Dispermic androgenesis in sturgeons with the help of cryopreserved sperm: production of androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nuclear cytoplasmic hybrids (Siberian sturgeon, A. baerii x Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Development of the skull and pectoral girdle in Siberian sturgeon,Acipenser baerii,and Russian sturgeon,Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The head is considered the major novelty of the vertebrates and directly linked to their evolutionary success. Its form and development as well as its function, for example in feeding, is of major interest for evolutionary biologists. In this study, we describe the skeletal development of the cranium and pectoral girdle in Siberian (Acipenser baerii ) and Russian sturgeon (A. gueldenstaedtii ), two species that are commonly farmed in aquaculture and increasingly important in developmental studies. This study comprises the development of the neuro‐, viscero‐ and dermatocranium and the dermal and chondral components of the pectoral girdle, from first condensation of chondrocytes in prehatchlings to the early juvenile stage and reveals a clear pattern in formation. The otic capsules, the parachordal cartilages, and the trabeculae cranii are the first centers of chondrification, at 8.4mm TL. These are followed by the mandibular, then the hyoid, and later the branchial arches. Teeth form early on the dentary, dermopalatine, and palatopterygoid, and then appear later in the buccal cavity as dorsal and ventral toothplates. With ongoing chondrification in the neurocranium a capsule around the brain and a strong rostrum are formed. Dermal ossifications start to form before closure of the dorsal neurocranial fenestrae. Perichondral ossification of cartilage bones occurs much later in ontogeny. Our results contribute data bearing on the homology of elements such as the lateral rostral canal bone that we regard homologous to the antorbital of other actinopterygians based on its sequence of formation, position and form. We further raise doubts on the homology of the posterior ceratobranchial among Actinopteri based on the formation of the hyoid arch elements. We also investigate the basibranchials and the closely associated unidentified gill‐arch elements and show that they are not homologous. J. Morphol. 278:418–442, 2017. © 2017 Wiley Periodicals, Inc.     

Evidence of mitochondrial DNA clones of Siberian sturgeon, Acipenser baerii, within Russian sturgeon, Acipenser gueldenstaedtii, caught in the River Volga

Eleven of 34 sturgeons caught in the River Volga classified morphologically as Acipenser gueldenstaedtii were identified as Acipenser baerii from sequence analysis of the mitochondrial cytochrome- b gene. The Caspian Sea and its tributaries including the Volga are not native habitats of A. baerii . No A. baerii haplotype was observed in A. gueldenstaedtii from the Sea of Azov or the South Caspian Sea. Genetic contamination of A. gueldenstaedtii with A. baerii or A. baerii hybrids has occurred in the Volga. Crosses and backcrosses of these specimens with native A. gueldenstaedtii resulted in the loss of the morphological diagnostic A. baerii features. These findings are of special concern for conservation and management programmes, as well as for specimen identification for caviar trading control.     

Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition

In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P<0.05) to uterine horn insemination, there was no significant (P>0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.     

Morphology, chemical contents and physiology of chondrostean fish sperm: a comparative study between Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus)

The present study was carried out with two sturgeon species, Siberian sturgeon (Acipenser baerii) and sterlet (A. ruthenus) to compare spermatological parameters to better understand inter‐species differences. Significant differences between morphometric features were observed such as acrosome length, acrosome width, head length, midpiece width and flagellar length, while midpiece length did not reveal such differences. The sterlet has a shorter spermatozoon than the Siberian sturgeon. Ultrastructural parameters vary significantly in terms of length of the nucleus, diameter of the endonuclear canals (EC), size of posterolateral projections (PP) and diameter of flagellum. Mean values for density of spermatozoa in the semen, seminal plasma pH, osmolality (mOsmol kg^?1), along with Ca²⁺, Na⁺, K⁺, Cl^? ions concentrations (mm ) were determined to be 0.61 ± 0.37 × 10⁹, 8.16 ± 0.18, 77.20 ± 52.28, 0.24 ± 0.06, 31.39 ± 10.21, 3.51 ± 1.10, 14.00 ± 4.30 in A. baerii and 0.41 ± 0.32 × 10⁹, 8.13 ± 0.19, 50.74 ± 6.27, 0.16 ± 0.11, 20.11 ± 3.78, 1.26 ± 0.54, 6.11 ± 0.60 in A. ruthenus, respectively. Significant differences were observed in Na⁺, K⁺ and Cl^? concentrations in the seminal plasma as well as in sperm velocity. The percentage of motile spermatozoa did not show any significant difference between the two species. Comparing the results of this study with published literature data on sturgeon spermatozoa reveals that morphological and ultrastructural parameters of spermatozoa together with some parameters of the seminal fluid and spermatozoa velocity can be used in comparative spermatology to better understand inter‐species differences. The observed biochemical and physiological differences should be also considered for the development of methods for controlled reproduction and for sperm cryopreservation techniques.     

