본 연구에서는 토마토 MAB에 활용하고자 토마토 7 품종의 genome-wide SNPs 데이터베이스를 구축하고, MAB를 위한 분자마커 선발 프로그램을 개발하였다. 토마토 전사체 데이터를 NCBI-SRA에서 다운로드 하여 in silico 분석으로 SNP를 추출하였다. 전사체 데이터에서 추출된 SNP를 재료로 7 품종의 토마토 계통을 이용해 총 21개 교배조합별 SNP 분자마커를 선발하였고, primer가 이용 가능한 마커를 이용하여 데이터베이스를 구축하였다. 마커를 선발하기에 앞서 염색체의 분획으로 두 가지 방법을 사용하였는데, 물리적 거리에 따른 분획과 유전거리에 따른 분획 방법이다. 물리적 거리를 이용한 분획은 각 염색체를 동일한 크기의 5개의 구획으로 나누고, 한 구획 당 교배조합별 차이를 보이는 3개의 SNP를 선발하였다. 교배조합이 바뀔 때마다 이용 가능한 SNP가 자동으로 primer 정보와 함께 제공되도록 하였다. 유전거리를 반영한 분획 방법은 각 염색체의 유전적 거리를 측정하여 물리적 거리에 차등을 두어 염색체 구획을 설정하였다. 즉 재조합이 자주 일어나는 염색체 양끝 말단 부분은 구획을 조밀하게 나누어 MAB 마커 또한 많이 할당하여 자세히 조사하도록 구성하였다. 유전거리에 따른 마커 선발에는 1,924개의 tomato- EXPEN 2000 map 분자마커와 SNP 마커를 이용하였다. 교배조합별로 이용할 수 있는 마커를 12개 염색체 상에 그래픽적으로 제공함으로써 사용자가 쉽게 이해하고 이용할 수 있는 MAB 위한 마커 선발 프로그램을 개발하였다. 이러한 토마토 MAB용 분자마커를 제공하는 프로그램은 실제적인 여교잡 선발 육종에 적용하여 분자마커의 활용을 높이고, 육종효율을 증진시킬 것이다.

본 연구는 감 수집종의 분류 및 품종 육종을 위하여 EST-SSR 마커를 개발해 유전적 유연관계를 분석하고, 형태적 유연관계를 비교 분석하여 DNA 마커의 효율성을 극대화한 연구 결과이다. 경북농업기술원 상주감시험장에서 수집한 42품종을 대상으로 6가지의 양적형질(과실크기, 과고, 과경, 과경굵기, 과경길이, 종자크기)과 19가지의 질적형질(횡단면, 종단면, 골의 정도, 얕은 동심원 균열, 옆모양, 정부열과, 세로홈, 꽃받침 끝 주름, 배꼽 홈길이, 꽃받침 쪽의 홈, 꽃받침 크기)을 사용하여 형태적 유연관계를 분석하였다. 유전적 유연관계를 분석하기 위해 수집한 감에서 cDNA library를 만들어 sequence를 분석한 후, PCR을 통해 얻은 polymorphism이 인정되는 25개의 primer set에서 16개의 EST-SSR primer set를 선발하였다. 수집한 감 42품종의 형태적 유연관계와 개발한 14개의 EST-SSR 마커를 이용하여 유전적인 유연관계를 분석한 결과 형태적 유연관계에서는 여러 그룹이 형성되었지만 coefficient가 0.02 이하로 형성되어 형태적 특성을 사용해 분류하기는 어려웠다. 유전적 유연관계는 coefficient 0.77에서 3개 그룹으로 분류되어, 상주수수감과 상주수꽃감, 밀양반시와 밀양고동시, 영동반시와 영동수시는 각각 같은 그룹으로 분류되었다. 형태적 분석과 유전적 분석의 상관관계를 조사한 결과 형태적 분석의 유사도 거리와 유전적 분석의 유사도 거리 간의 값이 -0.03으로 유의성이 매우 낮게 나왔다. 본 실험에서 얻어진 분자마커는 (EST-SSR 마커) 국내 감육종 효율 증진뿐만 아니라 우수형질을 도입하는데 유용하게 이용할 수 있을 것으로 기대된다.

