Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781 bp per gene in 82.473 Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals.

Development of CACTA transposon derived SCAR markers and their use in population structure analysis in Zea mays.

Molecular marker technologies have proven to be an important breakthrough for genetic studies, construction of linkage maps and population genetics analysis. Transposable elements (TEs) constitute major fractions of repetitive sequences in plants and offer a wide range of possible areas to be explored as molecular markers. Sequence characterized amplified region (SCAR) marker development provides us with a simple and time saving alternative approach for marker development. We employed the CACTA-TD to develop SCARs and then integrated them into linkage map and used them for population structure and genetic diversity analysis of corn inbred population. A total of 108 dominant SCAR markers were designed out of which, 32 were successfully integrated in to the linkage map of maize RIL population and the remaining were added to a physical map for references to check the distribution throughout all chromosomes. Moreover, 76 polymorphic SCARs were used for diversity analysis of corn accessions being used in Korean corn breeding program. The overall average polymorphic information content (PIC) was 0.34, expected heterozygosity was 0.324 and Shannon's information index was 0.491 with a percentage of polymorphism of 98.67%. Further analysis by associating with desirable traits may also provide some accurate trait specific tagged SCAR markers. TE linked SCARs can provide an added level of polymorphism as well as improved discriminating ability and therefore can be useful in further breeding programs to develop high yielding germplasm.

Construction of genetic linkage map and identification of QTLs related to agronomic traits in maize using DNA transposon-based markers.

Transposable elements (TEs), are a rich source for molecular marker development as they constitute a significant fraction of the eukaryotic genome and impact the overall genome structure. Here, we utilize Mutator-based transposon display (Mu-TD), and CACTA-derived sequence-characterized amplified regions (SCAR) anchored by simple sequence repeats and single nucleotide polymorphisms to locate quantitative trait loci (QTLs) linked to agriculturally important traits on a genetic map. Specifically, we studied recombinant inbred line populations derived from a cross between dent corn and waxy corn. The resulting linkage map included 259 Mu-anchored fragments, 34 SCARs, and 614 SSR markers distributed throughout the ten maize chromosomes. Linkage analysis revealed three SNP loci associated with kernel starch synthesis genes (sh2, su1, wx1) linked to either Mu-TD loci or SSR markers, which may be useful for maize breeding programs. In addition, we used QTL analysis to determine the chromosomal location of traits related to grain yield and kernel quality. We identified 24 QTLs associated with nine traits located on nine out of ten maize chromosomes. Among these, 13 QTLs involved Mu loci and two involved SCARs. This study demonstrates the potential use of DNA transposon-based markers to construct linkage maps and identify QTLs linked to agronomic traits.

LTR-retrotransposons and inter-retrotransposon amplified polymorphism (IRAP) analysis in Lilium species.

LTR-retrotransposons are ubiquitous and highly abundant in plant genomes. Moreover, LTR-retrotransposons can often cause genome obesity in plants. Although Lilium species have been known carrying large genomes among flowering plants, reports on the LTR-retrotransposons in Lilium species are rather limited. We isolated a novel Ty3/gypsy-like retrotransposon, LIRE-del, and two Ty1/copia-like retrotransposons, a LIRE-del and an unclassified, from a fosmid clone of Lilium longiflorum. Decayed internal ORF sequences indicated that they were non-autonomous elements. IRAP protocol was developed based on the LTR sequences of the isolated LTR-retrotransposons. Fourteen primer combinations showed clear distinctive PCR amplification bands that were highly informative in the analysis of species relationship among Lilium species. The phylogenetic relationship based on the IRAP profile revealed some discordant with phylogenetic studies based on the ITS sequences of 45S ribosomal gene and matK gene variations in a few species. Thus, the phylogenetic relationship among Lilium species may need to be re-evaluated with other tools such as cross compatibility and selectively neutral genetic markers.

O- and N-Methyltransferases in benzylisoquinoline alkaloid producing plants.

