Yellow lupin (Lupinus luteus L.) transcriptome sequencing: molecular marker development and comparative studies

Abstract

Background: Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species.

Results: Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession's origin.

Conclusion: L. luteus deep transcriptome sequencing will facilitate the further development of genomic tools and lupin germplasm. Massive sequencing of cDNA libraries will continue to produce raw materials for gene discovery, identification of polymorphisms (SNPs, EST-SSRs, INDELs, etc.) for marker development, anchoring sequences for genome comparisons and putative gene candidates for QTL detection.

Figure 1

GO term annotations for L1L2. Isotigs were grouped under three categories: (a) molecular function, (b) biological processes, and (c) cellular components. Numbers between parentheses indicate the number of positive matches for each function.

Figure 2

Venn diagram summarizing the distribution of tBlastX matches between L. luteus and four model species (A. thaliana, M. truncatula, L. japonicus and G. max). Numbers following the model species correspond to the size of the respective data base. Numbers within the Venn diagram indicate the number of sequences sharing similarity using tBLASTx. Numbers within parenthesis indicate the percentage of matches in terms of the total number of L. luteus sequences.

Figure 3

Microsyntenic L. luteus DNA fragments mapped on the Medicago genome using a GBrowse platform. (a) L. luteus microsyntenic region 13 on M. truncatula chromosome 1; (b) L. luteus microsyntenic region 5 on M. truncatula chromosome 1; (c) L. luteus microsyntenic region 11 on M. truncatula chromosome 2.

Figure 4

Alignment of L. luteus, L. hispanicus and L. mutabilis containing several repeat motifs. (a) isotig03739 with GA and AGA motifs; (b) isotig16318 with a TAA motif; and (c) isotig21236 with a GAA motif.

Figure 5

Neighbour Joining tree relating the 64  L. luteus accessions included in the diversity study. Numbers above branches correspond to bootstrap values. Accessions are identified by a letter L followed by numbers. Letters around accessions identify country of origin based on seed bank or breeding histories (RUS: Russia, ISRL: Israel, HUNG: Hungary, CHIL: Chile, GER: Germany, SPN: Spain, PORT: Portugal, MORO: Morocco, POL: Poland, BYS: Belarus, UKR: Ukraine). The scale is in distance units.