The main objectives of IRRI’s variety development should meet the needs of customers/farmers from diverse rice sectors in each target region. The dynamic market change asks rapid variety development with highly valued QTLs/genes. Molecular breeding implemented through the efficient crossing, high throughput genotyping and rapid generation advancement will provide packages to breeders to develop new varieties quickly and more economically. The more efficient and cost-effective marker-assisted backcrossing service will provide the more opportunity for the success in molecular breeding platform. To make MABC system more successful, the development of molecular marker system for the high-throughput SNP genotyping is must. Currently Genotyping Service Lab (GSL) of IRRI provides high-throughput SNP genotyping service using BeadXpress and Fluidigm system. Meanwhile, the linked SNP markers for the specific traits are being developed. For abiotic stress tolerances, the markers for submergence, drought, heat, anaerobic germination, salinity, and phosphorus deficiency for Fluidigm system are being developed and tested in variety diversity panel and segregating populations. In MABC, due to the high number of crossings, the labor- and space-saving crossing system is being developed. As a result of an integrated MABC platform will speed up the development of pre-breeding line which are containing single or multiple QTLs/genes.

Rice genetic resources are composed of various species and ecotypes, and each accession reveals different genetic and phenotypic characters. For the managenment of diverse rice genetic resources, seed integrity is important factor in that the individuals of one accession in self-pollinating crop might be homogeneous. To elevate the management efficiency of rice germplasm contrary to the phenotypic distinction, we focused on applicable microsatellite markers because this markers are widly used for genetic evaluation in diverse genetic resources with a character of high reproducibility and polymorphism. In this regard, we selected microsatellite markers based on genotypes; diversity set including 150 accessions using 249 SSR markers. As SSR loci with high PIC(polymorphism information content) values usually revealed multi bands in one accession, proper genotyping were difficult in these loci. Therefor, we checked the band clarity in addition to PIC values and chose 12 and 6 SSR markers finally. All accessions of rice diversity set were distinguished with the first marker set comprising 12 SSR markers, and only 3 combinations of tested accessions(0.03%, 3/11,175) showed same genotype with second marker set comprising 6 SSR markers. The tested 142 Korean bred varieties revealed 0.19%(19/10,011 combinations) and 0.69%(69/10,011 combinations) genotypic identity using first and second marker set, respectively. These newly selected markers might be useful for the analysis of genetic homogeneity and relationship in rice genetic resources.

60년대와 70년대의 녹색혁명은 전통적인 육종의 성과이지만, 최근의 육종기술은 유용유전자의 유전자형을 활용한 분자표지 마커를 활용하고 있다. 목적 형질을 갖고 있는 계통을 선발하는 MAS(Marker assisted selection) 마커는 많은 육종가들에 의해 활용되고 있지만, 유전자의 SNP(Single nucleotide polymorphism)을 활용한 MAB(Marker assisted backcross) 마커는 거의 활용되고 있지 않다. SNP 마커는 단 하나의 염기서열의 차이를 구별할 수 있어, 유전적으로 매우 가까운 계통들도 구분할 수 있으며, 자동화 분석이 가능하다는 점에서 활용도가 높다. 솔젠트(주)에서는 내수 및 종자수출에서 차지하는 산업적 비중이 가장 큰 채소작물 중 고추 병저항성 관련 유전자를 활용한 multiplex 고추병 진단 제품을 개발했다. 이 제품에는 오이모자이크바이러스(CMV), 담배 etch 바이러스(TEV), 토마토반점위조바이러스(TSWV), 토바모바이러스(TMV), 세균성점무늬병(BS), Chilli venial mottle virus(ChiVMV), 고추역병에 관련된 저항성 유전자의 SNP을 활용해 개발했으며, 본 제품을 활용해 고추 육종가들이 쉽고 간편하게 육종 소재에 적용해 신품종 육종에 도움을 줄 것으로 기대한다.

