Marker-Assisted Backcrossing (MABC)을 이용한 토마토 웅성불임 엘리트 계통 육성

김동현,정유진,김종희,김희경,남기홍,이효주,김명권,노일섭,강권규 한국원예학회 2019 원예과학기술지 Vol.37 No.6

Recurrent backcrossing is a traditional breeding method that is commonly employed to transfer alleles at one or more loci from a donor to an elite variety. In order to develop male-sterile elite tomato lines, marker-assisted foreground and background selections were performed during backcross breeding. Compared to conventional backcrossing, marker-assisted backcrossing (MABC) is extremely useful for recovery of a recurrent parent’s genetic background. For foreground selection, the tomato mutant anthocyanin absent (aa) presents a green hypocotyl during the seedling stage. Lines carrying the Aa genotype were selected from BC1F1 and BC2F1 populations using indel markers derived from the unique aa mutation of T2-517. This trait has been utilized in marker-assisted selection of male sterile 10 (ms10) at the seedling stage because their corresponding loci are closely linked on chromosome 2. For background selection, a total of 48 single nucleotide polymorphism (SNP) markers obtained from resequencing data between the MR10-3211 and T2-517 lines evenly distributed in the tomato genome were finally selected. BC1F1 and BC2F1 plants carrying the heterozygous (Aa) genotype were subjected to background selection using a set of 48 SNP markers. Multiple genotype analysis was done using a high-throughput genotyping system. As a result, one plant, 87.5% similar to the recurrent parent genome in the BC1F1 generation, and three plants with a 95.5% recovery rate of the recurrent parent genome in the BC2F1 generation, were selected. These selected plants were fixed in the BC2F2 and BC2F3 generations for the male sterile tomato elite line. We therefore demonstrate the utility of the MABC method for the recovery of recurrent parent genomes in tomato breeding strategies. Recurrent backcrossing은 donor친으로부터 엘리트 품종에 하나 이상의 좌에서 대립유전자들을 도입하려고 할 때 일반적으로 사용되는 전통적인 육종 방법이다. 웅성불임 엘리트 토마토 계통을 육성하기 위해 마커 지원 foreground 및 background 선발은 여교잡 육종 과정에서 수행하였다. MABC(marker-assisted backcrossing)은 기존의 여교배와 비교할 때 반복친의 유전적 배경을 회복하는데 매우 유용하게 사용하고 있다. Foreground 선발을 위해서 육묘 단계에 녹색 배축을 보이는 안토시아닌 결손(aa) 토마토 변이체는 T2-517 계통 유래 InDel 마커를 사용하여 BC1F1 및 BC2F1 집단에서 선발하였다. 이들 특성은 웅성불임 ms1035 유전자와 2번 염색체상에 매우 밀접하게 연관되어 있으므로 MAS(marker-assisted selection)에 이용하였다. Background 선발을 위해서 MR10-3211과 T2-517 계통 간의 resequencing 데이터로부터 얻은 48개의 single nucleotide polymorphism(SNP) 마커는 토마토 게놈에 고르게 분포되도록 최종 선발하였다. 이형접합(Aa) 유전자형을 갖는 BC1F1 및 BC2F1 개체들은 48개의 SNP 마커 세트를 background 선발에 이용하였다. 다중 유전형 분석은 high-throughput genotyping system(EP1TM, Fluidigm®, USA)을 사용하여 수행되었다. 그 결과 BC1F1 세대에서 반복친 게놈과 87.5%가 유사한 1개의 식물체와 BC2F1 세대에서 반복친 게놈의 회복률이 95.5% 이상으로 보이는 3개의 식물체를 선발하였다. 선발된 개체들은 토마토 웅성불임 계통을 육성하기 BC2F2 및 BC2F3 세대로 고정하였다. 따라서 MABC 방법은 토마토 육종 프로그램에서 반복친 게놈을 회복시키기 위한 획기적인 방법으로 생각된다.

