Microsatellite-centromere mapping in the loach, Misgurnus anguillicaudatus

Primer sets for 15 polymorphic microsatellite loci were developed in the loach, Misgurnus anguillicaudatus (Cobitidae) by molecular cloning and sequencing techniques. Mendelian inheritance was confirmed for the 15 loci by examining the genotypic segregation produced with the primer sets in two full-sib families. The loci were mapped in relation to their centromere in four gynogenetic diploid lines, which were induced by inhibition of the second meiotic division after fertilization with genetically inert sperm. Microsatellite-centromere recombination rates ranged between 0.06 and 0.95 under the assumption of complete interference. Thus, these loci are distributed from the centromeres to the telomeres of their respective chromosomes. The success of mitotic gynogenesis, produced by suppression of the first cleavage, was verified by homozygosity at three diagnostic microsatellite loci that exhibited high gene-centromere meiotic recombination rates in the same family. The differences in heterozygosity levels observed with these markers were attributed to differences in the temporal application of heat shock following inert sperm activation.     

Genetic analysis of androgenetic rainbow trout.

We analyzed a number of genetic characteristics in androgenetic rainbow trout (Oncorhynchus mykiss) and their progeny. The androgenetic progeny of individual androgenetic males appeared genetically identical to each other based on eight enzyme loci. Their viability was no higher than that of androgenetic progeny of outbred males. Homozygous androgenetic female rainbow trout produced very poor quality eggs. When common eggs and sperm from outbred individuals were used to produce androgenetic and gynogenetic progeny, the yield of gynogenetic progeny was higher but some were heterozygous at protein loci, while no androgenetic progeny were heterozygous. Some androgenetic diploid rainbow trout were successfully produced from cryopreserved sperm. The progeny of some androgenetic males crossed to normal females were virtually all males, while the progeny of other males were virtually all females. This suggests that both XX and YY androgenetic individuals may develop as males. Androgenesis is likely to be useful for generating homozygous clones for research and for recovering strains from cryopreserved sperm.     

Microsatellite analysis of gynogenetic families in the Pacific oyster, Crassostrea gigas

Five families of gynogenetic diploid Pacific oyster (Crassostrea gigas) were induced by inhibiting the second polar body in meiotic cell division of eggs fertilized with UV-irradiated sperm. Segregation patterns of eight microsatellite loci were investigated in the gynogenetic diploid offspring; the proportion of heterozygous progeny was used to estimate microsatellite-centromere (M-C) distances. Mendelian inheritance was confirmed for the eight loci by examining the genotypic segregation in the control crosses. Three of the eight microsatellite loci showed the existence of null alleles in four control crosses. All gynogenetic offspring only possessed the alleles of the mother, indicating 100% success level for the five families. The M-C recombination frequency estimates ranged from 0.62 to 0.77 (0.72 mean), comparable to those in the oyster based on allozyme markers and suggesting that meiotic gynogenesis does not appear to be a very efficient inbreeding method in the oyster. Recombination frequencies observed were often higher than the theoretical maximum of 0.67, indicating the existence of positive interference after a single chiasma formation in some chromosomes. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution toward assembly of genetic maps in C. gigas.     

Gene-centromere mapping of 312 loci in pink salmon by half-tetrad analysis.

We estimated recombination rates between 312 loci and their centromeres in gynogenetic diploid pink salmon (Oncorhynchus gorbuscha) that we produced by initiating development with irradiated sperm and blocking the maternal second meiotic division. Amplified fragment length polymorphisms (AFLPs) were significantly more centromeric than loci identified by three other techniques (allozymes, microsatellites, and PCR using primer sequences from interspersed nuclear elements). The near absence of AFLPs in distal regions could limit their utility in constructing linkage maps. A large proportion of loci had frequency of second division segregation (y) values approaching 1.0, indicating near complete crossover interference on many chromosome arms. As predicted from models of chromosomal evolution in salmonids based upon results with allozyme loci, all duplicated microsatellite loci that shared alleles (isoloci) had y values of nearly 1.0.     

Gene segregation in induced tetraploid rainbow trout: genetic evidence of preferential pairing of homologous chromosomes

Gene segregation at six protein loci was analysed in progeny from tetraploid males and females obtained by suppression of first mitosis. The triploid full-sib families from five tetraploid males and the diploid gynogenetic lines from four tetraploid females were examined. The proportions of heterozygous gametes (0.83 on the average) were significantly higher than expected from tetrasomic inheritance (0.667) at all the loci studied. This was explained by preferential pairing of homologous chromosomes. The proportions of heterozygous gametes were significantly different between loci, but the variations were not correlated with the gene--centromere distances. Our results showed that, at least for one locus, the homozygous gametes mainly resulted from pairing of homologous chromosomes rather than from pairing of homologous chromosomes, quadrivalent formation, and chromatin exchanges between homologous chromosomes.     

