Sterile Triploids Research Articles

Polyploidy as a Management Strategy for Invasive Species

Most invasive species are prolific seed-producing landscape ornamental plants that have been introduced to non-native habitats with limited or no natural controls on their reproduction and spread. Techniques for converting prolific seed-producing landscape ornamentals into sterile or nearly sterile forms are available. Oryzalin and colchicine have been used to double chromosomes, resulting in autotetraploids with reduced fertility and potential parent plants of sterile triploids. Guard cell measurements and flow cytometry have been used to determine ploidy conversion and identify polyploids. Complete sterility has been achieved in three species of shower trees (Cassia sp.), and up to a 95% reduction in seed production has been achieved in royal poinciana (Delonix regia) and african tulip tree (Spathodea campanulata). Monkey pod (Albizia saman) crosses have produced triploid progeny to evaluate for sterility.

Differentiating diploid and triploid individuals using single nucleotide polymorphisms genotyped by amplicon sequencing.

Triploidy can occur naturally or be induced in fish and shellfish during artificial propagation in order to produce sterile individuals. Fisheries managers often stock these sterile triploids as a means of improving angling opportunities without risking unwanted reproduction of the stocked fish. Additionally, the rearing of all-triploid individuals has been suggested as a means to reduce the possibility of escaped aquaculture fish interbreeding with wild populations. Efficient means of determining if an individual is triploid or diploid are therefore needed both to monitor the efficacy of triploidy-inducing treatments and, when sampling fish from a body of water that has a mixture of diploids and triploids, to determine the ploidy of a fish prior to further analyses. Currently, ploidy is regularly measured through flow cytometry, but this technique typically utilizes a fresh blood sample. This study presents an alternative, cost-effective method of determining ploidy by analysing amplicon-sequencing data for biallelic single-nucleotide polymorphisms (SNPs). For each sample, heterozygous genotypes are identified and the likelihoods of diploidy and triploidy are calculated based on the read counts for each allele. The accuracy of this method is demonstrated using triploid and diploid brook trout (Salvelinus fontinalis) genotyped with a panel of 234 SNPs and Chinook salmon (Oncorhynchus tshawytscha) genotyped with a panel of 298 SNPs following the GT-seq methodology of amplicon sequencing.

Oocyte maturation and origin of the germline as revealed by the expression of Nanos-like in the Pacific oyster Crassostrea gigas

Nanos gene plays an important role in germline development in animals. However, the molecular mechanisms involved in germline development in Mollusca, the second largest animal phylum, are still poorly understood. Here we identified the Nanos orthologue from the Pacific oyster Crassostrea gigas (Cg-Nanos-like), and investigated the expression patterns of Nanos during gametogenesis and embryogenesis in C. gigas. Tissue expression analysis showed that Cg-Nanos-like was specifically expressed in female gonads. During the reproductive cycle, the expression of Cg-Nanos-like mRNA increased matching the seasonal development of the ovarian tissues in diploids, while the expression levels were significantly lower in the ovaries of sterile triploids compared to diploids. High expression of Cg-Nanos-like transcripts were detected in early embryonic stages, while the expression significantly dropped at gastrulation and was barely detectable in veliger stages. In situ hybridization showed that Cg-Nanos-like was expressed at different stages of developing oocytes, whereas positive signals were detected only in spermatogonia during the spermatogenic cycle. These findings indicated that Cg-Nanos-like was involved in the development of germ cells, and maintenance of oocyte maturation. In early embryogenesis, the transcripts were broadly expressed; following gastrulation, the expression was restricted to two cell clumps, which might be the putative primordial germ cells (PGCs) or their precursors. Based on the results, the formation of the PGCs in C. gigas was consistent with the model of transition from epigenesis to preformation.

