黄姑鱼异质雌核发育子代的遗传分析

为了解黄姑鱼（Nibea albiflora）异质雌核发育子代的基因纯合情况,利用微卫星标记（SSR）和扩增片段长度多态性标记（AFLP）对黄姑鱼异质雌核发育家系进行遗传鉴定和分析。结果显示：（1）雌核发育家系在4个SSR位点和5对AFLP引物组合扩增出的位点均未发现父本特异性等位条带,表明雌核发育体比率为100%。（2）用于遗传分析的7个SSR位点在雌核发育家系和正常交配家系中均未见完全纯合的情况,雌核发育家系7个SSR位点的平均纯合度为0.382,是正常交配家系（0.161）的2.37倍。雌核发育家系各个体的纯合位点数为0~6个,纯合位点所占比例为0~85.7%。（3）5对AFLP引物共扩增出182条清晰的扩增条带,其中有21条父本特异性条带和16条母本特异性条带。16条母本特异性条带中有7个条带在雌核发育家系中显著偏分离（P<0.05）。雌核发育家系和正常交配家系多态性条带比例分别为14.7%和20.3%。（4）雌核发育家系与母本的遗传相似度高于与正常交配家系的遗传相似度,正常交配家系同父本和母本的遗传距离大致相同。研究结果表明,黄姑鱼异质雌核发育二倍体家系的遗传纯合度显著高于正常交配家系,人工诱导雌核发育是促进基因纯合的一个有效途径,它不仅可以加速有利基因的纯合固定,还可以加速有害基因的淘汰,从而有效提高育种效率。     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

Chromosome set manipulation and sex control in common carp: a review

The development of techniques for production of gynogenetic, androgenetic, polyploid, and monosex progenies in common carp (Cyprinus carpio L.) is described from a chronological perspective. Gynogenetic progenies were obtained either by suppression of the second meiotic division in eggs (meiotic gynogenesis) or by suppression of the first mitotic division in haploid embryos (mitotic gynogenesis). As a rule, gynogenetic progenies of common carp were all-female, revealing female homogamety (females—XX, males—XY) in this species. Induced gynogenesis results in increased homozygosity; the rate of increase depends on the type of gynogenesis. Inbreeding coefficient (F) for one generation of meiotic gynogenesis in common carp is about 0.6, while diploids obtained by mitotic gynogenesis are homozygous for all genes (F = 1.0). Mitotic gynogenesis was used for production of clones in common carp. In androgenetic progenies of common carp, YY males were identified, that after crossing with normal females (XX) produced all-male progenies. Triploids of common carp are characterized by a significant reduction in gonad development (especially ovaries). However, the reduction in gonad development did not result in an increase of somatic growth rate of fish. The procedure for androgen treatment to induce phenotypic sex reversal in genotypic females (XX) was elaborated. All-female progenies of common carp were produced on a large scale by crossing normal females (XX) with hormonally sex-reversed males (XX). Rearing of all-female progenies in conditions when fish normally reach sexual maturity before reaching of market size increased production yield by 7–8%. In a few cases distant hybridization resulted in polyploidy of fish without application of any physical treatment. The ability of hybrid females between crucian carp (Carassius auratus) and common carp to produce diploid (with unreduced chromosome number) gametes resulted in opportunities to produce triploid and tetraploid hybrid progenies.     

Fast development of genetically uniform strains by gynogenesis in Japanese flounder (Paralichthys olivaceus)

The Japanese flounder is a major species in both aquaculture and research. Inbred strains of Japanese flounder were developed efficiently in our laboratory by meiotic and mitotic gynogenetic reproduction techniques. To determine the induction efficiency of gynogenesis and the rate at which full homozygosity is produced, six meiotic gynogenetic females (G1–G6) that had experienced meiotic gynogenesis once and three common females (C1–C3) were selected for production of mitotic gynogenesis in our experiments. Of the nine adult females, all six gynogenetic fish successfully produced viable offspring. However, only one of the three common fish did. Using microsatellite markers, we estimated the homozygosity of gynogenetic fish induced by mitotic gynogenesis. We found that the homozygosity of seven lines (C1, G1–G6) increased quickly, individually from 43%, 72%, 69%, 71%, 78%, 69% and 58% to 100%, 100%, 100%, 90%, 100%, 100% and 90% offspring complete homozygosity. Under mitotic gynogenesis, individuals with higher homozygosity had a higher induction rate. The G4 line showed the highest induction rate, achieving 44.59% convert hatching rate and 29.28% convert normality rate. We conclude that meiotic gynogenesis may be a feasible method to produce DH genetic material in Japanese flounder.     

