3.1 Mitochondrial genome sequencing and primer design
Mitochondrial genomes of the reference corn strain and reference rice strain of S. frugiperda were extracted from the genome publication (Gouin et al., 2017a). Another entire mitochondrial genome was reported in 2016 from China (15,365 bp) (Liu et al., 2016) and in 2019 from Korea (15,388 bp) (Seo et al., 2019). We sequenced individuals from cornfields in three Korean localities: Jeju (GenBank MN599981: 15,368 bp), Muan (MN599982: 15,368 bp), and Miryang (MN599983: 15,387 bp). Additionally, two African populations, one collected in Nigeria (MN599980: 15,400 bp) and the other in Benin (MN803322: 15,369 bp) were sequenced (Table 1). Among the five S. frugiperda populations, 15 indels and 232 SNPs were identified (Supplementary Information). Mitochondrial genome size varied from 15368 bp to 15400 bp and the sequence of the Jeju population was identical to the Muan population. Three indels and one SNP differed between Jeju and Miryang. The GC content was 18.7 % except for Benin (18.9 %). The three Korean populations grouped with the rice reference strain and the Nigerian population, while the Benin sample grouped with the corn reference strain (Fig. 1A).
Based on the global mVISTA alignment results, conserved regions amongSpodoptera species and variable regions were observed (Fig. 1B). Just as for other Lepidoptera, the mitochondrial genome includes 13 protein coding genes: NADH dehydrogenase components (complex Ⅰ, ND), cytochrome oxidase subunits (complex Ⅵ, COX), cytochrome oxidase b (CYPB) and two ATP synthases; two ribosomal RNA genes and 22 transfer RNAs (Fig. 2).
For primer design we focused on a region including part of HD5, the tRNA-R and short non-coding region between primers FAW_F3 and FAW_R3 (Fig. 2). Primer sequences are given in Table 2 and primer-binding regions shown in Fig. 3. Primer FAW_F3 is the main diagnostic primer and along with FAW_B3 (Fig. 4A) and FAW_UR in (Fig. 4B) will amplify a product only from S. frugiperda mtDNA. Primer BAW_DF along with FAW_UR will amplify a product only from S. exigua mtDNA (Fig. 4C). These primer pairs can distinguish S. frugiperda andS. exigua by conventional PCR (Fig. 4). Primers FAW_F3 and FAW_B3 amplify the larger region that participates in the LAMP reaction, only from S. frugiperda . The dumbbell structure that is further amplified has one strand generated by primer BIP, which consists of B1c fused to B2 and primes from B2c on the top strand (Fig. 3). The other strand is generated by primer FIP, which has F1c fused to F2 and primes from F2c on the bottom strand. Primers FAW_F3 and FAW_B3 then further amplify the region by strand displacement through these double-stranded products. The base-paired regions in the dumbbell structure can also prime extension along with the BIP and FIP primers. Additionally, loop primers (LF and LB) could potentially contribute even more to the amplification (Nagamine et al., 2002).