Development and Evaluation of Diploid and Polyploid Hibiscus moscheutos

Hibiscus moscheutos L. is an herbaceous hibiscus native to eastern North America that has been a popular landscape and container plant exhibiting large and colorful flowers in the summer. However, unsightly fruit develop and remain on the stalks at the end of the blooming season, which greatly decreases the ornamental value. Thus, breeding for sterility was attempted through ploidy level manipulation to reduce formation and growth of seed stalks, and to improve blooming vigor and longevity. Colchicine and oryzalin were used as mitotic inhibitors to induce tetraploid breeding lines that could be used to develop sterile triploids. Germinated seedlings of ‘Luna Red’ were soaked in three concentrations of each doubling agent for three different durations. Exposure to a low concentration of colchicine solution for a long time or to a low concentration of oryzalin for a short period was found to be effective in yielding a high number of tetraploids with a low rate of mortality. Triploids were obtained from the traditional method of crossing tetraploids with diploids. Triploid and tetraploid plants showed a decrease in height with a more compact form. Leaves of tetraploid plants were more ruffled, with an increase in overall leaf thickness, but were not different from leaves of diploids and triploids in regard to leaf mass per area (LMA). Triploid plants bloomed longer but had smaller flowers than diploid plants. Although the whole planting was infected by aerial phytophthora, diploid, tetraploid, and triploid plants were significantly different in their tolerances: all diploid branches were infected, but only a minor infection occurred on one triploid branch, and the transmission remained slow. Flowers of tetraploid plants failed to produce pollen, whereas flowers of triploid plants produced only nonviable pollen grains and fruits aborted after pollination, which led to infertility of induced triploids.

Ploidy elicits a whole-genome dosage effect: growth of triploid Atlantic salmon is linked to the genetic origin of the second maternal chromosome set

BackgroundThe Atlantic salmon aquaculture industry is investigating the feasibility of using sterile triploids to mitigate genetic interactions with wild conspecifics, however, studies investigating diploid and triploid performance often show contrasting results. Studies have identified dosage and dosage-compensation effects for gene expression between triploid and diploid salmonids, but no study has investigated how ploidy and parent-origin effects interact on a polygenic trait in divergent lines of Atlantic salmon (i.e. slow growing wild versus fast growing domesticated phenotype). This study utilised two experiments relating to the freshwater growth of diploid and triploid groups of pure wild (0% domesticated genome), pure domesticated (100% domesticated genome), and F1 reciprocal hybrid (33%, 50% or 66% domesticated genome) salmon where triploidy was either artificially induced (experiment 1) or naturally developed/spontaneous (experiment 2).ResultsIn both experiments, reciprocal hybrid growth was influenced by the dosage effect of the second maternal chromosome, with growth increasing as ploidy level increased in individuals with a domesticated dam (from 50% to 66% domesticated genome), and the inverse in individuals with a wild dam (from 50% to 33% domesticated genome).ConclusionsWe demonstrate that the combined effect of ploidy and parent-origin on growth, a polygenic trait, is regulated in an additive pattern. Therefore, in order to maximise growth potential, the aquaculture industry should consider placing more emphasis on the breeding value of the dam than the sire when producing triploid families for commercial production.

Molecular characterization and expression analysis of Cyclin B and Cell division cycle 2 in gonads of diploid and triploid bighead catfish, Clarias macrocephalus Günther, 1864

This study investigated the differential expression of genes associated with reproduction in sterile triploid and normal diploid bighead catfish (Clarias macrocephalus Günther, 1864). The triploid fish were produced using cold shock and were reared in the same conditions as the diploid counterpart. The histomicrographs showed completely retarded triploid gonads across the samples aged 2–12 mth, whereas the gonads of the diploids were in developing stages during 2–4 mth, reached the early maturing stage at 6 mth, matured at 8 mth and showed signs of atresia at 10–12 mth. In parallel, the full-length cDNAs of cyclin B1 (CmCcnb1; 1539 bp in length with an open reading frame (ORF) of 1194 bp corresponding to 397 amino acids) and cell division cycle 2 (CmCdc2; 1355 bp, an ORF of 909 bp, 302 amino acids) of bighead catfish (C. macrocephalus Günther, 1864) were isolated. Phylogenetic analysis revealed that the newly characterized CmCcnb1 should be regarded as a member of cyclin B1 rather than cyclin B2. The expression level of CmCcnb1 mRNA was limited in different stages of the ovaries and testes of triploids. In diploid ovaries, its expression was significantly higher than that in triploid ovaries in fish aged 2 mth (513.43 ± 82.22 fold) and in fish aged 8 mth (2430.87 ± 900.06 fold). The CmCcnb1 level in the testes of diploids was significantly greater than that in triploids in fish aged 2 mth (928.85 ± 208.72 fold). Similarly, expression of CmCdc2 mRNA was also reduced in triploids. Its expression was significantly lower than that in diploid females aged 2 mth (7.66 ± 3.42 fold), 4 mth (59.42 ± 10.50 fold) and 8 mth (42.74 ± 8.36 fold). In males, significantly greater expression of CmCdc2 was observed at age 6 mth (58.61 ± 34.64 fold) and 8 mth (72.70 ± 4.36 fold) diploids compared to triploids. The results illustrated that CmCcnb1 and CmCdc2 are functionally involved in oogenesis and spermatogenesis and reduced expression levels of these transcripts affected the reproductive development of triploid C. macrocephalus.

