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SESSION GENETICS AND GERMPLASM ENHANCEMENT GABI - BEET: THE GERMAN SUGAR BEET GENOME INITIATIVE GEORG KOCH Strube-Dieckmann A. Dieckmann-Heimburg, Kirchhorster Straf3e 16, 31688 Nienstadt, Germany SUMMARY GABI-BEET will provide new technologies for the molecular breeding of sugar beet Marker technologies (RFLPs, RAPDs, SSRs, AFLPs) have already been employed successfully in practical beet breeding. Here, we aim to introduce new markers based on single nucleotide polymorphisms (SNPs) derived from both expressed genes and anonymous markers in combination with a high throughput system for marker analysis as a new tool for molecular breeding and research GABI-BEET is divided into four major projects project A (sequencing of ESTs and bioinformatics) is the requirement for project B (high density mapping and allelic variation detected by SNP technologies) Project B will be achieved through the integration of conventional and functional markers. This approach is complemented by the comparative mapping and genome analysis by in situ hybridization (project C) and the construction of representative large insert libraries (proJect D) The GABI-BEET project is focussing the following milestones A) Supply of extensive sequence information of transcribed (expressed sequence tags, ESTs) of the sugar beet genome, regions B) Establishment of an integrated data base for molecular and genetic information and the development of new marker systems based on single nucleotide polymorphisms (SNPs), C) Comparison of the genome structure of wild and cultivated beet with respect to repetitive and mapped low-copy sequences, D) Construction of a bacterial artificial chromosome (BAC) library from sugar beet genome. The presentation will give an overview of the network of partners in the project, the goals and current status of the project ZUSAMMENFASSUNG GABI-BEET wird neue Technologien fUr die molekulare Zuchtung der Zuckerrube zur VerfUgung stellen. Verschiedene Marker-Technologien (RFLPs, RAPDs, SSRs, AFLPs) werden bereits erfolgreich in der praktischen Zuckerrubenzuchtung eingesetzt Hier mbchten wir neue Marker, basierend auf Einzelnukleotidpolymorphismen (SNPs) von exprimierten Genen und anonymen 1st joint 1/RB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA) 249 SESSION GENETICS AND GERMPLASM ENHANCEMENT Sequenzen, als neues Werkzeug der molekularen Zuchtung und Forschung in Kombination mit einem Hochdurchsatz-System fUr die Markeranalyse einfUhren. GABI-BEET ist unterteilt in vier gror..e Projekte Projekt A (Sequenzierung von ESTs und Bioinformatik) als Voraussetzung fUr Projekt B (hochdichte Kartierung und Detektion der allelischen Variation mittels SNP-Technologien) mit Integration konventioneller und funktioneller Marker. Dieser Ansatz wird vervollstandigt durch eine vergleichende Kartierung und eine Genomanalyse mittels in situ Hybridisierung (Projekt C) und durch die Konstruktion einer reprasentativen Bibliothek mit gror..en lnsertionen (Projekt D) Das GABI-BEET ProJekt ist fokussiert auf folgende Meilensteine A) Bereitstellung umfangreicher Sequenzinformationen von transkribierten Regionen (expressed sequence tags, ESTs) des Zuckerrubengenoms, B) Erstellung einer integrierten Datenbank fUr molekulare und genetische lnformationen und die Entwicklung von neuen Markersystemen basierend auf Einzelnukleotidpolymorphismen (SNPs), C) Vergleich der Genomstruktur von Wildruben und Zuckerruben in Bezug auf repetitive und kartierte low-copy Sequenzen, D) Aufbau einer Bibliothek aus artifiziellen Bakterienchromosomen (BACs) des Zuckerrubengenoms Die Prasentation wird einen Uberblick vermitteln uber das Netzwerk der Partner im Projekt, die Ziele und der gegenwartige Status des Projektes ABREGE Le programme GABI-BEET a pour but Ia production de nouveaux outils pour Ia selection assistee par marqueurs moleculaires (SAM). Les RFLPs, RAPDs, SSRs et AFLPs ont deja ete utilises avec succes en selection de Ia betterave. L'objectif est