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Haematococcus pluvialis is the best resource for natural astaxanthin, which has multiple biological functions. Nitrogen depletion can result in astaxanthin accumulation in H. pluvialis. In order to explore the molecular mechanism of astaxanthin accumulation in H. pluvialis under nitrogen depletion stress, the growth rate of the algae was found to decrease during 0-72 h, the accumulation of astaxanthin mainly occured during 12-24 h, and then slowed down. MSAP analysis of stress time point at 0, 24 and 72 h got 291 methylation polymorphism loci, among which 29.90% of 0-24 h methylation loci and 53.95% of 24-72 h methylation loci changed. After 24 h of nitrogen depletion stress, the DNA semi-methylation rate was the highest(12.71%), and the full-methylation rate got the lowest(26.80%). On the contrary, full-methylation rate was the highest(28.52%)and the semi-methylation rate was the lowest(1.72%)after 72 h stress. DNA methylation changes appeared to be a vital regulation for astaxanthin accumulation. [ABSTRACT FROM AUTHOR]