Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter.
ABSTRACT: Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC?, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC?, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.
Project description:BACKGROUND:Bleeker's yellow tail (Xenocypris davidi Bleeker, YT) and topmouth culter (Culter alburnus Basilewsky, TC) are both famous and important economic freshwater fish in China. YT, a kind of omnivorous fish, has strong resistance. TC, a kind of carnivorous fish, has high-quality meat but poor resistance. Distant hybridization can integrate the advantages of both parents. There has been no previous report regarding hybrid fish derived from female YT?×?male TC. It is expected that hybridization of these two kinds of fish will result in F1 hybrids with improved characteristics, such as faster growth rate, stronger resistance, and high-quality meat, which are of great significance in fish genetic breeding. RESULTS:In this study, we investigated the main biological characteristics of diploid hybrid fish derived from female YT?×?male TC. The hybrids had an intermediate number of upper lateral line scales between those for YT and TC. The hybrids were diploids with 48 chromosomes and had the same karyotype formula as their parents. The hybrids generated variations in 5S rDNA (designated class IV: 212?bp) and lost specific 5S rDNA derived from the maternal parent (designated class II: 221?bp), which might be related to hybridization. In terms of reproductive traits, all the tested female hybrids exhibited normal gonadal development, and the two-year-old F1 females produced mature eggs. However, all the tested testes of the male hybrids could not produce mature sperm. It is possible that the hybrid lineage will be established by back-crossing the fertile female hybrids and their parents. CONCLUSIONS:Obtaining a fertile female hybrid fish made the creation of a new type of fish possible, which was significant in fish genetic breeding.
Project description:Interspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive.In the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division. The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies. To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe.Our study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.
Project description:This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.
Project description:To better understand genomic changes in the early generations after polyploidisation, we examined the chromosomal consequences of genomic merger in allotetraploid hybrids (4 nF1) (AABB, 4n = 148) of Carassius auratus red var. (RCC) (AA, 2n = 100) (♀) × Megalobrama amblycephala (BSB) (BB, 2n = 48) (♂). Complete loss of the paternal 5S rDNA sequence and the expected number of maternal chromosomal loci were found in 4 nF1, suggesting directional genomic changes occurred in the first generations after polyploidisation. Recent studies have reported instability of newly established allotetraploid genomes. To assess this in the newly formed 4 nF1 genome, we performed fluorescence in situ hybridisation on an allotetraploid gynogenetic hybrid (4 nG) (AABB, 4n = 148) and an allopentaploid hybrid (5 nH) (AABBB, 5n = 172) from 4 nF1 (♀) × BSB (♂) with 5S rDNA gene and centromere probes from RCC, the original diploid parent. The expected numbers of maternal chromosomal loci were found in 4 nG, while chromosomal locus deletions and chromosome recombinations were detected in 5 nH. These observations suggest that abnormal meiosis did not lead to obvious genomic changes in the newly established allotetraploid genomes, but hybridisation with the original diploid parent resulted in obvious genomic changes in the newly established allotetraploid genomes, as was found for the maternal genome.
Project description:KEY MESSAGE:Tetraploid `Moncada´ mandarin, used as male and female in interploidy hybridizations, displays mainly tetrasomic inheritance for most LGs, with slight variations according to the direction of the crossing. Triploid-breeding programs in citrus are key tool to develop seedless cultivars. Obtaining triploid citrus hybrids may be achieved through different strategies, such as the exploitation of female unreduced gamete in crosses between diploid parents and diploid by tetraploid sexual hybridizations, in which tetraploid genotypes can be used as male or female parents. Genetic configuration of triploid populations from interploid crosses greatly depends on the chromosomic segregation mode of the tetraploid parent used. Here, we have analyzed the inheritance of the tetraploid 'Moncada' mandarin and compared the genetic structures of the resulting gametes when used as male and as female parent. The preferential chromosome pairing rate is calculated from the parental heterozygosity restitution (PHR) of codominant molecular markers, indicating the proportion between disomic and tetrasomic segregation. Tetraploid 'Moncada' both as female and male parent largely exhibited tetrasomic segregation. However, as female parent, one linkage group (LG8) showed intermediate segregation with tendency towards tetrasomic inheritance, while another linkage group (LG4) evidenced a clear intermediate segregation. On the other hand, when used as male parent two linkage groups (LG5 and LG6) showed values that fit an intermediate inheritance model with tetrasomic tendency. Significant doubled reduction (DR) rates were observed in five linkage groups as female parent, and in six linkage groups as male parent. The new knowledge generated here will serve to define crossing strategies in citrus improvement programs to efficiently obtain new varieties of interest in the global fresh consumption market.