Water temperature and oxygen: The effect of triploidy on performance and metabolism in farmed Atlantic salmon (Salmo salar L.) post-smolts

The use of sterile triploids in Atlantic salmon aquaculture would mitigate the environmental risks associated with introgressive hybridization between escaped farmed and wild Atlantic salmon. However, production of farmed triploid salmon is limited due to reports of poorer growth and higher mortality when compared to diploids, in particular under sub-optimal environmental conditions. To address these concerns, we monitored triploid and diploid Atlantic salmon post-smolts at temperatures between 3 and 18°C and 100% oxygen saturation (O2 sat), and additional periods of 60% O2 sat (hypoxia) at 6 or 18°C, respectively. Feed intake and oxygen consumption rate were monitored throughout the experimental period. Muscle and blood samples were collected at 100 and 60% O2 sat at 6 and 18°C for analysis of white muscle energy phosphates (creatine phosphate, adenosine triphosphate) and carbohydrate fuels (glucose, glycogen) as well as blood clinical chemistry (whole blood: hematocrit; plasma: Na+, K+, Cl−, glucose, lactate, pH, triacylglycerol). Mortality was similar between ploidies, but higher in triploids compared to diploids during reduced O2 sat at 18°C. Compared to diploids, triploids had higher feed intake (% biomass) at ≤9°C, but lower feed intake at ≥15°C. Feed intake peaked at 12 and 15°C for triploids and diploids, respectively. Triploids progressively reduced feed intake with increasing temperature after peak feeding, indicating reduced scope for specific dynamic action with increasing water temperature. During hypoxia, triploids had lower feed intake than diploids at 6 and 18°C. The difference in feed intake was not associated with any ploidy effect on body weight gain or feed conversion ratio, but triploids had greater body length growth compared to diploids. At ≥15°C triploids consumed less oxygen than diploids. In the white musculature, the only observed difference between ploidies was a lower level of glycogen in triploids compared to diploids at 18°C and 100% O2 sat. In the blood plasma, the concentration of ions was lower and glucose level higher in triploids compared to diploids at 18°C and 60% O2 sat. The results of this study indicate that triploid Atlantic salmon post-smolts can substitute diploids, but are less tolerant to high seawater temperature and low O2 sat. For sea-cage farming of triploid salmon post-smolts, this would favour production areas with maximum temperatures of 15°C and sufficient oxygen. Statement of RelevanceThis study demonstrates that triploid Atlantic salmon post-smolts have lower temperature optima for feeding and growth than diploids, a similar physiological response to high temperatures, but higher mortality under suboptimal conditions. Consequently triploid Atlantic salmon post-smolts may be well suited for commercial farming in geographical regions with moderate water temperatures in the summer and autumn months.

