도라지에서 Expansin 유전자 familiy의 동정 및 분자생물학적 분석

박상익 충북대학교 2019 국내석사

Platycodon grandiflorum is called balloonflower because its flower bud looks like a balloon. In Korea, China and Japan, roots of balloonflower have been used for a long time as an edible or medicinal material. Balloonflower is an allogamous plant and has purple and white flowers. Expansin is a family gene consisting of four groups: EXPA, EXPB, EXLA, and EXLB. It is a non-enzymatic protein that loosens the cell walls of a plant under acidic condition. It is related to the growth of plant length, the maturation of fruit, and the development of root hair. We performed the genome sequencing of balloonflower using the Illumina HiSeq platform. A total of 4,911 scaffolds were assembled using SOAPdenovo2. We performed the Basic Local Alignment Search Tool (BLAST) with previously reported expansin genes and created a phylogenetic tree. Twenty PlgEXPA, 8 PlgEXPB, 5 PlgEXLA, and 2 PlgEXLB were grouped with reported expansin genes. The exon-intron structure, number of amino acids, molecular mass, and isoelectric points of expansin protein were predicted using GSDS 2.0, MEME suite 5.0.2, and ProtParam, respectively, and the presence and location of domains constituting expansin were predicted using ScanProsite and SignalP 4.1 programs. RNA-Seq analysis of 8 tissues of balloonflower was performed to test the expression level of expansin genes obtained from the above process. The expression of each gene was calculated by performing a differentially expressed gene (DEG) analysis using the RNA-Seq results. By comparing the results with the previously reported genes, we could confirm the characteristics of the expansin genes of balloonflower. From the DEG analysis, the expression patterns of some genes were tissue-specific, and the results of DEG analysis were confirmed through reverse transcription quantitative PCR (RT-qPCR) analysis. Full length cDNA clones of 2 EXLB genes were obtained and transformed to Arabidopsis. These transformed plants would be used for the further studies on the function of balloonflower EXLB genes.

참당귀 분자마커 개발과 추대 및 개화 특성 분석

길진수 충북대학교 2020 국내박사

Quantitative Trait Loci (QTL) Analysis and Candidate Gene Selection in Watermelon (Citrullus lanatus) Fruit Traits Using Resequencing