Ontogenetic behavior of Siberian sturgeon, Acipenser baerii: A synthesis between laboratory tests and field data

We have reviewed field data and studies on the behavior and development of Siberian sturgeon at early-life intervals and related them to different ecologically relevant environmental factors that may play a role in the distribution, recruitment, and survival of young fish. Four behavioral phases (swimming-up, rheotactism, shoaling, and foraging) are observed from hatching to the juvenile phase. Each behavior is associated with an early-life interval and might allow fish to occupy different river habitats, directly influencing their distribution, survival, and recruitment. River current intensity, substrate typology, food resources, and predation pressure seem to be the most important factors affecting the distribution of Siberian sturgeon free embryos and larvae, while juveniles and adult fish disperse and migrate according to food abundance and reproduction. Mechanisms involved in regulating downstream migration during Siberian sturgeon early life stages are different than those observed in anadromous sturgeon species. In all large Siberian rivers, with the exception of the Lake Baikal, the Siberian sturgeon is represented by population continuums, and in many cases the foraging range also includes the spawning areas. Ontogenetic changes in Siberian sturgeon behavior could be interpreted as a species-specific mechanism to maintain the population continuums described in this species without significant mixture of local populations within the river.     

Endemic sturgeons of the Amur River: kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii

General biological characteristics and the contemporary status of the kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii, are described. Both inhabit the Amur River basin. Kaluga is the largest freshwater fish in this river system reaching more than 5.6 m in length and more than 1000 kg in weight. We recognize four populations of kaluga: the first is from the estuary of the Amur River and coastal brackish waters of the Sea of Okhotsk and Sea of Japan, the second is from the lower Amur River, the third is from the middle-Amur, and the fourth occurs in lower reaches of the Zeya and Bureya rivers. Freshwater and brackish water morphs exist in the estuary population, with the freshwater morph predominating in number. The number of individuals in the lower Amur River population at age 2 or greater was recently estimated to be 40 000, and in the middle Amur, 30 000. The population will continue to decline because of rampant overfishing. The Amur sturgeon is represented in the Amur River basin by two morphs: brown and gray. Brown morphs occur in the middle and lower parts of the Amur River; they grow more slowly than the gray ones. Today, the lower Amur River population of Amur sturgeon is made up of 95 000 fish at age 2 or greater and is approximately half as large as the population in the middle Amur River. Populations of kaluga and Amur sturgeon in the Zeya and Bureya rivers are extremely small and on the verge of extinction.     

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.     

Pregnancy rates in heifers and cows with cryopreserved sexed sperm: Effects of sperm numbers per inseminate, sorting pressure and sperm storage before sorting