Assessment of morphological and phytochemical attributes in triploid and hexaploid plants of the bioenergy crop Miscanthus × giganteus

Giant Miscanthus (Miscanthus×giganteus) is a perennial, warm-season C4 grass. It is a sterile triploid that grows rapidly, accumulates significant biomass, and has the potential to be a new bioenergy crop. Hexaploid Miscanthus×giganteus can be induced from triploid M×giganteus using colchicine. Here, we compared a range of parameters between triploid and hexaploid plants using phytochemical and histological analyses. Inductively coupled plasma-atomic energy spectrometry (ICP-AES) and high performance liquid chromatography (HPLC) showed significant differences in the amounts of 21 microelements and macroelements elements (Al, Ca, Co, Cr, Cu, Fe, Li, Mg, Mn, Ni, Pb, Se, Ag, Ti, Zn, P, Si, S, Na, K, and Sr) between triploid and hexaploid plants. Additionally, phenolic compounds such as pyrogallol, catechin, veratric acid, o-coumaric acid, and myricetin were present at higher concentrations in leaf extracts of hexaploid plants compared to triploid plants. Pyrogallol, gentisic acid, chlorogenic acid, and catechin were also higher in the stem extracts of hexaploid plants. The cellulose content of leaf extracts of hexaploid plants was significantly higher than that of triploid plants, while total lignin content was higher in triploid plants. Significant differences were found between triploid and hexaploid plants for plant height, number of leaves, leaf length, leaf width, number of nodes, stem diameter, tiller number, fresh weight during both pre-flowering and post-flowering stages, and photosynthetic rate. Our analyses indicate that hexaploid M×giganteus exhibited superior agronomical traits and improved biomass yield compared to triploid plants and thus could be suitable for future large-scale biomass production.

Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, Nibe croaker (Nibea mitsukurii)

Intra-peritoneal spermatogonial transplantation could become important in the production of allogenic or xenogenic donor-derived offspring from surrogate parent fish in commercial aquaculture. However, the rates at which surrogates with chimeric germ-lines are produced and contribute donor-derived gametes and offspring to subsequent generations must be improved for this technology to become practical, especially for marine fishes. Here, we performed intra-peritoneal spermatogonial transplantation into triploid Nibe croaker Nibea mitsukurii to evaluate the suitability of functionally sterile triploids as surrogates. Donor testicular cells were collected from hemizygous pHSC-GFP transgenic (gfp/−) individuals and then transplanted into the peritoneal cavities of 12-day-old larvae (diploid or triploid). By 6months after transplantation, 37% (male) and 29% (female) of triploid recipients had produced gfp-positive, donor-derived gametes; these rates were 7- and 4-fold higher, respectively, than the rates for diploid male and female recipients, respectively. Sperm and eggs from triploid recipients were artificially fertilized with eggs or sperm, respectively, from wild-type fish; approximately 50% of the resulting offspring were gfp-positive, suggesting that nearly all of offspring originated from donor-derived gametes that had descended from transplanted gfp/− germ cells. The fecundity and spawning frequency of surrogate triploid recipients were not significantly different to those of non-transplanted diploids, and the triploid recipients produced subsequent donor-derived offspring for at least two spawning seasons. Our results demonstrated that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Further, the use of triploid recipients resulted in exclusive production of donor-derived gametes in this marine teleost. Statement of relevanceIntra-peritoneal spermatogonial transplantation using allogenic triploid recipients was first demonstrated in marine teleosts. In addition to efficient and exclusive donor-derived gametogenesis, we found that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Consequently, triploid surrogates were more suitable than diploid surrogates and that use of triploid surrogates may accelerate the application of surrogate broodstock technology in aquaculture-targeted, genetically valuable, or endangered marine teleosts.

Induction and viability of tetraploids in brook trout (Salvelinus fontinalis)

Brook trout (Salvelinus fontinalis) populations are threatened by introduction of invasive species, habitat loss, and habitat degradation in their native range and are a problem invasive species in western Unites States and Canada and in Europe. Stocking sterile triploids has been promoted as an approach to reduce negative effects of stocking of brook trout for recreational fishing on native fish populations. Crossing a tetraploid with a diploid is a method of triploid production that may help hatcheries meet demand. We induced tetraploidy in brook trout by application of 633 kg·cm−2 of hydrostatic pressure for 8 min at 70%–72.5% of the first cleavage interval. Yields of above 50% tetraploid progeny at hatching were readily achieved, although few animals reached 1 year of age. We crossed a male tetraploid with female diploid fish and produced interploid-triploids with eyeing rates in excess of 50%, demonstrating male tetraploids are fertile and capable of siring triploid progeny. Female tetraploid fish were reared to 16 months posthatching and possessed follicles in secondary vitellogenesis, suggesting tetraploid females are also fertile. Tetraploid induction rates in excess of 96% were achieved applying the same hydrostatic pressure treatment to zygotes of tetraploid × diploid crosses at 30 min postfertilization.

Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP).

Saffron (Crocus sativus) is a sterile triploid (2n = 3x = 24) cultivated species, of unknown origin from other diploid and polyploid species in the genus Crocus (Iridaceae). Species in the genus have high morphological diversity, with no clear phylogenetic patterns below the level of section Crocus series Crocus. Using DNA markers, this study aimed to examine the diversity and relationships within and between species of Crocus series Crocus. Eleven inter-retroelement amplified polymorphism (IRAP) primers were used in 63 different combinations with 35 single-plant accessions of C. sativus and related Crocus species in order to determine genetic variability and to conduct phylogenetic analysis. A total of 4521 distinct polymorphic bands from 100 bp to approx. 4 kb were amplified; no fragment specific to all accessions of a single species was amplified. The polymorphic information content (PIC) values varied from approx. 0·37 to approx. 0·05 (mean 0·17 ± 0·1) and the major allele frequency had a mean of 0·87. High levels of polymorphism were identified between accessions of the six species of Crocus series Crocus related to C. sativus, with further variation between the species. In contrast, no polymorphisms were seen among 17 C. sativus accessions obtained in the region from Kashmir through Iran to Spain. In contrast to the intraspecific variability seen in other Crocus species, C. sativus has minimal genetic variation, and it is concluded that the triploid hybrid species has most probably arisen only once. The data show that saffron is an allotriploid species, with the IRAP analysis indicating that the most likely ancestors are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics, and show the need for conservation and collection of wild Crocus.

Non-structural carbohydrate profiles and ratios between soluble sugars and starch serve as indicators of productivity for a bioenergy grass.

There is a pressing need to find a sustainable alternative to fossil fuels that will not compromise food security or require extensive use of agrochemicals. Miscanthus is a perennial energy grass predominantly used for combustion but with the current advancement of ligno-cellulosic fermentation technologies there is an interest in using Miscanthus for bioethanol production. Currently, the only commercially grown genotype of Miscanthus is M.× giganteus; a high yielding, interspecific hybrid of M. sacchariflorus and M. sinensis. As M.× giganteus is a sterile triploid, it cannot be used as a parent so Miscanthus breeding effort is focused on producing new interspecific varieties that out-perform M.× giganteus. The carbohydrate profiles of four genotypes of Miscanthus, including M. sacchariflorus (Sac-5), M.× giganteus (Gig-311), M. sinensis (Sin-11) and M. sinensis (Goliath), were characterized at replicated field sites in Aberystwyth, West Wales and Harpenden, south-east England. Our hypothesis was that a distinctive carbohydrate profile underlies enhanced biomass accumulation. Biomass accumulation is greatest when day-lengths and solar intensity are highest; so, observations were made in the middle of UK summer (July) for 2 years. Gig-311 had a greater abundance of fructose in its stems at both sites, and both Gig-311 and Sac-5 had low abundance of starch. At both sites, the highest yielding genotype was Gig-311 and Sac-5 was also high yielding at Harpenden, but performed comparatively poorly at Aberystwyth. At both sites Gig-311 had a distinctly high concentration of fructose, low starch and a high ratio of soluble sugars: starch, and at Harpenden, Sac-5 was similar. We conclude that the abundance of starch and fructose and a greater partitioning of soluble sugars, relative to starch, are candidate biomarkers of productivity in Miscanthus.