Induction of meiotic gynogenesis in the stinging catfish Heteropneustes fossilis (Bloch) and evidence for female homogamety

This study reports the results on induced meiotic diploid gynogenesis and female homogametic nature in the Indian catfish, Heteropneustes fossilis. The eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm mL⁻¹ in Hanks balanced salt solution. Sperm were irradiated under UV light, with the exposure time ranging from 15 to 360 s (7500 ergs mm⁻² for 60 s). The genetic inactivation of paternal chromosomes was confirmed by chromosome counting from the larval cells and the larvae also had a characteristic haploid syndrome. A typical ‘Hertwig effect’ in the yield of hatched larvae was observed with doses of UV exposure >75 s (9375 ergs mm²). Larvae resulting from sperm UV irradiated above 120 s (15 000 ergs mm²) were 100% haploids. Application of heat shock to the activated eggs was effective in suppressing the release of the second polar body (meiotic gynogenesis) and resulted in diploid gynogenetic larvae morphologically identical to those of the control. The best yield of diploid gynogens (49.3% with respect to the control) was found to be at 6 min after egg activation and the heat shock at 41 °C for a 1-min duration, at an ambient water temperature of 27 °C. A total of 113 diploid gynogenetic fry from seven different female fish were reared and subjected to sexing. All gynogenetic fish were female in contrast to the control, which had a mean sex ratio of 56.7% females (which was not significantly different from 50% female). From these results, the sex determination mechanism in H. fossilis was presumed to be female homogamety.     

Spontaneous mosaicism occurred in normally fertilized and gynogenetically induced progeny of the kokanee salmon Oncorhynchus nerka

ABSTRACT:   In normally fertilized progeny of the kokanee salmon Oncorhynchus nerka , DNA content flow cytometry revealed that all the externally normal embryos were diploid, whereas abnormal embryos exhibited haplo-diploid, diplo-tetraploid and haplo-diplo-tetraploid mosaicisms, together with a few haploid and diploid individuals. When gynogenetic development was artificially induced by fertilization of eggs obtained from a female of the same kokanee brood stock with UV-irradiated sperm, haplo-diploid mosaics appeared most frequently. These mosaics were likely to happen by certain cytological events, such as meiotic or mitotic errors during the process of maturation, fertilization or early cleavage.     

Artificial gynogenesis and assessment of homozygosity in meiotic gynogens of spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm⁻²) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H _O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.     

牙鲆有丝分裂雌核发育二倍体的制备

本研究采用紫外线灭活的真鲷(Pagrus major)精子激活牙鲆(Paralichthys olivaceus)卵子, 经过静水压机处理诱导有丝分裂雌核发育二倍体。优选施压起始时间、持续时间和压力大小3个方面参数, 获得最佳参数组合为: 起始时间60 min、持续时间6 min、施加压力650 kg/cm²。在此条件下的受精率最高为67.80%, 孵化率54.23%, 与其他处理组间达显著性差异(P<0.05)。用流式细胞仪检测未经静水压处理的胚胎, 其DNA含量约为普通二倍体的1/2, 即单倍体; 检测有丝分裂雌核发育胚胎, 其DNA含量与普通二倍体大体一致。结果表明, 应用该方法可成功诱导获得牙鲆有丝分裂雌核发育二倍体。

     

Use of microsatellite loci and AFLP markers to verify gynogenesis and clonal lines in Nile tilapia Oreochromis niloticus L.

To develop an effective system for parentage analysis in gynogenetic and clonal progeny of Nile tilapia, Oreochromis niloticus L., polymorphic microsatellite loci and amplified fragment length polymorphisms (AFLPs) were investigated in several gynogenetic families and clonal lines. Six microsatellite loci were screened in two meiotic gynogenetic families to look for loci with high gene–centromere recombination rates, which can be used to discriminate meiotic from mitotic gynogenetics. Microsatellite loci UNH189 and UNH211 showed 96.7% and 92.0% heterozygosity, respectively, in these families, while other loci showed lower recombination frequencies. Scoring both UNH189 and UNH211 would give a very low probability of an individual meiotic gynogenetic being homozygous for both loci. Multiplex polymerase chain reaction of microsatellite loci was used to verify parentage in four families of mitotic gynogenetics and five fully inbred clonal lines. The genotype of each clonal line should serve as a unique identifier. Twelve AFLP primers were also investigated and 26 diagnostic AFLP bands were identified to follow inheritance in mitotic gynogenetic individuals. Amplified fragment length polymorphisms were found to be effective for this purpose but microsatellites were more appropriate since they are co‐dominant, while AFLPs are dominant markers. A multiplex of the microsatellite loci used in this study would be useful for general parental assignment as well as for the analysis of the products of chromosome set manipulations.     