Field trials were conducted to increase fertility with AI of flow-sorted, sexed bovine sperm. In the first trial, a novel competitive fertilization approach was used to compare pressures (30 psi vs 50 psi) for sorting sperm. Both X- and Y-sperm were sorted to approximately 95% purity at 30 and at 50 psi; X-50 + Y-30 (and the converse) were mixed in equal numbers for AI of heifers. Fetal sex divulged which treatment produced the pregnancy; 82% of pregnancies resulted from the 30 psi treatment (P < 0.05). Based on a similar approach, a new-pulsed laser did not damage sperm any more than the previous standard continuous wave laser. In a large field trial, sorting sperm at 40 psi increased pregnancy rates in heifers relative to 50 psi (42.3% vs 34.1%, n = 367/group, P < 0.05). Storing sperm for 20 h before sorting at 40 psi decreased pregnancy rates from 42.3% (n = 367) to 36.8% (n = 368; P < 0.05). Breeding heifers with sexed sperm 55-56 h after CIDR removal and PGF_2α resulted in 34% (n = 32) pregnant, compared to 49% (n = 35) with fixed-time insemination 67-68 h after CIDR removal (P > 0.1). Lactating dairy cows pre-screened for normal reproductive tracts when OvSynch injections (GnRH, prostaglandin, GnRH) were initiated, had similar (P > 0.1) pregnancy rates to timed AI, with 10 × 10⁶ sexed sperm (43.9%, n = 57), 2 × 10⁶ sexed sperm (40.5%, n = 57) and 10 × 10⁶ unsexed control sperm (55.6%, n = 58). A final field trial with unselected, lactating dairy cows resulted in similar pregnancy rates for 2 × 10⁶ sexed sperm in 0.25 mL straws (25.0%, n = 708) and 0.5 mL straws (24.4%, n = 776), but lower (P < 0.05) than unsexed control sperm (37.7%, n = 713). Younger cows and those >84 days in milk had the highest pregnancy rates for both sexed and unsexed sperm. These studies improved sperm sexing procedures, and provided insight into appropriate commercial use of sexed sperm.     

Isolation and preliminary characteristics of β-N-acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)

The aim of this study was to characterize the enzyme β- N -acetyglucosaminidase (β-NAGase) in the milt and spermatozoa extracts from Siberian sturgeon and rainbow trout. After ion exchange chromatography one protein peak showed β-NAGase activity in sturgeon milt plasma and sperm extracts of both species. Surprisingly, two protein peaks showing β-NAGase activity were found in rainbow trout milt plasma. The molecular mass of β-NAGase was estimated by gel filtration as 127 kDa for rainbow trout spermatozoa, 271 kDa for sturgeon spermatozoa, and 74 kDa for milt plasma from both species. The kinetic parameters were determined for milt plasma and sperm extracts. The optimum pH of the β-NAGases was 3.8 for sturgeon milt plasma, 4.4 for sturgeon sperm extract, and 4.4–4.8 for milt plasma and sperm extract from rainbow trout. K _m value of the β-NAGases was 0.212, 0.563, 0.779 m m for sturgeon milt plasma, sturgeon sperm extract or rainbow trout extract, respectively. The β-NAGase from sperm extracts in both species showed 100% activity even after incubation at 56°C by 20 min, whereas its activity was decreased to 23% in sturgeon milt plasma and to 2% in trout milt plasma.     

First evidence of hybridization between endangered sterlets (Acipenser ruthenus) and exotic Siberian sturgeons (Acipenser baerii) in the Danube River

Most natural populations of Eurasian sturgeons have declined dramatically during recent decades, reaching historic low levels today. During the same period, sturgeon has become very popular in European aquaculture. Because many hatcheries are located near rivers, their unintentional escape is often documented, especially during floods. Until now, no cases of successful hybridization of these escaped fish with wild stocks have been reported. In this study, the genetic structure of a highly threatened population of sterlets (Acipenser ruthenus) from the Upper Danube was analysed as a requirement for their conservation. Surprisingly, we observed genotypes and morphotypes of Siberian sturgeon (Acipenser baerii), as well as hybrids between this species and native sterlets. This hybridization poses a serious threat for the survival of this isolated sterlet population in the upper part of the Danube. For the first time, natural reproduction is documented for Siberian sturgeon outside their natural range in Europe. This finding demonstrates the risk of extinction by hybridisation of endangered populations. We would like to stress that taking into consideration the risks for native sturgeon populations, farmed sturgeon should not be released into the wild, and all measures should be taken to prevent their accidental escape.     

