1. Comparative mapping of Brassica oleracea using sequence-based markers derived from other Brassica relatives and transcriptome sequences generated from three parental lines using GS-FLX-Titanium Brassica oleracea L. is the most important vegetable crops over the world and its consumption increase because of its healthy food compounds. In order to develop map-based molecular breeding system in Brassica oleracea, we are trying to construct genetic map and to identify quantitative trait locus controlling black rot and club root resistance to support conventional breeding. Sixteen B. oleracea breeding lines, which consisted of 5, 2, and 9 lines resistant to black rot and club root disease, and susceptible to both diseases, respectively, were investigated to select best parental combinations. A total of 105 polymorphic primers which consist of intron-based polymorphism(IBP) markers, SSR markers and NBS domain related markers were found to estimate the genetic diversity of those lines. Two best parental combinations were selected for genetic mapping; IMO-005 x IMO- 010 for black rot resistance and IMO-010 x IMO-018 for clubroot resistance. Polymorphism survey on those parental combinations was conducted with more than 2,000 DNA markers derived from Brassicas genome map. We found more than 220 polymorphic markers in each combination. Additionally, transcirptome sequencing of three parental lines was carried out using GS-FLX-Titanium. We have obtained approximately 40Mbp transcriptome sequence from each accession. Based on comparison of sequences of three parental lines, large numbers of SNPs will be mapped by high resolution melting (HRM) analysis. High resolution genetic frame map and DNA markers related to two diseases will be practically applied to cabbage breeding for marker assisted foreground and background selection to support conventional breeding. *Corresponding author: Tel. 82-2-880-4547, Email: tjyang@snu.ac.kr Brassica oleracea L. is the most important vegetable crops over the world and its consumption increase because of its healthy food compounds. In order to develop map-based molecular breeding system in Brassica oleracea, we are trying to construct genetic map and to identify quantitative trait locus controlling black rot and club root resistance to support conventional breeding. Sixteen B. oleracea breeding lines, which consisted of 5, 2, and 9 lines resistant to black rot and club root disease, and susceptible to both diseases, respectively, were investigated to select best parental combinations. A total of 105 polymorphic primers which consist of intron-based polymorphism(IBP) markers, SSR markers and NBS domain related markers were found to estimate the genetic diversity of those lines. Two best parental combinations were selected for genetic mapping; IMO-005 x IMO- 010 for black rot resistance and IMO-010 x IMO-018 for clubroot resistance. Polymorphism survey on those parental combinations was conducted with more than 2,000 DNA markers derived from Brassicas genome map. We found more than 220 polymorphic markers in each combination. Additionally, transcirptome sequencing of three parental lines was carried out using GS-FLX-Titanium. We have obtained approximately 40Mbp transcriptome sequence from each accession. Based on comparison of sequences of three parental lines, large numbers of SNPs will be mapped by high resolution melting (HRM) analysis. High resolution genetic frame map and DNA markers related to two diseases will be practically applied to cabbage breeding for marker assisted foreground and background selection to support conventional breeding. *Corresponding author: Tel. 82-2-880-4547, Email: tjyang@snu.ac.kr Tae-Jin Yang: tjyang@snu.ac.kr (http://im-crop.snu.ac.kr/) tjyang@snu.ac.kr Jonghoon Lee 1, Nur Kholilatul Izzah 1, JeeYoung Park 1, Shailendra Karki 1, Sampath Perumal 1, Mina Gene 2, Beom-Seok Park 2, Jinling Meng 3, Kyung Gu Ahn 4, and Tae-JinYang 1* 1 Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Korea 2 National Academy of Agricultural Science, 249 Suin-Ro Kweonseonku, Suwon, 441-857, Korea. 