Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing.

Jinsu Gil,Yurry Um,Yi Lee,Ho Kim,Chang Hong,Serim Kim,Chinreddy Reddy,Dong Lee,Sung Koo,Jong-Wook Chung,Seong-Cheol Kim,Sin-Gi Park,Ok Kim,Byung-Hoon Jeong

doi:10.3390/genes8100238

Abstract

Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity (HO) values, and the expected heterozygosity (HE) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

Highlights

The genus Angelica, which includes more than 60 species, is an important medicinal herb inFar East countries, especially in Korea, Japan, and China
Young leaves or fresh seedlings were used for extraction of the genomic DNA, which is required for library construction and sequencing
The libraries were constructed from five A. gigas accessions: Manchu variety (MV), Hwangje variety (HV), Gangwon local variety (GLV), Jecheon local variety (JLV), and Sancheong local variety (SLV)

Summary

Introduction

The genus Angelica, which includes more than 60 species, is an important medicinal herb in. Due to the scarcity of efficient molecular markers, there is a dearth of information about the genetic relationships and genetic diversity among breeding populations of Angelica crops [9]. The Angelica species used in conventional medicine varies by country according to specific regulations, i.e., A. gigas in Korea, A. sinensis in China, and A. acutiloba in Japan. SSR markers are especially useful to distinguish closely related genotypes This is the reason that SSR markers are widely used in genetic studies and identification of closely related cultivars. By using these SSR markers, various problems of Angelica genus can be solved [21]. The SSR markers developed in this study could be used for genetics and the breeding of Angelica genus

Plant Material and DNA Isolation

Genomic DNA Sequencing and Sequence Assembly

SSR Findings and Primer Designs

In Silico SSR Polymorphism Screening

PCR Amplification and Genotyping

Results and Discussion