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

Abstract

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.

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

“…Both purified Sonet amplicons were used to screen the Sonet BAC clone library (Kasprzak et al ., ) for clones containing LanFTc1 and LanFTc2 using the methods described by Książkiewicz et al . (). In brief, radiolabelled 231‐ and 293‐bp Sonet amplicons were hybridized to high‐density DNA macroarrays containing 55 296 BAC clones.…”

“…Of all the known species of genistoid legume (> 2400; Cardoso et al ., ), it has the most advanced set of genomic resources including a comprehensive transcriptome‐based unigene assembly, two genome survey sequences, two large insert genomic libraries and extensive collections of wild and domesticated germplasm (Kasprzak et al ., ; Gao et al ., ; Berger et al ., ; Yang et al ., ; Cannon et al ., ; Kamphuis et al ., ; Smýkal et al ., ). Gene‐rich regions of L. angustifolius revealed well‐conserved microsyntenic links to five reference legume species, which facilitated gene annotation by sequence similarity (Książkiewicz et al ., , ). It is therefore a highly suitable model system for investigating the FT clade in legumes.…”

Validation of Diaporthe toxica resistance markers in European Lupinus angustifolius germplasm and identification of novel resistance donors for marker-assisted selection

“…Nevertheless, deciphering the molecular background underlying the Ku locus took more than decade of extensive research involving various techniques, from BAC library screening via DNA hybridization, restriction siteassociated physical and linkage mapping, fluorescent in situ hybridization of DNA probes in metaphase chromosomes, Sanger, 454 and Hi-seq sequencing to gene expression profiling (Książkiewicz et al 2016;Nelson et al 2017Nelson et al , 2006. Although a similar BAC library approach was applied to L. angustifolius Phomopsis stem blight resistance markers PhtjM2 (linked with PhtjR) and Ph258M2 (Phr1), it did not yield resistance gene identification or the development of tightly linked diagnostic markers (Książkiewicz et al 2013(Książkiewicz et al , 2015. Those studies showed convincingly that MFLP-derived probes are not suitable for positional cloning of particular genes because they hybridize to numerous loci dispersed in the genome, localized both in repetitive and gene-rich regions.…”

“…Highdensity linkage maps carrying thousands of markers, including gene-based sequence tagged sites (STS), were constructed and aligned to the draft genome sequence (Hane et al 2017;Kamphuis et al 2015;Nelson et al 2006;Yang et al 2013b). Exploitation of BAC resources for cytogenetic mapping resulted in the integration of all linkage groups with the corresponding chromosomes, as well as in the identification of several gene-rich regions (Książkiewicz et al 2013(Książkiewicz et al , 2015Leśniewska et al 2011;Przysiecka et al 2015;Wyrwa et al 2016). The release of reference transcriptomes for wild and domesticated lupin accessions constituted a platform for targeting particular genes of interest (Kamphuis et al 2015).…”

Feeding behavior of aphids on narrow‐leafed lupin (Lupinus angustifolius) genotypes varying in the content of quinolizidine alkaloids

“…As nitrogen-fixing plants, lupins are of special interest for organic farming, where the utilization of nitrogen fertilizer is prohibited (Jensen et al, 2004). Its contribution of fixed nitrogen and organic matter to soil results in increased yields of successive crops and has therefore excellent potential for sustainable crop-rotation systems (Kurlovich, 2002;Ksiazkiewicz et al, 2013). Because of their high protein content of 27-40% (Jansen et al, 2013), the use of narrow-leafed lupins in human and animal nutrition is of great interest.…”