d'introduire, pour Ia SAM et Ia recherche en general, un nouveau type de marqueurs les SNPs dont le principe reside dans Ia detection de polymorphisme simple nucleotide, ceci a partir de genes exprimes ou de marqueurs anonymes combines avec un systeme complexe d'analyse de marqueur GABI-BEET est divise en 4 projets principaux . le projet A (sequen~age d'ESTs et bioinformatique) necessaire au projet B (cartographie haute densite et variation allelique detectee par SNPs) ceci avec /'integration de marqueurs conventionnels et fonctionnels Cette approche est renforcee par Ia cartographie comparative et /'analyse des genomes par hybridation in situ (proJet C) et Ia construction de banques representatives a grands inserts (projet D). Le programme GABI-BEET se deroule suivant les etapes suivantes • A) Production en masse de sequences de regions transcrites (ESTs) du genome de Ia betterave, B) Realisation d'une banque de donnees integrees contenant des informations moleculaires et genetiques et le developpement nouveaux outils de marquage moleculaire (SNPs), 250 1st joint /IRB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA) SESSION GENETICS AND GERMPLASM ENHANCEMENT C) Comparaison de Ia structure des genomes de betteraves sauvages et cultivees en reference a !'utilisation de sequences repetees et de sequences faiblement repetees cartographiees. D) Construction d'une banque BAC (Banque sur chromosome artificiel de bacterie) du genome de Ia betterave. La presentation donnera une vue generale du reseau de partenaires participants aux projets, les objectifs et Ia situation actuelle des projets INTRODUCTION GABI is an abbreviation for Qenom 6nalysis in the Plant Biological System (www gabi de). This national genome programme collects and focuses all German plant genome projects funded by the BMBF, the German federal ministry of education and research GABI phase I started end of 1998 and ends in 2003 Currently preparations for a second four year round of the project is underway Joint projects with the French plant genome initiative, genoplante (www genoplante org) are already installed and will be extended in GABI II GABI I is focused to the model crops, Arabidopsis thaliana and barley, and a few other crops with model characters for specific traits and applications, e g rape seed, sugar beet, potato. wheat, rye, maize and poplar Additionally, GABI I is funding resource centres to provide key technologies and materials and bioinformatics projects facilitating intelligent access to genomics data from within and from outside of the project The funding of GAB I I is approximately 50 Mio €. GABI II w1ll continue with the basic ideas of GABI I but while applying more focus to bridging projects between the model plants and crops and placing more emphasis on applied research in crops with model traits of agronomic importance The funding structure of GABI touches two distinct areas of research (I) Fundamental research is applied to research area I with up to 100 % federal funding: (II) Applied research is organised in research area II giving public institutes and life science companies more freedom to develop proprietary foreground rights related to the substantial contribution given by companies or institutes to the specific project with their background rights GABI-BEET is the only research area I project in GABI where companies are participating (www gabi-beetde) The partners in the project are given in Tab. 1 Three further sugar beet projects has started so far in GABI research area II. • GABI-BOL T Cloning of the boltmg gene B from sugar beet • SWEET-GAB! Crosslinking structural and functional genomics with regards to metabolism and development in sugar beet • BREATH-LESS GABI Molecular physiology of storage organs All three projects are based in part on GABI-BEET by using tools and material developed in GABI-BEET, sharing material and genomic information and in some cases by overlap in stuff 1st joint 1/RB-ASSBT Congress. 26t 11 Feb.