Genetic characterization of asymmetric reciprocal hybridization between the flatfishes Paralichthys olivaceus and Paralichthys dentatus

Interspecific reciprocal crosses between the two flatfishes Paralichthys olivaceus and P. dentatus yielded hybrids with different viabilities. Specifically, the hybrids of P. olivaceus female and P. dentatus male (HI) were found to be viable, while the reciprocal hybrids from P. dentatus female and P. olivaceus male (HII) were completely inviable. All the HII individuals showed morphological deformities and died before first feeding. The chromosome analysis showed that HI individuals had the same chromosome number as parents. However, two chromosomes were missing in HII offspring indicating that the latter were aneuploids. Genomic inheritance from the parents to F-1 progeny was also examined by amplified fragment length polymorphism (AFLP) analyses, and the results showed differences between reciprocal hybrids. Almost all AFLP bands (97.71%) observed in parents were passed on to HI individuals. In contrast, only 86.64% of the AFLP bands from parents were scored in HII individuals. Frequency of lost parental bands was thus significantly higher in HII than that in HI and intraspecific crosses, which was probably associated with chromosomal elimination. In addition, higher segregation distortions were found in hybrids than in controls, although these differences were not significant. The present study indicates that chromosomal elimination and loss of AFLP loci occurred in inviable HII individuals, while such genomic changes were not found in viable HI individuals. Possible implications of such difference on genomic changes for asymmetric viability in reciprocal hybrids are discussed.

Identification of Porphyra lines (Rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique AFLP,fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germplasm identification-AFLP) was designed for identification of the 27 Porphyra lines. In addition, 21 specific AFLP markers from 15 Porphyra lines were identified; 6 AFLP markers from 4 Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of the Porphyra lines.

Construction of AFLP-based genetic linkage map for Zhikong scallop, Chlamys farreri Jones et Preston and mapping of sex-linked markers

Zhikong scallop (Chlamys farreri) is an economically important aquaculture species in China; however, frequent mass mortality seriously affects the development of its industry. Genetic linkage map is useful for genetic improvement and selective breeding of C. farreri. Linkage maps were constructed using an intraspecific F-1 cross and amplified fragment length polymorphism (AFLP) markers. Thirty-two selected AFLP primer combinations produced 545 AFLP markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 166 were mapped to 19 linkage groups of the female framework map, covering a total of 1503.9 cM, with an average marker spacing of 10.2 cM; and 197 markers were assigned to 20 linkage groups of the male map, covering a total of 1630.7 cM, with 9.2 cM per marker. A sex-linked marker was mapped on the female map with zero recombination and a LOD of 27.3. The genetic length of C farreri genome was estimated as 1889.0 cM for the female and 1995.9 cM for the male. The coverage of the framework map was calculated as 79.6% for the female and 81.7% for the male. When the triplets and doublets were considered, the observed length of the map was calculated as 1610.2 cM with coverage of 85.2% for the female, and 1880.5 cM with coverage of 94.2% for the male. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map and mapping of economically important genes. (C) 2004 Published by Elsevier B.V.

A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked microsatellite markers and high recombination rates

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.

Effects of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate on the Mytilus galloprovincialis biomarker system

The effects of in vivo exposure of Mytilus galloprovincialis to two anionic surfactants (SDBS and SDS) on the molecular biomarker system were studied. After continuous exposure for 72 days, activities/levels of GST, GPx and GSH were significantly higher than in corresponding control groups following exposure to 3.000 mg/L SDS and SDBS. Activities of SOD and CAT were significantly inhibited by experimental SDBS (except CAT in 0.100 mg/L group), but not by SDS. Statistical analysis of enzyme activities/levels suggested that there were significant positive relationships between GST and GPx, and negative relationships were found between GSH and CAT, GSH and SOD. Amplified fragment length polymorphism (AFLP) results showed that a greater genotoxic effect was observed for SDBS than for SDS. Based on the above results, the biomarker system of mussels can be affected by the two anionic surfactants (>= 3.000 mg/L); it was more easily affected by SDBS than by SDS. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.

