SummaryAdaptation of Lupinus angustifolius (narrow-leafed lupin) to cropping in southern Australian and northern Europe was transformed by a dominant mutation (Ku) that removed vernalization requirement for flowering. The Ku mutation is now widely used in lupin breeding to confer early flowering and maturity. We report here the identity of the Ku mutation.We used a range of genetic, genomic and gene expression approaches to determine whether Flowering Locus T (FT) homologues are associated with the Ku locus.One of four FT homologues present in the narrow-leafed lupin genome, LanFTc1, perfectly co-segregated with the Ku locus in a reference mapping population. Expression of LanFTc1 in the ku (late-flowering) parent was strongly induced by vernalization, in contrast to the Ku (early-flowering) parent, which showed constitutively high LanFTc1 expression. Cosegregation of this expression phenotype with the LanFTc1 genotype indicated that the Ku mutation impairs cis-regulation of LanFTc1. Sequencing of LanFTc1 revealed a 1.4-kb deletion in the promoter region, which was perfectly predictive of vernalization response in 216 wild and domesticated accessions. Linkage disequilibrium rapidly decayed around LanFTc1, suggesting that this deletion caused the loss of vernalization response. This is the first time a legume FTc subclade gene has been implicated in the vernalization response.

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.

White lupin (Lupinus albus L.) is a valuable source of seed protein, carbohydrates and oil, but requires genetic improvement to attain its agronomic potential. This study aimed to (i) develop a new high-density consensus linkage map based on new, transcriptome-anchored markers; (ii) map four important agronomic traits, namely, vernalization requirement, seed alkaloid content, and resistance to anthracnose and Phomopsis stem blight; and, (iii) define regions of synteny between the L. albus and narrow-leafed lupin (L. angustifolius L.) genomes. Mapping of white lupin quantitative trait loci (QTLs) revealed polygenic control of vernalization responsiveness and anthracnose resistance, as well as a single locus regulating seed alkaloid content. We found high sequence collinearity between white and narrow-leafed lupin genomes. Interestingly, the white lupin QTLs did not correspond to previously mapped narrow-leafed lupin loci conferring vernalization independence, anthracnose resistance, low alkaloids and Phomopsis stem blight resistance, highlighting different genetic control of these traits. Our suite of allele-sequenced and PCR validated markers tagging these QTLs is immediately applicable for marker-assisted selection in white lupin breeding. The consensus map constitutes a platform for synteny-based gene cloning approaches and can support the forthcoming white lupin genome sequencing efforts.

The legume genus, Lupinus, has many notable properties that make it interesting from a scientific perspective, including its basal position in the evolution of Papilionoid legumes. As the most economically important legume species, L. angustifolius L. (narrow-leafed lupin) has been subjected to much genetic analysis including linkage mapping and genomic library development. Cytogenetic analysis has been hindered by the large number of small morphologically uniform chromosomes (2n = 40). Here, we present a significant advance: the development of chromosome-specific cytogenetic markers and assignment of the first genetic linkage groups (LGs) to chromosomal maps of L. angustifolius using the bacterial artificial chromosome (BAC)-fluorescence in situ hybridization approach. Twelve clones produced single-locus signals that "landed" on 7 different chromosomes. Based on BAC-end sequences of those clones, genetic markers were generated. Eight clones localized on 3 chromosomes, allowed these chromosomes to be assigned to 3 LGs. An additional single-locus clone may be useful to combine an unassigned group (Cluster-2) with main LGs. This work provides a strong foundation for future identification of all chromosomes with specific markers and for complete integration of narrow-leafed lupin LGs. This resource will greatly facilitate the chromosome assignment and ordering of sequence contigs in sequencing the L. angustifolius genome.

BackgroundThe narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome.ResultsThe genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs.ConclusionsIn general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that not only was the gene nucleotide sequence conserved between soybean and lupin GRRs, but the order and orientation of particular genes in syntenic blocks was homologous, as well. These findings will be valuable to the forthcoming sequencing of the lupin genome.

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10577-016-9526-8) contains supplementary material, which is available to authorized users.