-1 5t March 2003. San Antonio (USA) 251 SESSION GENETICS AND GERMPLASM ENHANCEMENT Tah. 1:Partncr stmcturc o(GABI-RFFT. lthtttut.: of( 'rop Science and Plant llrccding. ( '.-\11 1\.icl Suh.:<1ntract<1rs. Prof. I )r. Christian Jung. (oordinatln Dr I othar I res.: !'rut' Dr llcnm ard l\1iir!Umkr ~II'! ( i·\lllinlll. tiS!. Munich !'rut' Dr \\ crncr t(H Plant llrccding Research. ColognL· I)r Katharina 'ichnetdcr 'iuhcontractor .-\1 )J <; I'D Dr llcrnd W.:isshaar lthlilutc of Plant llrccding. 1\11 1 II aile l'rof Ilr I· b.:rlwrd \\ cbcr I )r habit k \\ S S.\.\ I.\( i. I in beck l\k11CS ~!arlin ( i;ul<li Dr llnlla 'ichul; Strube-l )tccklllann ..·\ Iliccklllann-llcilllhurg. l'iicnstiidl I)r ( 'Jaudia licllllllcrl ing OBJECTIVES GABI-BEET is a sugar beet project developing and providing major tools and access infrastructure to large scale genome analysis in sugar beet Major objectives are A) Supply of extensive sequence information of transcribed (expressed sequence tags, ESTs) of the sugar beet genome, regions B) Establishment of an integrated data base for molecular and genetic information and the development of new marker systems based on single nucleotide polymorphisms (SNPs), C) Comparison of the genome structure of wild and cultivated beet with respect to repetitive and mapped low-copy sequences, D) Construction of a bacterial artificial chromosome (BAC) library from sugar beet genome. PROJECT A) UNRAVELLING THE TRANSCRIPTOME OF SUGAR BEET The genome is build up by the linear sequence of the four nucleotides; A, T, C, G. The transcriptome describes the subset of sequence that is expressed via mRNAs, essentially the raw basic genes themselves Putative gene fragments identified by sequencing such transcribed sequences are called ESTs, expressed sequence tags ESTs are cloned in eDNA libraries (Fig 1) The KWS project partner provided the consortium with 14,778 ESTs. Putatively unique sequences have been selected in the absence of sequence data from large collections of cDNAs by the oligonucleotide fingerprinting methodology (HERWIG eta!. 2002). eDNA sequence clustering was achieved by correlating patterns of cross-hybridisation with a variety of oligonucleotide probes The 14,778 ESTs for just over 10,000 distinct cDNAs were sequenced within GABI-BEET by AD IS 252 1st joint 1/RB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA) SESSION GENETICS AND GERMPLASM ENHANCEMENT producing a unigene set of 9, 730 sequences. This collection of oligonucleotide fingerprinted EST sequences is meanwhile publicly accessible in GenBank (http/ /www. ncbi. n lm. nih. gov/Gen bank/index. htm 1). Fig. 1: Cloning tile tmnscripto111c in ESTs 11 c[)NA li/lmry. Cloning the Transcriptome DNA eDNA ( < 1 kbp) makes mRNA copied in eDNA cloned in plasmids transfected to bacteria ordered eDNA library in libraries spotted on filter and chips Ill RNA, lllf",c.,erlqer RNA trdrlc,crrbed genec, eDNA, cumplemenlary or copy lJNA. ,,.., rnRNA copreclrr1 ch DNA [_5 I, t \f't''~'~'d _c,P,7Ut'lll c' T.1y dor1ed mRNA lranc,cr r!Jerl qene', rla..,mrcl extr.l-chrollloc,ornnl hacterrdl DNA uc.ed ac, vector The GSF proJeCt partner was and remains one of the maJor contributors to annotation of the Arabidopsis genome The functional catalogue (FunCat) developed by the group provides a resource that can be used to transfer sequence annotation between heterologous sequences Based on the bioinformatic tools developed during the Arabidopsis genome proJect GSF was able to annotate putative functions to the ESTs of the unigene set and grouped them according to different metabolic pathways of the plant cell. Fig. 2 shows the distribution of the annotated functions of the unigene ESTs The functional annotation of the beet ESTs is available from the Ml PS website (http •Ilm ips. gsf d e/proj/sputn i k/). PROJECT B) TOWARDS A FUNCTIONAL MAP OF THE SUGAR BEET GENOME Molecular markers are the technological basis of molecular plant breeding. Molecular markers are landmarks on a genetic map of anonymous or preferable on genes of known function, e.g. candidate genes for agronomically important traits. Previously mapped RFLP markers serve as the framework on which the newly characterized functionally annotated ESTs can be mapped. According to the functional classification, each of three partners will map to the sugar beet 15 t JOint 1/RB-ASSBT Congress, 2611' Feb.