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 insertion–deletion (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 2–3 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

Root variability among yellow lupin (Lupinus luteus L.) accessions grown at low temperatures in an undisturbed substrate

Abstract Root variation has become an important target for geneticists, ecologists, and breeders, given its direct influence on plant adaptation, resilience to climate change, and biomass and seed yields. However, the underground nature of roots has limited the assessment of root variability in plant species. In this study, we evaluated several root traits in a sample of distinct yellow lupin accessions grown under cold conditions and at three time points. Analyses of variance showed a highly significant accession (genotype) effect on primary root length (PRL), primary root area (PRA), and total root area (TRA), indicating that at least part of the root variation was explained by a genetic component. Significant accession*time interactions for PRL and PRA suggested that root growth rates (assessed using these traits) may change over time across genotypes; however, a more extensive study including a larger number of accessions and growing times must be conducted to confirm this finding. Differences among L. luteus accessions in PRL, PRA and TRA suggest the existence of favorable variation in plantlet root traits and the possibility of breeding stronger and better-established yellow lupin plants when grown under cold conditions.Resumen La variación fenotípica de las raíces se ha convertido en un objetivo importante para genetistas, ecólogos y mejoradores, dada su influencia directa sobre la adaptación de las plantas, la resistencia al cambio climático, biomasa y el rendimiento de las semillas. Sin embargo, su naturaleza subterránea ha limitado la evaluación de la variabilidad contenida en las especies vegetales. En este estudio se evaluaron caracteres de la raíz en una muestra de accesiones diversas de lupino amarillo, cultivadas bajo condiciones de frío y durante tres puntos de su desarrollo. Análisis de varianza mostraron que la longitud de raíz primaria (LRP), el área de raíz primaria (ARP) y el área de raíz total (ART) fueron altamente significativas para el efecto accesión (genotipo), señalando que al menos parte de la variación observada se explicaría por un componente genético. Las interacciones significativas de accesión*tiempo, para LR y ARP, sugirieron que las tasas de crecimiento de raíz para los distintos genotipos pueden cambiar con el tiempo; sin embargo, más estudios, incluyendo un mayor número de accesiones y tiempos de crecimiento, deben realizarse para confirmar este hallazgo. Diferencias entre las accesiones de L. luteus para LR, ARP y ART sugieren la existencia de variación favorable para los caracteres de raíz y la posibilidad de mejorar plantas de lupino amarillo más robustas y mejor establecidas cuando se cultiven bajo condiciones frías

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

Yellow lupin (<it>Lupinus luteus</it> 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.</p

2D-PAGE gel images of yellow lupin digested products.

<p>Meal, the dehulled seed meal extracts; Blank, only digestive enzymes. The leftward-pointing arrow and the leftward-pointing dotted arrow point the 12 kDa and other minor indigestible yellow lupin proteins, respectively. The circle shows a protein from the digestive enzymes.</p

Protein digestibility for yellow lupin meal and protein isolate.

<p>Meal, the dehulled seed meal extracts; Isolate, the protein isolate extracts. (*) significantly different (p = 0.043).</p

Protein band profiles of non-digested and digested yellow lupin products.

<p>Meal, the dehulled seed meal extracts; Isolate, the protein isolate extracts; Casein, casein extracts; N, non-digested product; D, digested product; B Blank, only digestive enzymes; E, Blank, empty lane. The position of the main low digestibility lupin protein is shown by a leftward-pointing arrow.</p