Draft Genome Sequence for the Symbiotic Pine Mushroom Tricholoma matsutake.

We report the high-quality genome sequence of Tricholoma matsutake strain 2001, which was isolated from a mushroom fruiting body in South Korea. The genome has 80 contigs, a size of 162.6 Mb, and an N50 value of 5,103,859 bp and will provide insight into the symbiotic association between T. matsutake and Pinus densiflora.

Broussonetia × hanjiana (Moraceae) microsatellite markers for species and ploidy determination developed using next-generation sequencing.

BACKGROUND: Broussonetia × hanjiana has been considered a hybrid owing to its morphology, which is intermediate between that of B. papyrifera (L.) L'Her. ex Vent. and B. kazinoki Siebold. A recent study demonstrated the hybrid origin of B. × hanjiana in Korea using molecular markers. In this study, we developed microsatellite markers for B. × hanjiana using next-generation sequencing and cross-species transferability analysis. METHODS AND RESULTS: A total of 432 primers were designed from 205,819 contigs. Among them, 24 microsatellite markers showing polymorphisms were used to evaluate the population genetic characteristics. The observed heterozygosity (HO) and expected heterozygosity (HE) were 0.835 and 0.628, respectively. The cross-species transferability of these markers was evaluated in two closely related species of Broussonetia; all 24 markers showed cross-species amplification. Using flow cytometry, diploid and triploid individuals were identified in B. × hanjiana. In particular, the BR137 marker showed evidence of two parent species (B. papyripera and B. kazinoki), with a hybrid pattern observed in B. × hanjiana, demonstrating its utility for species identification and ploidy assessment. CONCLUSIONS: The new B. × hanjiana microsatellite markers can be useful in genetic studies of closely related B. papyripera, B. kazinoki, and B. × hanjiana.

Development and characterization of novel microsatellite markers in Tilia amurensis Rupr. using next-generation sequencing.

BACKGROUND: Tilia amurensis (Malvaceae) is a deciduous broad-leaved tree distributed in Korea, China, and Japan. T. amurensis is used as a honey tree and also as a material for furniture, carving, and pulp. This study aimed to develop and characterize novel microsatellite markers using next-generation sequencing (NGS) of T. amurensis. METHODS AND RESULTS: NGS analysis using GS-FLX Titanium obtained 629,273 reads, of which 15,795 contigs were assembled with an average length of 830 bp. A total of 4774 microsatellite regions were detected in 3602 of 15,795 contigs. In total, 360 primer sets were designed based on the microsatellite regions. Among them, 15 primer sets were selected as reproducible polymorphic markers and were characterized for three populations of T. amurensis in Korea. The average number of alleles (NA) was 3.5 in Mt. Hambaek (HB), 3.7 in Mt. Odae (OD), and 3.8 in Mt. Sobaek (SB). The average observed heterozygosity (HO) and expected heterozygosity (HE) values were 0.497 and 0.370 in the HB population, 0.470 and 0.372 in the OD population, and 0.524 and 0.410 in the SB population, respectively. The average polymorphic information content (PIC) value of the 15 microsatellite markers was 0.686. CONCLUSIONS: The novel microsatellite markers will be useful for further studies on genetic diversity evaluation to conserve the genetic resources and natural populations of T. amurensis.

Technical note: Polyvinylpyrrolidone (PVP) and proteinase-K improve the efficiency of DNA extraction from Japanese larch wood and PCR success rate.

Illegal distribution of timber disrupts the timber market and depletes forest resources. DNA markers are used to verify the legal distribution of wood. However, it is difficult to obtain the quantity and quality of DNA suitable for genetic analysis because of the physicochemical properties of wood; therefore, an efficient wood DNA extraction method is required. In this study, to prepare an efficient DNA extraction method from Japanese larch (Larix kaempferi) wood, we investigated the ability of polyvinylpyrrolidone (PVP) and proteinase-K to improve DNA extraction efficiency and PCR success rate. It was found that the addition of PVP resulted in a significant increase in the DNA concentration of the treatment group compared to that of the control group, while the purity (A260/A280) showed no difference. Moreover, the addition of proteinase-K significantly increased both the DNA concentration and purity of the treatment group compared to those of the control group. Further analysis showed that the PCR success rate of psbC (approximately 350 bp) was higher than 90% in the control, PVP treatment, and proteinase-K treatment groups. However, in the PCR success rate of rbcL (approximately 1.3 kb) was higher in the proteinase-K and PVP treatment groups than in the control group. The addition of PVP and proteinase-K increased the success rate of PCR amplification for long regions by preventing DNA damage caused by phenolic compounds and proteins in the wood. The results of this study can thus develop DNA extraction methods to identify the species and origin of woods.