-1st March 2003. San Antonio (USA) 253 SESSION GENETICS AND GERMPLASM ENHANCEMENT of tlic unigcnc of t/tc )lfllllf eel/. Fig. 2: I1istrihutwu c'( tlic 1111/lototcdfiinctions IICCOrtfillg difttTCiltfilllCfiollllf COIIIJllll'fllll'llfS FST set mtcgori:cd METABOLISM 1014 eNERGY 340 UNCLASSIFIED PROTEI~JS 1981 Cell I~ROWTH CELL DIVISIOfJ AND DNA SYNTHESIS 414 TRAfiSCRIPTION 505 CLASSIFICMION NOT YET Clci\RC'UTB17 PROT! IN SYNTHESIS 263 ORGANISM SPECIFIC PROTEINS •, PROTEIN Pl '\SMill f~'ROTEINS El ION ;,Q~, IN I R;,Cl-1 LULAR DEVELOPMENT '16 TR/1NSPCJR f :J/3 MOl lillY 0 C::ELLlJLAR DESTI~~,\! TR,\NSflQRT FACILITATION 72:) RETROlRANSPOSONSAND OF~t3f\NI.~·/, CELL lJLA.R 81Q(;F Nl S:S 36C liON 1:~nA CELLlJLAR C:OMMIJNICATIUf'J/SIC~N/\L TRMNSDUC I IO~J ,~2b Cl II RESI'tJE DEFEWil Ct ll DEATH ;-~j\J[! ,\C_;F INC C01 genetic genome an equal proportion of selected 1,000 ESTs Partner CAU will map ESTs with a putative function in pathogen resistance or genes related to known resistance genes, so called reststance gene analogues or RGAs. TraitGenetics is focused towards stress-related sequences, and MPI selected candidate genes involved in carbohydrate metabolism and beet development Several approaches to map these ESTs onto the sugar beet map are possible. The most advanced and sophisticated technology uses SNPs. single nucleotide polymorphisms Fig 3 shows an example of such a SNP \/ \, 'l!'.l I', I' '1'-"-1 I I' 'I' T!','l 1',\'l r' I'.''. I ·r· 1'1' I 'I 1'/r'l'l •1. l'i._l 'I ,1 ['• 'I . I!'.. \'Ti1 '1 "l:~·!·l!'\'i~'l'/>1 'I ~.1 T 254 1'/\'1 'I "I'< E'. '1"'J <'·'!'!':·,I'. 1'.' I ·,"I • 'I,Yl 'I /1'1'= Tt· r,, "! •\ I l"' 1''1 ·1 i'.l '·, 'l'r [, "j ir_ j, 'r /\1 t\ 1'"1' r r: I I' I/\ l'/1_1 nl't···l'iH I j' I I I' j'("'['i"; I '1'1' rr T1 I ('\ I, 'I< 'r T ·c,--.,,\r l'r ~ '"I '·1:' r\!'T·.TTrf .., ['r': ~t~Tr\Ti\ r I'= ' ,/1./\! l I ' '( 'I : 1\(~11' .i\, :·1 T ',-, ''j'( i'' "I'• Tr l ' 1st joint 1/RB-ASSBT Congress. 26u' Feb.-1st March 2003. San Antonio (USA) SESSION GENETICS AND GERMPLASM ENHANCEMENT A SNP is a single base pair difference between two sequences derived of the same genetic locus, e g a gene, from two different plants. Fig. 3 shows part of a sequence from the same locus of 9 different plants differing by only one base in certain plants, a G/C SNP. GABI-BEET has adopted a panel of 16 different plants provided by the breeders that includes wild beets These sugar beets largely represent the sugar beet gene pool and provide the opportunity to uncover many of the SNPs that exist in sugar beet Sequencing of all these plants for each of the 1,000 targets, ESTs and some RFLP framework markers, is a laborious and time consuming task but will be finished by end of 2003. More than 400,000 bases have been sequenced and analysed already and more than 1,500 SNPs detected; approx. 1 SNP every 70 base pairs SNP mapping is another milestone hard to achieve The specific advantages of using SNPs as the markers of the future are the potential of highly automated and high-throughput marker detection systems like MALDI-TOF, a chip-based method or the various very efficient primer-extension based scoring methods. Partner MPI will screen the available methods and make them available to the consortium. Model studies are already finished and large scale mapping can be started soon. However, only a small fraction of the detected SNPs are already mapped. The vast maJority of the ESTs will be mapped as final task at the end of the project The first 50 SNPs are mapped on the growing functional map of the sugar beet shown in Fig 4. Progress reports were presented at Plant and Animal Genome Conferences 2003 and 2002 (www pag-intl.org). A first publication out of the GABI-BEET consortium towards a functional map of the sugar beet genome was published by HUNGER eta/. (2003) rig. 4: 1\u iuft'gmtt'tlfiull"tioullilllllJt o/1/tc ~ugor/tcct gcuoutc. GABI BEET Integrated functional map of • sugar beet \) II Ill = 1:::=:1 c:::J = = = = = = = ~ El C.:..:l = == = = = = = = = \ II = C:-=-:1 = = == = = = ~ = = \"1 I I = ~ ~ = == !J'.G2.J = c=J = L:::J \I I\ = = = = = = = c::.