BACKGROUND: Secondary metabolites such as benzylisoquinoline alkaloids (BIA) have attracted considerable attention because of their pharmacological properties and potential therapeutic applications. Methyltransferases (MTs) can add methyl groups to alkaloid molecules, altering their physicochemical properties and bioactivity, stability, solubility, and recognition by other cellular components. Five types of O-methyltransferases and two types of N-methyltransferases are involved in BIA biosynthesis. OBJECTIVE: Since MTs may be the source for the discovery and development of novel biomedical, agricultural, and industrial compounds, we performed extensive molecular and phylogenetic analyses of O- and N-methyltransferases in BIA-producing plants. METHODS: MTs involved in BIA biosynthesis were isolated from transcriptomes of Berberis koreana and Caulophyllum robustum. We also mined the methyltransferases of Coptis japonica, Papaver somniferum, and Nelumbo nucifera from the National Center for Biotechnology Information protein database. Then, we analyzed the functional motifs and phylogenetic analysis. RESULT: We mined 42 O-methyltransferases and 8 N-methyltransferases from the five BIA-producing plants. Functional motifs for S-adenosyl-L-methionine-dependent methyltransferases were retained in most methyltransferases, except for the three O-methyltransferases from N. nucifera. Phylogenetic analysis revealed that the methyltransferases were grouped into four clades, I, II, III and IV. The clustering patterns in the phylogenetic analysis suggested a monophyletic origin of methyltransferases and gene duplication within species. The coexistence of different O-methyltransferases in the deep branch subclade might support some cases of substrate promiscuity. CONCLUSIONS: Methyltransferases may be a source for the discovery and development of novel biomedical, agricultural, and industrial compounds. Our results contribute to further understanding of their structure and reaction mechanisms, which will require future functional studies.

Isolation and characterization of novel Ty1-copia-like retrotransposons from lily.

Species of the genus Lilium are well known for their large genomes. Although expansion of noncoding repeated DNA is believed to account for this genome size, retroelement del Ty3-gypsy is the only one described so far in the genus Lilium. We isolated Ty1-copia elements from Lilium longiflorum and named them LIREs (lily retrotransposons). The long terminal repeats, primer binding site, and polypurine tract sequences are highly similar among the LIRE elements, indicating that they are in the same lineage. Although the protein-coding regions were highly decayed, the sequence motifs of the integrase, reverse transcriptase, and RNase H domains were identifiable as belonging to the order of Ty1-copia elements. Phylogenetic analysis and primer binding site sequences revealed that these elements belonged to the Ale lineage among the six lineages of plant Ty1-copia elements. Base substitutions in the long terminal repeats estimated that the integration times of the LIRE Ty1-copia elements were between 0.7 and 5.5 mya. In situ hybridization showed that the LIRE elements were present in all the chromosomes of L. longiflorum and L. lancifolium, but absent in centromeres, telomeres, and 45S rRNA sites in both species. The LIRE elements were present very abundantly in species of the genus Lilium, but absent in other genera of the family Liliaceae, implying that the LIRE elements might have contributed to the expansion of the genome in the genus Lilium.

Identification and Characterization of Key Genes Responsible for Weedy and Cultivar Growth Types in Soybean.

In cultivated plants, shoot morphology is an important factor that influences crop economic value. However, the effects of gene expression patterns on shoot morphology are not clearly understood. In this study, the molecular mechanism behind shoot morphology (including leaf, stem, and node) was analyzed using RNA sequencing to compare weedy (creeper) and cultivar (stand) growth types obtained in F7 derived from a cross of wild and cultivated soybeans. A total of 12,513 (in leaves), 14,255 (in stems), and 11,850 (in nodes) differentially expressed genes were identified among weedy and cultivar soybeans. Comparative transcriptome and expression analyses revealed 22 phytohormone-responsive genes. We found that GIBBERELLIN 2-OXIDASE 8 (GA2ox), SPINDLY (SPY), FERONIA (FER), AUXIN RESPONSE FACTOR 8 (ARF8), CYTOKININ DEHYDROGENASE-1 (CKX1), and ARABIDOPSIS HISTIDINE KINASE-3 (AHK3), which are crucial phytohormone response genes, were mainly regulated in the shoot of weedy and cultivar types. These results indicate that interactions between phytohormone signaling genes regulate shoot morphology in weedy and cultivar growth type plants. Our study provides insights that are useful for breeding and improving crops to generate high-yield soybean varieties.

