Characterization of Newly Bred Cordyceps militaris Strains for Higher Production of Cordycepin through HPLC and URP-PCR Analysis

Abstract

<i>Cordyceps militaris</i>, a member of Ascomycota, a mushroom referred to as caterpillar Dongchung-ha-cho, is commercially valuable because of its high content of bioactive substances, including cordycepin, and its potential for artificial cultivation. Cordycepin (3'-deoxyadenosine) is highly associated with the pharmacological effects of <i>C. militaris</i>. <i>C. militaris</i> is heterothallic in that two mating-type loci, idiomorph <i>MAT1-1</i> and <i>MAT1-2</i>, exist discretely in two different spores. In this study, nine <i>C. militaris</i> strains were mated with each other to prepare newly bred strains that produced a larger amount of cordycepin than the parent strains. Nine strains of <i>C. militaris</i> were identified by comparing the internal transcribed spacer sequence, and a total of 12 single spores were isolated from the nine strains of <i>C. militaris</i>. After the MAT idiomorph was confirmed by PCR, 36 mating combinations were performed with six single spores with <i>MAT1-1</i> and the others with <i>MAT1-2</i>. Eight mating combinations were successfully mated, producing stroma with perithecia. Cordycepin content analysis of all strains by high-performance liquid chromatography revealed that the KASP4-bred strain produced the maximum cordycepin among all strains, regardless of the medium and stroma parts. Finally, universal rice primer-PCR was performed to demonstrate that the bred strains were genetically different from the parental strains and new <i>C. militaris</i> strains. These results may be related to the recombination of genes during mating. The newly produced strains can be used to meet the industrial demand for cordycepin. In addition, breeding through mating suggests the possibility of producing numerous cordycepin-producing <i>C. militaris</i> strains.