PHASEOMICS EST WORKGROUP
IntroductionMolecular techniques are radically altering the way
that plant breeding is being performed. In a sense this is surprising
for the individual methods that derive from biochemistry, physiology,
genetics, structural biology, and informatics are hardly new. What
has changed however is the scale at which genes can be sequenced, their
expression analysed, and proteins identified. Genomics, transcriptomics,
and proteomics (when applied to beans we call them Phaseomics) permit
the study of many (and sometimes all) genes of a particular organism.
Significant discoveries concerning the inter-relationships between some
of the basic metabolic functions of an organism have been made this way.
As a consequence, an integrated, almost holistic view of the organism
is evolving. What were once thought to be separate, unrelated functions
are now seen as part of a complex network of interacting genes and their
products.
Perhaps the most important information necessary to address both fundamental and applied questions in the agricultural and biological sciences is the basic DNA sequence. There are several ways of obtaining molecular markers and one of the cheaper is to sequence messenger RNA’s extracted from tissues of interest. These so-called expressed sequence-tags (ESTs) are short (450-600 bp) sequences that are like milestones on a chromosome. Breeders can use them to position other genes. Judicious selection of the type of tissue from which to isolate the mRNA (and hence prepare a cDNA library) provides valuable information not only on the type of genes found in a particular plant, but also on the conditions in which they are expressed. EST projects thus permit “skimming” of the genome. How much information they gather is dependent on pre-existing information as well as on the abundance of mRNAs, their stability and so on. Nevertheless, they are a cheap and efficient way of generating data that can be directly applied in traditional breeding programmes.
Why this web site During the 2nd Phaseomics meeting in Geneva (May
2002), it was suggested that different working groups should be formed
to concentrate on certain of the Phaseomics goals. One of these was a
workgroup on EST sequencing and transcriptome analysis with the aim of
continually exchanging information amongst the participants of the EST
sequencing projects, and to avoid duplications. Gina Hernández
of CIFN-UNAM in Cuernavaca, Mexico will coordinate these efforts. The
website will provide links to related sites.
Members of the EST workgroupinclude: Gina Hernández,
Miguel Lara, Mario Ramírez, Carroll P.Vance, Jos Vanderleyden,
Jan Michiels, Ellen Luyten, Carla Snoeck, Maeli Melotto, Bob Haselkorn,
Mathew Blair, Steeve Beebe, Joe Tohme, Jean-Jacques Drevon, Farida Shah,
Francesca Sparvoli, and William Broughton. Anyone, either within (or outside)
Phaseomics that wants to contribute is welcome to join. Simply E-mail
Gina to have your name added to
the list.
StatusThe article containing the Phaseomics global project
(“Beans (Phaseolus spp.) - Model Food Legumes” W.J. Broughton, G.
Hermández, M. Blair, S. Beebe, P. Gepts and J. Vanderleyden. 2002.
Plant & Soil. In press) provides a detailed description of the specific
EST projects that the groups from the consortium plan to do or are currently
doing. This information is summarised in Table 4.1 of that article.
Below is a summary of current funded projects
that are being developed by different Phaseomics groups. In contrast to
Table. 4.1, the data listed below summaries progress and/or the short-term
goals of each project. This information will be updated periodically.
Current Phaseomics EST sequencing projects from cDNA
libraries:
Matthew Blair, Steve Beebe and Joe Tohme from CIAT (Colombia) have prepared a cDNA library from leaves from G19833 genotype, 64 000 clones have been picked and approximately 2,000 clones have been sequenced; approximately 500 EST´s have been submitted to Genebank. From the DOR364 genotype they have prepared cDNA libraries from young lateral and basal roots treated with limited or unlimited P concentrations. From each treatment, +/- P, 32,000 clones have been picked of which 1,000 clones have been sequenced. In addition another two cDNA libraries are in preparation; one from anthracose infected leaf tissue (GA19833 genotype) and the other, in collaboration with group of J.-J. Drevon from INRA-Montpellier (France), from nodular cortex tissue from the BAT477 genotype P treatment, inoculated with R. tropici 899. EST sequencing from this library is being done in CIAT, in collaboration with the group of M. Blair. In relation with the later, a study of aquaporin diversity in BAT477 nodules is under progress in INRA- Montpellier. M. Melotto et al. from ESALQ/U. Sao Paulo (Brasil) are preparing two cDNA libraries from seedling shoots of the SEL1308 genotype +/- inoculation with C. lindemuthianum 20,000 clones have been picked, and they will sequence a total of 10,000 ESTs 5,000 from each library J. Vanderleyden, J. Michiels, E. Luyten and C. Snoeck from CPM-U. Leuven (Belgium) are preparing a cDNA library from nodules (10 days post-inoculation) of the BAT477 genotype inoculated with R. etli CNAPF512. cDNA will be sequenced AFLPs generated and will micro-array analyses performed. G. Hernández, M. Lara and M. Ramírez from CIFN-UNAM, Cuernavaca (Mexico) in collaboration with C.P. Vance from U. Minnesota-USDA (USA) have sequenced 3,000 ESTs from a cDNA library of nodules (18 days pot-inoculation)(Jamapa 81 variety) inoculated with R. tropici 899. Nodule ESTs were grouped and their expression measured in roots, leaves, and nodules using macro-arrays. Libraries were also prepared from mRNA isolated from developing pods and phosphorus-stressed roots. 3,000 cDNA clones will be sequenced from both pods and roots. W.J. Broughton, LBMPS – U. Geneva is currently isolating mRNA from root-hairs (BAT93) treated with Rhizobium sp. NGR234 and its Nod-factors. F. Sparvoli and R. Bollini, IBBA-CNR, Milan (Italy), are currently isolating mRNAs from developing cotyledons of Phasolus lunatus (PHA8152) treated or not with the glycosylation inhibitor tunicamycin. Subtracted cDNA libraries will be prepared and searched for up- and down-regulated cDNAs Faridah Shah, UKM, Malaysia will prepare cDNA libraries
and sequence ESTs of flowers and pods from P. vulgaris plants that have
been challenged with pathogens such as the white mould, and also of roots
from plants that have been grown on different soil conditions. The possibility and advantages of having a common database
on EST sequencing data for the Phaseomics consortium was discussed in
Geneva, but no decision was taken. Each group has or will have their internal
database and will share the EST data with the public through Genebank.
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