Kim, Moonkyo 충북대학교 2023 국내석사

수박(Citrullus lanatus)은 전 세계적으로 상업적으로 생산되는 박과 식물로 매우 중요한 작물이다. 유전체 크기는 대략 425 Mb이며 lycopene, beta carotene, citrulline과 같은 식물 화학물질을 제공함으로써 인간 건강에도 상당히 유익하다. 아프리카가 원산지인 것으로 알려져 있으며, 경제적이나 원예학적으로 중요하기 때문에 육종에 대한 관심이 높아지고 있다. 본 연구에서는 국내산 수박 유전자원인 CBARES1과 CBARES2에서 유래한 F2 집단의 유전자형과 표현형 데이터를 이용하여 quantitative trait loci (QTL) 분석을 수행하였다. 국내 수박 품종인 CBARES1은 흑색 과피, 황색 과육, 타원형의 과형을 가지며, CBARES2는 황색 과피, 적색 과육, 원형의 과형을 가지는 독특한 수박이다. CBARES1과 CBARES2 품종에 대해 long-read sequencing과 short-read sequencing을 실시하여 고품질의 유전체를 생산하였고 assembly 과정을 거쳐 각 품종 별 유전체를 완성하였다. 완성된 CBARES1과 CBARES2의 유전체를 비교하여 최종적으로 1,539개의 single nucleotide polymorphism (SNP)와 80개의 insertion or deletion (InDel) 마커를 개발하였으며, 이 중 SNP 마커는 resequencing을 이용하여 유전자형을 분석하였고 InDel 마커는 전기영동을 이용하여 유전자형을 분석하여 데이터를 수집하였다. 총 1,619개의 분자마커를 사용하여 길이 3036.90 cM 길이의 연관지도를 작성하였다. 유전자형 데이터를 이용하여 작성된 연관지도는 F2 집단의 표현형 데이터를 추가하여 과일과 관련된 QTL 분석을 실시하였다. QTL 분석 방법은 ICIM (inclusive composite interval mapping)을 이용하였으며 분석 결과 15개의 유효한 QTL 값을 찾았다. QTL 결과를 근거로 과일 형질과 관련된 유전자 302개를 선발하였으며, 이 유전자들의 coding sequence (CDS) 서열이 P세대인 CBARES1과 CBARES2의 유전체에서 변화를 보이는 유전자 33개를 선발하였다. 이 33개의 유전자 중 염기서열의 변화가 아미노산까지 변화시키는 유전자 22개를 선발하였으며 gene ontology (GO) 분석을 통해 과실 형질과 관련되어 있을 것으로 예상되는 유전자 Cla97C02G038270, Cla97C01G008760를 선발하였다. 또한 후보유전자의 F2 집단에서의 유전체형을 비교하여 형질과 관련 있을 것으로 예상되는 Cla97C04G070840를 선발하였다. 유전자 Cla97C04G070840는 아미노산 수송체 관련 유전자로 예상되는데 이 유전자의 염기서열의 SNP 변이에 따라 과피의 색과 과육의 색이 변하는 것이 확인되었다. 이 연구는 한국의 수박 품종의 유전체를 완성시키는 것을 시작으로 대량의 분자마커 개발, 고밀도 연관 지도 작성, QTL 분석을 진행하여 형질과 관련된 유전자를 발견했습니다. 이러한 연구 결과는 국산 수박을 이용하여 고품질 및 기능성 수박 품종 개발로 이어질 것으로 기대한다. Watermelon (Citrullus lanatus) is a very important gourd crop grown commercially worldwide. It has a genome size of approximately 425 Mb and is of great benefit to human health by providing phytochemicals such as lycopene, beta carotene, and citrulline. It is known to be native to Africa, and interest in breeding is increasing because it is economically and horticulturally important. In this study, Quantitative trait loci (QTL) analysis was performed using the genotypic and phenotypic data of the F2 population derived from Korean watermelon genetic resources, CBARES1 and CBARES2. The CBARES1 (black rind, yellow flesh, oval shape) and CBARES2 (yellow rind, red flesh, round shape) are Korean watermelon cultivars with unique colors and fruit shapes. Long-read sequencing and short-read sequencing were performed on the CBARES1 and CBARES2 cultivars to produce high-quality genomes, and the genomes for each cultivar were completed through the assembly process. By comparing the completed CBARES1 and CBARES2 genomes, 1,539 single nucleotide polymorphism (SNP) and 80 insertion or deletion (InDel) markers were finally developed. Among them, SNP markers were genotyped using resequencing, and InDel markers were genotyped using electrophoresis to collect data. An association map with a length of 3036.90 cM was prepared using a total of 1,619 molecular markers. The constructed linkage map was used for fruit-related QTL analysis using the phenotypic data of the F2 population. The QTL analysis method used ICIM (Inclusive Composite Interval Mapping), and as a result of the analysis, 15 valid QTL values were found. Based on the QTL results, 302 genes related to fruit traits were selected, and 33 genes whose coding sequence (CDS) showed changes in the genomes of P generation CBARES1 and CBARES2 were selected. Among these 33 genes, 22 genes whose nucleotide sequence changes even change amino acids were selected, and genes Cla97C02G038270 and Cla97C01G008760, which are expected to be related to fruit traits, were selected through gene ontology (GO) analysis. In addition, Cla97C04G070840, which is expected to be related to the trait, was selected by comparing the genotypes of the candidate genes in the F2 population. The gene Cla97C04G070840 is expected to be an amino acid transporter-related gene, and it was confirmed that the color of the skin and flesh of the fruit changes according to the SNP mutation of the base sequence of this gene. Our research started with completing the genome of Korean watermelon cultivars and then proceeded with the development of large-scale molecular markers, the creation of high-density association maps, and QTL analysis to discover genes related to traits. These research results are expected to lead to the development of high-quality and functional watermelon cultivars using Korean watermelons.

