Sequence Repeat Regions Research Articles

Forensic analysis and sequence variation of 133 STRs in the Hakka population.

Introduction: Short Tandem Repeats (STRs) are highly valuable genetic markers in forensic science. However, the conventional PCR-CE technique has limitations, and the emergence of massively parallel sequencing (MPS) technology presents new opportunities for STR analysis. Yet, there is limited research on Chinese population diversity using MPS. Methods: In this study, we obtained genotype data for 52 A-STRs and 81 Y-STRs from the Hakka population in Meizhou, Guangdong, China, using the Forensic Analysis System Multiplecues SetB Kit on the MGISEQ-2000 platform. Results: Our findings demonstrate that these 133 STRs are highly efficient for forensic applications within the Meizhou Hakka population. Statistical analysis revealed Hobs values ranging from 0.61306 to 0.91083 and Hexp values ranging from 0.59156 to 0.91497 for A-STRs based on length polymorphism. For sequence polymorphism, Hobs values ranged from 0.61306 to 0.94586, and Hexp values fluctuated between 0.59156 and 0.94487. The CPE values were 1-5.0779620E-21 and 1-3.257436E-24 for length and sequence polymorphism, respectively, while the CPD values were 1-1.727007E-59 and 1-5.517015E-66, respectively. Among the 80 Y-STR loci, the HD values for length and sequence polymorphism were 0.99764282 and 0.99894195, respectively. The HMP values stood at 0.00418102 and 0.00288427, respectively, and the DC values were 0.75502742 and 0.83363803, respectively. For the 52 A-STR loci, we identified 554 and 989 distinct alleles based on length and sequence polymorphisms, respectively. For the 81 Y-STR loci, 464 and 652 unique alleles were detected at the length and sequence level, respectively. Population genetic analysis revealed that the Meizhou Hakka population has a close kinship relationship with the Asian populations THI and KOR based on length polymorphism data of A-STRs. Conversely, based on length polymorphism data of Y-STRs, the Meizhou Hakka population has the closest kinship relationship with the Henan Han population. Discussion: Overall, the variation information of repeat region sequences significantly enhances the forensic identification efficacy of STR genetic markers, providing an essential database for forensic individual and paternity testing in this region. Additionally, the data generated by our study will serve as a vital resource for research into the genetic structure and historical origins of the Meizhou Hakka population.

The complete chloroplast genome of Hibiscus syriacus using long-read sequencing: Comparative analysis to examine the evolution of the tribe Hibisceae.

Hibiscus syriacus, a member of the tribe Hibisceae, is considered an important ornamental and medicinal plant in east Asian countries. Here, we sequenced and assembled the complete chloroplast genome of H. syriacus var. Baekdansim using the PacBio long-read sequencing platform. A quadripartite structure with 161,026 base pairs was obtained, consisting of a pair of inverted repeats (IRA and IRB) with 25,745 base pairs, separated by a large single-copy region of 89,705 base pairs and a short single-copy region of 19,831 base pairs. This chloroplast genome had 79 protein-coding genes, 30 transfer RNA genes, 4 ribosomal RNA genes, and 109 simple sequence repeat regions. Among them, ndhD and rpoC1, containing traces of RNA-editing events associated with adaptive evolution, were identified by analysis of putative RNA-editing sites. Codon usage analysis revealed a preference for A/U-terminated codons. Furthermore, the codon usage pattern had a clustering tendency similar to that of the phylogenetic analysis of the tribe Hibisceae. This study provides clues for understanding the relationships and refining the taxonomy of the tribe Hibisceae.

Expression characteristics of piRNAs in ovine luteal phase and follicular phase ovaries.