분자육종법과 관행육종법을 활용한 고식미계통 선발효율성 비교분석

서정필,조영찬,원용재,이정희,안억근,전재범,이점식,김명기,정응기,김보경 한국육종학회 2014 한국육종학회지 Vol.46 No.3

지금까지 밥맛특성이 우수한 품종을 육성하려는 노력이 부단히 진행되고 있으나, 저세대에서 미질을 평가하는 기준과 지표가 미흡한 실정이다. 고식미 품종육성과정 중 저세대에서 고식미계통을 선발하기에는 어려운 실정이다. 본 연구에서는 DNA분석에 의한 식미회귀식값을 자포니카 간의 교잡에서 유래된 저세대 계통선발에 적용하여 선발효율성을 관행육종법과 비교분석하였다. 9개 교배모본들을 군집분석하여 그룹간에 식미회귀식값과 밥의 윤기치의 연관분석을 실시한 결과, 유의성이 있었다. 분자육종법과 관행육종법으로 계통을 선발한 결과 선발집단규모는 각각 34.4%, 38.6%로 비슷하였고, 분자육종법과 관행육종법을 병행하였을 때는 19.5%로 집단규모가 상당히 줄었다. 밥의 윤기치와 식미회귀식값은 집단간에는 차이가 있었으나, 육종방법에 따라서는 차이가 없었다. 식미회귀식값은 13개 DNA마커를 가지고 값을 구하게 되는데, 본 실험에 사용된 교배조합에서 양친 간에 다형성을 보이는 DNA마커는 3~7개로 상당히 적어, 식미회귀식값을 구하여 표현형을 설명하기에는 부족한 것으로 생각된다. 식미회귀식값에 의한 고식미계통선발을 하려면 교배조합별로 DNA마커조합과 식미회귀식값을 다시 산출해서 사용을 하거나, 식미회귀식에 활용되는 모든 DNA마커에 다형성을 보이는 교배조합을 선정해서 적용한다면 어느 정도 효과가 있을 것으로 생각된다. 본 실험에 활용된 식미연관 13개 DNA마커에 의한 식미회귀식값은 모든 조합의 육종집단에서 선발에 활용하기는 어려울 것으로 생각된다. This paper compares selection efficiency for high palatability breeding lines using marker-assisted selection and conventional selection methods in rice. A total 4 cross combinations of japonica cultivars were selected using marker-assisted selection with a set of 13 DNA markers associated with grain quality and conventional selection methods in F3 and F5 generations assessing palatability using the Toyo taste meter. The multiple regression value with a set of 13 DNA markers was utilized as the marker-assisted selection index. The number of polymorphic markers among 13 DNA markers ranged from 3 to 7 between the parental cultivars. Among these cross combinations, there was no significant difference between marker-assisted selection and conventional selection methods for selection of lines with high palatability. This demonstrates that marker-assisted selection by marker-based regression value might not be a good method for selection to apply the all breeding populations for high palatability line selection. While each method allowed equally effective selection of high palatability lines, the regression analysis using polymorphic markers will need to be re-calculated for each cross combination.

Marker Assisted Selection-Applications and Evaluation for Commercial Poultry Breeding

Sodhi, Simrinder Singh,Jeong, Dong Kee,Sharma, Neelesh,Lee, Jun Heon,Kim, Jeong Hyun,Kim, Sung Hoon,Kim, Sung Woo,Oh, Sung Jong The Korean Society of Poultry Science 2013 韓國家禽學會誌 Vol.40 No.3

Poultry industry is abounding day by day as it engrosses less cost of investment per bird as compared to large animals. Poultry have the most copious genomic tool box amongst domestic animals for the detection of quantitative trait loci (QTL) and marker assisted selection (MAS). Use of multiple markers and least square techniques for mapping of QTL affecting quality and production traits in poultry is in vogue. Examples of genetic tests that are available to or used in industry programs are documented and classified into causative mutations (direct markers), linked markers in population-wide linkage disequilibrium (LD) with the QTL (LD markers), and linked markers in population wide equilibrium with the QTL (LE markers). Development of genome-wide SNP assays, role of 42 K, 60 K (Illumina) and 600 K (Affymetrix$^{(R)}$ Axim$^{(R)}$) SNP chip with next generation sequencing for identification of single nucleotide polymorphism (SNP) has been documented. Hybridization based, PCR based, DNA chip and sequencing based are the major segments of DNA markers which help in conducting of MAS in poultry. Economic index-marker assisted selection (EI-MAS) provides platform for simultaneous selection for production traits while giving due weightage to their marginal economic values by calculating predicted breeding value, using information on DNA markers which are normally associated with relevant QTL. Understanding of linkage equilibrium, linkage dis-equilibrium, relation between the markers and gene of interest are quite important for success of MAS. This kind of selection is the most useful tool in enhancing disease resistance by identifying candidate genes to improve the immune response. The application of marker assisted selection in selection procedures would help in improvement of economic traits in poultry.