잔대의 작물학적 특성 조사 및 NGS를 이용한 SSR마커 개발

박기찬 충북대학교 2017 국내석사

본 연구는 농촌진흥청 국립원예특작과학원 인삼특작부에서 수집된 국내 잔대 유전자원의 작물학적 특성을 조사하고, 잔대의 분자 육종을 위한 근간을 마련하기 위한 SSR 마커 개발을 위해 수행되었다. 본 연구에서는 전국에서 수집한 약 1,000개 개체를 대상으로, 생육 조사 기본 자료에 기초하여 생육 조사를 수행한 후, 최종적으로 생육이 우수한 잔대 유전자원으로써 2년생 35개체를 선발하였다. 또한, 초장, 잎의 크기, 경수 등의 항목에서 상대적으로 높은 수치를 보였으며, 잎의 거치가 적은 개체, 개화시기가 상대적으로 늦었던 개체, 개체에 털이 없는 개체를 중심으로 ES-003, ES-008, ES-014, ES-018, ES-023, ES-025, ES-026, ES-035 8개의 개체를 3년생 계통 선발을 위한 개체로 선발하였다. 또한, NGS를 이용한 잔대의 유전체 정보기반 SSR마커 개발을 통해 수집한 유전자원의 유전적 다양성을 분석하고, 분자 육종을 위한 기틀을 마련하고자 하였다. 잔대의 유전체 정보 획득을 위하여 우리나라 대표적 잔대 재배지인 평창에서 수집한 생육 우수 개체 AP001번의 genomic DNA를 추출하여 NGS를 성공적으로 수행하였다. Illumina 2500 기계에 의해 생산되어진 염기의 개수는 총 455,402,955 bp이였으며, 이를 바탕으로 잔대의 유전체 크기를 추정하기 위한 k-mer 분석을 수행한 결과, 잔대는 최소 약 5.6 Gbp 이상의 거대한 유전체로 추측되었다. 또한, SSR구간을 탐색하기 위해 de novo assembly하여, 총 3,316,165 bp (0.73%)이 탐색되었으며, 총 9개의 SSR motif의 약 83,000 개가 탐색되었고, 총 14,133개의 SSR의 프라이머가 디자인 되었다. 잔대 개체의 적용을 위하여 tri-nucleotide, tetra-nucleotide, penta-nucleotide의 3가지 motif를 가지는 SSR을 중심으로 무작위로 총 95개의 프라이머를 선발하고 제작하였으며, 이를 잔대 AP001개체에 적용한 결과 모두 밴드가 증폭되었음을 확인하였다. 이를 바탕으로 수집한 개체간의 다형성을 나타내는 프라이머를 선발하기 위해, 평창, 보은, 나주, 임실, 화순 지역에서 수집한 총 5개체의 gDNA를 pooling하여 PCR을 수행한 후, 3 - 4개 밴드 사이즈를 보이는 40개의 프라이머를 SSR 마커 후보로 선발하였다. 이를 다시, 7개 지역에서 수집한 총 23개체에 각각 적용하고, 다형성을 분석하였다. 그 결과, 개발한 총 40개 마커의 PIC value는 0.64였다. 또한, 수집한 국내 잔대 유전자원의 다양성을 분석한 결과, 국내 수집되어진 유전자원은 크게 2가지 그룹으로 나뉘어졌다. 그룹 1에는 임실, 곡성, 화순에서 수집한 개체가 속하였으며, 그룹 2에는 진안, 평창, 보은에서 수집한 개체들 속하였으며, 나주는 두 그룹 모두에 존재하였다. 위 결과로 미루어보아, 국내의 잔대 유전자원은 임실과 진안 지역을 기점으로 하여 크게 두 가지로 분류됨을 알 수 있었다.

