Earticle

To develop a method for the differentiation of Pleurotus ostratus cultivars, the multiplex-simple sequence repeat (SSR) primer set based on the SSRs obtained from whole genomic DNA sequence analysis was designed with two polymerase chain reaction (PCR) primer sets. These SSR primer sets were employed to distinguish 10 cultivars and strains. Twenty polymorphic markers were selected based on the genotyping results. PCR with each primer produced 1–4 distinct bands ranging in size from 150 to 350 bp, which was within the expected range. However, since a sole SSR marker was unable to detect polymorphisms in every cultivar, multiplex PCRs with composite PCR primer sets were employed. The multiplex primer, “166+115,” completely discriminated 12 cultivars and strains with 40 loci, which were 12 more than the simple arithmetic addition of each locus of the primers 115 and 166. These results might be useful to provide an efficient method for the differentiation of P. ostreatus cultivars with separate PCRs for the quality control of spawn and protection of breeders’ rights.

Simple sequence repeats (SSRs) were isolated from the genome sequences of “Heuktari” and “Miso” those were sequenced by Pac-Bio sequencing technology. The total numbers of SSRs detected in the “Heuktari” and “Miso” genomes were 949 and 968, respectively. These SSR numbers the highest among five mushrooms including P. ostreatus var. florida (PC9 and PC15) and P. eryngii. These are 133% and 135% of PC9 and PC15, and 145% of P. eryngii, respectively. The similar pattern of SSR distributions were found in all mushrooms, which Tri-, hexa- and octanucleotide motifs accounted for the top three fractions of SSR distributions. These SSRs could be used as a primer for genotyping of segregation population and screening for the trait linked markers. In SSR distribution patterns, all mushrooms studied showed similar results. Tri-, hexa-, and octanucleotide motifs accounted for the top three fractions of all SSRs. Over Three repeats or more of 10-mers accounts for 9.8 in “Heuktari” and 6.5% in “Miso”. [Supported by a grant from the IPET (213003-04-3-SBY20), MIFAFF, Republic of Korea.]

Simple sequence repeats (SSRs) were isolated from the genome sequences of ‘Heuktari’ and ‘Miso’ those were sequenced by Pac-Bio sequencing technology. The total numbers of SSRs detected in the ‘Heuktari’ and ‘Miso’ genomes were 949 and 968, respectively. These SSR numbers the highest among five mushrooms including P. ostreatus var. florida (PC9 and PC15) and P. eryngii. These are 133%와 135% of PC9 and PC15 and 145% of P. eryngii, respectively. The similar pattern of SSR distributions were found in all mushrooms, which Tri-, hexa- and octanucleotide motifs accounted for the top three fractions of SSR distributions. These SSRs could be used as a primer for genotyping of segregation population and screening for the trait linked markers. In SSR distribution patterns, all mushrooms studied showed similar results. Tri-, hexa-, and octanucleotide motifs accounted for the top three fractions of all SSRs. Over Three repeats or more of 10-mers accounts for 9.8 in Heuktari” and 6.5% in “Miso.

The genotyping of segregation population, 110 monokaryotic progenies derived from F1 Pleurotus ostreatus hybrid between ‘Heuktari’ and ‘Miso’ (HMS012 × JHH021), was performed by PCR amplification with SSR primer sets. ‘Heuktari’ and ‘Miso’ showed distinct traits in fruiting body including cap color, yield and length. Those monokaryotic progenies were selected by median values in their fruiting body which made from selfing of 50 monokaryons of ‘Heuktari’ and ‘Miso’. A total of 546 SSR primers which were designed based on the genome sequences of two P. ostreatus monokaryons, JHH021 (from Heuktari) and HMS012(from Miso) were screened on HMS012 and JHH021. Of the 546 primers tested, more than 300 primers showed polymorphism on the parental strains. The hybrids made by crossing 110 monokarotic progeny from HMS012 x JHH021 strain with a monokarotic progeny from KNR2247 to access the quantitative trait. This study will be useful reference for QTL analysis of P. ostreatus.

Twenty Inter simple sequence repeat (ISSR) and 30 SSR primers were used to assess genetic diversity of 64 Agaricus strains including 45 A. bisporus strains and other 19 Agaricus spp. Of them, four ISSR primers, (GA)₈T, (AG)₈YC, (GA)₈C and (CTC)₆and seven SSR markers produced PCR polymorphic bands between the Agaricus species or within A. bisporus strains. PCR polymorphic bands were inputted for UPGMA cluster analysis. Forty five strains of A. bisporus are genetically clustered into 6 groups, showing coefficient similarity from 0.75 to 0.9 among them. The varieties, Saea, saedo, Saejeong and Saeyeon that have recently been developed in Korea were involved in the same group with closely genetic relationship of coefficient similarity over 0.96, whereas, other strains were genetically related to A. bisporus strains that were introduced from USA, Eroupe and Chinese.

