Development and Characterization of New Microsatellite Markers for Perilla frutescens (L.) Britton

Based on RNA sequences using transcriptome analysis, 37 new simple sequence repeat (SSR) primer sets were developed for Perilla species. These new SSR markers were applied to analyze the genetic diversity among 15 accessions of Perilla species. A total of 182 alleles were confirmed in 37 loci, with an average of 4.9 alleles per locus and from 2 to 9 alleles per locus. The MAF (major allele frequency) per locus varied from 0.200 to 0.733, with an average of 0.463. The gene diversity (GD) ranged from 0.391 to 0.853, with an average of 0.670. The average polymorphic information content (PIC) was 0.624, ranging from 0.315 to 0.838. The new SSR markers of Perilla species reported in this study may provide potential markers to analyze the genetic diversity and genetic relationships of Perilla species. In addition, new Perilla SSR markers developed from transcriptome analysis can be useful for the identification of cultivars, conservation of Perilla germplasm resources, and genetic mapping and designating of important genes/QTLs for future Perilla crop breeding programs.

[1] [2] [3]. Today, P. frutescens var. frutescens and var. crispa are extensively cultivated and used in Korea and Japan [2] [3] [4]. 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 [2] [3] [5]. 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 [1] [2] [3]. In East Asia, the weedy plants naturally grow in wastelands, roadsides, around farming fields or farmhouses [2] [3] [5] [6]. 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 [2] [7]. The weedy type of var. crispa is occasionally recognized and used as cultivated var. crispa by farmers because of their morphological similarities [2] [3].
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 [1] [5] [8] [9] [10]. 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 [11]. 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 [11] [12]. Our previous study successfully isolated SSRs for Perilla crop [13] [14] [15] and analyzed Perilla accessions collected from various regions [9] [14] [16] [17] [18]. 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 [19]. 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.

SSR Marker Development
To construct the transcriptome reference set in a previous study [19], 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.    Table 1).

Results and Discussion
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. 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.