In this study, we identified and characterized 160 microsatellite loci from an expressed sequence tag (EST) database generated from immune-related organs of turbot (Scophthalmus maximus). A final set of 83 new polymorphic microsatellites were validated after the analysis of 40 individuals of Atlantic origin including both wild and farmed individuals. The allele number and the expected heterozygosity ranged from 2 to 18 and from 0.021 to 0.951, respectively. Evidences of null alleles at moderate-high frequencies were detected at six loci using population data. None of the analysed loci showed deviations from Mendelian segregation after the analysis of five full-sib families including approximately 92 individuals/family. The markers are used to consolidate the turbot genetic map, and because they are mostly EST-derived, they will be very useful for comparative genomic studies within flatfishes and with model fish species. Using an in silico approach, we detected significant homologies of microsatellite sequences with the EST databases of the flatfish species with highest genomic resources (Senegalese sole, Atlantic halibut, bastard halibut) in 31% of these turbot markers. The conservation of these microsatellites within Pleuronectiformes will pave the way for anchoring genetic maps of different species and identifying genomic regions related to productive traits.
In this study we have developed protocols for induced triploidy and gynogenesis of Senegalese sole (Solea senegalensis), a promising flatfish species for marine aquaculture, in order to: 1) identify the sex-determination mechanism; and 2) to improve its production by generating a) sterile fish, avoiding problems related with sexual maturation, and b) all-female stocks, of higher growth rate. Triploidy was induced by means of a cold shock. Gynogenesis was induced by activating eggs with UV-irradiated sperm, and to prompt diploid gynogenesis, a cold-shock step was also used. Ploidy of putative triploid larvae and gynogenetic embryos were determined by means of karyotyping and microsatellite analysis. Haploid gynogenetic embryos showed the typical "haploid syndrome". As expected, triploid and gynogenetic groups showed lower fertilization, hatching, and survival rates than in the diploid control group. Survival rate, calculated 49 days after hatching, for haploid and diploid gynogenetic groups was similar to those observed in other fish species (0% and 62.5%, respectively), whereas triploids showed worse values (45%). Sex was determined macroscopically and by histological procedures, revealing that all the diploid gynogenetic individuals were females. In conclusion, we have successfully applied chromosomal-manipulation techniques in the flatfish species Senegalese sole in order to produce triploid, haploid, and diploid gynogenetic progenies.
In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.
Transporters of the NRAMP family are ubiquitous metal-transition transporters, playing a key role in metal homeostasis, especially in Mn and Fe homeostasis. In this work, we report the characterization of the NRAMP family members (RiSMF1, RiSMF2, RiSMF3.1 and RiSMF3.2) of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. Phylogenetic analysis of the NRAMP sequences of different AM fungi showed that they are classified in two groups, which probably diverged early in their evolution. Functional analyses in yeast revealed that RiSMF3.2 encodes a protein mediating Mn and Fe transport from the environment. Gene-expression analyses by RT-qPCR showed that the RiSMF genes are differentially expressed in the extraradical (ERM) and intraradical (IRM) mycelium and differentially regulated by Mn and Fe availability. Mn starvation decreased RiSMF1 transcript levels in the ERM but increased RiSMF3.1 expression in the IRM. In the ERM, RiSMF1 expression was up-regulated by Fe deficiency, suggesting a role for its encoded protein in Fe-deficiency alleviation. Expression of RiSMF3.2 in the ERM was up-regulated at the early stages of Fe toxicity but down-regulated at later stages. These data suggest a role for RiSMF3.2 not only in Fe transport but also as a sensor of high external-Fe concentrations. Both Mn- and Fe-deficient conditions affected ERM development. While Mn deficiency increased hyphal length, Fe deficiency reduced sporulation.
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