Perilla frutescens (L.) Britton is widely cultivated in East Asia. There are two varieties based on the uses and morphology, P. frutescens var. frutescens and var. crispa. Generally, var. frutescens is used as an oil crop (ren in Chinese, dlggae in Korean and egoma in Japanese), whereas var. crispa is used as a vegetable crop or Chinese medicine (zisu in Chinese, cha-jo-ki in Korean and shiso in Japanese)    . Today, P. frutescens var. frutescens and var. crispa are extensively cultivated and used in Korea and Japan    . Var. frutescens is used as both a leafy vegetable and an oil crop in Korea. In contrast, var. crispa is used for vegetables or pickles, using the leaves in Japan, and is also used for Chinese medicine in China    . In East Asia, the wild species of these two varieties of Perilla crop are unknown but weedy plants of two cultivated types of P. frutescens have been identified    . In East Asia, the weedy plants naturally grow in wastelands, roadsides, around farming fields or farmhouses     . The two cultivated types of var. frutescens and crispa in East Asia have several distinguishing morphological characters including the seed size, stem and leaf color, and plant fragrance. The weedy type of var. frutescens has a same stem and leaf color and fragrance as cultivated var. frutescens but its seeds are smaller and harder than those of cultivated var. frutescens   . The weedy type of var. crispa is occasionally recognized and used as cultivated var. crispa by farmers because of their morphological similarities   .
Information on the genetic diversity and genetic relationships among Perilla crop and their weedy types is very important for successful Perilla crop breeding programs, the use of the germplasm resources and conservation. In previous studies, RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism), and SSR (simple sequence repeat) analyses showed that the two weedy types of Perilla crop were each grouped with the two cultivated types of var. frutescens and var. crispa      . Although these studies were performed to distinguish cultivated types of var. frutescens and crispa and their weedy types at the DNA level, they did not present a clear classification for these two Perilla varieties and their weedy types. Among many DNA molecular markers, SSRs are very abundant in eukaryotic genomes and show a highly variable number of repeats among individuals in a given population  . SSRs are often selected for genetic studies such as genetic diversity and relationship analyses because they have advantages such as high reproducibility, polymorphisms, abundance, and codominance in plant genomes   . Our previous study successfully isolated SSRs for Perilla crop    and analyzed Perilla accessions collected from various regions      . However, the number of SSR markers for clear classification is still lacking.
In our previous study, we obtained 15,991 SSR loci from transcriptome sequencing by RNA-seq in one cultivated type (PF98095) of P. frutescens var. frutescens  . In this study, we successfully developed SSR primers from Perilla species, and these novel additional SSR markers can be used to analyze the genetic diversity and genetic relationships and to perform QTL mapping among two cultivated types of Perilla crop and their weedy types.
2. Materials and Methods
2.1. Plant Materials and DNA Extraction
This study used 15 accessions including the cultivated type of var. frutescens, cultivated type of var. crispa and weedy type of var. frutescens to evaluate polymorphisms and identify new SSR markers. Total DNA was extracted from the leaf tissues of a representative individual plant for each accession following the Plant DNAzol Reagent protocol (GibcoBRL Inc., Grand Island, NY, USA).
2.2. SSR Marker Development
To construct the transcriptome reference set in a previous study  , de novo assembly of the PF98095 RNA-seq data was performed using Trinity software. The raw reads from NGS sequencing with a Phred quality score of at least 20 and read length of at least 50 bp of HiSeq 2000 data were filtered before assembly. A Perl script MISA tool (https://pgrc.ipk-gatersleben.de/misa) was used to search microsatellite sites in the assembled transcriptome sequences of PF98095. The SSRs with di-, tri-, and tetra-nucleotide repeat units were identified. Based on the SSR flanking sequences, PRIMER 3 software was employed to design the primer pairs. We searched all unigenes in the cultivated type of var. frutescens (PF98095) and detected 15,991 SSR loci. In this study, we selected 200 SSR primer sets based on the 80 di-, 60 tri- and 60 tetra-nucleotide types and the number of repeat units.