차세대 염기서열 분석법을 이용한 더덕의 SSR마커 개발

김세림 충북대학교 2017 국내석사

Codonopsis lanceolata is an herbaceous perennial plant. It is predominantly cultivated in East Asia, and is used as a medicinal plant as well as a vegetable. However, there is a lack of research about genetic studies of C. lanceolata. Therefore, we sequenced genomic DNA using next generation sequencing (NGS) and searched the simple sequence repeats (SSRs) for marker development in C. lanceolata. A total of 250,455 SSRs were identified and dinucleotides and trinucleotides accounted for the majority of all the SSRs. A total of 26,334 primer sets were designed from dinucleotide to octanucleotide motifs. We investigated 2,626 SSRs (trinucleotide to pentanucleotide motifs) and found 573 SSRs showed in silico polymorphism. One-hundred seventy-seven SSR primer sets were selected to confirm that the 573 SSRs showed polymorphism in silico. As a result, 160 primer sets amplified expected amplicons and all of them showed polymorphism. From the results of genotyping using 39 SSR markers, the number of alleles ranged from 2 to 13, and the mean value of PIC was 0.54. In addition, 33 SSR markers were applied to 26 C. lanceolata collections (95 individual plants), and genetic diversity and population structure analysis were performed. Genetic relationship analysis showed that C. lanceolata collected from South Korea were not grouped by collection sites and showed genetic diversity. The results of population structure analysis showed that the members of most of the C. lanceolata collections were not grouped into one population and were considered an admixture. Consequently, we developed SSR markers from C. lanceolata that were polymorphic enough to analyze genetic diversity even though the markers did not distinguish the C. lanceolata by collection sites.