Effect of parental stock size on F-1 genetic structure in the bay scallop, Argopecten irradians (Lamarck, 1819)

Three F-1 families of the bay scallop, Argopecten irradians, were produced from one, two and 10 individuals. The genetic changes in these populations, which suffered recent and different levels of bottleneck, were analysed using amplified fragment length polymorphism (AFLP) techniques. In the parental stock, a total of 330 bands were detected using seven AFLP primer pairs, and 70% of the loci were polymorphic. All F-1 groups had a significantly lower proportion of polymorphic loci when compared with the initial stock, and loss of the rare loci and reduction in heterozygosity both occurred. The progeny of the larger population (i.e., N=10) exhibited a lesser amount of genetic differentiation compared with the progeny from N=2, which showed lesser differentiation than progeny from N=1. The effective population sizes (N-e) in N=1, 2 and 10 were estimated as 1.50, 1.61 and 2.49. Based on regression analysis, we recommend that at least 340 individuals be used in hatchery populations to maintain genetic variation.

A preliminary genetic linkage map of the pacific abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F, family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (alpha = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.

Otimização de protocolo para análise de AFLP em Lolium multiflorum Lam.

ESTABELECIMENTO DE METODOLOGIA PARA ANÁLISE MOLECULAR DE AZEVÉM ANUAL COM MARCADORES AFLP. O uso de marcadores moleculares no manejo de bancos de germoplasma tem sido cada vez mais expressivo. Entre os diferentes tipos de marcadores moleculares, o AFLP, Amplified Fragment Length Polymorphism, apresenta algumas vantagens para uso na caracterização de recursos genéticos, como a detecção de grande número de bandas informativas por reação, com ampla cobertura do genoma e considerável reprodutibilidade, além de não necessitar de dados de seqüenciamento prévio da espécie para a construção de primers. Embora a análise de AFLP seja freqüentemente utilizada em estudos de variabilidade genética em diferentes espécies, o uso da técnica em Lolium multiflorum ainda é incipiente. Com a finalidade de estabelecer um protocolo para o emprego da técnica de AFLP em azevém anual foi conduzido este trabalho. Foram avaliadas as concentrações iniciais de DNA genômico de 100 e 250 ng, a digestão do DNA com 1,25 e 1U das enzimas EcoRI e MSe, e os respectivos tempos de reação de digestão: 3, 6 e 12 horas. Também foram avaliadas quatro concentrações da solução resultante da ligação dos adaptadores: solução sem diluição; diluída 1:5; 1:10 e 1:20 e duas diluições após a reação de pré-amplificação, de 1:25 e 1:50. Como resultado, foi estabelecido como melhor protocolo, no qual foi obtido um maior número e qualidade de fragmentos, o que utiliza a concentração inicial de DNA genômico de 100 ng, num volume final de reação de digestão 10 ?l, com 1U de cada enzima EcoRI e MseI e tempo de reação de 12h a 37°C, com reação de ligação de adaptadores realizada com a adição da solução de ligação de adaptadores, do Kit AFLP? Analysis System I (InvitroGen Life Technologies, Carlsbad, Calif., USA), e 0,4 U de T4 DNA ligase em um volume final de 10?l, por 2h a 20°C. Após a ligação de adaptadores a diluição deverá ser de 1:5. A reação de pré-amplificação deverá ocorrer a partir de 1?l desta última solução (diluída 1:5), 1,0 X PCR buffer com Mg Plus [Tris-HCl (pH 7.6) 20 mM, MgCl2 1,5 mM, KCl 50 mM], BSA 0,003% e 1 U de Taq DNA polimerase, completando com mix de pré-amplificação do Kit AFLP? Analysis System I até alcançar o volume final de 11?l. O produto da pré-amplificação deverá ser diluído 1:25 antes de ser procedida à amplificação seletiva, a qual deve ser realizada utilizando 2,5 ?l da solução de DNA pré-amplificado (diluído 1:25), 1 X PCR buffer com Mg Plus [Tris-HCl (pH 8,4) 20 mM, MgCl2 1,5 mM, KCl 50 mM], BSA (0,003%), 1 U de Taq DNA polimerase, 10 ng de primer EcoRI, 1,5 ng de primer MseI, 0,4mM de DNTps e H2O MilliQ? até completar o volume final de 10?l. Com este protocolo uma única combinação de primers permitiu identificar 58 bandas polimórficas na análise de duas populações de azevém anual.

High levels of genetic structuring as a result of population fragmentation in the tropical tree species Caesalpinia echinata Lam.

