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\u2013deletion (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\u20133 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

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

How Does Diurnal and Nocturnal Warming Affect the Freezing Resistance of Antarctic Vascular Plants?

The Antarctic Peninsula has rapidly warmed up in past decades, and global warming has exhibited an asymmetric trend; therefore, it is interesting to understand whether nocturnal or diurnal warming is the most relevant for plant cold deacclimation. This study aimed to evaluate the effect of diurnal and nocturnal warming on Antarctic vascular plant’s freezing resistance under laboratory conditions. This was studied by measuring the lethal temperature for 50% of tissue (LT50), ice nucleation temperature (INT), and freezing point (FP) on Deschampsia antarctica and Colobanthus quitensis plants. Additionally, soluble carbohydrates content and dehydrin levels were analyzed during nocturnal and diurnal temperatures increase. Nocturnal warming led to a 7 °C increase in the LT50 of D. antarctica and reduced dehydrin-like peptide expression. Meanwhile, C. quitensis warmed plants reduce their LT50 to about 3.6 °C. Both species reduce their sucrose content by more than 28% in warming treatments. Therefore, nocturnal warming leads to cold deacclimation in both plant species, while C. quitensis plants are also cold-deacclimated upon warm days. This suggests that even when the remaining freezing resistance of both species allows them to tolerate summer freezing events, C. quitensis can reach its boundaries of freezing vulnerability in the near future if warming in the Antarctic Peninsula progress

The distribution of transgene insertion sites in barley determined by physical and genetic mapping.

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome

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