더덕(Codonopsis lanceolata) 소기관 게놈의 유전적 다양성 분석 및 유전자원 식별을 위한 분자마커의 개발

조남수 충북대학교 2021 국내박사

Codonopsis lanceolata is one of the few valuable plants among the 42 species of genus Codonopsis that is used as a medicinal herb for traditional medicine practice and as an ingredient in traditional foods such as Deodeok gui (grilled Deodeok) and Jangajji (pickled vegetables) in Republic of Korea. The dried root of C. lanceolata is known as Yangyu (羊乳) or Yangyugeun (羊乳根), and its representative ingredient is lancemaside, one of the triterpenoid saponins. In particular, it is called 'deodeok' in Republic of Korea and is widely used in various fields due to its excellent pharmacological effects such as expectorant, antipyretic, and drainage, as well as unique flavor and taste, but research on its development as a genetic resource is very insufficient. In addition, the herbal medicine name of Adenophora triphylla var. japonica Hara, which is known to have the effect of protecting the liver and inhibiting damage, is Sasam (沙蔘), but the dried root of C. lanceolata was incorrectly labeled as Sasam and marketed, causing problems of mixing and misuse in the use of herbal medicines. Therefore, the aim of this study is to identify the origin of wild and distributed C. lanceolata through genetic analysis, to establish a molecular biology database and to promote development and utilization as a useful genetic resource by using molecular markers to verify incorrect product labeling of oriental medicine resources. Genomic DNA information of 43 C. lanceolata collected nationwide was collected using next generation sequencing (NGS), and 43 chloroplast genomes and 37 mitochondrial genomes were completed based on the obtained libraries. We performed a genetic diversity analysis by applying 10 chloroplast-based insertion-deletion (InDel) markers to 93 C. lanceolata wild genetic resources collected nationwide and 18 herbal medicine resources collected from 4 herbal medicine markets (54 individual plants; 16 C. lanceolata [48 individual plants] and 2 outgroups, A. triphylla [6 individual plants]). As a result of generating the unweighted pair group method with arithmetic mean (UPGMA)-based phylogenetic tree, wild collections were not classified by region, nor did they form a specific community, and thus it was judged that the genetic diversity was very rich. In addition, it has been found that some C. lanceolata products are being sold as Sasam, which is an incorrect product label, rather than Yangyu or Yangyugeun in resources collected from the herbal medicine market. Nucleotide polymorphisms were detected in the complete organelle genomes (43 chloroplast and 37 mitochondrial genomes) in this study. In chloroplast genomes, 190 single nucleotide polymorphism (SNP) loci and 43 InDel loci were found, and 140 SNP and 13 InDel loci were found in mitochondria. Based on the double chloroplast genome search results, 37 SNP-based and 7 InDel-based kompetitive allele specific PCR (KASP) markers were developed. Genetic diversity analysis was performed using 44 KASP markers for wild, herbal medicine, distribution, and foreign resources. Analysis was performed on 144 collections including wild, herbal medicine, distribution, and foreign resources, and when only wild genetic resources were analyzed, three clusters were identified as shown in the results; it was shown that Gyeongsangnam-do, Jeollabuk-do, and Jeollanam-do resources were concentrated in a specific cluster with 66.7%, 71.4%, and 75.0%, respectively. In addition, distribution resources were included in only two out of three clusters, and it was shown that foreign resources had a deeper relationship in a specific cluster. Analysis of the genetic diversity confirmed the close relationship, such as the distribution state and the degree of regional collections. Research in a category similar to this study should be continuously expanded because it can prevent misuse of herbal medicines and increase the value and trust of natural medicines through precise classification of herbal medicines using molecular biological approaches. In addition, the construction of chloroplast and mitochondrial genomes of C. lanceolata, which are highly valuable as genetic resources, and the study of phylogenetic relationships through biological approaches using them will further increase the value of these resources in C. lanceolata breeding. In particular, the KASP markers developed in this study will be helpful in molecular breeding studies for the development of new varieties of C. lanceolata such as genome-wide association study (GWAS), marker-assisted selection (MAS), quantitative trait locus (QTL) analysis, DNA barcoding, and genome mapping. In the meantime, it will establish a basis for molecular breeding of C. lanceolata, which has been insufficiently researched for the development of new varieties, and will greatly contribute to molecular breeding and industrial growth of more domestic medicinal crops.