PIWI-interacting RNAs (piRNAs), as a novel class of small non-coding RNAs that have been shown to be indispensable in germline integrity and stem cell development. However, the expressed characteristics and regulatory roles of piRNAs during different reproductive phases of animals remain unknown. In this study, we investigated the piRNAs expression profiles in ovaries of sheep during the luteal phase (LP) and follicular phase (FP) using the Solexa sequencing technique. A total of 85,219 and 1,27,156 piRNAs tags were identified in ovine ovaries across the two phases. Most expressed piRNAs start with uracil. piRNAs with a length of 24 nt or 27–29 nts accounted for the largest proportion. The obvious ping-pong signature appeared in the FP ovary. The piRNA clusters in the sheep ovary were unevenly distributed on the chromosomes, with high density on Chr 3 and 1. For genome distribution, piRNAs in sheep ovary were mainly derived from intron, CDS, and repeat sequence regions. Compared to the LP ovary, a greater number of expressed piRNA clusters were detected in the FP ovary. Simultaneously, we identified 271 differentially expressed (DE) piRNAs between LP and FP ovaries, with 96 piRNAs upregulated and 175 piRNAs downregulated, respectively. Functional enrichment analysis (GO and KEGG) indicated that their target genes were enriched in reproduction-related pathways including oocyte meiosis, PI3K-Akt, Wnt, and TGF-β signaling pathways. Together, our results highlighted the sequence and expression characteristics of the piRNAs in the sheep ovary, which will help us understand the roles of piRNAs in the ovine estrus cycle.

Characterization of the complete chloroplast genome of Euphorbia pekinensis Rupr. (Euphorbiaceae)

Euphorbia pekinensis Rupr. 1859 is a medicinal herb endemic to China and distributed throughout the country, particularly across the northern part of the mainland. However, the systematic classification of Euphorbiaceae remains controversial. Therefore, studying the chloroplast genome of E. pekinensis is crucial for the resolution of this taxonomic dispute, clarification of the systematic status of Euphorbia, and establishment of an accurate classification system for Euphorbiaceae. In this study, we sequenced the complete chloroplast genome of E. pekinensis using Illumina sequencing technology and annotated it using GeSeq. The complete chloroplast genome was 162,002-bp-long with a guanine–cytosine (GC) content of 35.7%. It included one large single-copy (LSC), one small single-copy (SSC), and two inverted repeat sequence regions (IRa and IRb), which were 90,225 bp, 18,067 bp, and 26,855 bp in length, respectively, and are indicative of a typical tetrad structure. The genome encoded 129 functional genes, comprising 85 protein-coding genes, 36 tRNA genes, and eight rRNA genes. According to the maximum-likelihood phylogenetic tree that was constructed using 16 complete chloroplast genomes, E. pekinensis was found to be closely related to E. ebracteolata. Therefore, the complete chloroplast genome of E. pekinensis provides a better understanding of Euphorbia genetics.

Molecular diversity and genetic relationships between Carpinus betulus and C. orientalis with Inter Simple Sequence Repeat (ISSR) regions

A successful management and preservation of the natural populations depend on accurate assessment of genetic diversity. Knowing the genetic diversity within a population is important for choosing the conservation strategies for the species. The genus Carpinus belonging to Coryloideae, Betulaceae, has significant economic and ornamental importance. Determination of the taxa in the genus Carpinus in Iran is one of the most controversial issues among the researchers; for example, we can see this claim in the recent botanical literatures such as Sabeti and Browicz. However, two good species namely C. betulus L. and C. orientalis Mill. are the main species in Iran, adjacent regions and also in Europe. In general, taxonomic and biosystematics studies of the Carpinus are not known in Iran, moreover, in few cases, inter-specific hybrids and intermediate forms are recognised. A detailed molecular (ISSR) study of the Carpinus is done here with the following objectives: 1) to delimitate the species; 2) to carry out population genetic study and produce information on genetic structure, genetic variability within each population in Carpinus betulus and C. orientalis. In present study, 85 randomly collected plants from 17 geographical populations of two Carpinus species were considered. Our results indicated that ISSR markers can be used as a reliable and informative technique for evaluation of genetic diversity and relationships among Carpinus species.