3 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China 4 Joeun Seeds, #174, Munbang-Ri, Cheonhan-Myun, Goesan-Gun, Chungcheongbuk-Do, 367-833, Korea Marker typeAmplifiedPolymorphicMonomorphic No specific band Total SSR164854633170 IBP marker6014331371 NBS-based marker30622230 Total25410510148271 Table 1. Polymorphic survey of DNA markers on 16 B. oleracea breeding lines Phylogenetic analysis of 16 B. oleracea breeding lines to select best parental combinations Fig. 1 Cluster analysis of 16 B. oleracea breeding lines based on the similarity matrix S to black rot R to club root R to black rot S to black rot Construction of high density genetic map related to black rot and club root disease resistance Table 2. Preliminary result of primer screening between 2 parents combination Parents combination Marker type The number of polymorphic markers Total IMO05xIMO10 SSR60 219 IBP159 IMO10xIMO18 SSR77 253 IBP176 This work was supported by Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries(IPET), Korea(Grant No.: 309008-05-1-cg000) Transcriptome sequencing & high throughput SNP detection using NGS technology Reads NoLength Avg read length Contigs No Length Avg ctg length Singles No Singles Length Avg sing- let length IMO10 92,25537,988,1264125,9243,758,14863428,65110,978,478383 IMO05 127,52253,250,4614187,8725,126,76565132,87912,404,186377 IMO18 92,24738,713,6374205,8063,655,60663029,35011,347,746387 Assembled by Newbler2.3 with 98% sequence similarity threshold Table 3. Summary of Transcriptome sequencing Tanscriptome sequencing of 3 parental lines for black rot and clubroot diseases were performed by GS-FLX-Titanium. Contigs were assembled to isotigs. IMO10IMO05IMO18 Number of Isogroups4,9086,3544,751 Avg Contig Cnt1.21.31.2 Largest Contig Cnt153411 Number With One Contig4,4535,7124,294 Avg Isotig Cnt1.1 Largest Isotig Cnt202612 Number With One Isotig4,4635,7274,300 IMO10IMO05IMO18 Number of Isotigs5,5157,2715,382 Avg Contig Cnt1.3 Largest Contig Cnt9156 Number With One Conitg 4,4605,7154,297 Number Of Bases4,111,9645,757,8804,049,457 Avg Isotig Size745791752 N50 Isotig Size760815764 Largest Isotig Size3,8208,2314,181 1,331 (1,525) 1,748 (1,935) 1,022 (1,087) 572 (719, 774) 811 (1,142, 1,042) IMO18 IMO05 1,290 (2,901, 3,425, 2,922) 284 (369, 334) IMO10IMO05IMO18 The numbers of ctgs on each accession347744213472 Unique ctgs in each accession (%)384031 Common ctgs in three accession (%)373137 Common ctgs between two accessions(%)252932 The numbers of paralogs1.6 IMO10 Isotigs showing SNPs Functional genes Isotigs of functional genes showing SNPs IMO05 X IMO10IMO10 X IMO18Both combination No.1815954848661330 Fig 3. Classification of homologous UniESTs Isotigs in each lines were compared in EST sequence level. The numbers whose color are same as the color of accession number and Black-colored numbers mean the number of isotigs and predicted genes in those region, respectively. From the number of isotig and of genes, we can estimate that paralogs number could be 1.6 in all 3 lines as followed in table 4. Table 5. Summary of isotigs which can be used for SNP marker development Based on the results of transcriptomes sequencing, SNP candidate regions can be detected m p 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 3 4 5 6 7 8 9 50 1 2 3 4 5 6 7 8 9 60 1 2 3 4 5 6 7 8 9 70 1 2 3 4 5 6 7 8 9 80 1 2 3 4 5 6 7 8 9 90 1 2 3 4 Fig 2. Genotype scoring for the F2 progenies for black rot disease to construct genetic map. 5% non-denaturing polyacrylamid gel electrophoresis was carried out. Prminer numbers; (a) B086M08_1, (b) H121P05_1. Lanes; m, maternal parent; p, paternal parent; 1~94, F2 population. Segregation ratio; m, maternal type; h, heterozygous type; p, paternal type (a) (b) m : h : p = 23 : 46 : 25 m : h : p = 38 : 38 : 18 Fig 4. HRM analysis among 3 lines whose transcriptomes were sequenced Primers were designed in isotigs of functional genes showing SNPs as described in table 4. Candidate SNP regions can be confirmed by HRM technology in short time, and genotyping of large F2 population will be carried out fast. Table 4. Summary of homologous UniESTs and paralog estimation