July 2017  Vol. 27  No. 7 J. Microbiol. Biotechnol. (2017), 27(7), 1223–1232 https://doi.org/10.4014/jmb.1701.01043 Research Article jmb Review Characterization of Newly Bred Cordyceps militaris Strains for Higher Production of Cordycepin through HPLC and URP-PCR Analysis Hyun-Hee Lee 1† , Naru Kang 1,2† , Inmyoung Park 1,3† , Jungwook Park 1 , Inyoung Kim 1 , Jieun Kim 1 , Namgyu Kim 1 , Jae-Yun Lee 2 , and Young-Su Seo 1 * Department of Microbiology, Pusan National University, Busan 46241, Republic of Korea CMG Biofarm, Busan 46958, Republic of Korea Department of Asian Food and Culinary Arts, Youngsan University, Busan 48015, Republic of Korea Introduction The genus Cordyceps is an entomopathogenic and endoparasitic fungus that belongs to the division Ascomycota, class Sordariomycetes, order Hypocreales, and family Clavicipitaceae and contains more than 750 species [1]. Cordyceps spp. are mainly distributed in subtropical regions with high temperatures and humidity and parasitize arthropods or insects from lepidopteran larva and pupae to imago to obtain nourishment from the host [2]. Infection begins by the adherence of conidia on the insect’s exoskeleton and germination occurs within a few hours. After several weeks, the species grows continuously and creates stroma supported by the host exocuticula [3]. Many Cordyceps spp. have been used as health foods or medicines in China and South-East Asia for a long time. Cordyceps sinensis contains numerous bioactive substances [4], but is very costly because it is extremely difficulty to artificially culture. The demand for C. militaris has gradually increased because of artificial culture and its numerous bioactive substances. The bioactive substances in C. militaris include polysaccharides, ergosterol, cordycepic acid, and Received: January 16, 2017 Revised: April 19, 2017 Accepted: April 23, 2017 First published online April 25, 2017 *Corresponding author Phone: +82-51-510-2267; Fax: +82-51-514-1778; E-mail: yseo2011@pusan.ac.kr These authors contributed equally to this work. pISSN 1017-7825, eISSN 1738-8872 Copyright © 2017 by The Korean Society for Microbiology and Biotechnology Cordyceps militaris, a member of Ascomycota, a mushroom referred to as caterpillar Dong- chung-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 C. militaris. C. militaris is heterothallic in that two mating-type loci, idiomorph MAT1-1 and MAT1-2, exist discretely in two different spores. In this study, nine C. militaris 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 C. militaris were identified by comparing the internal transcribed spacer sequence, and a total of 12 single spores were isolated from the nine strains of C. militaris. After the MAT idiomorph was confirmed by PCR, 36 mating combinations were performed with six single spores with MAT1-1 and the others with MAT1-2. 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 C. militaris 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 C. militaris strains. Keywords: Cordyceps militaris, cordycepin, MAT gene, mating, Dong-chung-ha-cho