fleshed kiwifruit selection were hand-pollinated with pollen from males of diploid and tetraploid Actinidia chinensis var. chinensis and hexaploid A. chinensis var. deliciosa. The resulting fruit were evaluated for key commercial traits after storage. All fruit had intense red colour in the centre, but those from pollination by the diploid or tetraploid males had significantly more intense red coloration than those from pollination by the hexaploid male. Malic acid, total acid, sucrose and soluble solids concentrations, titratable acidity and total seed weight were all significantly higher following pollination by the diploid male. There were no significant differences among pollen donors in fruit weight or seed number, or in fructose, glucose, total sugar or quinic acid concentrations. These results contribute to our general understanding of pollen donor effects on fruit development, and will be of practical interest to kiwifruit growers when choosing males suitable for pollination of red-fleshed female cultivars.

Viable diploid progeny induced from sperm of Chinese tetraploid pond loach by cold-shock androgenesis

Natural tetraploid loach (Misgurnus anguillicaudatus) living in the Changjiang River basin and adjacent area in China has been considered a true genetic tetraploidy (4n = 100) with four sets of homologous chromosomes. Here, we reported its further supporting evidence provided by the cold-shock androgenesis. Viable androgenetic progeny appeared when eggs were cold-shocked at 3°C for 60 min, starting 5 min after fertilization with sperm of natural tetraploid males, although the survival rate was significantly lower than control group. The majority (87%) of androgenetic embryos were diploid (2n = 50) and all-paternal inheritance was verified in larval stage by microsatellite genotypes. Microscopic observations confirmed the elimination of both egg nucleus and second polar body from a fertilized egg, followed by the cleavage exclusively with sperm nucleus. Thus, the appearance of normal diploid androgenetic progeny from sperm of natural tetraploid loach revealed the presence of four chromosome sets in tetraploid males.

Evidence of the sterility of allotetraploid Cobitis loaches (Teleostei, Cobitidae) using testes ultrastructure.

The diploid-polyploid populations of Cobitis distributed in Poland are usually composed of the spined loach Cobitis taenia or, less often, the Danubian loach C. elongatoides and their triploid (females) and tetraploid hybrids (females and males). The aim of this study was to determine whether tetraploid males participate in the reproduction process by analyzing their testis ultrastructure and the process of spermatogenesis in comparison with diploid males of both parental species. Tetraploid loaches were obtained from three different diploid-polyploid populations distributed in Poland. The structure of Cobitis testes are typical for most Teleostei fish with cystic-type spermatogenesis. The successive stages of developing germ cells are enclosed within cysts formed by the Sertoli cells. This paper morphologically describes the different germ cell stages of spermatogenesis (spermatogonia, spermatocytes, spermatids, and spermatozoa) of C. taenia and C. elongatoides and provides a pioneering ultrastructural analysis of tetraploid Cobitis testes which reveals their unusual structure for the first time. Thus, cysts with normal spermatogonia and spermatocytes (pachyten or leptoten stages) containing synaptonemal complexes were present and no spermatids or spermatozoa were observed. Moreover, in contrast to previously analyzed diploid species, single cells or all of the cells within the cysts displayed chromatin condensation and/or chromatin fragmentation. The obtained results clearly demonstrated that tetraploid males are sterile and diploids are fertile and are the only sperm donors in the reproduction processes of diploid-polyploid Cobitis populations.

Aneuploid progeny of the American oyster, Crassostrea virginica, produced by tetraploid × diploid crosses: another example of chromosome instability in polyploid oysters.

The commercial production of triploids, and the creation of tetraploid broodstock to support it, has become an important technique in aquaculture of the eastern oyster, Crassostrea virginica. Tetraploids are produced by cytogenetic manipulation of embryos and have been shown to undergo chromosome loss (to become a mosaic) with unknown consequences for breeding. Our objective was to determine the extent of aneuploidy in triploid progeny produced from both mosaic and non-mosaic tetraploids. Six families of triploids were produced using a single diploid female and crossed with three mosaic and non-mosaic tetraploid male oysters. A second set of crosses was performed with the reciprocals. Chromosome counts of the resultant embryos were tallied at 2-4 cell stage and as 6-hour(h)-old embryos. A significant level of aneuploidy was observed in 6-h-old embryos. For crosses using tetraploid males, aneuploidy ranged from 53% to 77% of observed metaphases, compared to 36% in the diploid control. For crosses using tetraploid females, 51%-71% of metaphases were aneuploidy versus 53% in the diploid control. We conclude that somatic chromosome loss may be a regular feature of early development in triploids, and perhaps polyploid oysters in general. Other aspects of chromosome loss in polyploid oysters are also discussed.