Characterization of transcription factor genes related to cold tolerance in Brassica napus.

Brassica napus is the third most important oilseed crop in the world; however, in Korea, it is greatly affected by cold stress, limiting seed growth and production. Plants have developed specific stress responses that are generally divided into three categories: cold-stress signaling, transcriptional/post-transcriptional regulation, and stress-response mechanisms. Large numbers of functional and regulatory proteins are involved in these processes when triggered by cold stress. Here, our objective was to investigate the different genetic factors involved in the cold-stress responses of B. napus. Consequently, we treated the Korean B. napus cultivar Naehan at the 4-week stage in cold chambers under different conditions, and RNA and cDNA were obtained. An in silico analysis included 80 cold-responsive genes downloaded from the National Center for Biotechnology Information (NCBI) database. Expression levels were assessed by reverse transcription polymerase chain reaction, and 14 cold-triggered genes were identified under cold-stress conditions. The most significant genes encoded zinc-finger proteins (33.7%), followed by MYB transcription factors (7.5%). In the future, we will select genes appropriate for improving the cold tolerance of B. napus.

Analysis of genome variants in dwarf soybean lines obtained in F6 derived from cross of normal parents (cultivated and wild soybean).

Plant height is an important component of plant architecture and significantly affects crop breeding practices and yield. We studied DNA variations derived from F5 recombinant inbred lines (RILs) with 96.8% homozygous genotypes. Here, we report DNA variations between the normal and dwarf members of four lines harvested from a single seed parent in an F6 RIL population derived from a cross between Glycine max var. Peking and Glycine soja IT182936. Whole genome sequencing was carried out, and the DNA variations in the whole genome were compared between the normal and dwarf samples. We found a large number of DNA variations in both the dwarf and semi-dwarf lines, with one single nucleotide polymorphism (SNP) per at least 3.68 kb in the dwarf lines and 1 SNP per 11.13 kb of the whole genome. This value is 2.18 times higher than the expected DNA variation in the F6 population. A total of 186 SNPs and 241 SNPs were discovered in the coding regions of the dwarf lines 1282 and 1303, respectively, and we discovered 33 homogeneous nonsynonymous SNPs that occurred at the same loci in each set of dwarf and normal soybean. Of them, five SNPs were in the same positions between lines 1282 and 1303. Our results provide important information for improving our understanding of the genetics of soybean plant height and crop breeding. These polymorphisms could be useful genetic resources for plant breeders, geneticists, and biologists for future molecular biology and breeding projects.

Identification of resurrection genes from the transcriptome of dehydrated and rehydrated Selaginella tamariscina.

Selaginella tamariscina is a lycophyta species that survives under extremely dry conditions via the mechanism of resurrection. This phenomenon involves the regulation of numerous genes that play vital roles in desiccation tolerance and subsequent rehydration. To identify resurrection-related genes, we analyzed the transcriptome between dehydration conditions and rehydration conditions of S. tamariscina. The de novo assembly generated 124,417 transcripts with an average size of 1,000 bp and 87,754 unigenes. Among these genes, 1,267 genes and 634 genes were up and down regulated by rehydration compared to dehydration. To understand gene function, we annotated Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The unigenes encoding early light-inducible protein (ELIP) were down-regulated, whereas pentatricopeptide repeat-containing protein (PPR), late embryogenesis abundant proteins (LEA), sucrose nonfermenting protein (SNF), trehalose phosphate phosphatase (TPP), trehalose phosphate synthase (TPS), and ABC transporter G family (ABCG) were significantly up-regulated in response to rehydration conditions by differentially expressed genes (DEGs) analysis. Several studies provide evidence that these genes play a role in stress environment. The ELIP and PPR genes are involved in chloroplast protection during dehydration and rehydration. LEA, SNF, and trehalose genes are known to be oxidant scavengers that protect the cell structure from the deleterious effect of drought. TPP and TPS genes were found in the starch and sucrose metabolism pathways, which are essential sugar-signaling metabolites regulating plant metabolism and other biological processes. ABC-G gene interacts with abscisic acid (ABA) phytohormone in the stomata opening during stress conditions. Our findings provide valuable information and candidate resurrection genes for future functional analysis aimed at improving the drought tolerance of crop plants.

