Assessing the Effectiveness of Sex-Linked Molecular Markers to Identify Neomale Breeders for the Production of All-Female Progenies of Rainbow Trout.

A cultured stock of masculinized rainbow trout was diagnosed with Y-linked markers (sdY and OmyY1) aiming to detect neomales before their use at the production level. To achieve a reliable diagnosis, the following steps were considered: (1) PCR amplification of the housekeeping ß-actin gene to determine the DNA quality of samples, (2) validation of the Y-linked markers by their PCR amplification in male and female samples with known sex, and (3) molecular sexing of the masculinized juveniles based on male-specific (XY genotype) and neomale-specific (XX genotype) PCR product band patterns visualized on agarose gel. The validity and concordance of the markers were assessed. The housekeeping gene identified samples with negative PCR amplification revealing a poor DNA quality. The OmyY1 marker presented a more distinctive PCR product band pattern between males and females than the sdY marker and identified a higher proportion of true males (sensitivity = 1.0 and 0.91, respectively). The OmyY1 marker accurately identified 105 neomales of the 198 masculinized individuals on account their consistent and distinctive PCR product band pattern. Among both markers, there was a medium high positive concordance (γ index = 0.7). It is concluded that the OmyY1 marker shows the best performance to reliably detect neomales, a step that is essential to have certified breeders for the production of all-female progenies in fish farming.

Effect of Different Culture Conditions on Gene Expression Associated With Cyst Production in Populations of Artemia franciscana.

Artemia franciscana inhabits hypersaline environments in the Americas and has a well-adapted reproductive system that allows it to survive in these extreme conditions, represented by the production of diapause cysts (oviparous reproduction). This reproduction mode is controlled by numerous genes that are expressed in response to different environmental stressors, enabling this species to avoid population extinction. However, to date, the expression of these genes has not been sufficiently studied to clarify their levels in response to a combination of different environmental factors under controlled conditions. We analyzed the expression of eight genes related to oviparous reproduction (SGEG, Arp-CBP, artemin, BRCA1, p8, ArHsp21, ArHsp22, and p26) to determine their association with cyst production in two populations of A. franciscana with contrasting phenotypes, one with high (Barro Negro, BNE, Chile) and one with low (San Francisco Bay, SFB, United States) cyst production. Populations were cultured under controlled conditions of salinity (SAL, 35 and 75 ppt), photoperiod (PHO, 12L:12D and 24L:00D), iron concentration (IC, 0[Fe] and 5[Fe]), and microalgae diet (DIE; Dunaliella tertiolecta (DUN) and Tetraselmis suecica (TETRA)). Sixteen treatments were performed by combining the two conditions of each of the four factors. Data on nine reproductive parameters per female were recorded, including the percent of offspring encysted (%) (POE). The gene expression levels were analyzed by semiquantitative RT-PCR. The mean POE was significantly greater in BNE than in SFB (32.40 versus 12.74%, Mann-Whitney's test, p < 0.05). Significantly upregulated expression of seven genes in BNE (more than twofold, p < 0.05) was observed in 38.28% of the treatments (e.g., DUN-75ppt-12L:12D-5[Fe] and TETRA-35ppt-12L:12D-5[Fe]). In SFB, seven genes showed significant differential expression, but most were downregulated in 29.69% of the treatments (e.g., DUN-75ppt-12L:12D-0[Fe] and DUN-75ppt-24L:00D-0[Fe]). Multiple regression analyses indicated that in BNE, five genes (SGEG, artemin, Arp-CBP, p8, and BRCA1) and three environmental factors (DIE, SAL, and IC) were important predictor variables for the POE response variable given that all of them were included in the highest-ranking models. In SFB, only two genes (ArHsp21 and artemin) and one environmental factor (SAL) were important explanatory variables in the highest-ranking models. It was concluded that the BNE population presented a characteristic gene expression pattern that differed from that of the SFB population. This pattern might be related to the marked oviparous reproduction of the BNE population. This gene expression pattern could be useful for monitoring the reproductive mode leading to diapause in Artemia and to assist with intensive cyst production in pond systems.

Revealing the biodiversity of Chilean birds through the COI barcode approach.

The mitochondrial cytochrome c oxidase subunit I (COI) gene is an effective molecular tool for the estimation of genetic variation and the identification of bird species. This molecular marker is used to differentiate among Chilean bird species by analyzing barcodes for 76 species (197 individuals), comprising 28 species with no previous barcode data and 48 species with sequences retrieved from the BOLD and GenBank databases. The DNA barcodes correctly identified 94.7% of the species analyzed (72 of 76 species). Mean intraspecific K2P distance was 0.3% (range 0-8.7%). Within the intraspecific divergence range, three species, Phrygilus gayi, Sephanoides sephanoides and Curaeus curaeus, showed relatively high intraspecific divergence (1.5-8.7%), possibly due to the presence of a species complex or geographic isolation of sub-populations. Mean interspecific K2P distance was 24.7% (range 1.3-43.5%). Consequently, the intraspecific K2P distance showed limited overlap with interspecific K2P distance. The mean intraspecific divergence in our study was similar to that found in temperate regions of South America (0.24%). However, it was approximately one order of magnitude lower than values reported for bird species in tropical regions of northern South America (1.8-2.13%). This result suggests that bird species from Chile show low levels of genetic structure and divergence. The small overlap between intra- and inter-specific distances implies that COI barcodes could be used as an effective tool to identify nearly all the Chilean bird species analyzed.