ISSRseq: An extensible method for reduced representation sequencing

Abstract The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution and conservation. However, the wet‐laboratory expertise and bioinformatics training required to conduct genome‐scale variant discovery remain limiting factors for investigators with limited resources. Here we present ISSRseq, a PCR‐based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, used to construct sequencing libraries with a commercially available kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R. Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet‐laboratory skills and commonplace instrumentation, is flexible in terms of the number of single primers used, and can generate genomic‐scale variant discovery on par with existing RRS methods which require more complex wet‐laboratory procedures. ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non‐model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful to those lacking advanced expertise in wet‐laboratory methods or bioinformatics.

Invasive Species as Hosts of Zoonotic Infections: The Case of American Mink (Neovison vison) and Leishmania infantum

Leishmania infantum produces an endemic disease in the Mediterranean Basin that affects humans and domestic and wild mammals, which can act as reservoir or minor host. In this study, we analyzed the presence of the parasite in wild American minks, an invasive species in Spain. We screened for L. infantum DNA by PCR using five primer pairs: Two targeting kinetoplast DNA (kDNA), and the rest targeting the ITS1 region, the small subunit of ribosomal RNA (SSU) and a repetitive sequence (Repeat region). The detection limit was determined for each method using a strain of L. infantum and a bone marrow sample from an infected dog. PCR approaches employing the Repeat region and kDNA (RV1/RV2 primers) showed higher sensitivity than the other PCR methods when control samples were employed. However, only PCR of the Repeat region and nested PCR of SSU (LnSSU) detected the parasite in the samples, while the other three were unable to do so. The majority of the analyzed animals (90.1%) tested positive. American mink may act as an incidental host of the disease for other mammals and should be further investigated, not only for their negative impact on the local fauna, but also as carriers of zoonotic diseases.

The complete chloroplast genome sequence of rare and endangered Camellia lungzhouensis (Theaceae) of South China

The complete chloroplast genome sequence of rare and endangered Camellia lungzhouensis (Theaceae) was reported in this study. The cp genome was 157,110 bp in length with a GC content of 62.70%, including LSC (86,653 bp), SSC (18,277 bp), and two reverse repeat sequence regions (26,090 bp). The genome encoded 136 functional genes, including 84 protein-coding genes, 44 tRNA genes, and eight rRNA genes. The phylogenetic analysis showed that C. lungzhouensis was closely related to C. petelotii and C. nitidissima.

Sequence-based U.S. population data for 7 X-STR loci

The National Institute of Standards and Technology (NIST) U.S. population sample set of unrelated individuals was used to determine allele and haplotype frequencies for seven X-chromosome short tandem repeat (STR) loci in four linkage groups. DXS7132, DXS7423, DXS8378, DXS10074, DXS10103, DXS10135, and HPRTB were sequenced using the ForenSeq DNA Signature Prep Kit on a MiSeq FGx instrument from Verogen. Capillary electrophoresis data produced using the Qiagen Investigator Argus X-12 was compared to ForenSeq length-based alleles and found to be 99 % concordant. For three loci (DXS10103, DXS10074, and HPRTB) the length-based allele call is affected by the extent of flanking region included in the reported sequence. Six of the seven loci gained alleles by sequencing compared to length-based determinations. The increase in alleles are found in both the repeat and flanking region sequences. All sequences for which frequencies are reported in this dataset were cataloged as GenBank records in the STRSeq NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject/380127). Frequency information for both the loci and linkage groups is reported, along with results of statistical tests including gene diversity, polymorphism information content, power of discrimination, and linkage disequilibrium. All supplemental files are available at the NIST Public Data Repository – U.S. population data for Human Identification Markers (https://doi.org/10.18434/t4/1500024).