Backcrossing is a plant breeding method most commonly used to incorporate one or a few genes into an adapted or elite variety. To facilitate MAB (marker-assisted backcrossing) in a practice breeding program, we developed a SNP database and a program for providing selected markers for background selection from genome-wide SNPs of seven tomato accessions downloaded from NCBI-SRA. We identified 425,935 SNPs among 21 parental combinations with data from seven transcriptomes and developed a SNP database. To select the optimized number of markers for background selection, we divided 12 chromosomes according to physical length and genetic length. Initially, each chromosome was equally divided into five blocks according to physical length, and three SNPs were positioned per block. Additionally, we applied the genetic distance calculated from the recombination rate because the frequency of recombination can vary greatly among chromosomal regions. When considering genetic distance, each chromosome was divided into fifteen blocks unequally and one marker composed of EXPEN-2000 was positioned per block. The program for background selection was designed to be simple and easy to use, and it is available at http://tgsol.seeders.co.kr/ index.php/tg/mab. When the user selects the parental combination, the program provides selected markers with primer information. The value of this program for tomato breeding will further increase if more accession numbers are added to the database.

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present study, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. The Dx5’s own promoter was used for protein expression. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 270 hygromycin-resistant transformants, we obtained 27 transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 gene into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Protein expression of the Dx5 was analyzed with Western blot using polyclonal antibody recognising x-type of glutenin subunits in T1 seeds. It was suggested that the protein-processing system was conserved between rice and wheat. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation.

For developing molecular markers linked to white rust resistance in chrysanthemum, RAPD and AFLP were carried out in ‘Puma White’ x ‘Dancer’ mapping population through Bulked Segregant Analysis (BSA) methods. 10 resistant and 10 susceptible individuals were selected and bulked. And then, these bulks were screened using 280 RAPD primers (10 mer) with two parents. As a result of BSA-RAPD, 25 Dancer/R-bulk specific bands in 21 primers and 22 Puma White/S-bulk specific bands in 18 primers were selected. These resistant or susceptible specific bands were screened in 10 resistant and 10 susceptible individuals. Except OPI-13520, all bands were confirmed as false positive. OPI-13520 band presumed as closely linked marker to white rust disease resistance was tested in whole population. Among 187 progenies, just six off-springs did not correspond with phenotypic data. Based on expected phenotypic segregation ratios in the pseudo F1 progenies, it was assumed that a duplex type of white rust resistance in ‘Dancer’ (RRrrrr) were in combination with a duplex type of OPI-13520 marker. As a result of x2-test of independence between resistance gene and OPI-13520 marker, x2 score is 76.08 and probability is 2.13x10-16. And resistance gene and OPI-13520 marker were assumed to be linked in coupling phase. The value of recombination fraction obtained by successive trials and second derivative of log likelihood was 0.03832±0.0271.

전통육종과 marker assisted selection(MAS)를 함께 활용하는 방법이 최근 국내 상업육종에서 활발히 사용되고 있다. 식물 유전학의 빠른 발달과 차세대 유전체 염기서열 분석(NGS) 기술이 발전함에 따라, 대량 분자표지를 저렴한 가격에 확보할 수 있는 시대가 되었기 때문이다. 본 연구에서는 고추의 전사체 정보를 이용하여 SNP를 분석하고 Fluidign probe를 자동으로 디자인할 수 있는 파이프라인을 작성하였다. 또한 SNP를 calling하기 위해 총 6개의 accession(5개의 C. annuum과 1개의 C. chinense)의 전사체 염기서열을 Illumina 플랫폼으로 분석하였고, reference 염기서열은 고추 EST DB를 사용하였다. 분석한 SNP의 유전적 위치를 알기 위하여, UC-DAVIS의 고밀도 고추 유전자 지도와 연계하였다. Fluidigm probe를 design할 수 있는 조건으로 SNP를 filtering한 결과, short read의 depth가 10 이상을 기준으로 총 567개의 SNP를 분석하였고, 총 412개의 probe를 디자인하였다. 제작한 412개의 porbe와 논문에 공개된 SSRs, COSII 분자표지들, 2개의 고추 내병성 형질을 이용하여, 현재 내병성 고추 품종을 육성하기 위한 marker assistant breeding(MAB)을 진행 중에 있다.