Unraveling the complexity of faba bean (Vicia faba L.) transcriptome to reveal cold-stress-responsive genes using long-read isoform sequencing technology.

Faba bean (Vicia faba L.), a globally important grain legume providing a stable source of dietary protein, was one of the earliest plant cytogenetic models. However, the lack of draft genome annotations and unclear structural information on mRNA transcripts have impeded its genetic improvement. To address this, we sequenced faba bean leaf transcriptome using the PacBio single-molecule long-read isoform sequencing platform. We identified 28,569 nonredundant unigenes, ranging from 108 to 9669 bp, with a total length of 94.5 Mb. Many unigenes (3597, 12.5%) had 2-20 isoforms, indicating a highly complex transcriptome. Approximately 96.5% of the unigenes matched sequences in public databases. The predicted proteins and transcription factors included NB-ARC, Myb_domain, C3H, bHLH, and heat shock proteins, implying that this genome has an abundance of stress resistance genes. To validate our results, we selected WCOR413-15785, DHN2-12403, DHN2-14197, DHN2-14797, COR15-14478, and HVA22-15 unigenes from the ICE-CBF-COR pathway to analyze their expression patterns in cold-treated samples via qRT-PCR. The expression of dehydrin-related genes was induced by cold stress. The assembled data provide the first insights into the deep sequencing of full-length RNA from faba bean at the single-molecule level. This study provides an important foundation to improve gene modeling and protein prediction.

Detecting Genetic Mobility Using a Transposon-Based Marker System in Gamma-Ray Irradiated Soybean Mutants.

Transposable elements (TEs)-major components of eukaryotic genomes-have the ability to change location within a genome. Because of their mobility, TEs are important for genome diversification and evolution. Here, a simple rapid method, using the consensus terminal inverted repeat sequences of PONG, miniature inverted-repeat transposable element (MITE)-Tourist (M-t) and MITE-Stowaway (M-s) as target region amplification polymorphism (TE-TRAP) markers, was employed to investigate the mobility of TEs in a gamma-irradiated soybean mutant pool. Among the different TE-TRAP primer combinations, the average polymorphism level and polymorphism information content value were 57.98% and 0.14, respectively. Only the PONG sequence separated the mutant population into three major groups. The inter-mutant population variance, determined using the PONG marker (3.151 and 29%) was greater than that of the M-t (2.209 and 20%) and M-s (2.766 and 18%) markers, whereas the reverse was true for the intra-mutant population variations, with M-t and M-s values, being 15.151 (82%) and 8.895 (80%), respectively, compared with the PONG marker (7.646 and 71%). Thus, the MITE markers revealed more dynamic and active mobility levels than the PONG marker in gamma-ray irradiated soybean mutant lines. The TE-TRAP technique associated with sensitive MITEs is useful for investigating genetic diversity and TE mobilization, providing tools for mutant selection in soybean mutation breeding.

The complete chloroplast genome of a fern genus Thelypteris interrupta.

In this study, we report the complete chloroplast (cp) genome of Thelypteris interrupta, a fern member, and comparative analysis with its related family members. The cp genome was 155,983 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (25,614 bp) separated by a large (82,769 bp) and small (21,986 bp) single-copy (SC) region. The genome encodes a total of 88 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Additionally, we identified 86 RNA editing sites in 52 genes; most of the substitution was U to C (52 sites), while C to U conversion occurred in 34 positions. The phylogenetic analysis strongly supported the relationship of T. interrupta with Ampelopteris prolifera and Christella appendiculata of Thelypteridoideae family.

The complete chloroplast genome of a Korean endemic species Sophora koreensis, Nakai.

In this study, we report the complete chloroplast (cp) genome of Sophora koreensis and its relation with other species within the Fabaceae family. The cp genome was 154,870 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (25,866 bp) separated by a large (85,037 bp) and small (18,101 bp) single-copy (SC) region. The genome encodes a total of 84 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Phylogenetic analysis suggested that S. koreensis is closely related to genus Sophora alopecuroides var. alopecuroides within Fabaceae.