Production and characterization of somatic hybrids between the Japanese radish and cauliflower

Summary Somatic hybrids between the Japanese radish and cauliflower (Brassica oleracea) were produced by protoplast electrofusion in order to introduce clubroot disease resistance in the Japanese radish (Raphanus sativus) into Brassica crops. After electrofusion of iodoacetamide-treated cauliflower protoplasts with untreated radish ones, culture was performed under conditions, that allowed only cauliflower protoplasts to regenerate. Out of 40 regenerated plants, 37 were morphologically of a hybrid type and 3 of a cauliflower type. On the basis of isozyme and RFLP analysis, all of the hybrid-type plants tested proved to be true hybrids. Of the 10 true hybrids tested, 9 were found to contain chloroplasts similar to those found in the Japanese radish, while only 1 contained those of the cauliflower. Using two mitochondrial genes as probes, we were able to show that 3 hybrids contained mitochondria of the Japanese radish, with some modification, while 7 hybrids had either parental or new patterns. All of the hybrid-type plants showed resistance to clubroot disease as high as that found in the Japanese radish. Some hybrids were self-fertile. All of the self-fertile hybrids were found to contain 36 chromosomes, indicating that they were amphidiploids. In addition, a few seeds were obtained from a backcross of the self-fertile hybrids to both parents.     

Features of inheritance of several morphological characters in the hybrids between Siberian sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> and beluga <Emphasis Type="Italic">A. huso</Emphasis> (Acipenseridae) and in gynogenetic individuals of Siberian sturgeon

Comparative analysis of several morphological characters is conducted in experimentally obtained hybrids between Siberian sturgeon Acipenser baerii and beluga A. huso, in parental species, and in gynogenetic progeny of Siberian sturgeon. The gynogenetic individuals are similar to Siberian sturgeon by all characters used for differentiation between Siberian sturgeon and beluga that supports gynogenetic origin of these individuals. Based on the data obtained on the hybrid specimens, the features of inheritance of the studied characters in sturgeons and their variation in postlarval ontogeny are discussed.     

Development of the muscles associated with the mandibular and hyoid arches in the Siberian sturgeon,Acipenser baerii (Acipenseriformes: Acipenseridae)

The skeleton of the jaws and neurocranium of sturgeons (Acipenseridae) are connected only through the hyoid arch. This arrangement allows considerable protrusion and retraction of the jaws and is highly specialized among ray‐finned fishes (Actinopterygii). To better understand the unique morphology and the evolution of the jaw apparatus in Acipenseridae, we investigated the development of the muscles of the mandibular and hyoid arches of the Siberian sturgeon, Acipenser baerii. We used a combination of antibody staining and formalin‐induced fluorescence of tissues imaged with confocal microscopy and subsequent three‐dimensional reconstruction. These data were analyzed to address the identity of previously controversial and newly discovered muscle portions. Our results indicate that the anlagen of the muscles in A. baerii develop similarly to those of other actinopterygians, although they differ by not differentiating into distinct muscles. This is exemplified by the subpartitioning of the m. adductor mandibulae as well as the massive m. protractor hyomandibulae, for which we found a previously undescribed portion in each. The importance of paedomorphosis for the evolution of Acipenseriformes has been discussed before and our results indicate that the muscles of the mandibular and the hyoid may be another example for heterochronic evolution.     

Polyphyly of mtDNA lineages in the Russian sturgeon, Acipenser gueldenstaedtii: forensic and evolutionary implications

Molecular species identification methods are an important component of CITES monitoring programs for trade in sturgeon and caviar. To date, obtaining molecular evidence for distinguishing caviar from four closely related Eurasian sturgeon species Acipenser baerii (Siberian sturgeon), A. gueldenstaedtii (osetra), A. persicus (Persian sturgeon), A. naccarii (Italian sturgeon) remains problematic. Using approximately 2.3 kb of mtDNA sequence data (cytochrome b, NADH5, control region), we find this to be attributable to the polyphyletic nature of these mitochondrial DNA markers in the Russian sturgeon, A. gueldenstaedtii. Two mitochondrial lineages are present within this species: one is phylogenetically affiliated with A. persicus and A. naccarii, while the other clusters with A. baerii. These findings have a direct impact on molecular testing of commercial caviar and demonstrate the necessity of using large sample sizes when constructing forensic databases. Furthermore, the results affect current taxonomic designations for these species as well as hypotheses concerning their evolutionary origins.     

