This case study shows how bioinformatics in general, and CLC Combined Workbench in particular, can help researchers gain deeper knowledge of the complex processes involved in flower development in Daucus carota.

The study focuses on how the alignment-based primer design functionalities of the CLC Combined Workbench make it possible to design degenerate primers to amplify a set of conserved genes in Daucus carota. The research has been carried out at the Laboratory of Gene Expression at the University of Aarhus, Denmark.

Early flowering in Arabidopsis thaliana

Flowering plants, or angiosperms, represent one of the most successful and diverse groups of organisms on the planet, with more than 250,000 species in the wild. Although angiosperms such as orchids, roses and snapdragons have very distinctive flowers, most flowers contain just four organ types and their development involves highly conserved molecular mechanisms [Krizek and Fletcher, 2005].

Flowers, like shoot and leaves, derive from the shoot apical meristem (SAM). Whereas leaves arise early in the plant life cycle, flowers are only produced after transition of the SAM from vegetative to reproductive growth. The timing of the transition is crucial to reproductive success and as such must take place at the exact right time.

In Arabidopsis thaliana the switch from vegetative to reproductive growth is governed by four main pathways (see figure 2). Signals transmitted by genes belonging to each pathway converge at the Floral Pathway Integrators. These activate and/or up-regulate the expression of the Floral Meristem Identity (FMI) genes responsible for the establishment of the floral meristem [Simpson et al., 1999]. The FMI genes in turn activate the Floral Organ Identity genes, required for specifying the identity of the floral organs [Krizek and Fletcher, 2005].

Of the 15 genes central to early flower development, nine belong to a group of homeotic genes known as MADS-box genes, sharing a conserved region of approximately 180 bp in the 5'-end. Due to the high degree of conservation, degenerate primers targeting this region can be designed. This enables a broad search for MADS-box genes in virtually any organism.

Expressed in vegetative as well as floral tissues, MADS-box genes are likely to be involved in many aspects of plant development. They have been successfully cloned from more than 40 different plant genera [Purugganan et al., 1995]. In the Arabidopsis thalianagenome alone, more than 100 sequences have been recovered and although less than 20% are characterized, many have been shown to be involved in several aspects of bolting and flowering [Parenicová et al., 2003], [Martinez-Castilla and Alvarez-Buylla, 2003].

MADS-box genes in carrot (Daucus carota)

Cytoplasmic male sterility (CMS) results from incompatibility between the nucleus and cytoplasm. CMS plants are not capable of normal pollen development, in that maturation of viable pollen is prevented. In addition, flower phenotype is altered. The phenotypes resemble homeotic flower mutants of Arabidopsis thaliana.

In carrot, five nuclear genes involved in the expression of two CMS phenotypes have been identified. The five clones were aligned with putative equivalents of other plant species, and due to their homology to homeotic genes of Antirrhinum majus and Arabidopsis thaliana the genes were named DcMADS1-5 [Linke et al., 2003].

The research performed in the Laboratory of Gene Expression at the University of Aarhus alternated between tasks performed in the lab and bioinformatics tasks using the CLC Combined Workbench. Below, the work performed is summarized.

Research

The purpose of the research was to identify genes involved in the floral initiation process in carrots (Daucus carota cv. 'Nantes') and focus was set on the MADS-box genes. The work flow is outlined in figure 3.

The CLC Combined Workbench was used to search for, import and align the cDNA sequences of DcMADS1-5. Then, a pair of degenerate primers were designed targeting the conserved MADS-box region. These primers were used for PCR amplification of genomic DNA isolated from leaves of 19 weeks old carrots. The resulting fragments were cloned into the TOPO vector and sequenced.

The trace files were imported into the CLC Combined Workbench, where the forward and reverse reads were assembled. BLAST searches against the NCBI database were performed using BLASTx which translates the sequence into amino acid sequences before BLASTing against a protein database. Based on the results of the BLAST searches, the clones suggested to be fragments of MADS-box genes were selected. Based on the nucleotide sequence, primers for inverse PCR (iPCR) were designed.

Back in the lab, the positive clones were labeled with ³²P-dATP and used to screen a carrot genomic library. Positive λ-clones were isolated, DNA extracted and subjected to iPCR. The resulting amplicons were cloned into the TOPO vector and processed as described above.

The screening of the genomic library remains a work in progress and at this point in time putative MADS-box genes have been selected and annotations describing the regions identified in each clone were manually added. The images generated within the CLC Combined Workbench have been exported in high-quality graphics formats for use in presentations etc.

In the following sections, two of the steps in the work flow are described in further detail; The alignment-based primer design and export of images.

Zooming in on Alignment-based primer design

The CLC Combined Workbench makes it possible to align several sequences and design degenerate primer sets in regions of homology.

Annotations from the original nucleotide sequences or alignment are still visible within the primer design viewer, which is a big help when the primers are required to lie within specific regions. In figure 4, you can see the yellow annotations indicating the coding regions of the MADS-box genes.

The conservation is shown as a blue-to-red gradient background color making it easy to visually find the highly conserved regions of the alignment. These regions are defined as locations for the forward and the reverse primers as shown with the gray annotation in figure 4.

The parameters regarding the individual properties of the primers are adjusted in the Side Panel. In addition to normal primer settings, such as primer length, melting temperature, buffer properties and degree of self annealing, the alignment-based primer design makes it possible to select degree of degeneracy, or the number of mismatches acceptable.

When all the parameters are set, the CLC Combined Workbench calculates the best combinations of primer pairs for the selected regions. The properties for the primer pairs, such as melting temperature and self annealing, are inspected before deciding on a primer pair.

Zooming in on Export of images

In figure 5 you can see the graphical result of the first phase of the research described in this case study.

The graphics generated in the CLC Combined Workbench are easily exported in a variety of graphics formats such as gif, jpeg, png and pdf.

The CLC Combined Workbench graphics export is based on a WYSIWYG principle: What You See Is What You Get. This means that you can configure the layout of e.g. a sequence or alignment in the program and when you export this to a graphics file, it will look exactly the same as in the Workbench. This makes it easy to create good-looking illustrations of research findings for publications and presentations.

The annotations on the sequence shown in figure 5 illustrate putative MADS-box genes and the annotations furthermore describe the regions identified in each clone. This illustration is used for communicating the results of the research in reports and presentations.