Current Applicable DNA Markers for Marker Assisted Breeding in Abiotic and Biotic Stress Tolerance in Rice (Oryza sativa L.)

( Franz Marielle Nogoy ),( Jae-young Song ),( Sothea Ouk ),( Shadi Rahimi ),( Soon Wook Kwon ),( Kwon-kyoo Kang ),( Yong-gu Cho ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.3

Abiotic and biotic stresses adversely affect rice (Oryza sativa L.) growth and yield. Conventional breeding is a very effective method to develop tolerant rice variety; however, it takes a decade long to establish a new rice variety. DNA-based markers have a huge potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection (MAS). The large number of quantitative trait loci (QTLs) mapping studies for rice has provided an abundance of DNA marker-trait associations. The limitations of conventional breeding such as linkage drag and lengthy time consumption can be overcome by utilizing DNA markers in plant breeding. The major applications of DNA markers such as MAS, QTL mapping and gene pyramiding have been surveyed. In this review, we presented the latest markers available for some of the most important abiotic and biotic stresses in rice breeding programs. Achieving a significant impact on crop improvement by marker assisted breeding (MAB) represents the great challenge for agricultural scientists in the next few decades.

염색체 도식화와 imputation에 의한 GBS 기반 여교잡 회복률 계산 정확도 증진 방법

정혜리,최준경,이봉우,이보미,강윤주,이정희,김지은,남문,박영훈,박민우,박기림,조성환 한국육종학회 2020 한국육종학회지 Vol.52 No.4

Marker-assisted backcrossing is a powerful method for developing new cultivars. To develop genomic-wide markers, genotyping-by-sequencing(GBS) can be an efficient method. However, unrefined low-quality markers and missing data between markers can contribute to hamperingthe marker selection process, particularly in multi-way crosses. In this study, we aimed to calculate the recovery rate of offspring individualsand minimize errors that occur among a large number of markers. Initially, missing data were imputed by comparing samples using the k-nearestneighbor (k-NN) algorithm. Thereafter, low-quality single-nucleotide polymorphisms (SNPs) were corrected by applying the graphical representationmethod based on the k-NN algorithm in order of the SNPs in a chromosome designed for a multi-parental population. Four-way cross anddouble-backcrossed tomato BC1F1 (230 lines) and BC2F1 (96 lines) populations were genotyped by GBS. The genotype of samples of theBC1F1 and BC2F1 populations was determined based on the parental haplotype. Thus, the method of visualizing the genotype of offspringindividuals, generated via crosses of multiple parents, not only improves estimation of the recovery rate but also facilitates easier selectionin breeding programs.

Molecular Markers and Their Usefulness in Rice Breeding

( Neeraj Kumar Tyagi ),( Bandarupalli Ramesh ),( Kuldeep Tyagi ) 전북대학교 농업과학기술연구소 2009 농업생명과학연구 Vol.40 No.2

Molecular markers are extensively used for improving and sustaining the rice productivity. A variety of molecular genetic markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), microsatellites or simple sequence repeats (SSRs), expressed sequence tags (EST) and single nucleotide polymorphism (SNP) have been developed providing new tools for rice breeding. The major advantages of the molecular markers over the other classes of markers are their number is potentially unlimited, spanning across the genome, their expression is unaffected by the environment and their assessment is independent of the stage of plant development. Molecular markers are landmarks in the chromosome maps that can be used to monitor the transfer of specific chromosome segments known to carry useful agronomic traits. Breeders use these molecular markers to increase the precision of selection for the best trait combinations. Molecular markers have large number of applications ranging from diversity analysis to the improvement of rice varieties by marker assisted selection. This review describes the usefulness of some important DNA markers in rice improvement.

