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Nakano clocked a time of 1:36.24, with Colin Edwards third and world championship leader Nicky Hayden fourth.
BBC: SPORT | Motorsport | Motorbikes | Rossi to race despite hand injury
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Saturday's session was marred when French rider Randy de Puniet crashed into Colin Edwards, leaving the 38-year-old American with a broken collarbone.
CNN: Stoner on pole for Portugal MotoGP race
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Rossi's team-mate Colin Edwards was quickest in the two practice sessions, with another Yamaha rider, Carlos Checa, also on a Yamaha, second fastest.
BBC: SPORT | Motorsport | Motorbikes | Rossi aims to race despite crash
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Toni Elias, who will not be retained by San Carlo Honda Gresini for next season, was fourth ahead of American former world champion Colin Edwards and young Italian Andrea Dovizioso.
CNN: Rossi races to MotoGP pole despite Brno fall
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He managed to hold off a late challenge from Andrea Dovizioso, his replacement at Repsol Honda, while American Colin Edwards came home in fifth on his Monster Yamaha Tech 3.
CNN: Lorenzo capitalizes on Rossi's U.S. crash
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Team-mate Colin Edwards scored a season-best result in fifth.
BBC: Matt Roberts MotoGP column
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Dani Pedrosa was third quickest on his Repsol Honda, while the in-form Colin Edwards (Monster Yamaha Tech 3), Pedrosa's Italian team-mate Andrea Dovizioso and Spanish rider Toni Elias completed the top six.
CNN: Lorenzo pips Rossi in Czech MotoGP practice