Abstract
BackgroundBituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species.Results432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme.ConclusionsB. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.
Highlights
Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds
Sampling the B. bituminosa leaf transcriptome by Roche 454 sequencing Sequencing of the B. bituminosa leaf-derived cDNA library on the GS-FLX System resulted in 432,306 sequence reads with an average length of 345 bp (149.1 Mbp)
GS De Novo Assembler software assembled 266,461 (61.6%) of the reads into 4,542 contigs that were ≥ 100 bp in length. It grouped contigs into 2,929 “isogroups” and 3,798 “isotigs” with an average isotig length of 707 base pairs. 3657 isotigs and remaining singleton contig sequences that were ≥ 200 bp in length were deposited at NCBI Transcriptome a)
Summary
Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. Bituminaria bituminosa (L.) C.H. Stirt., commonly known as Tedera in the Canary Islands, is a perennial legume species widely distributed in the Mediterranean Basin and Macaronesia. Stirt., commonly known as Tedera in the Canary Islands, is a perennial legume species widely distributed in the Mediterranean Basin and Macaronesia It is a self-pollinated diploid species (2n = 20) with DNA content estimated to be between 0.998 and 1.094 pg DNA per diploid nucleus [1,2,3]. B. bituminosa shows high diversity in the Canary Islands, with three recognised botanical varieties: (i) var. Bituminosa is found [6] These botanical varieties were identified using morphological characteristics and were largely supported by preliminary molecular analyses using arbitrary DNA markers [7,8]. There is some ambiguity in botanical variety definitions that warrants further investigation
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