2.3. SSR Analysis and Data Analysis
SSR amplifications were conducted in a total volume of 20 µl consisting of 20 ng genomic DNA, 1 × PCR buffer, 0.5 µM of forward and reverse primers, 0.2 mM dNTPs, and 1 unit of Taq polymerase (Biotools, Madrid, Spain). The PCR profile consisted of an initial denaturation at 95˚C for 3 minutes followed by 36 cycles of 95˚C for 30 seconds, 55˚C for 30 seconds, and 72˚C for 1 minute 30 seconds with a final extension step of 5 minutes at 72˚C. After PCR analysis, the PCR products were resolved on a QIAxcel Screengel system (QIAGEN) with the 0M700 method according to the manufacturer’s protocol. The number of alleles, allele frequency, major allele frequency (MAF), gene diversity (GD), and polymorphic information content (PIC) for new SSR markers were calculated using PowerMarker 3.25  .
3. Results and Discussion
Among the selected 200 SSR primer pairs, 37 SSR primer pairs had good amplification and polymorphisms among 15 Perilla accessions (Table 1). However, the remaining 163 SSR primer pairs exhibited a monomorphic band (53) or ambiguous band pattern (33) and poor or no amplification (77) in the Perilla accessions. The new 37 SSR primer pairs were used to measure the genetic diversity index, such as the number of alleles, MAF, GD, and PIC among 15 Perilla accessions, including two cultivated types of Perilla crop and their weedy types in East Asia. 182 alleles were detected in the 15 Perilla accessions, with an average of 4.9 alleles per locus, ranging from 60 to 250 bp. The number of alleles per locus ranged from 2 (KNUPE-45 and KNUPE-57) to 9 (KNUPE-42). The MAF per locus varied
Table 1. Characteristics of the 37 SSR loci, including the primer sequence, repeat motif, annealing temperature, allele size range, and genetic diversity index among 15 Perilla accessions.
Ta: annealing temperature, MAF: major allele frequency, GD: genetic diversity, PIC: polymorphic information content.
from 0.200 (KNUPE-53) to 0.733 (KNUPE-73), with an average of 0.463. The GD ranged from 0.391 (KNUPE-45) to 0.853 (KNUPE-42), with an average of 0.670. The average PIC was 0.624, ranging from 0.315 (KNUPE-45) to 0.838 (KNUPE-42) (Table 1).
The analysis of the three groups of Perilla accessions (cultivated and weedy types of var. frutescens and cultivated type of var. crispa) using the 37 SSR primers showed that the average number of alleles ranged from 2.6 for the weedy type of var. frutescens to 3.4 for the cultivated type of var. crispa. The average GD were 0.586 and 0.461 for the cultivated and weedy types of var. frutescens, respectively, and 0.629 for the cultivated var. crispa. The average PIC were 0.524, 0.406 and 0.565 for the cultivated and weedy types of var. frutescens and cultivated var. crispa, respectively (Table 2). Perilla crop is widely distributed and cultivated in Korea, Japan and China. This information is about the genetic diversity of Perilla may be useful for the preservation of germplasm resources in East Asia.
Table 2. Estimates of allele number, MAF, gene diversity and PIC of 37 SSR primers among cultivated and weedy types of Perilla.
Group 1: Cultivated var. frutescens, Group 2: Weedy var. frutescens, Group 3: Cultivated var. crispa.
Our study results using new Perilla SSR primers validate the proposal that the weedy types of Perilla species are the key taxon in understanding the origin of the two cultivated types of var. frutescens and var. crispa. The new Perilla SSR primers described in this study should facilitate confirmation of the genetic diversity and could be used for the identification of cultivars, conservation of Perilla germplasm resources, and genetic mapping and designating of important genes/QTLs for Perilla crop breeding programs.
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (#2016R1D1A1B01006461).
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