The complete chloroplast genome of Betula costata (Betulaceae).

In the present study, we analyzed the complete chloroplast genome sequence of Betula costata using the Ion Torrent platform. The chloroplast genome of B. costata was found to be 160,547 bp in length, with a large single-copy (LSC) region of 89,385 bp, a small single-copy (SSC) region of 19,038 bp, and a pair of inverted repeats (IRs) of 26,062 bp each. The overall GC content of the chloroplast genome was 36.1%. It contained 132 genes, including 87 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. The phylogenetic analysis indicated that B. costata is closely related to Betula nana and Betula pubescens.

The complete chloroplast genome of Korean Gastrodia elata Blume.

The completed chloroplast genome of Gastrodia elata Blume (G. elata) from Korea was determined in this study. The cpDNA is 35,230 bp in length and lacked the large and small single copy (LSC and SSC) regions, due to the lost inverted repeat (IR). The overall AT content is 73.30%, and the cpDNA contains 20 protein-coding genes, 5 tRNA genes, and 3 rRNA genes. Remarkably, the Korean G. elata cp genome was 74 bp smaller than that of the Chinese G. elata. It revealed substantial sequence variants 495 SNPs and 75 InDels between the two G. elata genomes.

Development of microsatellite markers in Betula costata (Betulaceae) by next-generation sequencing and cross-species transferability test.

This study was conducted to develop the first species-specific microsatellite markers in Betula costata. A total of 178 primers were designed from 95,755 contigs and screened in two B. costata populations sampled from Mt. Hwaaksan and Mt. Gyebangsan. A total of 16 polymorphic microsatellite loci were selected and used for population genetic characterization. The average values of observed heterozygosity (HO) and expected heterozygosity (HE) of the Mt. Hwaaksan population were 0.488 and 0.493, respectively. The average values of HO and HE in the Mt. Gyebangsan population were 0.492 and 0.481, respectively. The null allele frequency was less than 0.2 in all loci. No significant linkage disequilibrium was detected in all combinations of loci. In addition, 26 polymorphic markers were selected by cross-species transferability test to B. costata using the microsatellite markers developed in four other Betula species. The cross-species transferability of the microsatellite markers developed in B. costata was conducted in two other Betula species. The transferability was 75% in B. ermanii and 100% in B. davurica. Therefore, the microsatellite markers developed and characterized in this study were expected to be useful for further genetic studies in B. costata and related species in the genus Betula.

The complete chloroplast genome of Chamaecyparis obtusa (Cupressaceae).

The complete plastid genome of Chamaecyparis obtusa was sequenced and analyzed in this study. It was found to be 126,821 bp long. The guanine-cytosine content of the whole genome was 35.1%, and there were 83 unique protein-coding genes, 33 tRNAs, and 4 rRNAs. Furthermore, an overlap of 120 bp was found (including trnQ-UUG). The order and organization of these genes were consistent with those of other complete plastid genomes from the Cupressaceae. A phylogenetic tree was constructed according to the 83 protein-coding genes found, which demonstrated that C. obusa had sister relationships within the genus Chamaecyparis.

The complete chloroplast genome of Torreya nucifera (Taxaceae) and phylogenetic analysis.

Torreya nucifera (L.) Siebold & Zucc. (Taxaceae) is a tertiary relict tree species with a distribution that is limited to South Korea and Japan. In the present study, the complete chloroplast (cp) genome of T. nucifera was sequenced and analyzed. The genome was 136,985 bp in length and contained 118 genes, including 82 protein-coding genes, 33 tRNA genes, and 4 rRNA genes. Fifteen of the genes contained a single intron, whereas ycf3 contained two introns and there were no inverted repeat sequences in the genome. Phylogenetic analysis supported the monophyly of Torreya species within the Taxaceae and T. nucifera was closely related to its congener T. grandis.

Complete chloroplast genome of Acer tegmentosum and phylogenetic analysis.

The aim of the present study was to sequence and analyze the complete plastid genome (i.e. plastome) of Acer tegmentosum Maxim. The plastome of A. tegmentosum was 156,435 bp in length and included both large (86,265 bp) and small (18,102 bp) single-copy regions, which were separated by a pair of identical inverted repeats (26,034 bp). The plastome contained 77 unique protein-coding genes, 30 tRNA genes, and four rRNA genes. In addition, the gene order and organization of the A. tegmentosum plastome were consistent with those of plastomes from other members of the Sapindaceae, and the overall GC content of the plastome was 37.8%. A phylogenetic tree that was based on 76 protein-coding genes demonstrated a sister relationship within genus Acer.