Purpose : To evaluate the genetic diversity and relationship of Polygonati Odorati Rhizoma and Polygonati Rhizoma in Polygonatum species, we carried out the single sequence repeat (SSR) analysis using 8 species 24 germplasm lines collected from different habitat in Korea and China. Materials and Methods : Materials were collected randomly from different geographic regions in Korea and China, and were analyzed simple sequence repeat (SSR) of 120 primer sets which were designed based on next generation sequence (NGS) data. Total bands amplified by SSR primer sets were used to analyze genetic relationship using the unweight pair group method with arithmetic averages (UPGMA). Results : SSR results of 17 primer sets were revealed 68 total band and 57 polymorphic band with a 83.8% ratio of polymorphism. 24 Polygonatum germplasm lines were ranged from 0.62 to 0.95 and clustered distinct three groups depending on the species with 0.66 coefficient value. Conclusion : In this study, 17 SSR primer sets proper to evaluate genetic diverstiy of 8 Polygonatum spp. were selected. 5 species belonging the original plant of Polygonati Odorati Rhizoma, except P. inflatum Kom., were clustered into Group II. P. falcatum A.Gray were classified into Group I and P. sibiricum F.Delaroche were classified into Group III, respectively. These result showed that original plant of Polygonati Rhizoma were genetically different compared to Polygonati Odorati Rhizoma.

SSR markers를 이용하여 벼의 품종간 유전적 유연관계 분석과 품종식별 방법에 대한 연구를 수행하여 얻어진 결과를 요약하면 다음과 같다. SSR primer 50개와 벼 보급종 21품종을 PCR 반응시킨 결과 다형성을 뚜렷하게 나타내는 primer는 23개였으며, 각 marker에 의해 발생된 대립유전자의 수는 $2{\sim}9$까지 검출되었고, 평균값은 3.00개로 나타났다. 유전적 다형성 정도를 나타내어 주는 SSR marker의 PIC 값은 최소 0.091에서부터 최대 0.839까지 다양하게 분석되었다. SSR marker를 이용하여 분석된 벼 21품종에 대한 전체 유전적 유사도는 $0.59{\sim}0.92$의 범위에 속하였고 유사도 지수 0.65를 기준으로 할 때 4개의 그룹으로 구분되었다. SSR marker중에서 RM206, RM225, RM418, RM478은 marker genotype에 의해 21 품종에 대해 각각 고유한 밴드 특성을 나타내어 품종판별이 가능한 것으로 나타났다. 금후 이 연구결과는 벼 보급종의 품종식별을 위해 효과적으로 이용될 수 있는 것으로 나타났다.

The objective of this study was carried out to evaluate the suitability of simple sequence repeat (SSR) markers for genetic diversity assessment and identification of rice varieties. The 23 primers selected from 50 SSR primers showed polymorphisms in the 21 rice varieties. The 2 to 9 SSR alleles were detected for each locus with an average of 3.00 alleles per locus. The polymorphism information content (PIC) ranged form 0.091 to 0.839. Based on band patterns, UPGMA cluster analysis was conducted. These varieties were separate into 4 groups corresponding to rice ecotype and pedigree information and genetic distance of cluster ranging from 0.59 to 0.92. The 4 SSR primer sets (RM206, RM225, RM418, RM478) selected form 23 polymorphic primers were differentiated all the rice variety from each other by markers genotypes. These markers may be used wide range of practical application in variety identification of rice.

국내에서 유통되고 있는 토마토 품종의 판별 방법에 SSR marker의 이용 가능성에 대한 연구를 수행하여 얻어진 결과를 요약하면 다음과 같다. 토마토 28품종을 18개의 SSR marker를 이용하여 분석하였을 때 대립유전자의 수는 $2{\sim}9$개로 비교적 다양한 분포를 나타내었으며 전체 60개의 대립유전자가 분석되었다. PIC 값은 $0.476 {\sim}0.800$ 범위에 속하였으며 평균값은 0.607로 나타났다. SSR marker를 이용하여 작성된 토마토 28품종의 품종간 유전적 거리는 $0.35{\sim}0.97$의 범위로 나타났고, 유사도 지수 0.36을 기준으로 할 때 28개 품종은 체리형 토마토 그룹과 일반형 토마토 그룹으로 나눌 수 있었으며, 공시 품종 모두 SSR marker의 genotype에 의해 뚜렷이 구분되었다. 이 연구결과는 토마토의 품종식별에 기초 자료로 유용하게 이용될 수 있는 것으로 나타났다.