Yield potential and stand establishment for 20 candidate bioenergy feedstocks

Here we assess 20 bioenergy crop accessions across 10 species, and examine the influence of weed management and annual harvest treatments over three growing seasons in Virginia and one season in Kentucky. Species in this study include: Andropogon gerardii, Arundo donax, Miscanthus × giganteus (sterile triploid and seeded tetraploid), Miscanthus sinensis (ornamental and naturalized), Miscanthus sacchariflorus, Panicum virgatum, Phalaris arundinacea, Saccharum sp., Sorghum bicolor and the unconventional choice of Sorghum halepense. There was a large difference in yield between locations at the end of the planting year, with all accessions producing equivalent or greater biomass in Kentucky. Weed management did not impact yield in the establishment year, as biomass was predictably low. After the third growing season, only A. gerardii and two accessions of naturalized M. sinensis had noticeable reductions in yield due to competition from weeds. After three growing seasons, we obtained the highest yields from M. × giganteus (63, 51 and 39 Mg ha−1 for ‘Illinois’, ‘PowerCane’ and ‘Nagara’ varieties, respectively), naturalized accessions of M. sinensis (54 Mg ha−1), and A. donax (39 Mg ha−1), using traditional cropping practices. Few accessions had low establishment rates limiting overall yields (<10 Mg ha−1); however, when aboveground biomass was calculated on an individual plant basis, yields were comparable to high yielding species, encouraging further examination at higher population densities. Our results indicate that site characteristics such as soil parameters and water availability may be more important for yield than weed competition, especially over time.

Effects of sexual maturation and feeding level on fatty acid metabolism gene expression in muscle, liver, and visceral adipose tissue of diploid and triploid rainbow trout, Oncorhynchus mykiss.

In many cultured fish species, such as salmonids, gonadal development occurs at the expense of stored energy and nutrients, including lipids. However, mechanisms regulating nutrient repartitioning during sexual maturation are not well understood. This study compared sexually maturing diploid (2N) and sterile triploid (3N) female rainbow trout to investigate effects of sexual maturation on expression of 35 genes involved in fatty acid metabolism, including genes within fatty acid synthesis, β-oxidation, and cofactors of the mTOR and PPAR signaling pathways, in liver, white muscle, and visceral adipose tissue. Diploid fish were fed at different rations (0.25% and 0.50% tank biomass, and satiation) to determine effects of ration on gene expression. Gene expression was affected by ration level only in white muscle; erk and acat2 had higher expression in fish fed higher rations. On the other hand, sexual maturation affected gene expression across all three tissue types. Data indicate 2N fish have higher expression of β-oxidation genes within white muscle and within visceral adipose tissue. These findings support enhanced fatty acid mobilization within these tissues during sexual maturation. Higher expression of fatty acid synthesis genes in 3N female liver is associated with higher expression of mTOR cofactors and pparγ, which reflects continued deposition of lipids in these fish. Furthermore, greater expression of genes involved in β-oxidation pathways across ration levels in 2N females suggests that sexual maturation and the associated maturation-related signals are stronger regulators of lipid metabolism-related genes rather the rations applied in the current study.

Genetic diversity and pedigree assignment in tetraploid Pacific oysters (Crassostrea gigas)

Abstract Tetraploid Pacific oysters (Crassostrea gigas) are commonly used within aquaculture to generate predominantly sterile triploids for commercial use. Unlike triploids, tetraploids are fertile and can be spawned with each other to maintain a commercial breeding population. The impacts this hatchery perpetuation has on the genetic diversity of the tetraploid population and subsequent commercial triploid product are currently unknown. This study determines the diversity (number of alleles and heterozygosity), pedigree assignment and effective population size over two generations of tetraploid production. A significant drop in diversity was observed over two successive generations of tetraploid oysters spawned in 2008 and in 2011, likely due to different numbers of broodstock used. Similar to diploids, pedigree assignment in tetraploids showed unequal parental contributions and an effective population size smaller than the census population, thus the potential for inbreeding to occur in the future is high.

Evolutionary significance of hybridization inOnosma(Boraginaceae): analyses of stabilized hemisexual odd polyploids and recent sterile hybrids