Methods of analysis of chloroplast genomes of C3, Kranz type C4 and Single Cell C4 photosynthetic members of Chenopodiaceae

BackgroundChloroplast genome information is critical to understanding forms of photosynthesis in the plant kingdom. During the evolutionary process, plants have developed different photosynthetic strategies that are accompanied by complementary biochemical and anatomical features. Members of family Chenopodiaceae have species with C3 photosynthesis, and variations of C4 photosynthesis in which photorespiration is reduced by concentrating CO2 around Rubisco through dual coordinated functioning of dimorphic chloroplasts. Among dicots, the family has the largest number of C4 species, and greatest structural and biochemical diversity in forms of C4 including the canonical dual-cell Kranz anatomy, and the recently identified single cell C4 with the presence of dimorphic chloroplasts separated by a vacuole. This is the first comparative analysis of chloroplast genomes in species representative of photosynthetic types in the family.ResultsMethodology with high throughput sequencing complemented with Sanger sequencing of selected loci provided high quality and complete chloroplast genomes of seven species in the family and one species in the closely related Amaranthaceae family, representing C3, Kranz type C4 and single cell C4 (SSC4) photosynthesis six of the eight chloroplast genomes are new, while two are improved versions of previously published genomes. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality and repeat region sequences. Comparison of the chloroplast genomes with previously sequenced plastid genomes revealed similar genome organization, gene order and content with a few revisions. High-quality complete chloroplast genome sequences resulted in correcting the orientation the LSC region of the published Bienertia sinuspersici chloroplast genome, identification of stop codons in the rpl23 gene in B. sinuspersici and B. cycloptera, and identifying an instance of IR expansion in the Haloxylon ammodendron inverted repeat sequence. The rare observation of a mitochondria-to-chloroplast inter-organellar gene transfer event was identified in family Chenopodiaceae.ConclusionsThis study reports complete chloroplast genomes from seven Chenopodiaceae and one Amaranthaceae species. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality, and repeat region sequences. Therefore, the use of high throughput and Sanger sequencing, in a hybrid method, reaffirms to be rapid, efficient, and reliable for chloroplast genome sequencing.

Cross-pollination effects on morphological, molecular, and biochemical diversity of a selected cinnamon (Cinnamomum zeylanicum Blume) seedling population

ABSTRACT Cinnamomum zeylanicum Blume, also known as true cinnamon, has gained more attention recently because of its proven medicinal properties. Having uniform quality raw materials is the key to the sustainability of pharmaceutical and other industries. Nevertheless, the majority of the cinnamon plantations in Sri Lanka and elsewhere have originated from highly cross-pollinated seeds. However, no reported studies exist on the variability of individuals caused by the natural cross-pollination of cinnamon. Therefore, our work focused on morphological, genetic, and biochemical characterization of a population of individuals that resulted from a single cross-pollination event of a known C. zeylanicum mother plant. The mother plant, the possible pollen donor and the selected progeny were assessed with several age- and environment-independent morphological traits and several Inter Simple Sequence Repeat (ISSR) regions. Progeny had significantly different leaf and apex shapes and ISSR regions were about 80% polymorphic compared to the parents. The morphological and genetic diversity of progeny was also represented in the cinnamaldehyde, cinnamyl alcohol and cinnamyl acetate content at the seedling stage.

The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

The complete chloroplast genome sequence of Ulmus lanceaefolia (Ulmaceae)

This study was conducted to sequence, assemble, annotate, and characterize the complete chloroplast (cp) genome sequence of Ulmus lanceaefolia. The result showed that the cp genome was 158,652 bp in length with 35.63% GC content, including a large single-copy (LSC) region of 87,119 bp, a small single-copy (SSC) region of 18,697 bp and an inverted repeat sequence (IRa/IRb) region of 26,418 bp, which was a typical tetrad structure. We had identified a total of 132 genes, including 87 protein-coding genes, eight rRNA genes, and 37 tRNA genes. The phylogenetic trees were constructed based on the cp genome sequences of U. lanceaefolia and 12 plants in the NCBI database, and the phylogenetic positions of U. lanceaefolia in the Ulmaceae were identified.

Population genetic study and barcoding in Iran saffron (Crocus sativus L.)