도라지에서 사포닌 생합성에 관여하는 CYP 유전자군 분석

박소현 충북대학교 2018 국내석사

본 연구에서는 도라지의 주요 성분인 사포닌의 생합성에 관여하는 효소 중의 하나인 Cytochrome P450(CYP)에 대하여 분석하였다. RNA-seq analysis와 de novo assembly를 통해 도라지에서 165,238 unigene을 확보하였으며, BLAST search를 이용하여 CYP로 추정되는 유전자 191 개를 확보하였다. 이들 유전자는 NCBI에서 확보된 reference 아미노산 서열과의 계통학적 분석을 통해 CYP716, CYP72, CYP708, CYP88, CYP93 family 등으로 나뉘어지는 것을 확인하였다. 그 중 사포닌 생합성에 연관되는 것으로 추정되는 family인 CYP716에서 8 개의 유전자와 CYP93에서 2 개의 유전자를 확보하였다. 확보된 유전자 중의 일부를 yeast 발현 vector인 pAG423GAL에 cloning 하였다. 도라지는 대부분 oleanane계 saponin을 생성하므로, oleanae계 전구체 물질이 되는 beta-amyrin을 yeast 내에서 생성하기 위하여 oxidosqualene cyclase인 PtbAS를 사용하였다. Co-expression을 유도하기 위하여, Saccharomyces cerevisiae cell line인 WAT21에 pYES2.1/PtbAS를 형질전환을 하였다. 이후, synthetic complete-uracil(SC-U) 배지로 형질전환이 된 clone들을 선별한 뒤에 CYP가 들어간 vector를 이용하여 형질전환 하였다. 형질 전환체를 synthetic complete-uracil-histidine(SC-U-H)를 이용하여 선별하였으며, contig104069에 대하여 5 clone, contig109688에 대하여 2 clone, contig489에 대하여 1 clone을 확보하였다. 형질 전환된 yeast 내에서 효소활성을 조사하기 전에 RNA의 발현으로 확인하기 위하여 RT-PCR을 진행하였다. 전기영동으로 분석한 결과, PtbAS와 CYP가 함께 형질 전환된 세포에서 PtbAS와 CYP가 발현되는 것을 확인하였다. Yeast 세포에 형질 전환된 유전자의 효소활성을 조사하기 위해 gas chromatography-mass spectrometry(GC-MS)를 이용하여 분석하였다. PtbAS만 형질 전환된 개체를 control로 하여 PtbAS와 plg_contig104069이 함께 형질 전환된 개체를 비교하였다. 그 결과, control에서 발생하지 않은 peak들이 CYP가 형질 전환된 개체에서 관찰할 수 있었다. 그러나, 확인 가능한 peak들은 monolinoleoylglycerol, octasiloxane, 6H-dibenzo pyran-1,8-diol로 판별되었다. 그 이외의 새로운 peak들은 물질을 동정하기에는 너무 작아서 형질 전환된 유전자의 분명한 활성을 확인할 수는 없었다. 형질 전환 체에서 RNA상으로는 발현을 확인하였으나 효소 활성에 대해서는 조건을 변경하여 활성 유도를 해야 할 필요성이 있다. 이를 토대로 기능까지 밝혀진다면 도라지 사포닌과 같은 유용 물질에 대한 증가에 많은 도움이 될 것이다. In this study, the cytochrome P450(CYP) genes putatively involved in saponin biosynthesis were studied from Platycodon grandiflorum. I obtained 165,238 unigenes using RNA-seq analysis and de novo assembly and found 191 CYP unigenes by BLAST search using the CYP gene sequences obtained from the NCBI database. CYP716, CYP72, CYP708, CYP88 and CYP93 families were identified using phylogenic analysis of the CYP genes and the reference genes obtained from NCBI. Among them, CYP716 (eight unigenes) and CYP93 (two unigenes) families were thought to be most closely related through saponin biosynthesis by reference study. I cloned the genes of the two families in pAG423GAL yeast expression vector. For co-expression, two vectors containing PtbAS, an OSC reference gene, and CYP, respectively, were transformed into yeast mutant WAT21. I got four clones of plg_contig104069, two clones of plg_contig109688 and one clone of plg_contig489 through yeast transformation. The RNA expression of PtbAS and CYP was confirmed by RT-PCR analysis. The transformed genes in yeast cells were analyzed for enzyme activity using gas chromatography-mass spectrometry (GC-MS) analysis. The extract of the yeast cells transformed with PtbAS and contig104069 was compared with that of the control transformed only with PtbAS. As a result, several of the peaks that did not form under the control were observed from the CYP transgenic cells. Two peaks were identified as octasiloxane and 6H-dibenzo pyran-1,8-diol. But, the other novel peaks were too small for the substance to be identified. Therefore I could not confirm the activity of the transformed CYP. If the function of the CYP genes are revealed, it would be very helpful to improve the amount of functional compounds such as saponin of P. grandiflorum.