=J = = = = = == = r:.;:s c:az, d'3 = = 1\ = = = c:::::f=l = = = f5l G:ZJ = = = r:=::J = c:::J ancho1 mad,cJ ltKI k.!cl c:::::J;lnchur marker luc1 c::::J r_s r mar!..er Colu~ne luCI Culogne 1st joint 1/RB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA) 255 SESSION GENETICS AND GERMPLASM ENHANCEMENT Touch stone of the SNP development and especially RGA mapping will be the mapping of the crown and root rot resistance genes. Disease resistance against the fungus Rhizoctonia so/ani is medium complex inherited. Several resistance genes may be involved but less than four major genes are expected (PANELLA & RUPPEL in SNEH eta/., 1996). Strube-Dieckmann produced several segregating plant populations for the specific needs of the GABI-BEET project (Fig. 5) and selected the most resistant one for the project (Fig. 6). Seed were handed over to lfZ that applied a newly developed, highly sophisticated bioassay to provide precise and reliable phenotypic scores for mapping the trait onto the functional map Finally mapped RGAs can be proved as candidate genes for the mapped resistance QTL, quantitative trait loci At present CAU is providing MLU with the corresponding marker information to fill up the existing map and to unravel Rhizoctonia resistance. Fig. 5: ncucloplllcnt u(a 111apping population scgrcgatiugfill' Rhizoctonia rcsistaucc at Strube-l! icckllltlllll. F2- .... PROJECT C) COMPARISON OF THE GENOME STRUCTURE OF WILD AND CULTIVATED BEET The genome of B vulgaris consists of approximately 60 % repeated DNA. The aim of this project performed by the CAU proJect partner is the identification, characterization and chromosomal localization of repetitive sequences The experimental work was focussed on the following research activities LTR Retrotransposons. The sugar beet Ty1-copia element Tbv1 (5.2 kb) is flanked by LTRs of 584-614 bp The variability of the LTR indicates a considerable divergence of Tbv1 within the B. vulgaris genome which is in line with the amplification and wide-spread distribution within Beta species. Fluorescent in situ hybridisation (FISH) revealed that Tbv1 is present on all B. vulgaris chromosomes and scattered throughout the genome (Fig. 7). 256 1st joint 1/RB-ASSBT Congress. 26th Feb.-1st March 2003, San Antonio (USA) SESSION GENETICS AND GERMPLASM ENHANCEMENT Fig. 6: Plrcnotypic cll!lmctcrization of three segregating F1 populations producedfor 1napping Rhizoctonia resistance- selection of tire best suited population for 111apping in CABI-BEET at Strubc-Diccklllmur. 50 40 Population-1 > c <) ~ Population-3 > 30 <) c ~ 20 25 0" 0" jg jg 10 0~~~~~~~~~~~~+-~ cy"'0 % disease root surtace 40 '),'0 c§' \)o(J v,'0 '0'0 '\'0 '0'0 cf' ~~'0 % disease root surface Populalion-2 ,--- > 30 g ~ 0" 20 t o jg 10 Jl.n.r-1 0 "'0 cy '),'0 "J(J \)o(J <f> '0'0 .,..-,,['l, '\'0 '0'0 o,C::, ~~'0 ('l'o d1sease root surface Fig. 7: Clrrolllosolllallomli:ation of Tlml. Mctaplrosc clrronrosonrcs of B. vulgaris ([)!\PI) B) Yellow-green lrylwidi:atiorr sigrroJ..., of"l/Jl•l C) ( )ucrlmj A) Class II Transposons. PCR assays were developed to detect En/Spm-like transposons in species of the genus Beta. Resulting PCR products have been cloned from B vulgaris, B corol/if/ora, B procumbens and B. nana Sequence analyses of 20 En!Spm fragments showed that these transposons are conserved (71.9 - 98.7 % identity at DNA level) Amplification of En/Spm-like transposons can also be demonstrated by FISH En/Spm-like transposons are highly amplified and dispersed on B. vulgaris chromosomes (Fig 8). Fig. 8: Clrrornosorrrol tlistrihution of En/Spnr. A) DAPI stained interplwsc and prornctapluN's of B. uulgaris Tire sar11c clrronwsonrcs after FISH zuitlr till Fn/Spm tmnsposasc fmgnzc 11 t B) 1st joint 1/RB-ASSBT Congress, 26t" Feb.