Triple hybrids obtained by artificial hybridization of the Russian sturgeon Acipenser gueldenstaedtii with the hybrid of the starred sturgeon A. stellatus and the great sturgeon A. huso (Acipenseridae): The kind of inheritance of some morphological characters and fertility of the parental hybrid form

Results are described of experimental crossing for obtaining of triple hybrids of the Russian sturgeon Acipenser gueldenstaedtii, the great sturgeon A. huso, and the starred sturgeon A. stellatus and of comparative analysis of some quantitative morphological characters in these hybrids, in the progeny of parental species and of reciprocal hybrids of starred sturgeon and great sturgeon. By most investigated characters, in triple hybrids, the states of the paternal species (Russian sturgeon) are inherited, by some other characters-of great sturgeon-the state of starred sturgeon is not inherited. Therefore, on the basis of external appearance, they may be identified as the hybrid of Russian sturgeon with great sturgeon. The observed segregation by the form of gill rakers into paternal, maternal, or intermediate variants leads to a conclusion of nonmonogenous inheritance of a wide form and a conical form of gill rakers in sturgeons. On the whole, the obtained results clearly demonstrate how, due to effects of matrocliny and partocliny, and different combination of dominant and recessive alleles of the genes determining the diagnostic characters, the hybrid individuals may acquire a different extent of similarity and differences from the parental species. The investigated triple hybrids are an interesting example of mutual leveling of divergent traits in the course of combination of different genomes within limits of one closely related Atlantic group of sturgeons. As a result, a certain averaged habitus is formed which might take place also in case of formation of polyploidy species in the process of evolution.     

Apoptosis and the processes of DNA fragmentation in native and cryopreserved human sperm cells with normo- and pathosperma

Cryopreserved human sperm is widely applied in the treatment of infertility using methods of assisted reproductive technologies. Comprehensive studies of the sperm condition after cryopreservation and subsequent cultivation will update the efficiency of the programs based on assisted reproductive technologies. The aim of this study was to investigate the effect of cryopreservation factors on the state of DNA and apoptotic processes in isolated human spermatozoa fractions in normo- and pathospermia. The analysis of DNA fragmentation and the assessment of apoptotic processes (human spermatozoa necrosis) were performed using the sperm chromatin dispersion test and the flow cytometry method. According to our study, sperm cryopreservation has not affected the DNA fragmentation index in normozoospermia and significantly increased it in oligoastenoteratozoospermia. The postcultivation increase of apoptotic and necrotic processes in oligoastenoteratozoospermia gives reason for choosing an alternative technique for oocyte fertilization— intracytoplasmic sperm injection.     

Comparison of the effects of gonadotropic preparations of the carp and stellate sturgeon pituitaries on in vivo and in vitro oocyte maturation in the Siberian sturgeon Acipenser baeri brandt

Injections of 2.5 mg/kg of stellate sturgeon (Acipenser stellatus Pall.) pituitary extract and 5 mg/kg of carp (Cyprinus carpio L.) pituitary extract in Siberian sturgeon (Acipenser baeri Brandt) females did not reveal significant differences in the effects of these preparations. There were no differences in the percentage of females that responded by ovulation, duration of the period from injection to ovulation, rate of ovulation, or quality of mature eggs as estimated by the percentage of fertilization or percentage of normal embryos at the small yolk plug stage. Thus, an insufficient efficiency in the artificial reproduction of the Siberian sturgeon grown in captivity is not related to the use of the carp pituitary preparation as a stimulus. Estimation of the ratio of specific activities of the pituitary extracts and purified gonadotropins of the stellate sturgeon and carp by in vitro oocyte maturation has shown that it varies within wide limits as a function of the medium composition and physiological state of follicles. Hence, the ratio of activities of the gonadotropins of different species as determined by in vitro maturation of sturgeon oocytes may markedly differ from that upon injection of these preparations in breeders.     

