*2.6. DNA–DNA Relatedness Tests*

Because of a lacking number of genome sequences of *Streptomyces aureoverticillatus* JCM4347T, *Streptomyces vastus* JCM4524T, *S. cinereus* DSM43033T, and *S. xiangluensis* NEAU-LA29T, DNA–DNA relatedness tests between strain NEAU-SSA 1T and those strains were carried out as described by De Ley et al. [40] under consideration of the modifications described by Huss et al. [41], using a model Cary 100 Bio UV/VIS-spectrophotometer (Hitachi U-3900, Hitachi Co., Tokyo, Japan) equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with in situ temperature probe (Varian). The genomic DNAs of strain NEAU-SSA 1<sup>T</sup> and its closely related species—*S. aureoverticillatus* JCM4347T, *S. vastus* JCM4524T, *S. cinereus* DSM43033T, and *S. xiangluensis* NEAU-LA29T—were extracted using the method of SDS-based DNA extraction [28]. The concentration and purity of these DNA samples were determined by measuring the optical density (OD) at 260, 280, and 230 nm. The DNA samples used for hybridization were diluted to OD260 around 1.0 using 0.1 × SSC (saline sodium citrate buffer), then sheared using a JY92-II ultrasonic cell disruptor (ultrasonic time 3 s, interval time 4 s, 90 times; Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China). The DNA renaturation rates were determined in 2 × SSC at 70 ◦C. The experiments were performed with three replications and the DNA–DNA relatedness value was expressed as a mean of the three values. Several genomic metrics are now available to distinguish between orthologous genes of closely related prokaryotes, including the calculation of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values [42,43]. In the present study, ANI and dDDH values were determined from the genomes of strain NEAU-SSA 1T and *S. flaveus* JCM3035T (JOCU00000000) using the ortho-ANIu algorithm from Ezbiotaxon and the genome-to-genome distance calculator (GGDC 2.0) at http://ggdc.dsmz.de.