Interspecific hybridization is an important evolutionary force promoting plant speciation. In the genus Onosma, one of three main evolutionary lineages presumably evolved by hybrid speciation. The assumed hybrid lineage (Heterotricha) consists of two species complexes with bimodal karyotypes containing different numbers of large (L) and small (S) chromosomes, the tetraploid Onosma pseudoarenaria (2n = 12 L + 14S) and the triploid Onosma arenaria (2n = 12 L + 8S). The latter represents a rare case of hemisexual, asymmetrically compensating allopolyploids. Representatives of the other two lineages of the genus, Haplotricha (2n = 12 L) and Asterotricha (2n = 14S), have been considered to be the ancestral taxa of O. pseudoarenaria and O. arenaria, although this has yet to be investigated critically. In the present study, we examined genetic [amplified fragment length polymorphism (AFLP), internal transcribed spacer (ITS) , and chloroplast (cp)DNA)], reproductive (pollen viability and seed production) and cytogenetic (chromosome counts, genome size assessment) patterns to resolve the hypothesized allopolyploid formations in the Heterotricha group, single or polytopic allopolyploid origins, as well as ongoing interspecific gene flow as one piece of evidence for understanding past hybrid speciation events in the genus. Discordant patterns in maternally inherited cpDNA (Heterotricha accessions bearing the haplotypes related to asterotrichous species) and the nuclear ITS and AFLP markers (Heterotricha clustering with haplotrichous Onosma fastigiata), as well as karyological features, support the hybrid origin of the stabilized Heterotricha lineage. Genetic variation that is both large and geographically correlated indicates multiple origins of Heterotricha allopolyploids or, less likely, a single origin with recurring introgression from the progenitor species. The nuclear markers and cytogenetic features also provide evidence for the ongoing hybridization between O. arenaria and Onosma echioides (2n = 14S), which gives rise to sterile triploids of 2n = 6 L + 15S. We contrast the two cases of triploids with LLS (hemisexual O. arenaria from the stabilized Heterotricha lineage) and LSS (recent sterile hybrids) karyotypes, which could help to understand the mechanisms ensuring the establishment and reproductive fitness of the odd allopolyploids in Onosma. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 89–107.

Elevated expression of Piwi and piRNAs in ovaries of triploid crucian carp

Increasing piRNAs provide RNA-interference pathways to regulate transposons and gene expression in germline cells. We demonstrate that Piwi transcripts are exclusively expressed in adult testes and ovaries in teleosts, with triploids showing the highest Piwi expression in the ovaries. Studies in vivo and in vitro showed that hCG and E2 treatment suppressed Piwi expression. We further cloned 200 small RNAs in the three kinds of fish. Seven piRNAs were obtained from all the three different ploidy fishes. During ovulation, five piRNAs showed significantly higher expression in the ovaries of sterile triploids than fertile diploids and tetraploids. Furthermore, E2 suppressed the expression of the six piRNAs at different levels in vivo and in vitro. The present study bridges the gap between the HPG axis and Piwi–piRNA pathway by suggesting that a dysfunctional HPG axis abrogated the piRNA suppression in triploid fish.

Family-specific differences in growth rate and hepatic gene expression in juvenile triploid growth hormone (GH) transgenic Atlantic salmon (Salmo salar)

Growth hormone transgenic (GHTg) Atlantic salmon (Salmo salar) have enhanced growth when compared to their non-transgenic counterparts, and this trait can be beneficial for aquaculture production. Biological confinement of GHTg Atlantic salmon may be achieved through the induction of triploidy (3N). The growth rates of triploid GH transgenic (3NGHTg) Atlantic salmon juveniles were found to significantly vary between families in the AquaBounty breeding program. In order to characterize gene expression associated with enhanced growth in juvenile 3NGHTg Atlantic salmon, a functional genomics approach (32K cDNA microarray hybridizations followed by QPCR) was used to identify and validate liver transcripts that were differentially expressed between two fast-growing 3NGHTg Atlantic salmon families (AS11, AS26) and a slow-growing 3NGHTg Atlantic salmon family (AS25); juvenile growth rate was evaluated over a 45-day period. Of 687 microarray-identified differentially expressed features, 143 (116 more highly expressed in fast-growing and 27 more highly expressed in slow-growing juveniles) were identified in the AS11 vs. AS25 microarray study, while 544 (442 more highly expressed in fast-growing and 102 more highly expressed in slow-growing juveniles) were identified in the AS26 vs. AS25 microarray study. Forty microarray features (39 putatively associated with fast growth and 1 putatively associated with slow growth) were present in both microarray experiment gene lists. The expression levels of 15 microarray-identified transcripts were studied using QPCR with individual RNA samples to validate microarray results and to study biological variability of transcript expression. The QPCR results agreed with the microarray results for 12 of 13 putative fast-growth associated transcripts, but QPCR did not validate the microarray results for 2 putative slow-growth associated transcripts. Many of the 39 microarray-identified genes putatively associated at the transcript expression level with fast-growing 3NGHTg salmon juveniles (including APOA1, APOA4, B2M, FADSD6, FTM, and GAPDH) are involved in metabolism, iron homeostasis and oxygen transport, and immune- or stress-related responses. The results of this study increase our knowledge of family-specific impacts on growth rate and hepatic gene expression in juvenile 3NGHTg Atlantic salmon. In addition, this study provides a suite of putative rapid growth rate-associated transcripts that may contribute to the development of molecular markers [e.g. intronic, exonic or regulatory region single nucleotide polymorphisms (SNPs)] for the selection of GHTg Atlantic salmon broodstock that can be utilized to produce sterile triploids of desired growth performance for future commercial applications.