雌核发育牙鲆与普通牙鲆的肌肉营养成分比较

测定了牙鲆Paralichthys olivaceus幼鱼肌肉的水分、粗蛋白、粗脂肪和粗灰分,分析了肌肉氨基酸和脂肪酸组成及含量,对普通牙鲆和雌核发育牙鲆肌肉的营养成分进行了比较。牙鲆肌肉中粗蛋白含量均较高,达到21.3%,鲜味氨基酸——谷氨酸在所有氨基酸中含量最高,占到总量的2.9%,且不饱和脂肪酸具有较高水平。测定结果表明,普通牙鲆雌、雄个体间肌肉营养组成和含量无显著性差异(P0.05),雌核发育牙鲆与普通牙鲆雌、雄间亦无显著差异(P0.05);雌核发育牙鲆与普通牙鲆肌肉相比,其营养组成无明显差别,同样具有营养丰富、组成均衡等优点,雌核发育对鱼类营养成分并无不良影响。     

Meiotic gynogenesis with heterologous sperm in the mandarin fish Siniperca chuatsi and evidence for female homogamety

The mandarin fish Siniperca chuatsi is a historically important aquaculture species in China and exhibits sexually dimorphic growth. However, sex determination of this fish remains unclear so far. In this study, we induced meiotic gynogenesis in S. chuatsi using irradiated heterologous sperm from spotted mandarin fish (Siniperca scherzeri) to uncover its mechanism of sex determination. Up to 7.52% diploid progeny were obtained among three gynogenetic families in this study. Molecular analysis of female and male donors and sampled young gynogens by seven microsatellite loci further confirmed no genetic contributions from the ‘father’ S. scherzeri. After 8 months of culture, external morphology of adult fish showed that all gynogens were cloned from their mothers. Gonads of the gynogenetic progeny were examined by histological observations and the sexing results showed that they were almost 100% females, strongly supporting an assumption of female homogamety in mandarin fish. By this study, we obtained pure lines of S. chuatsi and elucidated its genetic mechanism of sex determination, providing a basis for possible sex control breeding in this species.     

Microsatellite–centromere mapping in bighead carp ( Aristichthys nobilis) using gynogenetic diploid families

Gene–centromere mapping using half‐tetrad analysis is a powerful tool for understanding chromosomal behaviour and determining the position of centromeres in relation to genes or markers in fish. In this study, eight gynogenetic diploid families induced by inhibiting of the second meiotic division were genotyped at 66 microsatellite loci for microsatellite–centromere (M‐C) mapping in bighead carp ( Aristichthys nobilis). The absence of paternal alleles verified the success of gynogenetic development in all gynogenetic families. All loci were consistent with Mendelian segregation in control families. The estimated recombination frequency (y) ranged from 0.057 to 0.875 with an average of 0.477 ± 0.222. Seventeen loci (25.76%) showed high M‐C recombination frequencies of over 0.667. M‐C distances ranged from 2.85 to 43.75 cM under the assumption of complete interference. Thus, these loci are distributed from the centromeres to the telomeres of their respective chromosomes, while mainly in the intermediate region. Information on centromere mapping could serve as a starting point to consolidate the genetic linkage groups in bighead carp.     

Production of meiotic gynogenetic and triploid sea bass, Dicentrarchus labrax L.: 1. Performances, maturation and carcass quality

Meiotic gynogenetic and triploid sea bass were produced by pressure shocks according to a previously published protocol. Pressure-treated groups did not survive as well as controls during early development and larval rearing. Performances, sexual maturation and carcass quality were examined over a period of 34–45 months. At the age of 34 months, growth of the gynogenetic fish was comparable to that of the control but inferior in the triploid fish. A predominance of male fish was found within the triploid groups, while diploid and meiotic gynogenetic fish showed equal proportions of the sexes. Gonadal maturation in triploid fish was significantly impaired, particularly in the females that showed rudimentary ovaries. Triploid males exhibited primary maturation but proved to be gametically sterile. Pressure-induced triploids did not grow as well as diploids, but these results might be ascribed to specific on-growing conditions (communal rearing). The performance of gynogenetic sea bass was comparable to that of control. The superiority of diploid fish over their triploid counterparts was confirmed during the final growing period and more clearly so in females. Performances of triploids varied according to their maternal origin. Overall, striking qualitative differences between diploid and triploid fish were found at the age of 34 and 45 months, although the results varied in a gender-specific manner. A strong maternal effect was also observed. The potential advantages of triploid sea bass for aquaculture purposes are discussed.     

