Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes.

KEY MESSAGE: Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. Effective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5-5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40-85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.

The loss of vernalization requirement in narrow-leafed lupin is associated with a deletion in the promoter and de-repressed expression of a Flowering Locus T (FT) homologue.

Adaptation 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. Co-segregation 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.

Expansion of the phosphatidylethanolamine binding protein family in legumes: a case study of Lupinus angustifolius L. FLOWERING LOCUS T homologs, LanFTc1 and LanFTc2.

BACKGROUND: The 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. RESULTS: Bacterial 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. CONCLUSIONS: Numerous 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.

Integration of Lupinus angustifolius L. (narrow-leafed lupin) genome maps and comparative mapping within legumes.

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.

Remnants of the Legume Ancestral Genome Preserved in Gene-Rich Regions: Insights from Lupinus angustifolius Physical, Genetic, and Comparative Mapping.

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.

Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics.

BACKGROUND: The 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. RESULTS: The 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. CONCLUSIONS: In 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.