Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.).

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed-protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano-LC-MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty-seven of the 152 proteins were associated with KEGG-defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed-protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).

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

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.

FLOWERING LOCUS T indel variants confer vernalization-independent and photoperiod-insensitive flowering of yellow lupin (Lupinus luteus L.).

Ongoing climate change has considerably reduced the seasonal window for crop vernalization, concurrently expanding cultivation area into northern latitudes with long-day photoperiod. To address these changes, cool season legume breeders need to understand molecular control of vernalization and photoperiod. A key floral transition gene integrating signals from these pathways is the Flowering locus T (FT). Here, a recently domesticated grain legume, yellow lupin (Lupinus luteus L.), was explored for potential involvement of FT homologues in abolition of vernalization and photoperiod requirements. Two FTa (LlutFTa1a and LlutFTa1b) and FTc (LlutFTc1 and LlutFTc2) homologues were identified and sequenced for two contrasting parents of a reference recombinant inbred line (RIL) population, an early-flowering cultivar Wodjil and a late-flowering wild-type P28213. Large deletions were detected in the 5' promoter regions of three FT homologues. Quantitative trait loci were identified for flowering time and vernalization response in the RIL population and in a diverse panel of wild and domesticated accessions. A 2227 bp deletion found in the LlutFTc1 promoter was linked with early phenology and vernalization independence, whereas LlutFTa1a and LlutFTc2 indels with photoperiod responsiveness. Comparative mapping highlighted convergence of FTc1 indel evolution in two Old World lupin species, addressing both artificial selection during domestication and natural adaptation to short season environmental conditions. We concluded that rapid flowering in yellow lupin is associated with the de-repression of the LlutFTc1 homologue from the juvenile phase, putatively due to the elimination of all binding sites in the promoter region for the AGAMOUS-like 15 transcription factor.

Development and characterization of InDel markers for Lupinus luteus L. (Fabaceae) and cross-species amplification in other Lupin species

Background: Strong artificial selection and/or natural bottle necks may limit genetic variation in domesticated species. Lupinus luteus, an orphan temperate crop, has suffered diversity reductions during its bitter/sweet alkaloid domestication history, limiting breeding efforts and making molecular marker development a difficult task. The main goal of this research was to generate new polymorphic insertion­deletion (InDel) markers to aid yellow lupin genetics and breeding. By combining genomic reduction libraries and next generation sequencing, several polymorphic InDel markers were developed for L. luteus L. Results: A total of 118 InDel in silico polymorphic markers were identified. Eighteen InDel primer sets were evaluated in a diverse L. luteus core collection, where amplified between 2­3 alleles per locus. Observed heterozygosity (HO; 0.0648 to 0.5564) and polymorphic information content (PIC; 0.06 to 0.48) estimations revealed a moderate level of genetic variation across L. luteus accessions. In addition, ten and nine InDel loci amplified successfully Lupinus hispanicus Boiss & Reut, and Lupinus mutabilis Sweet, respectively, two L. luteus close relatives. PCA analysis identified two L. luteus clusters, most likely explained by the domestication species history. Conclusion: The development of InDel markers will facilitate the study of genetic diversity across L. luteus populations, as well as among closely related species.

A six nuclear gene phylogeny of Citrus (Rutaceae) taking into account hybridization and lineage sorting.

BACKGROUND: Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4-12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species. METHODOLOGY AND PRINCIPAL FINDINGS: (1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. CONCLUSIONS: We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches-gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus.

Identification of a low digestibility δ-Conglutin in yellow lupin (Lupinus luteus L.) seed meal for atlantic salmon (Salmo salar L.) by coupling 2D-PAGE and mass spectrometry.

The need of quality protein in the aquaculture sector has forced the incorporation of alternative plant proteins into feeding diets. However, most plant proteins show lower digestibility levels than fish meal proteins, especially in carnivorous fishes. Manipulation of protein content by plant breeding can improve the digestibility rate of plant proteins in fish, but the identification of low digestibility proteins is essential. A reduction of low digestibility proteins will not only increase feed efficiency, but also reduce water pollution. Little is known about specific digestible protein profiles and/or molecular identification of more bioavailable plant proteins in fish diets. In this study, we identified low digestibility L. luteus seed proteins using Atlantic salmon (Salmo salar) crude digestive enzymes in an in vitro assay. Low digestibility proteins were identified by comparing SDS-PAGE banding profiles of digested and non-digested lupin seed proteins. Gel image analysis detected a major 12 kDa protein band in both lupin meal and protein isolate digested products. The 12 kDa was confirmed by 2D-PAGE gels and the extracted protein was analyzed with an ion trap mass spectrometer in tandem mass mode. The MS/MS data showed that the 12 kDa low digestibility protein was a large chain δconglutin, a common seed storage protein of yellow lupin. Comparison of the protein band profiles between lupin meal and protein isolates showed that the isolatation process did not affect the low digestibility of the 12 kDa protein.

The reticulate history of Medicago (Fabaceae).

The phylogenetic history of Medicago was examined for 60 accessions from 56 species using two nuclear genes (CNGC5 and beta-cop) and one mitochondrial region (rpS14-cob). The results of several analyses revealed that extensive robustly supported incongruence exists among the nuclear genes, the cause of which we seek to explain. After rejecting several processes, hybridization and lineage sorting of ancestral polymorphisms remained as the most likely factors promoting incongruence. Using coalescence simulations, we rejected lineage sorting alone as an explanation of the differences among gene trees. The results indicate that hybridization has been common and ongoing among lineages since the origin of Medicago. Coalescence provides a good framework to test the causes of incongruence commonly seen among gene trees but requires knowledge of effective population sizes and generation times. We estimated the effective population size at 240,000 individuals and assumed a generation time of 1 year in Medicago (many are annual plants). A sensitivity analysis showed that our conclusions remain unchanged using a larger effective population size and/or longer generation time.