Authentication of Frozen Chilean Blue Mussel (Mytilus chilensis) Commercialized in the Town of Osorno, Southern Chile, Using PCR-RFLP Analysis.

BACKGROUND: DNA-based technologies are reliable authentication methods for food products, enabling the detection of fraud, non-intentional substitution and control of mislabeling. The Chilean blue mussel (Mytilus chilensis) is a seafood commercialized in Chile under different formats, including packages of frozen specimens. In this format, the valves of mussels are removed during processing, thus impeding identification of the product by the consumer due to the lack of external characters. OBJECTIVE: To assess the authenticity of frozen Chilean blue mussels commercialized in southern Chile, particularly in the town of Osorno. METHODS: Six commercial brands of frozen Chilean blue mussel were authenticated by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method, based on the analysis of an 18S rDNA fragment. RESULTS: Restriction patterns obtained indicate that three brands (50%) proved to be 100% authentic, given that all specimens contained in the package were Chilean blue mussels. The other three brands (50%) contained specimens of other commercial mytilids, particularly the cholga mussel (Aulacomya ater), in a variable percentage (12.5-50%). CONCLUSION: This study based on the PCR-RFLP method provides evidence that Chilean blue mussels commercialized in a town located in southern Chile lack authenticity. This finding highlights the necessity for national producers to improve the production and/or packaging processes of this seafood. The authentication of commercial mussels is a matter of consumer interest and has been described in a recent patent on this issue that proposes an alternative methodology.

Genetic divergence analysis of the Common Barn Owl Tyto alba (Scopoli, 1769) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile using COI sequence.

In this paper new mitochondrial COI sequences of Common Barn Owl Tyto alba (Scopoli, 1769) and Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile are reported and compared with sequences from other parts of the World. The intraspecific genetic divergence (mean p-distance) was 4.6 to 5.5% for the Common Barn Owl in comparison with specimens from northern Europe and Australasia and 3.1% for the Short-eared Owl with respect to samples from north America, northern Europe and northern Asia. Phylogenetic analyses revealed three distinctive groups for the Common Barn Owl: (i) South America (Chile and Argentina) plus Central and North America, (ii) northern Europe and (iii) Australasia, and two distinctive groups for the Short-eared Owl: (i) South America (Chile and Argentina) and (ii) north America plus northern Europe and northern Asia. The level of genetic divergence observed in both species exceeds the upper limit of intraspecific comparisons reported previously for Strigiformes. Therefore, this suggests that further research is needed to assess the taxonomic status, particularly for the Chilean populations that, to date, have been identified as belonging to these species through traditional taxonomy.

The New World Artemia species A. franciscana and A. persimilis are highly differentiated for chromosome size and heterochromatin content.

Chromosomal rearrangements have played a key role in the speciation of the New World sexual Artemia species (Crustacea, Anostraca) A. franciscana and A. persimilis. The species differ by a chromosome duplication (2n+2=44 in A. persimilis vs 2n=42 in A. franciscana), and a greater amount of heterochromatin (HCH) in A. franciscana. To investigate this difference in HCH, four parameters were compared for the first time in Artemia: 1) the absolute sizes of one A. persimilis and four A. franciscana karyotypes; 2) the relative lengths of all chromosome; 3) the number of heterochromatic bands and 4) the relative amounts of HCH per chromosome and its position. The two A.franciscana karyotypes with the largest HCH amount (26%), have twice (139.26 microm and 134.05 microm) the absolute size of the A.persimilis karyotype (64.91 microm; HCH: 1.97%). Interspecific and intraspecific (A. franciscana) differences in chromosome size and HCH were observed, although the two sets of information are not positively correlated. While A. persimilis shares plesiomorphic karyological traits with Old World species, A. franciscana has apomorphic features such as longer chromosomes and greater HCH content, mainly dispersed towards telomeres. The impacts of such chromosome rearrangements are discussed in relation to the wider geographic distribution, greater colonizing ability, and life history plasticity of A. franciscana. An additional, though preliminary, point of this paper is the observation that the female would be the heterogametic sex.

Karyotype and nuclear DNA content of Trichomycterus areolatus (Siluriformes, Trichomycteridae)

Cytogenetic analysis of Trichomycterus areolatus, collected from the Tijeral and Huilma Rivers in southern Chile has shown a diploid chromosome number of 2n = 54, a fundamental number of FN = 106, and a karyotypic formula of 44m + 8sm + 2st. Intra-individual polymorphism of chromosome number (2n = 54, 55 and 56) in specimens from the Huilma River has also been documented, providing further evidence of the occurrence of this phenomenon in Trichomycterus. The karyotype exhibited large chromosome pairs: metacentric pairs 1 (relative length 7.54 percent), 2 (5.75 percent) and 3 (5.09 percent), submetacentric pair 23 (5.25 percent), and subtelocentic pair 27 (5.28 percent). Nuclear DNA content analysis showed an average value of 5.04 ± 1.09 pg/nucleus. This DNA content is higher than the mean value described for other species in this genus.