Genetic differentiation of Mutator insertion polymorphisms and association with agronomic traits in waxy and common maize.

BACKGROUND: As waxy maize is considered a key economic crop in Korea, an understanding of its genetic variation and differentiation is fundamental for the selective plant breeding. The maize genome is primarily composed of transposable elements, for which large and stable insertions generate variations that reflect selection during evolution. OBJECTIVES: This study was to elucidate the genetic diversity based on the contribution of TEs and to investigate the effect of Mu transposition on the genetic divergence of waxy and common maize. We also performed an association analysis on these inbred lines to determine the Mu insertions associated with agronomic traits. METHODS: In this study, we utilized a Mutator-based transposon display method to study the genetic diversity and population structure of 40 waxy and 40 common inbred lines of maize in the Gangwon Agricultural Research and Extension Services collection at the Maize Research Institute. RESULTS: We detected polymorphisms in 86.33% of 278 Mutator (Mu) anchored loci, reflecting the activity of the Mu element and its contribution to genetic variation. Common maize showed a substantial amount of genetic diversity, which was greater than that observed in waxy maize. Principal-coordinate and neighbor-joining cluster analyzes consistently supported the presence of two genetically distinct groups. However, the distribution of genetic variation within the populations was much higher than the genetic differentiation among the populations. To explore the contribution of the Mu element to phenotypic variation, we analyzed the associations with ten important agronomical traits. On the basis of the combined results from two models (QGLM and Q + KLM), we found significant associations between seven Mu loci and four different traits. CONCLUSIONS: These results will assist waxy maize breeders in choosing parental lines and be useful for marker-assisted selection.

De novo assembly and characterization of transcriptome in the medicinal plant Euphorbia jolkini.

BACKGROUND: Euphorbia jolkini, a medicinal herb that grows on the warm beaches in Japan and South Korea, is known to be used for traditional medicines to treat a variety of ailments, including bruises, stiffness, indigestion, toothache, and diabetes. OBJECTIVE: It is to analyze the whole transcriptome and identify the genes related to the phenylpropanoid biosynthesis in the medicinally important herb E jolkini. METHODS: Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Public databases like TAIR (The Arabidopsis Information Resource), Swissprot and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used for annotations of unigenes obtained. RESULTS: The transcriptome of E. jolkini generated 139,215 assembled transcripts with an average length of 868 bp and an N50 value of 1460 bp that were further clustered using CD-HIT into 93,801 unigenes with an average length of 847 bp (N50-1410 bp). Sixty-three percent of the coding sequences (CDS) were annotated from the longest open reading frame (ORF). A remarkable percentage of unigenes were annotated against various databases. The differentially expressed gene analysis revealed that the expression of genes related to the terpenoid backbone biosynthesis pathway was higher in the flowers, whereas that of genes related to the phenylpropanoid biosynthesis pathway was both up- and downregulated in flowers and leaves. A search of against the transcription factor domain found 1023 transcription factors (TFs) that were from 54 TF families. CONCLUSION: Assembled sequences of the E. jolkini transcriptome are made available for the first time in this study E. jolkini and lay a foundation for the investigation of secondary metabolite biosynthesis.

The complete chloroplast genome of a rare species in Korea, Lilium Dauricum Ker Gawl.

Lilium dauricum Ker Gawler is a wild lily species that belongs to section Sinomartagon and is one of the ancestors of the Asiatic hybrid lilies. Unique traits such as disease resistance and early flowering make L. dauricum a desirable resource for interspecific hybridization. However, in Korea, the natural resources of L. dauricum are being exhausted by excessive exploitation and require urgent conservation. In this study, the complete chloroplast genome of L. dauricum was generated using Illumina paired-end sequencing technology, and its structure was compared with that of other Lilium species. The chloroplast genome was 152,063 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (26,492 bp) separated by a large (81,485 bp) and small (17,584 bp) single-copy (SC) region. The genome encodes 131 different genes, including 85 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. A phylogenetic analysis strongly supported the relationship of L. dauricum with other members of Sinomartagon and Martagon lilies.

