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IDENTIFICATION AND CLONING OF AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS LINKED TO THE MATING TYPE LOCUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)
Author(s) -
Werner Ralf,
Olschewski Jasna,
Mergenhagen Dieter
Publication year - 2001
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1046/j.1529-8817.2001.037003427.x
Subject(s) - amplified fragment length polymorphism , biology , chlamydomonas reinhardtii , genetics , chlamydomonas , locus (genetics) , mating type , sequence tagged site , gene , genetic marker , cloning (programming) , cleaved amplified polymorphic sequence , gene mapping , restriction fragment length polymorphism , polymerase chain reaction , chromosome , population , genetic diversity , demography , sociology , computer science , mutant , programming language
Amplified fragment length polymorphism (AFLP) markers have been widely used to generate molecular maps of plant species, including crops and cereals. We report on a useful protocol to identify AFLPs from Chlamydomonas reinhardtii Dangeard with digoxigenin labeled primers. Although Chlamydomonas has a small genome with a high GC content, we could detect polymorphic bands that led to the identification of several AFLP markers linked to the mating type locus of Chlamydomonas. Three of these markers were isolated from the gel, reamplified, and cloned. The clones were sequenced, and the insertion of the correct fragment was verified in AFLP gels and in Southern blots. One marker showed sequence identity to parts of the fus1 gene, known to be unique in the plus mating type. We also converted some of the AFLP markers into sequence tagged site markers, which allows a fast and convenient screening of progeny of crosses. This procedure will be a useful and fast alternative to the conventional generation of maps for the positional cloning of genes from Chlamydomonas.