Comparative genome anatomy reveals evolutionary insights into a unique amphitriploid fish.

Triploids are rare in nature because of difficulties in meiotic and gametogenic processes, especially in vertebrates. The Carassius complex of cyprinid teleosts contains sexual tetraploid crucian carp/goldfish (C. auratus) and unisexual hexaploid gibel carp/Prussian carp (C. gibelio) lineages, providing a valuable model for studying the evolution and maintenance mechanism of unisexual polyploids in vertebrates. Here we sequence the genomes of the two species and assemble their haplotypes, which contain two subgenomes (A and B), to the chromosome level. Sequencing coverage analysis reveals that C. gibelio is an amphitriploid (AAABBB) with two triploid sets of chromosomes; each set is derived from a different ancestor. Resequencing data from different strains of C. gibelio show that unisexual reproduction has been maintained for over 0.82 million years. Comparative genomics show intensive expansion and alterations of meiotic cell cycle-related genes and an oocyte-specific histone variant. Cytological assays indicate that C. gibelio produces unreduced oocytes by an alternative ameiotic pathway; however, sporadic homologous recombination and a high rate of gene conversion also exist in C. gibelio. These genomic changes might have facilitated purging deleterious mutations and maintaining genome stability in this unisexual amphitriploid fish. Overall, the current results provide novel insights into the evolutionary mechanisms of the reproductive success in unisexual polyploid vertebrates.

[Microsatellite analysis of genetic diversity of Aristichthys nobilis in China].

Seventeen microsatellite markers of Aristichthys nobilis previously discovered by our lab were selected to analyze the genetic diversity and characteristics of two populations of Aristichthys nobilis from Jiangxi and Sichuan provinces. The following parameters were calculated: heterozygosity, polymorphism information content (PIC), valid allele number, allele frequency, genetic distance, genetic similarity coefficient, Hardy-Weinberg balance deflection index and so on. Results show that there are 4 monomorphic and 13 polymorphic markers among the 17 selected microsatellite markers. The average of allele number in each microsatellite locus of the Jiangxi population and Sichuan populations is 3.325 and 3.882, respectively; the average valid allele number is 3.531 and 2.676, respectively; and the number of total alleles of these 17 microsatellite loci is 71. The PIC of polymorphic loci varies between 0.077-0.960, and the average PIC is 0.417. The average observed heterozygosity (Ho) of two populations is 0.385 and 0.360, respectively and the average expected heterzygosity (He) is 0.452 and 0.422, respectively. The genetic similarity coefficient of two populations of Aristichthys nobilis is 0.897 and the genetic distance of these populations is 0.109.

[Genetic diversity analysis of mitochondrial D-loop region of Chinese sucker (Myxocyprinus asiaticus)].

Chinese sucker, Myxocyprinus asiaticus, is an endemic species of China and also the only representatives of family Catostomidae in Asia. The fish was naturally distributed in Yangtze River and Mingjiang River and now few could be captured because of pollution and overexploitation. The fish has been listed in the second class of preserved animal in China. Studying and assessing its population structure is an imperative and fundamental work for making effective protection strategies. We amplified and sequenced the D-loop region of mtDNA of 8 samples. The size of the D-loop region is about 958 bp. A total of 32 variation loci were detected and the mutation rate was 0.033. All the mutation came from nucleotide substitution except one nucleotide deletion. Most of the nucleotide variations were found between the 55-365 bp region. The individual mutation rate varied from 0-1.36%, which exhibited nucleotide polymorphism to some extent among 8 samples. Compared with RAPD and other PCR-based methods, the directily sequencing of mtDNA D-loop region revealed much more genetic diversity. Meanwhile, the D-loop region of Moxostoma robustum derived from GenBank was aligned with that of Chinese sucker through CLUSTAL software. By comparison, we found that the mutation rate (0.033) of D-loop of Chinese sucker is higher than that of Moxostoma robustum (0.016).

Characterization of GHRs, IGFs and MSTNs, and analysis of their expression relationships in blunt snout bream, Megalobrama amblycephala.

In order to test the hypothesis that the expression levels of GH/IGF axis genes would be negatively related to the expression of the myostatin genes in fish species, we cloned six growth regulating genes including growth hormone receptors-1/-2 (GHRs), insulin-like growth factors-I/-II (IGFs) and myostatins-a/-b (MSTNs) from blunt snout bream (Megalobrama amblycephala). The contents of mRNA transcripts for the six genes were determined in the different tissues of adult and developmental stages for the embryonic and larval periods. The results of quantitative real-time PCR showed that GHRs, IGFs and MSTNs were widely expressed in the tissues we tested, with the relatively lower expression levels in mesonephros, gonad and spleen for the six genes. The analysis of expression correlation coefficients among these six genes showed that GHR 1, GHR 2 and MSTN b were correlated with each other in adult tissues (P<0.01). For the developmental stages, GHR 1 had a similar expression pattern to GHR 2 during the examined periods, both with the highest expression levels at 160 hpf (hours post-fertilization) (P<0.05). IGF-II had higher expression levels than that of IGF-I before 400 hpf (P<0.05), while IGF-I was active after 52 hpf. The maximum of MSTN a and MSTN b mRNA levels were at 24 hpf and 400 hpf, respectively. The analysis of expression correlation coefficients showed that GHR 1, GHR 2, IGF-I, IGF-II and MSTN b were positively correlated with each other during embryonic development (P<0.01). The results from this study suggested that the relationship between GH/IGF axis genes and MSTNs was complex and not absolutely negative correlated in fish species.

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp.

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.