Genetic and environmental influence on triploid Crassostrea virginica grown in Chesapeake Bay: Growth

Aquaculture of triploid Crassostreavirginica in the Chesapeake Bay, USA is popular with the majority of growers raising mostly triploids. This choice stems from improved growth and survival relative to diploids. Triploids are made commercially by crossing tetraploid males and diploid females, thus either or both parent could influence the performance of the triploid. We examined the influence of the female (diploid) parent in an experiment comparing nine diploid (2n×2n) and nine triploid (4n×2n) crosses across three environments. The diploid genotypes consisted of wild populations and selectively bred lines and the environments ranged from low salinity with no disease pressure to higher salinity with consistent disease pressure. Growth metrics (shell height and wet tissue weight) are reported. In the low salinity environment, triploidy seemed disadvantageous for any genotype at least when gauged by final tissue weight. In moderate salinity with low disease pressure, selectively bred diploids performed equivalently to their triploid counterparts showing that breeding efforts can improve diploid field performance to rival triploids. In high salinity with constant disease pressure, triploids offered the greatest advantage for growth. Genotype×environmental response of triploids indicates that breeders may have to tailor crosses to suit particular areas interested in using triploids. Statement of relevanceWe address factors of commercial triploid oyster performance.

Gamete quality in triploid Pacific oyster (Crassostrea gigas)

Triploidy induction in shellfish aims to obtain faster growth and sterility of reared individuals. Triploid Pacific oysters are most often not totally sterile, but have greatly reduced reproductive capacities compared to those recorded in diploid individuals. The description of gamete output in triploid aquatic animals is most often confined to the assessment of fecundity in females. The present work aims at further describing gamete quality of triploid Pacific oysters and comparing it to values observed in diploid individuals. Diploid and triploid oysters (produced by mating tetraploid males×diploid females) were reared in standard conditions and then transferred to the wild to allow the offspring to attain reproductive maturity. At the end of gametogenesis, the reproductive characteristics of both groups were estimated.After gonad sampling, gametes could be observed in 92.9% diploid oysters compared with 42.0% in triploids. A higher number of spermatozoa was produced in diploids (344±106×109 spermatozoa) compared with triploids (5±7×109 spermatozoa). Furthermore, the percentage of motile sperm and sperm swimming speed were higher in diploids compared with triploids. In contrast, a higher intracellular ATP content was found in triploids (99.1±34.0nmole10−9 sperm) than in diploids (63.6±20.7nmole10−9 sperm). A higher number of oocytes was collected from diploid oysters (19.1±3.8×106 oocytes), than from triploid ones (0.1±0.1×106 oocytes). The D-larval yield was 45% higher for crosses (female×male) triploid×diploid, than the control (diploid×diploid). Furthermore, the lowest D-larval yield was measured for triploid×triploid crosses.Considering the present data, a partial estimation of the reproductive potential of triploid Pacific oysters (triploid×triploid crosses) could be close to 0.06% of that of diploid individuals. However, this estimation is probably over-evaluated because it does not take into account the low sperm production of triploids (1.5% compared to diploids), the unknown frequency of spontaneous spawning in triploids and the low viability of the progeny. The consequences of the low reproductive potential of triploid Pacific oysters on natural populations and on hatchery practices are discussed. In conclusion, the present work confirms that triploidy leads to a limited reproductive potential as estimated by gamete characteristics and embryo developmental success. Statement of relevanceThis study is relevant to aquaculture because triploids are produced by aquaculture and the consequences of their low gamete quality, described in this paper, on the natural environment but also on hatchery practices are discussed.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae).

