Distant hybridization leads to different ploidy fishes

Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (♀)×common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (♂), and RCC (2n=100) (♀)×blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (♂) are described. In the distant hybridization of RCC (♀)×CC (♂), bisexual fertile F3–F18 allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (♀)×BSB (♂), different ploidy fishes were obtained, including diploid bisexual fertile natural gynogenetic fish (2n=100), sterile triploid hybrids (3n=124), and bisexual fertile tetraploid hybrids (4n=148). Furthermore, two kinds of pentaploid hybrids (5n=172 and 5n=198) were formed. The biological characteristics and the mechanisms of formation of the different ploidy fish were compared and discussed at the cellular and molecular level. The results indicated distant hybridization or the combination of this method with gynogenesis or androgenesis affects the formation of different ploidy fish with genetic variation.     

Formation and biological characterization of three new types of improved crucian carp

The improved tetraploids (G1×AT) were obtained by distant crossing and gynogenesis and the high-body individuals accounted for 2% among G1×AT. After mating with each other, the high-body in- dividuals produced three kinds of bisexual fertile diploid fishes: high-body red crucian carp, high-body fork-like-tails goldfish and gray common carp. The high-body red crucian carp mating with each other formed three types of improved crucian carp (ICC) including improved red crucian carp (IRCC), im- proved color crucian carp (ICCC) and improved gray crucian carp (IGCC). The phenotypes, chromo- some numbers, gonadal structure and fertility of the three kinds of ICC and their offspring were observed. All the three kinds of ICC possessed some improved phenotypes such as higher body, smaller head and shorter tail. The ratios of the body height to body length of IRCC, ICCC and IGCC were 0.54, 0.51 and 0.54, respectively. All of them were obviously higher than that of red crucian carp 0.41 (P<0.01). Three kinds of ICC had the same chromosome number as red crucian carp with 100 chromosomes. All the ICC possessed normal gonads producing mature eggs or sperm, which was important for the production of an improved diploid population. Compared with red crucian carp, all the ICC had stronger fertility such as higher gametes production, higher fertilization rate and higher hatchery rate. Three types of improved diploid fish population were generated from the three kinds of ICC by self-crossing, respectively. The ICC can serve as ornamental fish and edible fish. They are also ideal parents to produce triploids by mating with tetraploids. The new ICC plays an important role in biological evolution and fish genetic breeding.     

The formation of improved tetraploid population of red crucian carp × common carp hybrids by androgenesis

Bisexual fertile diploid androgenetic individuals (A0) (2n=100) were formed by androgenesis. In this way, the diploid spermatozoa from male allotetraploid hybrids (AT) (4n=200) of red crucian carp (Carassius auratus red var.) (♀) × common carp (Cyprinus carpio L.) (♂) were used to fertilize the UV-treated hap- loid eggs of goldfish (Carassius auratus), and living androgenetic diploid fish were developed. The A0 became sexually mature at the age of 2 years, and they fertilized with each other to form their offspring (A1). In this study, we observed the chromosomal number, gonadal structure and appearance of A1 fish. The results are as follows: (1) In A1, there were 85% tetraploids (A1-4n), 10% triploids (A1-3n) and 5% diploids (A1-2n), suggesting that diploid A0 could produce diploid gametes. It was concluded that the formation of diploid gametes generated from diploid A0 was probably related to the mechanism of pre-meiotic endoreduplication. (2) Among A1, only A1-4n possessed normal ovaries and testes. The mature males of A1-4n produced white semen. Under the electron microscope, the head of diploid sperm generated by A1-4n was bigger than that of haploid sperm generated by red crucian carp. In the testes of the A1-4n, there were many mature normal spermatozoa with a head bearing plasma mem- brane and a tail having the typical structure of "9 2" microtubules. Between the head and the tail, there were some mitochondria. The ovaries of A1-4n developed well and mainly contained II, III and IV-stage oocytes. The IV-stage oocytes were surrounded by inner and outer follicular cells. The micropyle was observed on the oolemma of follicular cells. There were abundant yolks and plenty of endoplasmic reticulum in the cytoplasm of IV-stage oocytes. Because A1-2n and A1-3n were distant crossing diploid hybrids and triploid hybrids respectively, they possessed abnormal gonads, and no mature semen and eggs were observed. (3) Compared with allotetraploids, the A1-4n fish not only had advantages such as fast growth rate and strong resistibility but also showed some new good performances such as high ratio of body width to body length, smaller heads and shorter tails. These results indicated that an- drogenesis could produce bisexual fertile tetraploids and improve the shape of allotetraploid hybrids as well, which will be of great significance in both the cell genetics research and fish breeding.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in- creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go- nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru- cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH) cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of al- lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp.MPF activity was measured by using histone H1 as phosphorylation substrate.There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes,but there existed some subtle difference between them.The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp.It was suggested that the difference may be related to the spindle behavior changes,such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.     