We have investigated levels of genetic diversity within and among seven remnant populations of Caesalpinia echinata Lam., an endangered species found as fragmented populations in three major areas around the coastal regions of Brazil. Using amplified fragment length polymorphism (AFLP) genetic markers, we detected levels of within-population genetic diversity ranging from 0.092 to 0.163, with the lowest values generally being found in the smallest populations. Estimates of between-population genetic differentiation were strongly correlated with geographical distance ( r = 0.884, p <0.001), which, along with a neighbour-joining phylogenetic analysis, strongly suggested high levels of genetic isolation by distance. Over half (62%) of the total genetic diversity was partitioned between populations, further highlighting the genetic distinctness of individual populations. Taken together, these results suggest that fragmentation has led to an increase in population differentiation between fragments of C. echinata. These formations will be of great value in the development of conservation plans for species exhibiting high levels of genetic differentiation due to fragmentation, such as indication of conservation unit size, which populations should be chosen as priority in conservation plans and which samples should be introduced in areas with a low number of individuals of brazilwood.

Conservation genetics of Ireland's sole population of the River water crowfoot (Ranunculus fluitans Lam.)

Populations of many freshwater species are becoming increasingly threatened as a result of a wide range of anthropogenically mediated factors. In the present study, we wanted to assess levels and patterns of genetic diversity in Ireland's sole population of the River water crowfoot (Ranunculus fluitans), which is restricted to a 12 km stretch of a single river, to assist the formation of conservation strategies. Analysis using amplified fragment length polymorphism (AFLP) indicated comparable levels of genetic diversity to those exhibited by a more extensive population of the species in England, and revealed no evidence of clonal reproduction. Allele-specific PCR analysis of five nuclear single nucleotide polymorphisms (SNPs) indicated no evidence of hybridization with its more abundant congener Ranunculus penicillatus, despite previous anecdotal reports of the occurrence of hybrids. Although the population currently exhibits healthy levels of genetic diversity and is not at risk of genetic assimilation via hybridization with R. penicillatus, it still remains vulnerable to other factors such as stochastic events and invasive species. © 2013 Elsevier B.V. All rights reserved.

Cadmium hyperaccumulation and genetic differentiation of Thlaspi caerulescens populations

Although the knowledge on heavy metal hyperaccumulation mechanisms is increasing, the genetic basis of cadmium (Cd) hyperaccurnulation remains to be elucidated. Thlaspi caerulescens is an attractive model since Cd accumulation polymorphism observed in this species suggests genetic differences between populations with low versus high Cd hyperaccumulation capacities. In our study, a methodology is proposed to analyse at a regional scale the genetic differentiation of T. caerulescens natural populations in relation to Cd hyperaccumulation capacity while controlling for different environmental, soil, plant parameters and geographic origins of populations. Twenty-two populations were characterised with AFLP markers and cpDNA polymorphism. Over all loci, a partial Mantel test showed no significant genetic structure with regard to the Cd hyperaccumulation capacity. Nevertheless, when comparing the marker variation to a neutral model, seven AFLP fragments (9% of markers) were identified as presenting particularly high genetic differentiation between populations with low and high Cd hyperaccurnulation capacity. Using simulations, the number of outlier loci was showed to be significantly higher than expected at random. These loci presented a genetic structure linked to Cd hyperaccumulation capacity independently of the geography, environment, soil parameters and Zn, Pb, Fe and Cu concentrations in plants. Using a canonical correspondence analysis, we identified three of them as particularly related to the Cd hyperaccumutation capacity. This study demonstrates that populations with low and high hyperaccurnulation capacities can be significantly distinguished based on molecular data. Further investigations with candidate genes and mapped markers may allow identification and characterization of genomic regions linked to factors involved in Cd hyperaccumulation.

Genetic diversity and differentiation processes in the ploidy series of Olea europaea L.: a multiscale approach from subspecies to insular populations.

Geographical isolation and polyploidization are central concepts in plant evolution. The hierarchical organization of archipelagos in this study provides a framework for testing the evolutionary consequences for polyploid taxa and populations occurring in isolation. Using amplified fragment length polymorphism and simple sequence repeat markers, we determined the genetic diversity and differentiation patterns at three levels of geographical isolation in Olea europaea: mainland-archipelagos, islands within an archipelago, and populations within an island. At the subspecies scale, the hexaploid ssp. maroccana (southwest Morocco) exhibited higher genetic diversity than the insular counterparts. In contrast, the tetraploid ssp. cerasiformis (Madeira) displayed values similar to those obtained for the diploid ssp. guanchica (Canary Islands). Geographical isolation was associated with a high genetic differentiation at this scale. In the Canarian archipelago, the stepping-stone model of differentiation suggested in a previous study was partially supported. Within the western lineage, an east-to-west differentiation pattern was confirmed. Conversely, the easternmost populations were more related to the mainland ssp. europaea than to the western guanchica lineage. Genetic diversity across the Canarian archipelago was significantly correlated with the date of the last volcanic activity in the area/island where each population occurs. At the island scale, this pattern was not confirmed in older islands (Tenerife and Madeira), where populations were genetically homogeneous. In contrast, founder effects resulted in low genetic diversity and marked genetic differentiation among populations of the youngest island, La Palma.

