ABSTRACT
Background: Also known as Simple Sequence Repetitions (SSRs), microsatellites are profoundly informative molecular markers and powerful tools in genetics and ecology studies on plants. Objective: This research presents a workflow for developing microsatellite markers using genome skimming. Methods: The pipeline was proposed in several stages that must be performed sequentially: obtaining DNA sequences, identifying microsatellite regions, designing primers, and selecting candidate microsatellite regions to develop the markers. Our pipeline efficiency was analyzed using Illumina sequencing data from the non-model tree species Pterodon emarginatus Vog. Results: The pipeline revealed 4,382 microsatellite regions and drew 7,411 pairs of primers for P. emarginatus. However, a much larger number of microsatellite regions with the potential to develop markers were discovered from our pipeline. We selected 50 microsatellite regions with high potential for developing markers and organized 29 microsatellite regions in sets for multiplex PCR. Conclusion: The proposed pipeline is a powerful tool for fast and efficient development of microsatellite markers on a large scale in several species, especially nonmodel plant species.
ABSTRACT
Ecotoxicity of glyphosate (GLY) and its metabolite aminomethylphosphonic acid (AMPA) was investigated in guppies, Poecilia reticulata. We tested the effects of these chemicals on the gills and liver of both male and female guppies using qualitative and quantitative histopathological analyses associated with histopathological condition indexes. Both genders showed similar median lethal concentration (LC50 ) at 96 h for GLY (68.78 and 70.87 mg l-1 ) and AMPA (180 and 164.32 mg l-1 ). However, the histopathological assessment of both fish organs exposed to sublethal concentrations of GLY (35 mg l-1 ) and AMPA (82 mg l-1 ) for 96 h showed a tissue- and gender-specific histopathological response. In both exposure assays, fish presented mainly progressive changes, such as proliferation of the interlamellar epithelium, partial and total fusion of secondary lamellae. The liver showed mainly regressive changes, such as steatosis, pyknotic nuclei and high distribution of collagen fibers. Unusually large hepatocytes as degenerated cells were also detected. Histopathological changes in gills were similar for the males and females, but the liver response was different between the genders. The hepatic inflammatory changes were more common in males. The increase in the area of hepatocyte vacuoles is gender dependent with higher values in the male compared to the female guppies exposed to GLY and AMPA. Multiparametric analysis indicated that the male guppies are more sensitive than females, particularly in the presence of AMPA. Our study shows that the histopathological assessment associated with gender-specific response can be successfully used in ecotoxicological assessment of GLY and the metabolite AMPA. Copyright © 2017 John Wiley & Sons, Ltd.