The cloning of Dmc1 cDNAs and a comparative study of its expression in different ploidy cyprinid fishes

Dmc1 (disrupted meiotic cDNA) is a functionally specific gene, which was firstly discovered in yeast and then found to encode a protein required for homologous chromosome synapsis during the process of meiosis. In this investigation, we cloned the partial cDNAs of Dmc1 of diploid red crucian carp, Japanese crucian carp, common carp, triploid crucian carp and allotetraploid hybrids by using a pair of degenerate primers based on the conservative sequence of amino acids of the DMC1 protein in yeast, mouse and human. The full length cDNAs were then obtained by rapid amplification of cDNA ends (RACE). Our data showed that the full length cDNAs of Dmc1 in the three diploid fishes are all 1375 bp long, while it is 1383 bp long in triploids and 1379 bp long in allotetraploids. And despite of the variation in length, all the cDNAs encode a protein of 342 amino acids. A high homology of 97.3% of the DMC1 protein can be drawn by comparing the amino acid sequences in the three diploids, which is also of 86%, 86% and 95% similarity to human, mouse and zebrafish, respectively. A comparative study of the expression pattern of Dmc1 was carried out by RT-PCR using specific primers against the same se-quences of coding regions in different ploidy cyprinid fishes, from which it was showed that Dmc1 was expressed only in gonads of these five kinds of fishes. The expression pattern of Dmc1 in both ovaries and testes from different ploidy fishes within breeding season was also studied by Real-time PCR, and the results showed that the expression of this gene was greatly different among the three different ploidy fishes, which was the highest of triploid and lowest of allotetraploids. The histological sections data showed matured gonads of both diploid red crucian carp and allotetraploids in breeding season, although the latter demonstrated a higher maturation, and no gonadal maturation could be observed in triploids. In conclusion, we suggest that Dmc1 is specifically expressed in the period of meiosis in all the ploidy cyprinid fishes and directly related with the development of gonad in a manner of ploidy-independent way. And further, the high expression of Dmc1 in female triploids might be associ-ated with abnormal meiosis and sterility.     

Evidence for the paternal mitochondrial DNA in the crucian carp-like fish lineage with hybrid origin

In terms of taxonomic status, common carp(Cyprinus carpio, Cyprininae) and crucian carp(Carassius auratus, Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish(2n=100)(2nNCRC) lineage(F1–F3) was established from the interspecific hybridization of female common carp(2n=100)×male blunt snout bream(Megalobrama amblycephala, Cultrinae, 2n=48). The phenotypes and genotypes of 2 n NCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial(mt) genomes of the 2n NCRC lineage from F1 to F3. The paternal mt DNA fragments were stably embedded in the mt-genomes of F_1–F_3 generations of 2n NCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1–F3 generations of 2 n NCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mt DNA organization and nucleotide composition of 2n NCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2nNCRC provided important evidence of the mt DNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mt DNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mt DNA in the mt DNA inheritance.     

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts fxom gynogenetic gibel carp (Carassius auratus gibelio)or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei coulddecondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei couldalso decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nucleicould not decondense sufficiently in the same extracts. The significant differences of morphological changeswere further confirmed by ultrastructural observation of transmission electron microscopy. The data furtheroffer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. Inaddition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decon-densed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of eggextracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts preparedfxom the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism ongynogenesis and reproduction mode diversity in fish.     

Genomic variation in the hybrids of white crucian carp and red crucian carp: evidence from ribosomal DNA

In this study, we conducted a cross of white crucian carp(♀)×red crucian carp(♂)(WR), and characterized the morphology, reproduction and genetics of the progeny. Different from parents, WR with the gray color showed the hybrid morphological traits of both parents. WR possessed normal gonads producing mature eggs or sperm, and exhibited high fertilization rate(90.2%) and high hatchery rate(80.5%), which contributed to produce and enlarge the population. WR with the same DNA content as parents was a diploid fish with 100 chromosomes(2n=100). Amplified ITS of 45 S r DNA, in WR the sequences consisting of 884 bp bases of the entire ITS-1 region, 5.8 S region, and entire ITS-2 region. The sequences showed high similarity between WR and its parents and leaned towards male inheritance. In WR, NTS of 5S r DNA consisted of three length types with total 654 bp bases. From sequence analysis of NTS, WR shared 94.2% and 95.1% similarities with their female and male parent, respectively. Sequence analysis of ITS and NTS revealed that there existed recombination and variation in the hybrid progeny, which was the genetic base for adaptation and speciation. In conclusion, we obtained WR from hybridization and it exhibited hybrid traits in morphology and variation in genetic composition showing essential difference with its parents. The obtainment of WR has important significance in fish genetic breeding.     