Segregation of Microsatellite Alleles in Gynogenetic Diploid Pacific Abalone (Haliotis discus hannai)

Inheritance of 9 microsatellite loci was examined in 3 families of gynogenetic Pacific abalone Haliotis discus hannai produced by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division. The proportion of heterozygous progeny was used to estimate marker-centromere (M-C) distances. All loci conformed to Mendelian segregation in the control crosses when null alleles were accounted for. The absence of paternal alleles confirmed the gynogenetic origin of the offspring and indicated 100% success for 3 families. Estimated recombinant frequencies ranged from 0.10 to 0.60, which is lower than those observed in other gynogenetic diploid animals. The mean recombination frequency was 0.22, corresponding to a fixation index of 0.78 in one generation. This is 3.12 times the increase in homozygosity expected after one generation of sib mating (0.25), suggesting meiotic gynogenesis may be an effective means of rapid inbreeding in the abalone. M-C map distances for the 9 loci varied between 5 and 30 cM under the assumption of complete interference. The information about M-C distances will be useful for future gene mapping in H. discus hannai.     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2)of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes.Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp, and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gynogenetic progeny of allotetrapioid hybrids of red crucian carp × common carp had important significances in both biological evolution and production application.     

Gene-Centromere Mapping in Rainbow Trout: High Interference over Long Map Distances

Ten enzyme loci were mapped in relation to their centromeres in gynogenetic diploid rainbow trout. Gene-centromere map distances, calculated under the assumption of complete interference, range from 1.1 cM for Ldh4 to 50 cM for Sod1. The Idh2 and Est1 loci are linked on the same chromosome arm.—The observation of close to 100% heterozygous gynogenetic diploids for the Sod1 and Mdh3,4 loci suggests that near-complete interference occurs on the chromosome arms carrying these loci. The high interference observed in this study and in several other species of fish may be related to the small size of fish chromosome arms.—Comparisons of map locations for the Ldh3 and Ldh4 and the Mdh3 and Mdh4 loci, which were duplicated by a tetraploid event in the evolution of salmonid fish, reveal that they are located at similar distances from their centromeres. Comparative mapping of loci duplicated longer ago shows more variation in map location.—The high proportion of heterozygotes for some loci after gynogenesis involving second polar body retention demonstrates that this is not a practical method for producing homozygous inbred lines in rainbow trout; treatments suppressing the first cell division are more promising for this purpose.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳检测,结果表明:1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

人工雌核发育鲢的遗传多样性及异源遗传物质整入的RAPD分析

运用RAPD技术对连续二代人工雌核发育鲢的遗传多样性及异源遗传物质的整入进行了分析 ,结果表明 :一代雌核发育鲢 ,个体间遗传相似度为 0 94 5— 0 995 6 ,多样性指数为 0 175 ;二代雌核发育鲢 ,个体间遗传相似度为0 96 15— 1 0 0 ,平均为 0 985 2 ,多样性指数为 0 0 6 2。研究揭示经过连续二代人工雌核发育后 ,其遗传多样性明显减少 ,种质进一步纯化。通过对雌核发育鲢二代、亲本鲢和雄鲤的RAPD扩增比较 ,发现雌核发育鲢含有少数与父本相同的特异DNA扩增带 ,而亲本鲢没有 ,在基因水平上表明雌核发育鲢整入了雄鲤的遗传物质     

Centromere mapping in the Pacific abalone (Haliotis discus hannai) through half-tetrad analysis in gynogenetic diploid families

Centromere mapping is an essential prerequisite for our understanding of the composition and structure of genomes. For centromere mapping, in two meiogynogenetic families of the Pacific abalone (Haliotis discus hannai), we screened 97 microsatellite markers that cover all linkage groups from a currently available abalone linkage map. Microsatellite analysis showed that no unique paternal allele was found in all gynogenetic progeny, which confirmed 100% success of induction of gynogenesis. In the control crosses, all 97 microsatellite loci were compatible with Mendelian inheritance, while in meiogynogenetic progeny, 5.2% of the microsatellite loci showed segregation distortions from an expected 1:1 ratio of two homozygote classes. The second division segregation frequency of the microsatellites ranged from 0.037 to 0.950 with a mean of 0.399, indicating the existence of interference. Heterogeneity among linkage groups in the crossover distribution was observed. Centromere location was mostly in accordance with the abalone karyotype, but differences in marker order between linkage and centromere maps occurred. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in the commercially important abalone species.     