Production and cytogenetics of intergeneric hybrids between the three cultivated Brassica diploids and Orychophragmusviolaceus

It has been proposed that both complete and partial separation of the parental genomes during mitosis and meiosis occurs in the intergeneric hybrids between Orychophragmus violaceus (2n=24) and the three cultivated Brassica tetraploids (B. napus, B. carinata and B. juncea). The hypothesis has been that this and the variations in chromosome numbers of these hybrids and their progenies result from the different roles of the A, B and C genomes originating from Brassica. To test this hypothesis, we produced hybrids between O. violaceus and the cultivated Brassica diploids. The hybrids with B. oleracea (2n=18, CC) had an intermediate morphology, but their petals were purple like those of O. violaceus. They were sterile and had the expected chromosome number (2n=21) in their mitotic and meiotic cells. The hybrid with B. campestris (2n=20, AA) was morphologically intermediate, except for its partial fertility and its yellow petals, which were similar to those of B. campestris. It was mixoploid (2n=23–42), and cells with 2n=34 were most frequent. Partial separation of parental genomes during mitosis, leading to the addition of O. violaceus chromosomes to the B. campestris complement, was proposed to explain the findings in the mitotic and meiotic cells of the hybrid and its progeny. In crosses with B. nigra (2n=16, BB), the majority of the F₁ plants were of the maternal type (2n=16), a small fraction had B. nigra morphology but were mixoploids (2n=16–18), predominantly with 2n=16 cells and three plants, each with a specific morphology, were mixoploids consisting of cells with varying ranges of chromosome numbers (2n=17–26, 11–17 and 14–17). The origin of these different types of plants was inferred to be a result of the complete and partial separation of parental genomes and the loss of O. violaceus chromosomes. Our findings in the three crosses suggest that the A genome was more influential than the C genome with respect to complete genome separation during mitosis and meiosis of the hybrids with B. napus. Possible complete and partial genome separation during mitotic divisions of the hybrids with B. carinata was mainly attributed to the role of the B genome. The combined roles of the A and B genomes would thus contribute to the most variable chromosome numbers of mitotic and meiotic cells in the hybrids with B. juncea and their progenies. The possible cytological mechanisms pertaining to these hybrids and the potential of genome separation in the production of Brassica aneuploids and homozygous plants are discussed. Received: 8 February 1998 / Accepted: 12 March 1999     

ISSR and isozyme characterization of androgenetic dihaploids reveals tetrasomic inheritance in tetraploid somatic hybrids between Solanum melongena and Solanum aethiopicum group Gilo

Gene exchanges between Solanum melongena and its allied relative Solanum aethiopicum are a crucial prerequisite for introgression of useful traits from the allied species into the cultivated eggplant. In order to evaluate the extent of genetic recombination between the 2 species, biochemical and molecular markers were employed. A dihaploid population obtained through anther culture of the corresponding tetraploid somatic hybrids was genetically analyzed. The extent of disomic/tetrasomic inheritance and segregation ratios of 3 isozyme systems and intersimple sequence repeat (ISSR) markers were evaluated. The dihaploids, being derived from microspores, allowed for simple, complete, and accurate analyses. The segregation of 280 ISSR markers (110 aethiopicum-specific, 104 melongena-specific, and 66 monomorphic) were evaluated in 71 dihaploids. According to the genetic constitution (simplex/duplex/triplex), almost 64% of the fragments revealed the tetrasomic and/or disomic inheritance. With regard to the assigned species-specific fragments, 68% and 4% were unambiguously the result of tetrasomic and disomic inheritance, respectively. Twenty-four of the 66 monomorphic ISSRs were inherited according to random chromatid segregation. The phenotypes of glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and shikimate dehydrogenase (SKDH) were studied in 70 dihaploids and inferences were made about the allelic state of their 5 loci. The isozyme markers segregated in the dihaploids in a distorted manner, their segregations did not fit in with any of the expected segregation ratios. However, tetrasomic inheritance might be suggested for G-6-PDH 2 and SKDH 1 loci. Our results demonstrated that gene exchanges occurred readily in the somatic hybrids between S. melongena and S. aethiopicum gr. Gilo.