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n=48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n=98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2% triploids and 44.8% tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2days after hatching, approximately 65.1% of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2% on average during 2-5 and 10-15months of life, respectively. The karyotype of tetraploid progeny (4n=98) included 3n=74 chromosomes of the parental female and n=24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66% of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Heteroploid mosaic tetraploids of Crassostrea virginica produce normal triploid larvae and juveniles as revealed by flow cytometry

Abstract Advantages associated with sterility in triploids have made them the standard for commercial aquaculture of Crassostrea virginica in the Chesapeake Bay. Current hatchery practices for commercial production of triploids employ tetraploids as broodstock, and thus tetraploids are a core asset for oyster culture. Tetraploids undergo reversion, losing entire sets of chromosomes, and become heteroploid mosaics comprised of triploid and tetraploid cells. The possible effects of using mosaic tetraploids for triploid production are a practical concern for commercial oyster culture. We crossed mosaic and non-mosaic tetraploids, males and females, to a reference diploid oyster and compared relative DNA content and phenotypic qualities of triploid progeny. Relative DNA content was measured in somatic and gametic tissue of tetraploid broodstock, as well as in resulting triploid larvae via flow cytometry. Size, abnormality, and survival of larvae were recorded through the rearing process. For six crosses, triploids were raised for 1 year and shell metrics, size, and somatic DNA content were compared. Flow cytometry analysis of broodstock revealed sperm from mosaic tetraploids were consistently di-haploid and virtually indistinguishable from sperm of non-mosaics. For resulting triploid progeny, there were no detectable differences between larvae or juveniles produced from mosaic and non-mosaic tetraploids. Differences existed, however, between triploid larvae produced from di-haploid sperm and those produced from tetraploid eggs: triploids from tetraploid eggs were significantly more abnormal, less viable, and grew slower. Our findings support current commercial practices of using tetraploid males as the principal parent for triploid production and demonstrate no practical consequence from using mosaic tetraploid broodstock for commercial production of triploid C. virginica . Further analysis via cytogenetic techniques may provide insight into long term consequences of using mosaic tetraploids, for example, in tetraploid × tetraploid crosses.

Meiotic chromosome pairing behaviour of natural tetraploids and induced autotetraploids of Actinidia chinensis

Non-preferential chromosome pairing was identified in tetraploid Actinidia chinensis and a higher mean multivalent frequency in pollen mother cells was found in colchine-induced tetraploids of A. chinensis compared with naturally occurring tetraploids. Diploid and tetraploid Actinidia chinensis are used for the development of kiwifruit cultivars. Diploid germplasm can be exploited in a tetraploid breeding programme via unreduced (2n) gametes and chemical-induced chromosome doubling of diploid cultivars and selections. Meiotic chromosome behaviour in diploid A. chinensis 'Hort16A' and colchicine-induced tetraploids from 'Hort16A' was analysed and compared with that in a diploid male and tetraploid males of A. chinensis raised from seeds sourced from the wild in China. Both naturally occurring and induced tetraploids formed multivalents, but colchicine-induced tetraploids showed a higher mean multivalent frequency in the pollen mother cells. Lagging chromosomes at anaphase I and II were observed at low frequencies in the colchicine-induced tetraploids. To investigate whether preferential or non-preferential chromosome pairing occurs in tetraploid A. chinensis, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of crosses between A. chinensis (4x) and A. arguta (4x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the tetraploid A. chinensis parent.

Genetic characterization of the progeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii

Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens’ cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males’ homospecific sperm.

Meiosis and sexual fertility of autotetraploid clones of greater yam Dioscorea alata L.

Meiosis in the tetraploid (2n = 80) males of Dioscorea alata L. was investigated for the first time. During metaphase I, the chromosomes were associated mostly in 6–8 quadrivalents and the remaining ones as bivalents with no trivalents or univalents. Anaphase I and subsequent stages of meiosis were normal. The observation of quadrivalents in the tetraploids provides cytological evidence for autotetraploidy. The autotetraploid males and females were highly fertile and they produced viable seeds on artificial pollination. Pollination between diploids (2n = 40) and tetraploids (2n = 80) were also successful via embryo rescue, producing triploid (2n = 60) progenies. The discovery of fertile autotetraploids could initiate polyploidy breeding in D. alata by conventional hybridization for the first time. The reduced number of quadrivalents and the high fertility of the autotetraploids are thought to be the result of partial diploidization of meiosis. The findings also refute the assumption of the allopolyploid origin of D. alata from two putative progenitors.