The complete chloroplast genome of a Woodwardia japonica.

Woodwardia japonica is one of the diverse members of the fern group and medicinally important genus. In Korea, the natural resources of W. japonica are being exhausted by excessive exploitation and require urgent conservation. In this study, the complete chloroplast genome of W. japonica was generated, and its structure was compared with that of other members of same family. The chloroplast genome was 153224 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (24591 bp) separated by a large (82480 bp) and small (21562 bp) single-copy (SC) region. The genome encodes a total of 88 protein-coding genes, 35 tRNA genes, and eight rRNA genes. Additionally we identified 87 RNA editing sites in 52 genes; most of the substitution was U to C (50 sites), while C to U conversion occurred in 37 positions. The phylogenetic analysis strongly supported the relationship of W. japonica with W. unigemmata and. A. melanocaulon (Blechnoideae).

Comparative transcriptome analysis reveals higher expression of stress and defense responsive genes in dwarf soybeans obtained from the crossing of G. max and G. soja.

BACKGROUND: Plant height is an important component of plant architecture and significantly affects crop breeding practices and yield. Dwarfism in plants prevents lodging and therefore it's a desired trait in crops. OBJECTIVE: To find differentially expressed genes to classify and understand the regulation of genes related to plant growth in mutant dwarf soybeans, which appeared in the F5 generation. METHODS: We obtained a few segregated dwarf soybeans in the populations derived from the crossing of Glycine max var. Peking and Glycine soja var. IT182936 in an F5 RIL population. These dwarf soybeans may be useful genetic resources for plant breeders, geneticists and biologists. Using the Illumina high-throughput platform, transcriptomes were generated and compared among normal and dwarf soybeans in triplicate. CONCLUSION: We found complex relationship of the expressed genes to plant growth. There were highly significantly up-/downregulated genes according to the comparison of gene expression in normal and dwarf soybeans. The genes related to disease and stress responses were found to be upregulated in dwarf soybeans. Such over-expression of disease resistance and other immune response genes can be targeted to understand how the immune genes regulate the response of plant growth. In addition, photosynthesis-related genes showed very low expression in dwarf lines. The transcriptome expression and genes classified as related to plant growth may be useful resources to researchers studying plant growth.

Genetic diversity and population structure among accessions of Perilla frutescens (L.) Britton in East Asia using new developed microsatellite markers.

SSRs were successfully isolated from the Perilla crop in our current study, and used to analyze Perilla accessions from East Asia. Analyses of the clear genetic diversity and relationship for Perilla crop still remain insufficient. In this study, 40 new simple sequence repeat (SSR) primer sets were developed from RNA sequences using transcriptome analysis. These new SSR markers were applied to analyze the diversity, relationships, and population structure among 35 accessions of the two cultivated types of Perilla crop and their weedy types. A total of 220 alleles were identified at all loci, with an average of 5.5 alleles per locus and a range between 2 and 10 alleles per locus. The MAF (major allele frequency) per locus varied from 0.229 to 0.943, with an average of 0.466. The average polymorphic information content (PIC) value was 0.603, ranging from 0.102 to 0.837. The genetic diversity (GD) ranged from 0.108 to 0.854, with an average of 0.654. Based on population structure analysis, all accessions were divided into three groups: Group I, Group II and the admixed group. This study demonstrated the utility of new SSR analysis for the study of genetic diversity and population structure among 35 Perilla accessions. The GD of each locus for accessions of cultivated var. frutescens, weedy var. frutescens, cultivated var. crispa, and weedy var. crispa were 0.415, 0.606, 0.308, and 0.480, respectively. Both weedy accessions exhibited higher GD and PIC values than their cultivated types in East Asia. The new SSR primers of Perilla species reported in this study may provide potential genetic markers for population genetics to enhance our understanding of the genetic diversity, genetic relationship and population structure of the cultivated and weedy types of P. frutescens in East Asia. In addition, new Perilla SSR primers developed from RNA-seq can be used in the future for cultivar identification, conservation of Perilla germplasm resources, genome mapping and tagging of important genes/QTLs for Perilla breeding programs.