-1st March 2003, San Antonio (USA) 257 SESSION GENETICS AND GERMPLASM ENHANCEMENT Physical mapping of BACs. Towards the integration of genetic and physical maps, the CAU project partner used their expertise in FISH to provide a technical platform for the physical mapping of candidate SAC clones, cf. Project D) above. In a pilot study in cooperation with GABI-BOL T CAU performed the physical mapping of three SACs around the bolting gene B by FISH (Fig. 9). Fig. 9: HAC-FISH ora clone front 11 cltrotJ/USOI/1111 rcgiu11 u{ tltc gene B A) DAJJI stained lltctapluN' and JlrOlltctapluN' of B. vulgaris H) )d/o<l'-grn·n signals u(fl!\l~ (arrmucd) C) ( Jucrlay I ig. 7. 8 a11d L} 1111711/ISrTijll ill arc courtesy of 1. Sclnuidt; prcpamtion /~ V Plant Breeding K1el PROJECT D) CONSTRUCTION OF A BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY FROM SUGAR BEET GENOME Bacterial artificial chromosomes, SACs, are molecular vectors to clone large DNA inserts They are built from both plasmid and bacterial genetic elements. They are integrated and inherited in bacteria as wild type chromosomes This Cloning tltc gr'IIOJ/tc in ll!\Cs, bacterial cHtificial chromosomes. fig. I 0: BACs Cloning of the Genome DNA is cloned I in BAGs large p1eces of genomic DNA (0 120 kbp) ~ 57,600 ~ ~· ·~ ~ insert size (average): 114kb~ genome equivalents: 8.7 No. clones: (57,600 X 114 kb I 5 CCAT AGGCl A(_;GC rT AAT 3 GGTATCCGATCCGAATTA transfected to bacteria ordered in libraries BAC library spotted on filters 750Mb) BAC, Bactenal Ar11ficia/ Chromosome 258 1st joint 1/RB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA) SESSION GENETICS AND GERMPLASM ENHANCEMENT tool provides the means to clone complete plant genomes in manageable numbers of clones with realistic insert fragment sizes. Sugar beet DNA was cut into pieces using restriction enzymes and randomly cloned into such BACs. It is viable to store frozen BACs for the long-term in large arrays. In parallel BAC DNA was spotted on very dense ordered filter membrane grids for further hybridisation, characterization and identification (Fig 10) Partners CAU and TraitGenetics have put together their expertise and equipment to produce a genomic BAC library of 57,600 clones with an average insert size of 114 kbp (8,7 genome equivalents; HOHMANN eta!. submitted) The BAC-Iibrary exists in multiple copies and is already in use by the GABI consortium (Fig. 11) l .;;;() kh I I Ill kh .::;1) kh B Fig. 11: A Vu/ido!Jo/1 oftlir' !i!\C Jif,mn; A) Sr'lltill'm lllfl'rit!r:.ntion t'{t7 li/\C~tiltcr !Pill! r/JNA B) I mg111mf ~/:r' Sr'J'IIflll/rl/1 o( mntlo111 !i!\C /1/SrTis I JN/\. t'/gt'/IOII/ic sngnr l'l'!'i ACKNOWLEDGEMENTS The GABI-BEET proJect 1s supported by German ministry BMBF (GABI grant 0312283) and the involved companies. GABI-BEET is a consortium dnven proJect All partners have contributed irregardless whether they are mentioned in this paper or not I acknowledge to all the groups and group members their specific and un1que contributions Without the1r help this paper would not be made possible Many thanks to Stephen Rudd who corrected the English and Bruno Desprez for the French translation 151 joint 1/RB-ASSBT Congress. 26tt' Feb -1 51 March 2003. San Antonio (USA) 259 SESSION GENETICS AND GERMPLASM ENHANCEMENT REFERENCES 1. HERWIG, R et a/.: Construction of a 'unigene' eDNA clone set by oligonucleotide fingerprinting allows access to 25 000 potential sugar beet genes The Plant Journal, 32, 845-857, 2002. 2. HUNGER, S et a! Isolation and linkage analysis of expressed diseaseresistance gene analogues of sugar beet (Beta vulgaris L) Genome, 46, 70-82, 2003. 3 PANELLA, L and E. Kluwer 1996 4. HOHMANN, U et a!.· A bacterial artificial chromosome (BAC) library of sugar beet and a physical map comprising the bolting gene B. submitted 260 1st joint 1/RB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA) RUPPEL in SNEH et a!. (eds) Rhizoctonia species