基于M-C作图鉴定牙鲆不同二倍体的遗传特征

为了分析连锁群上不同位置的微卫星标记对鉴定牙鲆二倍体遗传特征的作用,实验以牙鲆选育基础群体为亲本,选择性腺发育良好的个体制备普通二倍体(ND),减数分裂雌核发育二倍体(MGD-1)、连续两代减数分裂雌核发育二倍体(MGD-2),并利用MGD-1发育达性成熟的雌鱼与同时诱导的性反转伪雄鱼交配制备近交二倍体(MGD1H)。从牙鲆遗传连锁图谱选择均匀分布于24个连锁群的72个微卫星标记,用4个MGD-1家系估计微卫星标记与着丝粒之间的相对距离。在假设无交叉干涉的情况下,17个标记位于着丝粒区域,19个标记位于连锁群中部,36个标记位于远着丝粒区域。分别选择上述区域的微卫星标记鉴定牙鲆4种二倍体的遗传特征。结果显示,4种二倍体的等位基因数(A)和多态信息含量(PIC)在不同区域变化范围较小,ND的A和PIC均为最高,MGD1H则表现为最低。随着标记与着丝粒之间距离的增加,4种二倍体的观测杂合度逐渐升高,纯合度逐渐降低。纯合个体比例在着丝粒区域最高,为8.8%～29.1%;在远着丝粒区域最低,为2.4%～23.2%。其中,MGD-1和MGD-2的变化幅度显著高于其余二倍体。结果表明,选择连锁群上不同位置的微卫星标记对鉴定牙鲆不同二倍体的遗传特征具有显著影响。     

Growth of triploid oyster, Crassostrea madrasensis (Preston)

The performance of I and II meiotic triploids and control oysters (Crassostrea madrasensis) reared at Tuticorin Bay was compared to determine if the improvements in the growth of edible oysters were additive to faster growth in triploids. After a grow‐out period of 12 months, both mean whole weights and shell heights were in order I meiotic triploid>II meiotic triploid>control. Mean whole weights and shell height of different oyster lines were all significantly different (P<0.05). On an average, larger morphological traits indicated that growth improvements from triploids were additive, and throughout the study triploid oysters maintained faster growth rate than their diploid siblings. Condition index and adductor muscle diameter of both triploids were higher than those of control.     

Artificial gynogenesis in Cynoglossus semilaevis with homologous sperm and its verification using microsatellite markers

Effective methods for induction of gynogenetic diploids in Cynoglossus semilaevis are needed to initiate monosex culture. An effective protocol to induce half‐smooth tongue sole gynogenesis using homologous sperm was developed in this study. A UV dose of 50 mJ cm^?2 was found to be the most effective for genetic inactivation of tongue sole sperm. Treatment optima for cold shocks were 5 °C for 20–23 min at 5 min after fertilization and the hatching rate of gynogenetic diploids was 10.0%. Microsatellite analysis at locus Csou 6 revealed that there was no genetic contribution from the paternal genome in 24 progenies of a meiotic gynogenetic family. Polymerase chain reaction demonstrated that only four individuals of 24 meiotic gynogenetic diploids produced the female‐specific band of about 205 bp. The female/male ratio of gynogenetic diploids was significantly different from the theoretical ratio of 1:1. It is possible that there are some recessive lethal genes in W chromosome.     

RAPD and microsatellite analysis of diploid gynogens from allotetraploid hybrids of red crucian carp (Carassius auratus)×common carp (Cyprinus carpio)

