Abstract
BackgroundCommon bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean.ResultsTwo full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes.ConclusionsThe present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.
Highlights
Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods
The libraries were based on totals of 3.789 mg and 4.258 mg of high-quality total RNA obtained from the six different irrigation × soil treatments for these two genotypes, respectively, which was sufficient for the highly complex process of full-length mRNA selection from polyA mRNAs
In our functional analysis of the unigenes discovered in the full-length cDNA library, we found that BLAST2GO found a range of hits from our low threshold of 1 × 10-10 up to 1 × 10-175 and similarity values ranging from 40 to 100% alignment within a range of nucleotide windows
Summary
Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. A large effort has gone into constructing many different cDNA libraries for major legume crops such as soybean [5] and model legume species such as Lotus japonicus [6] and barrel medic, Medicago truncatula [7]. For the legumes a total of over 3 million sequences have been generated with the largest numbers in soybean (1.5 million) and the model legumes barrel medic (280,000) and lotus (242,000). This compares to over 6 million sequences in the Gramineae and nearly 3 million in the Brassicaceae
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