Induction of meiotic gynogenesis in bagrid catfish (Pseudobagrus ussuriensis) with homologous sperm and its confirmation for female homogamety

The bagrid catfish, Pseudobagrus ussuriensis, exhibits significant sexual dimorphism in growth rate and body size with males growing faster than females. Therefore, an all‐male culture can dramatically increase the output and profitability of fishery products. According to the monosex breeding route, super‐male individuals’ acquirement by XY male sex reversal and artificial gynogenesis is the key step. An effective protocol to induce meiotic gynogenesis using homologous sperms has been developed in this study. The most effective UV irradiation for sperm genetic inactivation was found to be at a distance of 20 cm with 66 μW/cm² light intensity for 25 min. Optimal treatment for cold shock was 5 min post‐fertilization at 0‐4°C for 30 min, which gave the best survival rate of 13.65 ± 2.87%. The sex ratio in the meiotic gynogens showed a significant female‐biased deviation (p < .05); thirty meiogynogens and their parents were further analysed using a male‐specific AFLP marker, of which only the male parent produced a male‐specific DNA band of 412 bp. These results indicated the female homogametic XX/XY sex determination system in P. ussuriensis. The optimization of a protocol for the successful induction of meiogynogenesis in the bagrid catfish lays the basis for all‐male production and is useful in ascertaining the genetic sex determination system in this promising aquaculture species.     

雌核发育草鱼群体及两个普通草鱼群体的微卫星遗传分析

以草鱼(Ctenopharyngodon idellus)养殖群体(YZ)为母本,以紫外线灭活的鲤鱼精子激活草鱼卵子,冷休克抑制第二极体排出的方法诱导获得异精雌核发育草鱼群体(CH)。利用12对微卫星引物对YZ群体、YS (扬州广陵长江系家鱼原种场引进草鱼群体)群体、CH群体进行PCR扩增并分析,共检测出194个等位基因,其中75.8个有效等位基因。YZ群体、YS群体、CH群体的平均等位基因数依次为13.0、12.6、4.7;平均有效等位基因数依次是7.7、6.6、2.3;平均期望杂合度依次为0.87、0.82、0.56;平均多态信息含量依次为0.84、0.79、0.49。从每个个体在微卫星位点的纯合率看,YZ群体中个体的纯合度在0.00～0.33, YS群体r中个体的纯合度在0.00～0.42, CH群体中个体的纯合度在0.42～0.92。这表明与YZ群体和YS群体相比,CH群体的遗传多样性显著下降,并且在每个位点的纯合率CH群体均高于普通草鱼群体,表明人工诱导减数雌核发育可加速草鱼大多数基因位点的纯合,是快速建立高纯品系的有效手段。同时,本研究筛选并利用微卫星位点组合建立了雌核发育草鱼子代不同家系及其母本亲缘关系的简易、高效鉴别技术,旨在为雌核发育草鱼标记辅助育种打下基础。     

Genetic identification of a newly synthetic allopolyploid strain with 206 chromosomes in polyploid gibel carp

A newly synthetic allopolyploid strain (SAS) was selected and established from gynogenetic offspring of gibel carp clone A⁺ with 156 chromosomes induced by common carp sperm with 50 chromosomes. In this study, the allopolyploid strain was detected to contain 206 chromosomes, and the growth trait was evaluated to have 25.15% growth faster than that of clone A⁺. Genetic marker analyses of transferrin (Tf) alleles, ITS1 sequences and mtDNA D‐loop sequences indicated that the allopolyploid strain was synthetized from maternal gibel carp clone A⁺ and paternal common carp, and the synthetic chromosome sets were further confirmed by genomic in situ hybridization (GISH) and chromosome localization of 45S rDNA. Significantly, the synthetic allopolyploid strain has tended to be stable by five successive generations of gynogenesis, because it still keeps unisexual reproduction mode of gynogenesis. Therefore, it will become a novel variety for gibel carp aquaculture in future.     

鳙异源雌核发育子代的形态学研究

本研究在已成功利用鳙(Aristichthys nobilis)(♀)×兴国红鲤(Cyprinus carpio Var.Xingguo)(♂)远缘杂交诱导鳙异源雌核发育的基础上,测定了鳙异源雌核发育子代及其母本鳙和父本兴国红鲤3组实验鱼的7项可量性状,计算其框架系数,并采用单因素方差分析、判别分析、主成分分析和聚类分析对三组鱼框架系数的差异和距离进行了分析。结果显示3组鱼多项框架系数间均具显著差异,雌核发育子代较母本表现为体型更高,头更长。判别分析能将3组鱼明显判别分为3个区域,其中雌核发育子代和兴国红鲤的判别准确率均为100%,母本鳙群体判别准确率为94.4%,总判别准确率为97.9%。主成分分析结果显示,可提取2个主成分,其中主成分1能解释64.504%的变异,主成分2能解释21.292%的变异,二者合并能解释85.796%的变异。聚类分析表明雌核发育子代形态学可量性状指标更接近于母本鳙,与父本兴国红鲤距离较远。本研究结果表明雌核发育后,鳙异源雌核发育子代主要可量性状与双亲产生显著差异,但更接近于母本。     