Saffron (Crocus sativus L.) is an economical important plant species with High demand throughout the world. Population genetic study can produce data on genetic structure and genetic diversity in saffron populations to be used in breeding programs. We collected 50 samples in saffron farms of 8 different populations in South and West Khorasan states which is considered as the main geographical region of saffron production in Iran. Two types of molecular markers viz. Inter simple sequence repeat (ISSR) and Chloroplast DNA (trnH-psbA) region were used in this study. ISSR markers produced 18 reproducible bands which were used in further analyses. Analysis of molecular variance (AMOVA) produced significant genetic difference among the studied saffron populations (PhiPT = 0.72, P = 0.001). It revealed that 72% of total genetic difference is due to among population difference, while 28% is due to within population genetic variability. These indicate that saffron populations are genetically differentiated and we can use such genetic difference in future breeding programs of this valuable crop plant species. Principal coordinate analysis (PCoA) of ISSR data separated the studied saffron populations in different groups. Chloroplast DNA (trnH-psbA) region sequences determined in saffron genotypes of Iran as well as the other countries, could differentiate these two types saffron cultivars from each other. Nei's gene diversity index (GST) analysis produced significant molecular difference between the two groups of saffron. The method of templeton (TCS) revealed that 80 nucleotide mutations differentiate these saffron genotypes. However, within Iranian saffron genotypes as well as foreign genotypes, only 1 or 2 mutations occur. This result indicates that we can produce barcode for Iran saffron which in high demand in the world market. We therefore, provided barcode of Iran saffron for the first time.

Emergence of a Novel Ehrlichia minasensis Strain, Harboring the Major Immunogenic Glycoprotein trp36 with Unique Tandem Repeat and C-Terminal Region Sequences, in Haemaphysalis hystricis Ticks Removed from Free-Ranging Sheep in Hainan Province, China.

Ehrlichia minasensis, a recently described Ehrlichia species that is the most closely related to, but clearly distinct from, Ehrlichia canis, has been circulating in not only bovines, cervids, and dogs but also several tick species from Canada, Brazil, France, Pakistan, Ethiopia, and Israel. However, there are no reports of E. minasensis in China. The purpose of this study was to explore whether E. minasensis is present naturally in ticks in China. Through PCR targeting of the genus-conserved dsb gene, E. minasensis DNA was detected in Haemaphysalis hystricis ticks removed from free-ranging sheep in Hainan Province, South China in 2017. The partial sequence of the dsb, 16S rRNA, and groEL genes demonstrated that the Hainan strain shared 99% identity with the dsb gene of E. minasensis strain UFMG-EV (GenBank: JX629808), with the 16S rRNA of E. minasensis isolate E-2650 (MH500005) and with the groEL gene of E. minasensis strain UFMG-EV (JX629806), respectively. Moreover, sequence analysis of the major immunogenic tandem repeat protein (trp36) revealed that the Hainan strain harbored a unique tandem repeat sequence (APEAAPVSAPEAAPVSAPVS) and a C-terminal region that differed from those of other known E. minasensis strains. Additionally, phylogenetic analysis based on the entire amino acid sequence of trp36 revealed that the Hainan strain was closely related to a recently described E. minasensis strain from Brazil, of which the sister clade contained different strains of E. canis. The discovery of this novel Hainan strain in H. hystricis ticks represents the first known natural presence of E. minasensis in South China, highlighting the need for its constant surveillance.

Isolation and characterization of 15 SSR loci for the endangered European tetraploid species Gladiolus palustris (Iridaceae).

Premise Gladiolus palustris (Iridaceae) is an endangered European perennial tetraploid herb with special conservation interest in the European Union. Microsatellite markers can serve as effective tools for the conservation genetics of this species.Methods and ResultsWe utilized a 454 pyrosequencing approach to identify simple sequence repeat (SSR) regions in a microsatellite‐enriched library. Of all SSR regions, 46 were screened for specific PCR amplification, and 15 were found to be applicable in the target species. We found 1.62–3.08 alleles per population (effective alleles: 1.58–2.08) that indicated moderate to high genetic diversity values (0.28–0.44) in three pilot populations. Cross‐species amplification was less effective in G. imbricatus and G. tenuis.ConclusionsThe primers reported here can be used for the population genetic characterization of G. palustris. They will help us to better understand the conservation genetics of this highly endangered species.

