Complementary combination of multiplex high-throughput DNA sequencing for molecular phylogeny

The rapid development of DNA sequencing technology in recent years has provided new tools for phylogenetic data acquisition. By using high-throughput DNA sequencing technology, molecular phylogenetic information can be obtained more quickly and economically. Here, we describe a complementary combination of two multiplex high-throughput DNA sequencing methods. One is multiplexed phylogenetic marker sequencing (MPM-seq), and the other is multiplexed inter-simple sequence repeat (ISSR) genotyping by sequencing (MIG-seq), whose protocol is improved over that of the original one. Both MPM-seq and MIG-seq begin with multiplex polymerase chain reaction (PCR), each amplifying multiple phylogenetic markers and genome-wide ISSR regions, respectively. After another PCR using a second PCR primer set that is common in both methods, next-generation sequencing is used to simultaneously detect DNA sequences of multiple regions from multiple samples in each method. In this case study, we performed a molecular phylogenetic analysis of Japanese fir (Abies) and the closely related Abies species. MPM-seq revealed DNA sequences of three regions from chloroplast DNA and one nuclear internal transcribed spacer and created a partially informative phylogenetic tree for 13 Abies species. Whereas MIG-seq detected 6700 single-nucleotide polymorphisms and exhibited clear clustering of related species with 97%-100% bootstrap support for all branches of the phylogenetic tree. Hence, with a complementary combination, quick, simple, and economical analysis can be performed in a wide range of genomic studies, including molecular phylogeny, as well as for investigating genetic differentiation or genetic identification among species, hybrids, and populations, and even among clones and cultivars, as a DNA barcoding technique.