转录因子结合位点生物信息学研究进展

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid G.arboreum×G. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreum×G. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化

以雌核发育银鲫和两性生殖彩鲫的成熟卵为材料,分离卵壳,经处理得到卵壳可溶性蛋白组分。SDS－PAGE梯度凝胶电泳分析在雌核发育银鲫中揭示出3条较明显的差异蛋白带。同时,采用相同处理方法对受精前后卵壳蛋白组分进行比较分析后发现,这些差异蛋白带在受精后发生了变化,其带纹表现为减弱或消失,表明这些差异蛋白可能与受精过程相关。 Abstracts:Egg chorions were isolated from unfertilized and fertilized eggs of gynogenetic silver crucian carp and gonochoristic color crucian carp by homogenization and further purification techniques.Then,soluble proteins were extracted from the isolated egg chorions,and were analyzed by gradient SDS-PAGE.Three differential protein bands were revealed between the gynogenetic silver crucian carp and gonochoristic color crucian carp.Furthermore,these differential proteins were demonstrated to undergo obvious changes during fertilization.It was suggested that these differential proteins should be related to the special fertilization process in gynogenetic silver crucian carp.     

Embryonic and genetic manipulation in fish

Fishes,the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics.Nuclear transplantation in fish has been thoroughly studied in China since 1960s.Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults.Most importantly,nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish.This was the first case of cloned fish with somatic cells.Based on the technique of microinjection,recombinant MThGH gene has been transferred into fish eggs and the firsh batch of transgenic fish were produced in 1984.The behavior of foreign gene was characterized and the onsed of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis.This eventually led to the transgenic mosaicism.The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults.The transgenic common carp were more efficient in utilizing dietary protein than the controls.An “all-fish” gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (grGH) coding sequence.The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait.Combination of techniques of fish cell culture,gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21^st century.     

Genome evolution trend of common carp （Cyprinus carpio L.） as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication （FSGD） may have occurred before the radiation of the teleosts. Common carp （Cyprinus carpio L.） has been considered to be a tetraploid species, because of its chromosome numbers （2n=100） and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution （genome duplication and diploidization） of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs （HLJ021 and HLJ332） were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition （dynamic） and equilibrium （static） were proposed to explain the common carp genome evolution between genome duplication and diploidization.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Engineering stable heterosis

The ability to fix heterosis or hybrid vigor in crops without crossing holds great potential for crop yield improvement and global food security. Recent studies in Nature and Nature Biotechnology from two independent groups demonstrate the feasibility of clonal propagation of hybrids by engineering changes in meiosis and embryogenesis. These synthetic apomixis technologies represent a watershed moment for both understanding the fundamental basis of heterosis and plant breeding.     

Meiotic Studies of Diploid Perennial Teosinte and Its Hybrids with Maize

1. Open-pollinated diploid perennial teosinte Microsporocytes at pachytene stage from six different diploid perennial teosinte plants were investigated. It was found that they all had 10 pairs of chromosomes (2n = 20). All of the knobs and large chromomere were terminal (Plate Ⅰ,). The size of the knobs varied from medium to small. The short arms of chromosomes 1 and 2 and the long arms of chromosomes 2 and 3 had a small knob. A large chromomere     

Chromosome Transfer from Pink Salmon (Oncorhynchus gorbuscha) to Masu Salmon (O. masou)

Sperms from Pink salmon were subjected to incomplete irradiation by γ-ray to cause partial breakageof their chromosome. Normal eggs from masu salmon were fertilized by these damaged sperms andput under hydrostatic pressure to suppress the release of second polar bodies. The resultant eggscontained complete sets of genome from masu salmon and some chromosomes and chromosomefragments from pink salmon. Karyotype and isozyme of the embryos were analyzed. The resultsdemonstrate that the chromosomes from pink salmon showed genetic activities. Variations in hatchedindividuals were observed.     

Intergeneric Somatic Hybridization Between Brassica napus L. and Sinapis alba L.

Electrically induced protoplast fusion was used to produce somatic hybrids between Brassica napus L. and Sinapis alba L. Seven hybrids were obtained and verified by the simple sequence repeat and cleaved amplified polymorphic sequence analysis of the genefael, indicating that the characteristic bands from S. alba were present in the hybrids. The hybridity was also confirmed by chromosome number counting because the hybrids possessed 62 chromosomes, corresponding to the sum of fusion-parent chromosomes. Chromosome pairing at meiosis was predominantly normal, which led to high pollen fertility,ranging from 66% to 77%. All hybrids were grown to full maturity and could be fertilized and set seed after self-pollination or back-crosses with B. napus. The morphology of the hybrids resembled characteristics from both parental species. An analysis of the fatty acid composition in the seeds of F1 plants was conducted and the seeds were found to contain different amounts of erucic acid, ranging from 11.0% to 52.1%.     

Can overexpression of TGF—β1 gene change the sex ratio in transgenic mice？

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene.The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAs gave an integration efficiency of 33%.TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation.Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene.The most interestin finding of the present work is the striking eviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1.The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.