This study was carried out to evaluate the suitability of simple sequence repeat (SSR) markers for varietal identification and genetic diversity in 28 commercial tomato varieties. The relationship between marker genotypes and 28 varieties was analyzed. Of the 219 pairs of SSR primers screened against ten tomato varieties, 18 pairs were highly polymorphic with polymorphism information content (PIC) ranging from 0.467 to 0.800. Among the polymorphic loci, two to nine SSR alleles were detected for each locus with an average of 3.3 alleles per locus. Genetic distances were estimated according to Jaccard's methods based on the probability that the amplified fragment from one genotype would be present in another genotype. These varieties were categorized into cherry and classic fruit groups corresponding to varietal types and genetic distance of cluster ranging from 0.35 to 0.97. The phonogram discriminated all varieties by marker genotypes. The SSR markers proved to be useful variety identification and genetic resource analysis of tomato.

We have mined each of the five A. thaliana chromosomes for the presence of simple sequence repeats (SSRs) and developed custom perl scripts to examine their distribution and abundance in relation to genomic position, local G/C content and location within and around transcribed sequences. The distribution of repeats and G/C content with respect to genomic regions (exons, UTRs, introns, intergenic regions and proximity to expressed genes) are shown. SSRs show a non-random distribution across the genome and a strong association within and around transcribed sequences, while G/C density is associated specifically with the coding portions of transcribed sequences. SSR motif repeat number shows a high degree of variation for each SSR type and a high degree of motif sequence bias reflecting local genome sequence composition. PCR primers suitable for the amplification of identified SSRs have been designed where possible, and are available for further studies.

Molecular markers are useful tools for evaluating genetic diversity and determining cultivar identity. Present study was conducted to evaluate the genetic diversity within a diverse collection of rice accessions used for Korean breeding programs. Two hundred eighty-seven rice cultivars, composed of temperate japonica, tropical japonica, indica, and Tongil-type of Korean crossing parents were evaluated by means of 15 simple sequence repeat (SSR) markers. A total of 99 alleles were detected, and the number of alleles per marker ranged from 4 to 11, with an average of 6.6 per locus. Polymorphism information content (PIC) for each of the SSR markers ranged from 0.2924 to 0.8102 with an average of 0.5785. These results, with the result that use of only 15 SSR markers made all rice cultivars examined could be uniquely distinguished, imply the efficiency of SSR markers for analysis of genetic diversity in rice. Cluster analysis was performed on similar coefficient matrics calculated from SSR markers to generate a dendogram in which two major groups corresponding to japonica (Group I) and indica and Tongil type rice (group II) with additional subclasses within both major groups. The narrowness of the Korean breeding germplasm was revealed by the fact that most of the Korean-bred and Japan-bred temperate japonica cultivars were concentrated into only 2 of the sub-group I-1 (143 cultivars) and I-2 (58 cultivars) among six sub-groups in major group of japonica. This is because of the japonica accessions used in this study was a very closely related ones because of frequent sharing of the crossing parents with similar genetic background with synergy effect of the inherited genetic difference between indica and japonica. A rice breeding strategy with the use of molecular markers was discussed for overcoming of genetic vulnerability owing to this genetic narrowness.

Balloon flower (Platycodon grandiflorum A. DC) is widely distributed in South Korea and there are some local landraces that are cultivated as a vegetable crop or medicinal plant. Making use of the gene resources of wild-type and landraces is a way to increase the genetic diversity of the cultivars. However, few tools or information are available on an efficient identification system for maintaining and management of these landraces. To improve the genetic resources for balloon flower, 22 simple sequence repeat (SSR) markers, also known as microsatellite markers, were evaluated in a collection of 42 balloon flower landraces, 34 of which were from Korea and eight from China. All microsatellite markers produced the 107 alleles ranging from 2 to 10 with a mean of 4.864 alleles per each locus ($N_A$). The values of observed heterozygosity ($H_O$) and expected heterozygosity ($H_E$) ranged from 0.00 to 0.667 (mean of 0.285) and from 0.024 to 0.741 (mean of 0.416), respectively. An average value of polymorphic information contents (PIC) were 0.382 with a range of 0.023 to 0.703. Results of population structure and phylogenetic and principal coordinate analysis (PCoA) indicated that P. grandiflorum germplasm formed two largely distinct clusters according to their origins and the genetic differentiation. There was a high level of genotypic diversity at broad geographic regions between Korea and China, but the low genetic differentiation was found within the collections from Korea. The results of the genetic diversity will be useful for the selection of the parents for developing balloon flower breeding and the multi-locus SSR markers developed herein will be a valuable resource for germplasm assessments, evaluation of genetic diversity, and population genetic studies of balloon flower.