Complete chloroplast genome of Sorbaria sorbifolia var. stellipila.

The complete plastid genome of Sorbaria sorbifolia var. stellipila was sequenced and analyzed in this study. It was found to be 160,820 bp and consisted of a large (88,869 bp) and small (20,853 bp) single-copy regions, separated by a pair of identical inverted repeats (25,549 bp). The GC content of the whole genome was 37.8%, and there were 84 unique protein-coding genes, 37 tRNAs, and 8 rRNAs. The gene order and organization were consistent with those of other complete plastid genomes from the Rosaceae. The phylogenetic tree construct was based on 76 protein-coding genes and demonstrated a sister relationship with the Rosaceae.

Complete chloroplast genome of Corylopsis spicata and phylogenetic analysis.

The complete plastid genome of Corylopsis spicata was sequenced and analyzed in this study. It was found to be 159,507 bp and consisted of a large (88,243 bp) and small (18,716 bp) single-copy regions, separated by a pair of identical inverted repeats (26,274 bp). The GC content of the whole genome was 36.9%, and there were 85 unique protein-coding genes, 37 tRNA, and eight rRNA. The gene order and organization were consistent with those of other complete plastid genomes from the Hamamelidaceae. A phylogenetic tree was constructed based on 76 protein-coding genes that demonstrated a sister relationship within the genus Corylopsis.

The complete chloroplast genome sequence of Actinidia Rufa (Actinidiaceae).

The complete chloroplast (cp) genome sequence of Actinidia rufa was determined by next-generation sequencing in this study. The whole cp genome was 156,543 bp in length, containing a large single-copy (LSC) of 88,435 bp and a small single-copy (SSC) region of 20,307 bp, which was separated by a pair of 23,900 bp inverted repeat (IR) regions. The genome contained 131 genes, including 84 protein-coding genes, 39 tRNA genes, 8 ribosomal RNA genes. Two events were found in the Actinidiaceae chloroplast genome. One was the deletion of the clpP gene and the other was the trnfM-CAU gene in the same direction in LSC region. The phylogenetic position of A. rufa was closely clustered with A. chinensis, A. deliciosa as sister species.

The complete chloroplast genome of Fraxinus chiisanensis (Oleaceae).

Fraxinus chiisanensis is endemic to Korea and restricted to local habitats with an extremely limited population size. The chloroplast genomic information can be used to formulate a comprehensive conservation strategy. In this study, the complete chloroplast (cp) genome sequence of F. chiisanensis was determined using next-generation sequencing. The entire cp genome was determined to be 155,571 bp in length. It contained large single-copy (LSC) and small single-copy (SSC) regions of 86,412 of 17,767 bp, respectively, which were separated by a pair of 25,696 bp inverted repeat (IR) regions. The genome contained 132 genes, including 87 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Among the 132 genes, seven coding genes, seven tRNA genes, and four rRNA genes occur in the two IR regions. The phylogenetic position of F. chiisanensis in Oleaceae was closely clustered with Olea, Chionanthus retusus, and Hesperelaea palmeri as sister species.

The complete chloroplast genome sequences of Larix kaempferi and Larix olgensis var. koreana (Pinaceae).

The complete chloroplast (cp) genome sequence of Larix kaempferi and L. olgensis var. koreana were determined by Ion torrent Platform sequencing in this study. The L. kaempferi cp genome was 122,158bp consists of two inverted repeat (IR) regions of 436bp each, a large single-copy (LSC) region of 65,394bp, and a small single-copy (SSC) region of 55,892bp. The chloroplast genome sequence of L. olgensis var. koreana was 122,573bp in length, consisting of two IRs (436bp), one LSC (65,597bp), and one SSC (56,104bp), and is longer than that of L. kaempferi. The genome contained 110 genes, including 71 protein-coding genes, 35 tRNA genes, and 4 ribosomal RNA genes. The 13 genes contain introns, including 12 genes with a single intron each and ycf3 gene with two introns. And, the rps12 gene is a trans-spliced gene. The phylogenetic analysis revealed that all sampled species in Pinaceae formed a monophyletic clade with high bootstrap value. The genus Larix is closely related to Pseudotsuga.