Genetic variation was comparatively analyzed between the artificially induced diploid gynogen population from F₁₀ allotetraploid hybrids of red crucian carp (♀) (Carassius auratus red var., 2n=100)×common carp (♂) (Cyprinus carpio L., 2n=100) and the normal F₁₀ allotetraploid hybrid population used as the control, using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. The specific 600-bp fragment for diploid gynogen population was detected by S45 and the specific 900-bp fragment for allotetraploid F₁₀ hybrid population was detected by S134. The results from RAPD assay and microsatellite analysis were in agreement with each other, that is to say, the diploid gynogens presented lower level of polymorphism than allotetraploid F₁₀ hybrids. Furthermore, as expected, microsatellite analysis revealed more detailed information on genetic diversity than RAPD assay. The mean percentage of polymorphic loci (12.71%) and Shannon's index of phenotypic diversity (0.25) from RAPD data for diploid gynogen population were significantly lower than those (30.69% and 0.63, respectively) for F₁₀ allotetraploid hybrid population. The mean number of alleles per microsatellite locus (1.73) in diploid gynogen population was considerably lower than that (2.55) in F₁₀ allotetraploid hybrid population. The average observed (0.36) and expected heterozygosity (0.26) in diploid gynogen population were lower than those (0.58 and 0.40, respectively) in F₁₀ allotetraploid hybrid population, indicating that the diploid gynogens presented lower genetic diversity than the allotetraploids. In addition, the mean effective number of alleles at 11 microsatellite loci (1.60) in diploid gynogen population was lower than that (1.88) in F₁₀ allotetraploid hybrid population. The significant differences between two populations in the average observed and expected heterozygosity, mean number of alleles and effective number of alleles, suggested that the effect of gynogenesis resulted in rather higher genetic homogeneity in diploid gynogens. The comparative RAPD analysis of diploid gynogens and their parents was performed with 34 primers. The identical RAPD pattern was detected between diploid gynogens and their female parent, however, some clear specific RAPD bands were detected between diploid gynogens and their male parents, but not detected in their female parent. The result indicated that heterologous genetic material had incorporated into diploid gynogenetic fish (G₁).     

Survival,growth and fertility of gynogenetic diploids induced in the cyprinid loach, Misgurnus anguillicaudatus

Gynogenetic diploids were induced in the loach, Misgurnus anguillicaudatus, by applying cold shock to the egg after fertilization with spermatozoa genetically inactivated by ultraviolet-ray irradiation, to examine their survival, growth and fertility. Eggs were obtained from orange phenotype loach (homozygous for a recessive demelanogenesis gene). Spermatozoa were obtained from black phenotype loach (homozygous for a dominant melanogenesis gene), and from common carp, Cyprinus carpio. All hybrids between loach and carp were inviably abnormal.Eggs fertilized with loach or carp spermatozoa irradiated at doses of 6000 and 12000 erg/mm ² and then treated with cold water (1°C for one hour 4 and 5 min after fertilization developed into embryos with 61–78% survival. About 70–87% of newly-hatched fry were normal in appearance. Gynogenetic fish induced by both loach and carp spermatozoa showed lower rates of survival and growth than did the normal diploid controls. All gynogenetic fish from carp spermatozoa were female and 94.9% of the gynogenetic fish from loach spermatozoa were female. The second gynogenetic offsprings were produced from females selected from both gynogenetic groups.     

Genetic contribution of parents in sex determination of honmoroko Gnathopogon caerulescens

The honmoroko has been inferred to have an XX/XY sex determination system, but the parental genome can also affect the sex ratio of the offspring. The extent of parental effects on sex determination was examined by checking the sex ratios of F1 and F2 gynogenetic diploids and control diploids. Eleven gynogenetic broods from different females consisted of all or nearly all females, but eight broods showed a variable proportion of males (<50 %). One second-generation brood of gynogenetic diploids consisted wholly of females, but others produced some males. In crosses with a control diploid female, four males from a high-percentage male brood of gynogenetic diploids produced offspring with a balanced sex ratio. Sib-mating between a gynogenetic female and three gynogenetic males from the brood produced predominantly male progeny. These results suggest that there are at least four possible genotypes: genotypic female (XX), phenotypic female carrying a silent Y chromosome, genotypic male (XY), and genotypic supermale (YY). These inferences suggest that this fish has an XY system but a relatively high proportion of females possess a mutated, silent Y chromosome which does not lead to testis formation.     

Gynogenesis and sex determination in large-scale loach Paramisgurnus dabryanus (Sauvage)

Gynogenesis was induced using heterologous sperms in large-scale loach, Paramisgurnus dabryanus (Sauvage), in which a ZW/ZZ sex determination was previously proposed. Three microsatellite loci were used to monitor exclusive maternal inheritance of gynogenetic progenies. The results showed that high percentages of meiogynogens were produced at 4 min post-fertilization and mitogynogens were produced at 18 min post-fertilization by heat shocks, while meiotic gynogenesis was induced by cold shocks within a wide period and high heterozygosity was even observed in gynogens produced at 24 min post-fertilization. The sex ratios of the F₁ progenies in three gynogenetic families were significantly deviated from 1:1 expectation with a female bias in two families and a male bias in one family (P < 0.05), and the other four gynogenetic families showed approximate 1:1 sex ratios. Moreover, the self-mating between gynogenetic F₁ progenies and mating between gynogenetic F₁ progenies and normal individuals produced all-female progenies or identical proportions of females and males. The data of sex ratios generally confirmed that the sex determination in large-scale loach was determined by the putative ZW/ZZ system, and the possible reasons causing the biased sex ratios are discussed.