Project description:The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n?=?100, 2n?=?AA) (?)?×?common carp (Cyprinus carpio L. 2n?=?100, 2n?=?BB) (?), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies.The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies.We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.
Project description:The karyotypes of Allium, a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA3/DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers (x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species (A. moly, A. oreophilum, A. karataviense, A. nigrum, A. sphaerocephalon, A. porrum). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A. sphaerocephalon L.) or four chromosomes (tetraploid A. porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA3+ bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A. fistulosum L. in which positive CMA3+ bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa.
Project description:The offspring with 100 chromosomes (abbreviated as GRCC) have been obtained in the first generation of Carassius auratus red var. (abbreviated as RCC, 2n = 100) (?) × Megalobrama amblycephala (abbreviated as BSB, 2n = 48) (?), in which the females and unexpected males both are found. Chromosomal and karyotypic analysis has been reported in GRCC which gynogenesis origin has been suggested, but lack genetic evidence.Fluorescence in situ hybridization with species-specific centromere probes directly proves that GRCC possess two sets of RCC-derived chromosomes. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (abbreviated as NTS) reveals that three types of 5S rDNA class (class I; class II and class III) in GRCC are completely inherited from their female parent (RCC), and show obvious base variations and insertions-deletions. Fluorescence in situ hybridization with the entire 5S rDNA probe reveals obvious chromosomal loci (class I and class II) variation in GRCC.This paper provides directly genetic evidence that GRCC is gynogenesis origin. In addition, our result is also reveals that distant hybridization inducing gynogenesis can lead to sequence and partial chromosomal loci of 5S rDNA gene obvious variation.
Project description:21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid 'Freya' had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true.
Project description:The phenomenon of heterosis is critical to plant breeding and agricultural productivity. Heterosis occurs when F1 hybrid offspring display quantitative improvements in traits to levels that do not occur in the parents. Increasing the genome dosage (i.e. ploidy level) of F1 offspring can contribute to heterosis effects. Sugar beet (Beta vulgaris) provides a model for investigating the relative effects of genetic hybridity and genome dosage on heterosis. Sugar beet lines of different ploidy levels were crossed to generate diploid and triploid F1 offspring to investigate the effect of; (1) paternal genome dosage increase on F1 heterosis, and; (2) homozygous versus heterozygous tetraploid male parents on F1 triploid heterosis. A range of traits of agronomic and commercial importance were analyzed for the extent of heterosis effects observed in the F1 offspring.Comparisons of parental lines to diploid (EA, EB) and triploid (EAA, EBB) F1 hybrids for total yield, root yield, and sugar yield indicated that there was no effect of paternal genome dosage increases on heterosis levels, indicating that hybridity is the main contributor to the heterosis levels observed. For all traits measured (apart from seed viability), F1 triploid hybrids derived from heterozygous tetraploid male parents displayed equivalent levels of heterosis as F1 triploid hybrids generated with homozygous tetraploid male parents, suggesting that heterosis gains in F1 triploids do not arise by simply increasing the extent of multi-locus heterozygosity in sugar beet F1 offspring.Overall, our study indicates that; (1) increasing the paternal genome dosage does not enhance heterosis in F1 hybrids, and; (2) increasing multi-locus heterozygosity using highly heterozygous paternal genomes to generate F1 triploid hybrids does not enhance heterosis. Our findings have implications for the design of future F1 hybrid improvement programs for sugar beet.