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The green microalga  Botryococcus braunii  produces hydrocarbon oils at 25–75% of its dry weight and is a promising source of biofuel feedstock. Few studies have examined this species’ ecology in natural habitats, and few wild genetic resources have been collected due to difficulties caused by its low abundance in nature. This study aimed to develop a real-time PCR assay for specific detection and quantification of this alga in natural environments and to quantify spatiotemporal variations of wild  B .  braunii  populations in a tropical pond. We designed PCR primers toward the hydrocarbon biosynthesis gene  SSL-3  and examined amplification specificity and PCR efficiency with 70 wild strains newly isolated from various environments. The results demonstrated that this PCR assay specifically amplified  B .  braunii  DNA, especially that of B-race strains, and can be widely used to detect wild  B .  braunii  strains in temperate and tropical habitats. Field-testing in a tropical pond suggested a diurnal change in the abundance of  B .  braunii  in surface water and found  B .  braunii  not only in surface water, but also at 1–1.5 m deep and in bottom sediments. This method can contribute to efficient genetic resource exploitations and may also help elucidate the unknown ecology of  B .  braunii .

Detection of the oil-producing microalga Botryococcus braunii in natural freshwater environments by targeting the hydrocarbon biosynthesis gene SSL-3