Integration of AFLPs, SSRs and SNPs markers into a new genetic map of industrial chicory (Cichorium intybus L. var. sativum) C  ELINE M UYS 1,4,* ,C LAIRE -N O € ELLE T HIENPONT 2,* ,N ICOLAS D AUCHOT 1 ,O LIVIER M AUDOUX 3 , X AVIER D RAYE 2 and P IERRE V AN C UTSEM 1 1 Research Unit in Plant Biology, University of Namur, 61 rue de Bruxelles, 5000, Namur, Belgium; 2 Earth and Life Institute, Universit e catholique de Louvain, Croix du Sud 2, L7.05.11, 1348, Louvain-la-Neuve, Belgium; 3 Chicoline, Cosucra Groupe Warcoing SA, 1 rue de la sucrerie, 7740, Warcoing, Belgium; 4 Corresponding author, E-mail: celine.muys@unamur.be With 2 figures and 3 tables Received January 21, 2013/Accepted August 26, 2013 Communicated by T. Debener Abstract A chicory genetic map of 1208 cM has been created using 247 F 2 plants and 237 markers (170 AFLP, 28 SSR, 27 EST-SNP and 12 EST-SSR). This map covers 84% of the chicory genome. The chicory-genic-markers- associated sequences were used to find potential orthologs in mapped let- tuce ESTs from the Compositae Genome Project Database. Twenty-seven putative orthologous pairs were retained, pinpointing seven putative blocks of synteny that covered 11% of the chicory genome and 13% of the lettuce genome, opening new perspectives for the analysis of these two species. Key words: linkage map — AFLP — SSR — SNP — genic markers — chicory — comparative mapping Industrial chicory (Cichorium intybus L. var. sativum) is a biennial plant grown for the production of inulin, a fructan polymer stored in roots (Van Laere and Van Den Ende 2002). This β-(1,2)-linked fructan is a texture and functional ingredient in the food industry, whose prebiotic properties result into a general improvement of health and a reduction of the risks of many diseases (Roberfroid et al. 2010). C. intybus is subdivided into wild chicory (var. sylvestre), witloof chicory (var. foliosum, the Belgian endive) and industrial or root chicory (var. sativum). The species is diploid (2n = 18) and belongs to the Asteraceae, along with economically important crops such as lettuce and sunflower. Conventional breeding of the industrial chicory has been performed for decades to increase root yield, inulin content and inulin chain length, to reduce bolting and to improve diseases resistance. Recently, molecular tools (SSRs and InDel polymorphisms) have been developed on chicory and are now routinely used. The aim of this work is to create a genetic map of chicory that will then be suitable to initiate comparative mapping of chicory and lettuce. Comparative mapping relies on the location of orthologous sequences from two species. It allows detecting events that occurred since the divergence of the species from an unknown ancestral genome. The analysis of macrosynteny allows mutually anchoring two incomplete genome data sets such as in the case of chicory and lettuce. Lactuca sativa is a close relative of C. intybus (Koopman et al. 1998) and is one of the most studied species in the Compositae Genome Project (http://cgpdb.ucdavis.edu). The comparative mapping of these two species is expected to allow transferring lettuce resources to chicory, and conversely. So far, three genetic maps of chicory have been published (De Simone et al. 1997, Van Stallen et al. 2003, Cadalen et al. 2010). Two of these maps cannot be used in comparative mapping because they are entirely composed of anonymous and non-trans- ferable markers. The third is a consensus map derived from witloof and industrial chicory (Cadalen et al. 2010). Unfortunately, the crosses were not available for our study, and the STS sequences localized on this map were either too short to develop reliable probes or not polymorphic in our mapping populations. We therefore decided to develop new genic markers for chicory and to create a new genetic map to initiate our comparative studies. Using an F 2 population, a genetic map using AFLP markers for rapid saturation and genic markers for comparative mapping was elaborated. The sequences associated with the genic markers were used in a RBH (Reciprocal Best BLAST Hit) analysis, to search for highly similar sequences previously positioned on the genetic and physical maps of lettuce (Compositea Genome Project, CGP). The position of potential orthologous pairs on the two genomes could then be analysed. This represents the first comparative mapping between chicory and lettuce allowing preliminary considerations on the synteny between these two closely related species. Analysis of putative syntenic regions highlights areas of interest for future study in chicory. Material and Methods Plant material: Two partly inbred industrial chicory plants (C. intybus cv sativum) were crossed as part of a breeding scheme (Cosucra Belgium). Parents showed different root morphology and had different genotypes for 17 SSRs. From the progeny of this cross, a single F 1 plant was auto-pollinated and provided a large seed set. A series of 247 F 2 plants constituted the mapping population for this study. All plant material was supplied by Cosucra Belgium. DNA extraction: Genomic DNA was extracted from young leaves of individual seedlings of the F 2 plants by a procedure based on the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson 1980) with some modifications. Fresh leaf tissue was ground in liquid nitrogen, and then incubated for 30 min at 65°C in 1 ml of extraction buffer [2% w/v CTAB, 2% w/v polyvinylpolypyrrolidone (PVPP), 2 M NaCl, 20 mM EDTA-Na 2 , 100 mM Tris–HCl, pH 8.0]. Samples were then cooled on ice and centrifuged at 21 900 g for 5 min. * The first two authors contributed equally to this work. wileyonlinelibrary.com Plant Breeding doi:10.1111/pbr.12113 © 2013 Blackwell Verlag GmbH