유전체 수준에서 Dyella thiooxydans ATSB10T의 식물생장촉진 특징 확인

황보경 충북대학교 2017 국내석사

Dyella thiooxydans ATSB10T is a novel species that was isolated from the rhizosphere soil of sunflower (Helianthus annuus L.) fields in Republic of Korea. Genetic properties of strain ATSB10T were investigated using next-generation sequencing (NGS) technology. The genome of D. thiooxydans ATSB10T had 4,227,172 base pairs of circular chromosomes with 67.98% GC content, including 6 rRNA and 49 tRNA sequences. According to comparative genome analysis of the genus Dyella, the pan-genome contains 10,738 genes and the core-genome contains 1,341 genes. Strain ATSB10T has 243 singletons from the total of 2,199 coding DNA sequences (CDSs); only 50 CDSs are function predicted. A Phylogenetic tree was constructed based on OrthoANI values. Strain ATSB10T is close to D. ginsengisoli LA-4, with a value of 93.2. In this study, investigation of the plant growth-promoting (PGP) character of ATSB10T was focused on root elongation factors such as indole-3 acetic acid (IAA) and biosynthesis of cytokinins and volatile organic compounds (VOCs). Observation of seed bacterization tests with Sorghum bicolor showed that ATSB10T induced root elongation. In conclusion, the D. thiooxydans ATSB10T genome was completed and the bacterium was suggested as a biofertilizer through genes related to PGP factors such as IAA and biosynthesis of cytokinins and VOCs.

인삼 (Panax ginseng C.A. Meyer)로부터 Malonyl Ginsenoside의 분리 및 정량분석

신우철 충북대학교 2020 국내석사

The root of Panax ginseng C.A. Meyer were extracted with 70% aqueous EtOH and the concentrates were partitioned into MeOH and H2O fractions using Diaion HP-20. The repeated SiO2 or ODS column, and medium pressure liquid chromatography (MPLC) for the MeOH fraction led to isolation of four malonyl ginsenosides. The chemical structures of these compounds were determined as malonyl ginsenoside Rd, malonyl ginsenoside Rc, malonyl ginsenoside Rb2, and malonyl ginsenoside Rb1 based on spectroscopic analyses including NMR and HR-TOF/MS. The total ginsenoside content was highest in ginseng fine root and five-year-old main root showed higher ginsenoside contents than that of the six-year-old. Additionally, the malonyl ginsenoside Rd exhibited protective effect against ethanol-induced hepatotoxicity in HepG2 cell line.

닥나무속 식물의 유전적 다양성 분석을 위한 분자마커의 개발

이은지 충북대학교 2023 국내석사

There are three species of Broussonetia B. kazinoki, B. monoica, and B. papyrifera which are native to Korea. Plants belonging to the Broussonetia genus are used as ingredients for hanji and are effective in whitening and anti-inflammatory, so they are used in various products. However, it is difficult to classify the plants of the genus Broussonetia by morphological identification, and breeding studies on B. kazinoki, a hybrid species, are incomplete. Therefore, in this study, 27 plants of the genus Broussonetia that are native or cultivated in various parts of the country, were used to develop InDel markers and SSR markers. The genetic diversity of plants of the genus Broussonetia was analyzed using these markers. Eight plants of the genus Broussonetia were analyzed and assembled using next-generation sequencing. For InDel markers, 8 candidate markers were selected using the chloroplast genome, and 5 markers were developed through polymorphism verification. As a result of genotyping of InDel markers, plants of the genus Broussonetia formed a total of 5 groups. This result shows that the maternal lineage of plants of the genus Broussonetia in Korea is more complex than known in previous studies. For SSR markers, 18 candidate markers were selected using the nuclear genome, and 12 markers were developed through polymorphism verification. As a result of genotyping of SSR markers, plants of the genus Broussonetia were found to form four groups. Some individuals showed the same genotype without differences. Individuals showing the same genotype were cultivated as B. kazinoki in different farms. Some individuals in Cheongju Munui Hanji Village also had the same genotype, indicating that these individuals were bred for B. kazinoki cultivation. Taken together, the results of the analysis using the InDel marker and the SSR marker showed that plants of the genus Broussonetia in Korea had more diverse genotypes than expected based on previously research. The markers developed in this study are expected to help in species classification, hybrid identification, and genetic studies of the plants of the genus Broussonetia, and to help in breeding of B. kazinoki.