ALTERED MERISTEM PROGRAM1 has conflicting effects on the tolerance to heat shock and symptom development after Pseudomonas syringae infection.

An Arabidopsis thaliana ALTERED MERISTEM PROGRAM1 (AtAMP1), which encodes a putative glutamate carboxypeptidase, not only controls shoot apical meristem development, but also is involved in tolerance response to abiotic stresses. Here, we introduce a novel mutant; named amp1-32 that is a phenocopier to previously isolated different amp1 mutant alleles. Interestingly, tiny leaves were continuously developed at the bottom of pre-emerged leaves in the amp1-32. The amp1-32 mutant was less sensitive to heat shock treatment lasting for 3 h, whereas disease symptoms were severely developed in the mutant after Pseudomonas syringae infection. The mRNA levels of 171 genes were significantly altered in the mutant, as compared to wild-type plants. The transcription of genes involved in hormone signaling, post-embryonic development, and shoot development were up-regulated in the amp1-32 mutant, whereas expression of genes related to responsiveness to pathogens and (in)organic matters, were decreased in the mutant. Taken together, perturbation of CK- and ABA-related events by AMP1 mutation caused aberrant development phenotype and conflicting responses against abiotic and biotic stresses in Arabidopsis.

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer).

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.

Rapid Identification of Ginseng Cultivars (Panax ginseng Meyer) Using Novel SNP-Based Probes.

In order to develop a novel system for the discrimination of five ginseng cultivars (Panax ginseng Meyer), single nucleotide polymorphism (SNP) genotyping assays with real-time polymerase chain reaction were conducted. Nucleotide substitution in gDNA library clones of P. ginseng cv. Yunpoong was targeted for the SNP genotyping assay. From these SNP sites, a set of modified SNP specific fluorescence probes (PGP74, PGP110, and PGP130) and novel primer sets have been developed to distinguish among five ginseng cultivars. The combination of the SNP type of the five cultivars, Chungpoong, Yunpoong, Gopoong, Kumpoong, and Sunpoong, was identified as 'ATA', 'GCC', 'GTA', 'GCA', and 'ACC', respectively. This study represents the first report of the identification of ginseng cultivars by fluorescence probes. An SNP genotyping assay using fluorescence probes could prove useful for the identification of ginseng cultivars and ginseng seed management systems and guarantee the purity of ginseng seed.

Construction of genomic DNA library of Korean ginseng (Panax ginseng C. A. MEYER) and development of sequence-tagged sites.

This study describes an efficient approach for developing sequence tagged sites (STS) for Panax ginseng C.A. MEYER, and their applications for line discrimination. By using the methylation filtering (MF) technique, a genomic library was constructed, in which clone inserts were derived from the hypomethylated regions of ginseng genome. A methylation unfiltered genomic library was also constructed and the clone inserts were compared to those from the MF library in terms of sequence characteristics. Sequence analysis revealed that MF efficiently enriched the protein coding region of P. ginseng, for which the repetitive DNA appeared to be as little as 2.5 fold lower than clones in the unfiltered library, and also indicated that the P. ginseng genome may contain a large fraction of methylated repetitive DNA elements. A total of 99 and 100 highly stringent STS primer sets were designed from the filtered and unfiltered library, respectively. Amplification products were tested for latent polymorphism across six cultivars of P. ginseng and other 2 Panax species using six endonucleases recognizing four-bases. STS primer sets described here will be useful for marker-assisted selection, genome mapping and line discrimination of P. ginseng or its cultivars from other Panax species.

Molecular authentication of 21 Korean artemisia species (Compositae) by polymerase chain reaction-restriction fragment length polymorphism based on trnL-F region of chloroplast DNA.

The present study describes the molecular authentication of 21 Korean Artemisia species using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique based on the trnL-F sequences in chloroplast DNA. Five different banding patterns were generated from 21 Artemisia species using HinfI restriction enzyme. A. apiacea, A. keiskeana and A. sieversiana have specific banding patterns. The remaining 18 species had shared two banding patterns. Phylogenetic analysis based on trnL-F sequence variations showed results similar to PCR-RFLP banding patterns. It suggested that the trnL-F region does not have sufficient variations to identify the 21 Artemisia species. However, the specific banding patterns for A. apiacea, A. keiskeana and A. sieversiana can be utilized as a DNA marker for discriminating them from other Artemisia species. These markers will be also useful for developing A. apiacea, A. keiskeana and A. sieversiana into new medicine and food based on their efficacy.