The transfer of a biotic resistance gene from indica rice cultivars into japonica cultivars by conventional breeding methods often difficult due to high sterility of the progenies, poor plant type, and linkage drag. Molecular markers provide opportunities to map resistance genes and accelerate the application of marker-assisted backcross(MAB) breeding through the precise transfer of target genomic regions into the recurrent parent. The basis of MAB breeding is to transfer a specific gene/allele of the donor parent into the recurrent parent genome while selecting against donor introgressions across the rest of the genome. The effectiveness of MAB breeding depends on the availability of closely linked DNA markers for the target locus, the size of the population, the number of backcrosses and the position and number of markers for background selection. We have successfully developed Bph18 version of the commercially cultivated japonica elite cultivar by using MAB and incorporating the resistance gene Bph18 that conferred enhanced resistance to BPH. MAB breeding provides a new opportunity for the selective transfer of biotic resistance genes into elite indica rice cultivars devoid of linkage drag. In additon, molecular markers precisely estimate the introgression of chromosome segments from donor parents and can speed up the recipient genome recovery via background selection.

The fruits of Schisandra chinensis have been used as an edible ingredient and traditional medicine in Korea. Due to morphological similarities of dried mature fruits, the correct identification of S. chinensis from other closely related Schisandrae species is very difficult. Therefore, molecular biological tools based on genetic analysis are required to identify authentic Schisandrae Fructus. Random amplifed polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop an easy, reliable and reproducible method for the authentication of these four species. In this paper, we developed several RAPD-derived species specific SCAR markers and established a multiplex-PCR condition suitable to discriminate each species. These genetic markers will be useful to distinguish and authenticate Schisandrae Fructus and four medicinal plants, S. chinensis, S. sphenanthera, S. repanda and K. japonica, in species level.

Despite a variety of accounts proposed to capture the grammatical properties that the so-called dummy plural maker (DPM) -tul in Korean displays, it has been commonly taken for granted that a DPMed phrase is to be c-commanded by its associate plural element (APE). This paper observes, however, that the APE itself can host DPM -tul. This fact calls for a novel licensing mechanism, as any account that resorts to a direct c-command relation between a DPMed phrase and its APE will fail. This paper proposes that there is a functional category that Agrees with the APE. This is plausible because the semantic interpretation of the DPM construction has more to do with a semantically plural element, i.e., the APE, rather than a DPMed category. Then DPM -tul on the APE can be naturally considered as a phonological realization of the Agree relation. DPM -tul on a non-APE element is viewed merely as a copy due to an operation called Spread, much like the distribution of negative morphemes in negative concord languages, except for the optionality of the phonological realization.

토마토 과실의 숙성기간은 운송기간과 상품성에 상당한 영향을 미친다. 특히, 고온에서는 과실숙성이 빨라지므로, 숙성기간은 열대지방에 수출하는 토마토 품종에서 반드시 고려되어야 한다. 토마토 과실숙성을 효과적으로 차단하는 RIPENING-INHIBITOR (Rin) 유전자는 열성 유전하는 돌연변이체에서 보고되었고, 교잡후대(Rin/rin)에서는 숙성이 느리게 진행되어 저장성이 상당히 향상된다고 알려졌다. 따라서, 본 연구에서는 수출용 토마토 품종의 육성을 위해, 과실 숙성의 저해와 관련한 Rin 유전자 특이적인 분자 표지를 개발하고자 하였다. Rin과 rin유전자의 mRNA와 genomic DNA의 염기서열 정보를 확보하고 다형성을 나타내는 프라이머 조합을 선발하여, 공우성 SCAR 분자표지로 개발하였다. 토마토 육성 라인 24주에서 과실숙성 정도와 PCR밴드를 비교한 결과, 공우성 마커는 동형(Rin/Rin, rin/rin)과 이형(Rin/rin)의 유전자형을 구별하기에 적합하였으며 이를 이용하여 저장성이 좋은 토마토 품종개발에 도움이 될 것으로 기대한다.