BackgroundThe Arabidopsis FLOWERING LOCUS T (FT) gene, a member of the phosphatidylethanolamine binding protein (PEBP) family, is a major controller of flowering in response to photoperiod, vernalization and light quality. In legumes, FT evolved into three, functionally diversified clades, FTa, FTb and FTc. A milestone achievement in narrow-leafed lupin (Lupinus angustifolius L.) domestication was the loss of vernalization responsiveness at the Ku locus. Recently, one of two existing L. angustifolius homologs of FTc, LanFTc1, was revealed to be the gene underlying Ku. It is the first recorded involvement of an FTc homologue in vernalization. The evolutionary basis of this phenomenon in lupin has not yet been deciphered.ResultsBacterial artificial chromosome (BAC) clones carrying LanFTc1 and LanFTc2 genes were localized in different mitotic chromosomes and constituted sequence-specific landmarks for linkage groups NLL-10 and NLL-17. BAC-derived superscaffolds containing LanFTc genes revealed clear microsyntenic patterns to genome sequences of nine legume species. Superscaffold-1 carrying LanFTc1 aligned to regions encoding one or more FT-like genes whereas superscaffold-2 mapped to a region lacking such a homolog. Comparative mapping of the L. angustifolius genome assembly anchored to linkage map localized superscaffold-1 in the middle of a 15 cM conserved, collinear region. In contrast, superscaffold-2 was found at the edge of a 20 cM syntenic block containing highly disrupted collinearity at the LanFTc2 locus. 118 PEBP-family full-length homologs were identified in 10 legume genomes. Bayesian phylogenetic inference provided novel evidence supporting the hypothesis that whole-genome and tandem duplications contributed to expansion of PEBP-family genes in legumes. Duplicated genes were subjected to strong purifying selection. Promoter analysis of FT genes revealed no statistically significant sequence similarity between duplicated copies; only RE-alpha and CCAAT-box motifs were found at conserved positions and orientations.ConclusionsNumerous lineage-specific duplications occurred during the evolution of legume PEBP-family genes. Whole-genome duplications resulted in the origin of subclades FTa, FTb and FTc and in the multiplication of FTa and FTb copy number. LanFTc1 is located in the region conserved among all main lineages of Papilionoideae. LanFTc1 is a direct descendant of ancestral FTc, whereas LanFTc2 appeared by subsequent duplication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3150-z) contains supplementary material, which is available to authorized users.

The narrow-leafed lupin (Lupinus angustifolius) was recently considered as a legume reference species. Genetic resources have been developed, including a draft genome sequence, linkage maps, nuclear DNA libraries, and cytogenetic chromosome-specific landmarks. Here, we used a complex approach, involving DNA fingerprinting, sequencing, genetic mapping, and molecular cytogenetics, to localize and analyze L. angustifolius gene-rich regions (GRRs). A L. angustifolius genomic bacterial artificial chromosome (BAC) library was screened with short sequence repeat (SSR)-based probes. Selected BACs were fingerprinted and assembled into contigs. BAC-end sequence (BES) annotation allowed us to choose clones for sequencing, targeting GRRs. Additionally, BESs were aligned to the scaffolds of the genome sequence. The genetic map was supplemented with 35 BES-derived markers, distributed in 14 linkage groups and tagging 37 scaffolds. The identified GRRs had an average gene density of 19.6 genes/100 kb and physical-to-genetic distance ratios of 11 to 109 kb/cM. Physical and genetic mapping was supported by multi-BAC-fluorescence in situ hybridization (FISH), and five new linkage groups were assigned to the chromosomes. Syntenic links to the genome sequences of five legume species (Medicago truncatula, Glycine max, Lotus japonicus, Phaseolus vulgaris, and Cajanus cajan) were identified. The comparative mapping of the two largest lupin GRRs provides novel evidence for ancient duplications in all of the studied species. These regions are conserved among representatives of the main clades of Papilionoideae. Furthermore, despite the complex evolution of legumes, some segments of the nuclear genome were not substantially modified and retained their quasi-ancestral structures. Cytogenetic markers anchored in these regions constitute a platform for heterologous mapping of legume genomes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11105-014-0730-4) contains supplementary material, which is available to authorized users.