Paternity testing using massively parallel sequencing and the PowerSeq™ AUTO/Y system for short tandem repeat sequencing.

Massively parallel sequencing (MPS) is gaining attention as a new technology for routine forensic casework, including paternity testing. Recently released MPS multiplex panels provide many more loci compared to CE methods, plus provide sequence-based alleles that together improve the statistical power of the genetic testing. Here, an MPS system (PowerSeq™ AUTO/Y) was applied for STR sequencing in the study of first-degree STR sequence allele inheritance from families in Southern Brazil. In 29 trios (mother-child-father) analyzed, the paternity index values generally increased when data from sequence-based analysis were used in comparison to length-based data. Further, allele inconsistencies (e.g., single repeat mutation events) between child and parents could be resolved with MPS by assessing the core repeat and flanking region sequences. Lastly, the sequence information allowed for identification of isoalleles (alleles of the same size, but different sequence) to determine specific paternal and maternal inheritances. The results from this study showed advantages of implementing sequence-based analysis, MPS, in paternity testing with improved statistical calculations and a greater resolution for the trios/families tested.

EST-SSR marker characterization based on RNA-sequencing of Lolium multiflorum and cross transferability to related species

Simple sequence repeat (SSR) markers are considered very useful molecular markers for genetic and molecular breeding, specifically for non-model species. To develop a set of whole genome-wide distributed SSR marker for annual ryegrass (Lolium multiflorum L.), we obtained the first transcriptome dataset of annual ryegrass subjected to drought stress and identified a total of 11,254 EST-SSR. Fifty EST-SSR markers were selected to test the amplification and polymorphism rates in 23 accessions of Lolium and Festuca diploid species. The results showed that all of the 50 functionally relevant markers were polymorphic, with allele number and polymorphism content averaging 7.4 and 0.839, respectively, and fully transferable across 23 accessions of Lolium and Festuca species. Sequencing of PCR products showed that the nucleotide sequences of Lolium and Festuca species had a high level of conservation. Different types of tri-repeats as abundant SSR motifs were observed in repeat sequence regions. A number of EST-SSRs were mapped to genome sequences of Lolium perenne and chromosomes of Brachypodium distachyon. Among them, 973 and 1375 Lolium SSR markers were located on the five chromosomes of B. distachyon and L. perenne scaffolds, respectively. In addition, we confirmed that EST-SSRs as well as SRAP markers could clearly identify varieties of Lolium and Festuca species, and the two sets of molecular markers had a significant correlation (r = 0.74). This set of EST-SSR markers are valuable genetic tools for genetic diversity, evolutionary, and association mapping studies in Lolium, Festuca, and many other grass species.

Complete Genome Sequence of the Circular Extrachromosomal Element of Naegleria gruberi Strain EGB Ribosomal DNA.

ABSTRACTThe circular extrachromosomal element of Naegleria gruberi strain EGB was linearized, molecularly cloned, and fully sequenced. The sequence comprises 14,007 bp and encodes the organism’s rRNA genes, two potential open reading frames, and numerous repeated sequence regions.

차세대염기서열분석법을 이용한 잔대의 SSR 마커 개발

Background: Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth with radical leaves during the first year and shows reproductive growth with cauline leaves and bolting during the second year. In addition, the shape of the plant varies within the same species. For this reason, there are limitations to classifying the species by visual examination. However, there is not sufficient genetic information or molecular tools to analyze the genetic diversity of the plant.Methods and Results: Approximately 34.59 Gbp of raw data containing 342,487,502 reads was obtained from next generation sequencing (NGS) and these reads were assembled into 357,211 scaffolds. A total of 84,106 simple sequence repeat (SSR) regions were identified and 14,133 primer sets were designed. From the designed primer sets, 95 were randomly selected and were applied to the genomic DNA which was extracted from five plants and pooled. Thirty-nine primer sets showing more than two bands were finally selected as SSR markers, and were used for the genetic relationship analysis.Conclusions: The 39 novel SSR markers developed in this study could be used for the genetic diversity analysis, variety identification, new variety development and molecular breeding of A. triphylla.