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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),
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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
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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
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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
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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
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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.
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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
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arc courtesy of 1. Sclnuidt;
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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
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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)
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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
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1st joint 1/RB-ASSBT Congress, 26th Feb.-1st March 2003, San Antonio (USA)
RUPPEL
in
SNEH
et a!. (eds) Rhizoctonia species