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be explored in the future within our laboratory, both in Laternula and economically important model temperate mollusc spe cies, RGFP966 such as Crassostrea gigas and Mytilus galloprovin cialis. Methods Animal sampling All animals used in experimental work were collected at Rothera Research Station, Adelaide Island, Antarctic Pen insula by SCUBA divers dur ing the austral summer at depths of 10 15 m. The animals were immediately returned to the laboratory where they were maintained in a through flow aquarium with a tem perature of 0. 6 0. 3 C, under a simulated natural light. dark cycle. All animals were mature adults, with a range of shell sizes between 50. 1 83. 5 mm. As shell length is related to animal age. surface aging estimates using growth rings produced an mean age of 8.
3 years with a range from 6 14 years and a median of 8 years, Mantle tissue was dissected from the animals and cross sections comprising all 3 folds and the Combretastatin A-4 periostracum were immediately flash frozen in liquid nitrogen for later RNA extraction. RNA isolation and cDNA production Mantle RNA was extracted PP1 from 24 animals using a mod ified TRI reagent protocol. After homogenization in Tri Reagent and chloroform extraction, the samples were subjected to a lithium chloride precipitation step. RNA was precipitated using a 1. 1 isopropanol. saline solu tion and after resuspension, the RNA was subjected to a further precip itation using 250 ul 7. 5 M LiCl. The extractions were fur ther cleaned using RNeasy mini kit columns following manufacturer instruc tions in order to eliminate LiCl and salt residues.
5 ug of RNA was PCR amplified using the protocol described in prior to preparation for the 454 run. Samples were nebulised at 30psi for one minute and subsequently puri fied with Ampure to produce fragments 300 bp and above. The ends were polished and Erythropoietin the 454 tita nanium adapters containing specific MID sequences were attached. Fragments containing both a and b adapters were selected and quantified. Libraries were amplified by emulsion PCR, beads recovered and enriched and placed on a picotiter plate for sequencing by the 454 procedure. The rapidly accumulating complete genomes in databases provide unique opportunities to study relationships among organisms. Since DNA sequences are conserved between closely related organisms, comparative genomic analyses are a powerful tool for understanding the com plex evolutionary events in specific phylogenetic lineages.
R. solanacearum, DBeQ formerly known as Pseudomonas solanacearum and Burkholderia solanacearum, is the causal agent of bacterial wilt, This soil RGFP966 borne vascular pathogen is widely distributed in tropical and subtropical climates and affects an unusually broad range of crops, including both monocot and dicot plants, Many affected hosts are critical for developing countries because of their strategic importance as cash crops or as subsistence foods like potato, tomato, eggplant, cook ing banana and peanut, In the 1990s, potato brown rot strains of R. solanacearum historically known as race 3 biovar 2 were intro duced in Europe and North America, Due to their adaptation to tropical highland climates, these strains, which are more virulent at cool temperatures than tropical strains, may pose major threats in tem perate zones.
Therefore, R. solanacearum was listed as a quarantine DBeQ organism in Europe and Canada and as a Biot errorism Select Agent in the U. S, R. solanacearum and the closely related RGFP966 species R. syzy gii and the banana blood disease bacterium form a complex in the R. picketii lineage, This species complex includes thousands of genetically distinct strains that can DBeQ differ from each other by more than 30%, and thus do not belong in the same species by conventional definition, This species complex includes strains with broad and narrow host ranges, which are ecologically different as well. potato strains are cold tolerant and banana strains are insect transmitted, and with different geo graphic origins. Because R. so