큰느타리 품종구분을 위한 마커의 개발을 위하여 큰느타리 전체 유전자 염기서열을 바탕으로 제작한 484개의 SSR마커를 사용하여 다형성 분석을 실시하였다. 그 결과 각 275개의 primer에서 다형성이 관찰되었다. 이 중 품종간에 다양한 패턴을 나타내는 5개의 마커를 최종 선발하였다. 이들 마커의 PIC 값은 0.6627에서 0.6848로 나타났고, 평균값은 0.6775였다. 이 결과를 밴드 이미지 인식 방법으로 dendrogram을 작성하였다. UPGMA 집괴분석 결과, 큰느타리 품종은 크게 Cluster 1과 Cluster 2로 구분되었다. SSR primer를 이용한 PCR 결과 나타나는 품종별 고유의 DNA 밴드를 품종특이적 마커로 개발하기 위하여, 선발된 마커중에서 SSR312과 SSR366, SSR178과 SSR 277 마커를 조합하여 초위성체 유래 다중 표지 세트를 개발하였다. Multiplex-SSR 마커의 사용을 통해 두번의 PCR 반응만으로 본 연구에서 사용된 12개의 큰느타리 품종을 구분할 수 있었다.

For development of a method for differentiation of Pleurotus eryngii cultivars, simple sequence repeats (SSR) from whole genomic DNA sequence analysis was used for genotyping and two multiplex-SSR primer sets were developed. These SSR primer sets were employed to distinguish 12 cultivars and strains. Five polymorphic markers were selected based on the genotyping results. PCR using each primer produced one to four distinct bands ranging in size from 200 to 300 bp. Polymorphism information content (PIC) values of the five markers were in the range of 0.6627 to 0.6848 with an average of 0.6775. Unweighted pairgroup method with arithmetic mean clustering analysis based on genetic distances using five SSR markers classified 12 cultivars into two clusters. Cluster I and II were comprised of four and eight cultivars, respectively. Two multiplex sets, Multi-1 (SSR312 and SSR366) and Multi-2 (SSR178 and SSR277) completely discriminated 12 cultivars and strains with 21 alleles and a PIC value of 0.9090. These results might be useful in providing an efficient method for the identification of P. eryngii cultivars with separate PCR reactions.

국내외에서 재배되고 있는 감귤속 식물 108 품종 및 유전자원과 SSR 마커를 활용하여 유전적 유사도 분석을 통한 품종식별력 검정 등에 대한 연구를 수행하였다. 감귤 8품종을 203개의 SSR 마커로 검정하여 반복 재현성이 높은 뿐만 아니라 다형성 정도가 높은 18개를 선정하였다. 이들 마커와 국내외에서 재배되고 있는 감귤 108품종을 검정하였을 때 마커당 평균 대립유전자수는 9.28개로 나타났고, 5-14개까지 다양한 분포를 나타내었다. PIC 값은 분자표지에 따라 0.417-0.791 범위에 속하였으며 평균값은 0.606으로 나타났다. 감귤류 108품종에 대하여 계통도를 작성하였을 때 감귤류 식물의 분류학적 특성 및 품종 육성의 계보도에 따라 13개의 그룹으로 크게 나누어졌다. 감귤류 식물 품종중 오렌지나 온주 밀감의 경우 대부분의 품종이 SSR 마커의 유전자형에 의해 구분이 되지 않은 것으로 나타났다. 본 연구에서 개발된 감귤속 식물의 품종별 SSR DNA 프로파일 데이터베이스는 감귤속 식물의 유전자원 특성평가와 육종가의 지식재산권 보호의 수단으로 유용하게 활용될 수 있을 것이다.

The present study investigated identification of cultivars through phylogenetic analysis of 108 Citrus varieties and related cultivars using simple sequence repeat (SSR) markers. Two hundred three SSR primer pairs were used to detect polymorphic markers among 8 Citrus cultivars consisting of 4 mandarins, 1 orange, 1 tangor, 1 tangelo, and 1 pumelo. Eighteen SSR primer pairs were reproducible and showed highly polymorphic alleles. These markers were applied to assess genetic variations of the 108 varieties. Each marker detected 5-14 alleles, with an average of 9.28. The polymorphism information content varied from 0.417 to 0.791 with an average of 0.706. Cluster analysis with SSR markers resulted in 13 major groups reflecting cultivar types and pedigree information. Twelve orange cultivars in the $I-1^{st}$ sub-cluster and 23 mandarin cultivars in the $II-1^{st}$ sub-cluster, respectively, were not discriminated using the SSR markers. This could be due to narrow genetic backgrounds originated through bud mutation or nucellars seedlings. The SSR profile database of Citrus cultivars will be useful as a tool for protection of plant breeders' intellectual property rights in addition to assessing genetic diversity in Citrus cultivars and germplasms.