BoCAPS: Rapid screening of chemical races in Botryococcus braunii with direct PCR-CAPS

The colony-forming microalga Botryococcus braunii produces petroleum-like hydrocarbons and is a promising source for biofuel production. B. braunii is classified into three chemical races (A, B, L) plus a tentative class (S), based on the structure of its hydrocarbons. The determination of hydrocarbon structure in a newly isolated strain requires several months for culturing and analysis. As potential for biofuel production differs among races, a rapid method for estimating race is needed for the effective bioprospecting. This study aimed to develop a DNA-based rapid estimation method (BoCAPS) of the chemical races of B. braunii and to test the feasibility of direct PCR from a single colony. A total of 247 wild strains were isolated from 29 ponds in Japan and Indonesia, their 18S rRNA sequences determined, and a phylogenetic tree constructed based on the sequences of standard strains with known chemical races. Polymorphisms between clades of the phylogenetic tree corresponding to chemical races were mapped, and a cleaved amplified polymorphic sequence (CAPS) marker was developed for race discrimination. Direct PCR was tested on a single colony as a template. We demonstrated that HaeIII restriction of the PCR product enables race discrimination on agarose gels, and that direct PCR amplifies the 18S rRNA gene from a single colony without any pre-treatment. With BoCAPS, we investigated race compositions of the isolated strains and determined that the B-race was dominant (74 %). Neither the A-race nor S-race strains were isolated from tropical regions. In addition, we observed a few ponds in which minor races (L or A) were dominant and some ponds with a mixture of different races. The BoCAPS allows us to estimate chemical race without the time-consuming and expensive analysis of hydrocarbons and will be useful for high-throughput screening and ecological studies of wild populations.