Fine Mapping of the Rice Bph1 Gene, which Confers Resistance to the Brown Planthopper (Nilaparvata lugens Stal), and Development of STS Markers for Marker-assisted Selection

Cha, Young-Soon,Ji, Hyeonso,Yun, Doh-Won,Ahn, Byoung-Ohg,Lee, Myung Chul,Suh, Seok-Cheol,Lee, Chun Seok,Ahn, Eok Keun,Jeon, Yong-Hee,Jin, Il-Doo,Sohn, Jae-Keun,Koh, Hee-Jong,Eun, Moo-Young Korean Society for Molecular Biology 2008 Molecules and cells Vol.26 No.2

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.

Fine Mapping of the Rice Bph1 Gene, which Confers Resistance to the Brown Planthopper (Nilaparvata lugens Stal), and Development of STS Markers for Marker-assisted Selection

차영순,지현소,윤도원,안병옥,이명철,서석철,이준석,안억근,전용희,진일두,손재근,고희종,은무영 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.2

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.

Development of Resistant Gene-Pyramided Japonica Rice for Multiple Biotic Stresses Using Molecular Marker-Assisted Selection

( Jung Pil Suh ),( Young Chan Cho ),( Yong Jae Won ),( Eok Keun Ahn ),( Man Kee Baek ),( Myeong Ki Kim ),( Bo Kyeong Kim ),( Kshirod K. Jena ) 한국육종학회 2015 Plant Breeding and Biotechnology Vol.3 No.4

Advances in plant molecular techniques have dramatically widened the applicability of gene identification and pyramiding valuable genes. This study was carried out to pyramid five resistance genes for biotic stress into the japonica rice cultivar using marker-assisted selection (MAS) and marker-assisted background analysis of selected progenies using SSR markers. The Pi40, Xa4, xa5, Xa21 and Bph18 genes were combined in Jinbubyeo, a Korean japonica rice variety using MAS. Gene specific co-dominant PCR-based markers were used to select for homozygous recombinant lines in a segregating population derived from a cross between the parental homozygous resistant gene introgression lines. We had successfully developed multiple gene pyramided breeding lines (GPLs) for bacterial blight, blast, and brown planthopper using MAS in rice. The GPLs exhibited high resistance against biotic stress and had around 93% of the genetic background of the recurrent parent Jinbubyeo based on SSR graphical mapping. The yield and agronomic traits of the GPLs were similar to those of the recurrent parent, indicating that there is no apparent agronomic trait penalty associated with the presence of the resistance genes. The strategy of simultaneous foreground and phenotypic selection to introduce multiple R genes is very useful to reduce the cost and the time required for the isolation of desirable recombinants with target resistance genes in rice. The GPLs could be useful to enhance effective resistance for biotic stress and produce stable grain yield in japonica rice breeding programs.

Marker-Assisted Foreground and Background Selection of Near Isogenic Lines for Bacterial Leaf Pustule Resistant Gene in Soybean

Kim, Kil-Hyun,Kim, Moon-Young,Van, Kyu-Jung,Moon, Jung-Kyung,Kim, Dong-Hyun,Lee, Suk-Ha The Korean Society of Crop Science 2008 Journal of crop science and biotechnology Vol.11 No.4

Bacterial leaf pustule (BLP) caused by Xanthomonas axonopodis pv. glycines is a serious disease to make pustule and chlorotic haloes in soybean [Glycine max (L). Merr.]. While inheritance mode and map positions of the BLP resistance gene, rxp are known, no sequence information of the gene was reported. In this study, we made five near isogenic lines (NILs) from separate backcrosses (BCs) of BLP-susceptible Hwangkeumkong $\times$ BLP-resistant SS2-2 (HS) and BLP-susceptible Taekwangkong$\times$ SS2-2 (TS) through foreground and background selection based on the four-stage selection strategy. First, 15 BC individuals were selected through foreground selection using the simple sequence repeat (SSR) markers Satt486 and Satt372 flanking the rxp gene. Among them, 11 BC plants showed the BLP-resistant response. The HS and TS lines chosen in foreground selection were again screened by background selection using 118 and 90 SSR markers across all chromosomes, respectively. Eventually, five individuals showing greater than 90% recurrent parent genome content were selected in both HS and TS lines. These NILs will be a unique biological material to characterize the rxp gene.