This study describes the identification of Panax species using mitochondrial consensus primers. Initially, a total of thirty primers were tested in ten Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, three primers (cox1, nad1/2-3 and nad2/1-2) generated co-dominant polymorphic banding patterns discriminating Korean ginseng cultivars from P. quinquefolius and P. notoginseng. However, these primers could not generated polymorphisms among the Korean ginseng cultivars, and simply represented species-specific polymorphisms for P. quinquefolius and P. notoginseng. Primers PQ91 and PN418 were designed from the consensus sequence of nad1/2-3 region. Two banding patterns (A or B) were detected in PQ91. Korean ginseng cultivars and P. notoginseng shared the same banding pattern (A type) and P. quinquefolius was identified another banding pattern (B type). In the case of PN418, two banding patterns (A or B) were detected in the Korean ginseng cultivars and two foreign Panax species. Korean ginseng cultivars and P. quinquefolius shared the same banding pattern (A type) and P. notoginseng was identified another banding pattern (B type). The combination banding patterns of three Panax species, Korean ginseng cultivars (Panax ginseng C. A. Mey.), P. quinquefolius and P. notoginseng, was identified as 'AA', 'BA' and 'AB', respectively. Consequently, PQ91 and PN418 primer sets can be used to distinguish among Panax species.

The Korean plural marker tul can be used in a peculiar way. It can not only attach to a nominal expression functioning as a usual plural marker, but it can also, rather unusually, attach to other types of phrasal category. Semantically, when used in the latter way, it brings to the discourse context a sense of distributivity which is rather weak – weak to the extent that truth conditions are sometimes unaffected by its presence. In this article, I derive these properties of extrinsic tul from two proposals: (i) extrinsic tul comes with a silent anaphoric element; and (ii) it conventionally implicates the existence of a distributive relation between the host phrase of tul and its plural antecedent.

Molecular genetic markers have been widely used as powerful tools for analyzing the genome. In particular, SSR markers have practical applications in breeding systems because they can be used in high-throughput analyses for genetic mapping, heritable diversity testing, purity analysis, and marker-assisted selection. Currently, due to technical advances in DNA sequencing, large sequence databases are available for large-scale SSR mining and marker development. Here, we describe an automated method, the SSR Finder program, for SSR discovery in the onion sequence database, and primer design for amplifying the detected SSRs. A total of 1,049 SSR primers were obtained for genetic purity testing, and 100 SSR sets were analyzed in 14 bulb onion breeding lines using clustering analysis. A total of 20 selected markers from screening of all 1,049 SSR primers, were finally applied for genetic purity testing in three breeding lines, NW1, NW9, and NW10. The initial tests showed that 15%, 71%, and 97% of individuals within NW1, NW9, and NW10, respectively, were genetically homogeneous. These markers produced using the SSR Finder will be useful for investigating the genetic purity of onion breeding lines.

In this study, Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR) analyses were used to clarify the genetic polymorphisms among Korean ginseng cultivars and breeding lines and to classify them into distinct genetic groups. Polymorphic and reproducible bands were produced by 14 primers out of total 30 primers used in this study. Fourteen EST-SSR loci generated a total of 123 bands. Amplified PCR products showed the highly reproducible banding patterns at 110~920 bp. The number of amplified bands for each EST-SSR primers ranged from 2 to 19 with a mean of 8.8 bands. P26 and P35 primers showed 13 and 12 banding patterns, respectively. The number of alleles for each EST-SSR locus ranged from 1.67 to 2.00 with a mean of 1.878 alleles. P34 and P60 primers showed the highest and the lowest genetic polymorphism, respectively. Cluster analysis based on genetic similarity estimated by EST-SSR markers classified Korean cultivars and breeding lines into 4 groups. Group included Gopoong and Chunpoong and 9 breeding lines (55%), group included 2 breeding lines (10%), group included 3 breeding lines (15%), group included Gumpoong and 3 breeding lines (20%). Consequently, the EST-SSR marker developed in this study may prove useful for the evaluation of genetic diversity and differentiation of Korean ginseng cultivars and breeding lines.