Genetic variability in meiotic gynogenetic muskellunge,Esox masquinongy (Mitchell), estimated from segregation of microsatellite alleles

Muskellunge, Esox masquinongy, is an important recreational freshwater fish native to North America. Since muskellunge populations are often maintained through stocking efforts, advances in muskellunge reproductive technologies are of direct relevance to fishery enhancement. We evaluated the efficiency of inbreeding through induced meiotic diploid gynogenesis. Eggs from six female muskellunge were manually stripped and activated using ultra violet‐irradiated yellow perch, Perca flavescens, sperm. Hydrostatic pressure shocking regimes (48 263 kPa) were then applied to the eggs to prevent second polar body expulsion producing unambiguous meiotic gynogens. Six female dams and samples of 12–20 of their gynogenetic progeny were genotyped at seven polymorphic microsatellite loci. Chromosomal recombination frequencies of microsatellite loci based on retention of heterozygosity among gynogens ranged from 0.043 to 0.839 (0.576 ± 0.237). There were no statistical differences in recombination frequency among females at any of the loci. The average inbreeding coefficient (F‐value) ranged from 0.581 to 0.979, equivalent to three to fourteen generations of full‐sibling crosses respectively. The average F‐value overall was 0.712, equivalent to between five and six generations of full‐sibling crosses. Centromere map distances of the seven microsatellite loci ranged from 2.15 to 41.95 cM and meiotic gynogenesis was useful in eliminating heterozygosity at loci proximal to the centromere, but not distal. Since the age at maturity of female muskellunge is approximately 5 years, gynogenesis may pose an expeditious alternative to traditional breeding strategies for creation of homozygous pedigrees for some loci that may be outcrossed to introduce positive heterozygosity effects in the offspring.     

牙鲆连续两代减数分裂雌核发育家系的遗传特征

对牙鲆(Paralichthys olivaceus)减数分裂雌核发育二倍体(Meio-G1)再次诱导减数分裂雌核发育,获得连续两代雌核发育二倍体家系(Meio-G2),以Meio-G1亲本与1尾普通牙鲆雄鱼人工授精获得的家系作为对照组(control)。利用15个微卫星标记对3个家系进行遗传特征分析。结果显示,15个微卫星位点在Meio-G1、Meio-G2和对照组3个家系中,分别扩增到30、28、50个等位基因,平均等位基因数为2.0、1.9、3.3,平均观测杂合度(Ho)分别为0.875 3、0.774 2、0.908 3,平均纯合度分别为0.124 7、0.221 5、0.091 7。3个家系个体间的平均遗传相似系数分别为0.891 7、0.923 8、0.520 2,亲代与子代之间的平均相似系数分别为0.916 6、0.930 4、0.560 3。高重组率的Poli9-8tuf、Poli18tuf、Poli107tuf 3个位点在Meio-G1和Meio-G2中观测杂合度均为1.0,低重组率的Poli33tuf、Poli24MHFS两个位点在Meio-G1和Meio-G2中均全部纯合,观测杂合度为0。结果表明,Meio-G2的纯合度、个体间平均相似系数以及亲子之间的平均相似度均略高于Meio-G1,显著高于对照组家系,证明连续两代诱导减数分裂雌核发育,能提高鱼类纯合度和遗传相似度,具有固定母本遗传性状的作用。     

3种染色体组加倍方法诱导黄河鲇雌核发育的比较

用经紫外线照射后遗传失活的黄河鲇精子、刺激性成熟的黄河鲇卵子, 进行雌核发育, 采用冷休克、热休克和秋水仙素浸泡等3种染色体组加倍方法诱导黄河鲇卵进行二倍体雌核发育。实验结果表明: 在人工诱导黄河鲇二倍体雌核发育过程中, 以冷休克处理组的效果最好, 胚胎孵化率达到8. 1%; 其次为秋水仙素处理组, 胚胎孵化率达到6. 3%; 热休克处理组诱导效果较差, 胚胎孵化率仅为4. 0%。     

AgNO_3对甜瓜离体胚囊发育的影响

许多作物的花药、小孢子培养技术被广泛的应用到实际育种工作中。与花药培养相比,离体培养子房或胚珠诱导单倍体植株具有独特的优势,在某些作物上诱导率和绿苗率较高。早在80年代,水稻、大麦、玉米、青稞和马铃薯等都已建立了未受精胚珠单倍体离体培养技术,而对于葫芦科植物至今尚未找到一条高效的单倍体诱导方法。1999年,FICCADENTI进行甜瓜未授粉胚珠离体培养,得到了单倍体植株。2003年,LOTFI采用FICCADENTI的培养方法,对不同甜瓜品种进行诱导,结果只得到了3棵再生植株,在未进行倍性鉴定之前死亡。至今,国内甜瓜离体雌核发育诱导单倍体研究尚未报道。