Broad-scale adaptive genetic variation in alpine plants is driven by temperature and precipitation.

Identifying adaptive genetic variation is a challenging task, in particular in non-model species for which genomic information is still limited or absent. Here, we studied distribution patterns of amplified fragment length polymorphisms (AFLPs) in response to environmental variation, in 13 alpine plant species consistently sampled across the entire European Alps. Multiple linear regressions were performed between AFLP allele frequencies per site as dependent variables and two categories of independent variables, namely Moran's eigenvector map MEM variables (to account for spatial and unaccounted environmental variation, and historical demographic processes) and environmental variables. These associations allowed the identification of 153 loci of ecological relevance. Univariate regressions between allele frequency and each environmental factor further showed that loci of ecological relevance were mainly correlated with MEM variables. We found that precipitation and temperature were the best environmental predictors, whereas topographic factors were rarely involved in environmental associations. Climatic factors, subject to rapid variation as a result of the current global warming, are known to strongly influence the fate of alpine plants. Our study shows, for the first time for a large number of species, that the same environmental variables are drivers of plant adaptation at the scale of a whole biome, here the European Alps.

Diversity among bacteria causing blotch disease on the commercial mushroom, agaricus bisporus /

A total of 251 bacterial isolates were isolated from blotched mushroom samples obtained from various mushroom farms in Canada. Out of 251 stored isolates, 170 isolates were tested for pathogenicity on Agaricus bisporus through mushroom rapid pitting test with three distinct pathotypes observed: dark brown, brovm and yellow/yellow-brown blotch. Phenotypic analysis of 83 isolates showed two distinct proteinase K resistant peptide profiles. Profile group A isolates exhibited peptides with masses of 45, 18, 16 and 14 kDa and fiirther biochemical tests identified them as Pseudomonasfluorescens III and V. Profile group B isolates lacked the 16-kDa peptide and the blotch causing bacterial isolates of this group was identified as Serratia liquefaciens and Cedecea davisae. Comparative genetic analysis using Amplified Fragment Length Polymorphism (AFLP) on 50 Pseudomonas sp. isolates (Group A) showed that various blotch symptoms were caused by isolates distributed throughout the Pseudomonas sp. clusters with the exception of the Pseudomonas tolaasii group and one non-pathogenic Pseudomonas fluorescens cluster. These results show that seven distinct Pseudomonas sp. genotypes (genetic clusters) have the ability to cause various symptoms of blotch and that AFLP can discriminate blotch causing from non-blotch causing Pseudomonasfluorescens. Therefore, a complex of diverse bacterial organisms causes bacterial blotch disease

Impact des polluants agricoles sur la génétique des populations d’une espèce sentinelle : le ouaouaron (Rana catesbeiana)

L’expansion agricole ne cesse d’agir sur la perte d’habitats essentiels et nécessaires au développement des espèces. Bien que plusieurs espèces réussissent à survivre dans ces habitats peu adéquats, la persistance et la santé de plusieurs populations semblent compromises par l’utilisation souvent intensive de polluants chimiques agricoles et de fertilisants. Cette étude a pour but de déterminer l’impact des contaminants et de l’écologie du paysage sur la diversité génétique des populations de ouaouarons retrouvées en milieu agricole. Notre hypothèse de départ stipule qu’une exposition chronique aux polluants agricoles induira des différences génétiques au niveau des populations exposées. Le bassin versant de la rivière Yamaska a été désigné comme site d’étude puisqu’il fait partie de la région agricole la plus importante du Québec et parce qu’on y retrouve un gradient d’utilisation des terres pour l’agriculture (faible, moyen, élevé). Le ouaouaron a été choisi à titre de modèle biologique puisque ses caractéristiques physiologiques et écologiques en font une espèce sentinelle capable de rendre compte de l’état de santé global des écosystèmes. La caractérisation génétique des populations a été effectuée à partir de marqueurs d’AFLP (Amplified Fragment Length Polymorphism). Les résultats montrent que la diversité génétique est liée à la colonisation à partir de l’embouchure de la rivière Yamaska et que quelques populations sont génétiquement différenciées. De plus, nous avons démontré une relation positive entre le nombre de locus polymorphes et l’atrazine, l’indice de contamination et le métolachlore et la concentration en azote ainsi qu’entre l’hétérozygotie attendue et la concentration en phosphate.