Clonal Atlantic salmon × brown trout hybrids produced by gynogenesis

Clonal full-sib progeny groups of Atlantic salmon Salmo salar × brown trout Salmo trutta hybrids were produced by gynogenesis. Eggs obtained from two 3-year-old Atlantic salmon (female) × brown trout (male) F₁ hybrids were activated with UV-irradiated rainbow trout Oncorhynchus mykiss sperm. Fecundity, percentage egg activation and percentage survival to completion of yolk-sac absorption were similar for the two females, and averaged 800 eggs kg⁻¹, 90 and 65%, respectively. Flow cytometric and protein electrophoretic analyses confirmed the progeny to be diploid hybrids. Isogenicity within progeny groups and to the maternal parent was indicated by identical DNA fingerprint patterns detected with multilocus oligonucleotide probes–GATA₍₅₎ and ACTG_(n). Isogenicity was also observed in the gynogenetic progeny of a third female spawned the following year. It appeared that a large portion of the oocytes in females of this hybrid underwent a premeiotic chromosome doubling, or possibly a complete suppression of meiosis. The result was ovulation of diploid eggs, each possessing a full set of both Atlantic salmon and brown trout chromosomes identical to those in the maternal somatic cells. Lines of clonal hybrids could therefore be perpetuated by gynogenesis and would have potential both as experimental animals and in commercial aquaculture.     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Survival, growth and sex ratios of gynogenetic diploid honmoroko

Survival, growth and sex ratios of gynogenetic diploid honmoroko Gnathopogon caerulescens induced by blocking the release of the second polar body were examined. Mean survival of gynogenetic juveniles at 130 days after hatching was about 33% lower than that of the controls. No significant difference was seen in early growth between control and gynogenetic diploids. Standard length and body weight in six groups of gynogenetic progeny were significantly greater but in two groups were significantly smaller than in the controls. Although 69% of gynogenetic diploids had well-developed gonads, the remaining 30% had undeveloped gonads (small in size or thread-like), and those gonads were divided into four types. The mean proportion of females in the 10 gynogenetic groups was 87·2% which was significantly ( P <0·01) higher than in the controls (44·7%). Gynogenetic diploids included 3·0–35·3% males. Most of those males produced a high proportion of female progeny, but the proportion of male offspring varied widely. From these results, the sex determining mechanism in honmoroko was presumed to be female homogamety, but other factors resulted in the production of males.     

Diploid gynogenesis induced by hydrostatic pressure in rainbow trout, Salmo gairdneri Richardson

Diploid gynogenetic fry of rainbow trout, Salmo gairdneri , were produced by treating eggs activated by UV-irradiated sperm with hydrostatic pressure. Treatments for l0 min at 40 min after activation with 55 000 kPa (8000 psi) pressure resulted in high hatching rates and yields of heterozygous diploid gynogenomes of up to 81%. Attempts to produce homo-zygous diploid gynogenomes by the suppression of the first mitotic division ofthe eggs failed. Ether treatment alone did not induce diploid gynogenesis.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Comparison of genetic variability and parentage in different ploidy classes of the Japanese oyster Crassostrea gigas

Chemical treatments with cytochalasin B were used to induce triploidy in the progeny of a mass fertilization of 3 male and 7 female Crassostrea gigas parents. Triploids were produced either by retention of the first (meiosis I (MI) triploids) or the second (meiosis II (MII) triploids) polar bodies. These animals, together with their diploid siblings, were divided for two experiments. One set was used to compare physiological performance, and the other set deployed to compare growth in two different natural environments. For both experiments, genetic variability in different ploidy classes was estimated using three microsatellite loci and eight allozyme loci. The microsatellite loci were highly polymorphic, allowing independent confirmation of ploidy status and the unambiguous identification of parentage for each oyster. Significant differences in parentage were found between ploidy classes, despite the fact they originated from the same mass fertilization. This indicates that the assumptions of a common genetic background among random samples of animals taken from the same mass fertilization may not be generally valid. Knowledge of parentage also allowed the more accurate scoring of allozyme loci. As expected, triploids were found to be significantly more polymorphic than diploids. However, MI triploids were not significantly more polymorphic than MII triploids. MII triploid genotypes were used to estimate recombination rates between loci and their centromeres. These rates varied between 0.29 and 0.71, indicating only moderate chiasma interference.     

Evidence for the Formation of the Male Gynogenetic Fish

The females and unexpected males of gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (RCC; ♀, 2n?=?100) × blunt snout bream (BSB; ♂, 2n?=?48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC₁) and another variational gray crucian carps (GGCC). The GRCC and their offspring were proved to be diploids (2n?=?100) with one to three microchromosomes by examining the chromosomal metaphases. The evidences for the male’s genetic effect in GRCC were provided by means of fluorescence in situ hybridization, Sox-HMG DNA markers, and microsatellite DNA markers. The genotypic variances of GRCC resulted in their phenotypic variances which were quite different from their maternal parent. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish. The formation of the bisexual GRCC and their progeny indicated that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish, which is of great significance in both fish genetic breeding and evolutionary biology.     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2) of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes. Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gyno- genetic progeny of allotetraploid hybrids of red crucian carp × common carp had important signifi- cances in both biological evolution and production application.