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

Abstract

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 (MIGseq), 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.

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

“…To evaluate the performance of MIG-seq, in the case of the experiment of first PCR of MIG-seq for the 12 species, the concentration of template DNAs to 20-ng/μl was adjusted using NanoDrop One (Thermo Fisher, Waltham, USA). Using 16 multiplexing primers developed by Suyama and Matsuki 17 ( Supplementary Table S4 ) and Multiplex PCR Assay Kit ver.2 (TAKARA Bio Co. Ltd., Japan), the first PCR was performed using the following profile, referring to the method in Suyama et al 24 : 94°C for 2 min, followed by 25 cycles at 94°C for 30 s, 38°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 10 min. We performed the second PCR using Prime Star GXL DNA polymerase (TAKARA Bio Co. Ltd., Japan), the first PCR products diluted 50-fold, and the second PCR primers ( Supplementary Table S5 ) with the following profile: 98°C for 30 s, followed by 20 cycles at 98°C for 10 s, 54°C for 15 s, 68°C for 30 s, and a final extension at 72°C for 10 min.…”

A new subspecies of Stellaria alsine (Caryophyllaceae) from Yakushima, Japan

“…2 (Takara Bio, Kusatsu, Japan) (first PCR). The first set of primers consisted of tail sequences and locus-specific primers ( Suyama et al 2022 ). Subsequently, the first PCR products were purified and used for the second PCR.…”

Molecular phylogeny and taxonomy of the Hydrangea serrata complex (Hydrangeaceae) in western Japan, including a new subspecies of H. acuminata from Yakushima

“…Multiplex ISSR genotyping by sequencing (MIG-seq, Suyama and Matsuki 2015) was used for de novo SNP detection. Briefly, a MIG-seq library was prepared by a two-step PCR amplification process based on the protocol detailed by Suyama et al (2022). The amplicons in the size range of 300-800 bp were purified and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) using an MiSeq Reagent Kit v3 (150 cycles, Illumina).…”

“…The chloroplast and nuclear genomic regions were sequenced using the next generation sequencing (NGS) technique (Suyama et al 2022). First, three chloroplast genomic regions, rbcL, trnL intron, and psbA-trnH, and two nuclear genomic regions, ITS1 and ITS2, were simultaneously amplified using the Multiplex PCR Assay Kit Ver.…”

“…2 (Takara Bio, Kusatsu, Japan) (first PCR reaction). The first primers consisted of tail sequences and locus-specific primers (Suyama et al 2022). Second, the products from the first PCR reaction were purified and used for the second PCR.…”