Confirmation of cytotype stability in autotetraploid black wattle (Acacia mearnsii) trees using flow cytometry and size differences of the reproductive gametes

Acacia mearnsii (black wattle) is grown commercially in South Africa for its timber and bark. However, the invasive nature of the species has resulted in it being considered an alien invader and for this reason research has been aimed at producing a sterile triploid variety that would be highly desirable for the South African commercial forestry industry. Tetraploids were successfully induced by soaking germinating diploid seeds in colchicine. Seed from these tetraploids was used to establish a field trial, where crossing diploid with tetraploid parent plants only produced diploid and tetraploid progeny and failed to produce any triploid progeny. Control-crossed seed set between diploids is generally low in A. mearnsii and, together with the possibility of an unstable tetraploid population, this could be reducing the chances of producing triploid seed. Thus identification and confirmation of stability within the existing advanced-generation tetraploid population (aged 10–11 years) was critical to ensure the production of sterile triploids. Flow cytometry was used to determine the stability of the ploidy of leaf vegetative tissues, whereas polyad and ovule size measurements were used to determine the stability of ploidy of the reproductive tissues. Results from the study revealed that the tetraploidy of within the leaf vegetative tissue was stable. For both the ovule and polyad size measurements, a size range was determined for diploids and tetraploids and, within the population under investigation, no overlap was apparent. This allowed for the conclusion that the advanced-generation tetraploid population was stable and that the absence of triploid progeny must be because of post-zygotic reproductive barriers within the ovary.

Relative Performance of Diploid and Triploid Catchable Rainbow Trout Stocked in Idaho Lakes and Reservoirs

AbstractIdaho Department of Fish and Game hatcheries stock predominantly sterile triploid (3n) rainbow trout Oncorhynchus mykiss to provide sportfishing opportunities while minimizing the genetic risks to wild stocks. Triploid catchable‐sized rainbow trout are stocked in over 500 water bodies across Idaho annually, but there remains some uncertainty regarding the performance of triploid rainbow trout relative to their diploid (2n) counterparts. We examined the relative survival, growth, and returns of diploid and triploid all‐female catchable rainbow trout across 13 lakes and reservoirs. Most reservoirs showed higher returns of 2n rainbow trout to anglers. In 2008, 3n rainbow trout returned on average at only 72% and 81% of the rates of 2n trout in gill nets and snout collection boxes, respectively, and the difference for both methods was statistically significant. Carryover of marked rainbow trout from 2008 was low or zero in most reservoirs. Where there was carryover, snout collection boxes suggested that 3n rainbow trout returned to anglers at 71% of the rate of 2n rainbow trout in the second year after planting, but the difference was not statistically significant. Triploid rainbow trout did not show any growth advantages over 2n rainbow trout but were similar in length, weight, and dressed weight. The disparity in returns between 2n and 3n trout varied across reservoirs but was more pronounced in locations subjected to greater drawdown and with greater species diversity. While 2n rainbow trout may grow and survive better in reservoirs subject to low water levels, triploid rainbow trout may perform equally well under good habitat conditions while not having genetic impacts on native stocks. These findings are rather fortuitous for fisheries managers, as triploids probably perform better in higher‐quality habitats where native trout often exist, whereas diploids are better suited to reservoirs with degraded habitats where native stocks have usually been extirpated.Received September 14, 2010; accepted May 18, 2011