Soto-Cerda BJ, Peñaloza E, Montenegro A, Rupayán A, Gallardo M, Salvo-Garrido H. (2013). An efficient marker-assisted backcrossing strategy for enhancing barley (Hordeum vulgare L.) production under acidity and aluminium toxicity. Molecular Breeding. 31; 4; 855-866.
To feed the predicted extra two billion people by 2050, crop production must increase on existing cultivated land at a rate that challenges our current capability. Acid soils and aluminium (Al3+) toxicity restrict productivity worldwide but also offer the greatest opportunity for increases in global food production. Our understanding of the physiology, genetic control and the identification of genomic regions underlying Al resistance in important staple crops has increased greatly over the past 20 years, enabling the application of molecular breeding. In this study, we report the application of an efficient marker-assisted backcrossing (MAB) strategy for the introgression of the HvAACT1gene which confers Al resistance in barley (Hordeum vulgare L.). We conducted foreground and background selection using microsatellite (SSR) markers linked to HvAACT1 and SSR-based linkage maps, along with embryo rescue and a cost-effective DNA preparation method shortening the breeding cycle to ~18 months. The MAB strategy enabled the development of homozygous (BC3F2) Al-resistant lines with the smallest introgressed region and 98.7 % of the recurrent parent genome. The Al-resistant line yielded significantly more seeds (121 %) than its isogenic line in soil-based assays containing 12 % of Al saturation. This MAB strategy could be extended to other staple crops with similar molecular toolboxes, expanding their cultivation onto acid soils, and contributing to greater yield stability and food security, particularly in developing countries.
Soto-Cerda BJ, Cloutier S. (2013). Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.). Plant Molecular Biology Reporter. 31; 4; 978-990.
Genomic microsatellites (gSSRs) and expressed sequence tag-derived SSRs (EST-SSRs) have gained wide application for elucidating genetic diversity and population structure in plants. Both marker systems are assumed to be selectively neutral when making demographic inferences, but this assumption is rarely tested. In this study, three neutrality tests were assessed for identifying outlier loci among 150 SSRs (85 gSSRs and 65 EST-SSRs) that likely influence estimates of population structure in three differentiated flax sub-populations (F ST = 0.19). Moreover, the utility of gSSRs, EST-SSRs, and the combined sets of SSRs was also evaluated in assessing genetic diversity and population structure in flax. Six outlier loci were identified by at least two neutrality tests showing footprints of balancing selection. After removing the outlier loci, the STRUCTURE analysis and the dendrogram topology of EST-SSRs improved. Conversely, gSSRs and combined SSRs results did not change significantly, possibly as a consequence of the higher number of neutral loci assessed. Taken together, the genetic structure analyses established the superiority of gSSRs to determine the genetic relationships among flax accessions, although the combined SSRs produced the best results. Genetic diversity parameters did not differ statistically (P > 0.05) between gSSRs and EST-SSRs, an observation partially explained by the similar number of repeat motifs. Our study provides new insights into the ability of gSSRs and EST-SSRs to measure genetic diversity and structure in flax and confirms the importance of testing for the occurrence of outlier loci to properly assess natural and breeding populations, particularly in studies considering only few loci.
Soto-Cerda BJ, Diederichsen A, Ragupathy R, Cloutier S. (2013). Genetic characterization of a core collection of flax (Linum usitatissimum L.) suitable for association mapping studies and evidence of divergent selection between fiber and linseed types. BMC Plant Biology. 13; 78.
Flax is valued for its fiber, seed oil and nutraceuticals. Recently, the fiber industry has invested in the development of products made from linseed stems, making it a dual purpose crop. Simultaneous targeting of genomic regions controlling stem fiber and seed quality traits could enable the development of dual purpose cultivars. However, the genetic diversity, population structure and linkage disequilibrium (LD) patterns necessary for association mapping (AM) have not yet been assessed in flax because genomic resources have only recently been developed. We characterized 407 globally distributed flax accessions using 448 microsatellite markers. The data was analyzed to assess the suitability of this core collection for AM. Genomic scans to identify candidate genes selected during the divergent breeding process of fiber flax and linseed were conducted using the whole genome shotgun sequence of flax.
Combined genetic structure analysis assigned all accessions to two major groups with six sub-groups. Population differentiation was weak between the major groups (FST = 0.094) and for most of the pairwise comparisons among sub-groups. The molecular coancestry analysis indicated weak relatedness (mean = 0.287) for most individual pairs. Abundant genetic diversity was observed in the total panel (5.32 alleles per locus), and some sub-groups showed a high proportion of private alleles. The average genome-wide LD (r2) was 0.036, with a relatively fast decay of 1.5 cM. Genomic scans between fiber flax and linseed identified candidate genes involved in cell-wall biogenesis/modification, xylem identity and fatty acid biosynthesis congruent with genes previously identified in flax and other plant species.
Based on the abundant genetic diversity, weak population structure and relatedness and relatively fast LD decay, we concluded that this core collection is suitable for AM studies targeting multiple agronomic and quality traits aiming at the improvement of flax as a true dual purpose crop. Our genomic scans provide the first insights into candidate regions affected by divergent selection in flax. In combination with AM, genomic scans have the ability to increase the power to detect loci influencing complex traits.
Barbara Demmig-Adams, Christopher M. Cohu, Véronique Amiard, Guusvan Zadelhoff, Gerrit A. Veldink, Onno Muller and William W. Adams III. (2013). Emerging trade-offs – impact of photoprotectants (PsbS, xanthophylls, and vitamin E) on oxylipins as regulators of development and defense. New Phytologist. 197; 3; 720-729.
This review summarizes evidence for a mechanistic link between plant photoprotection and the synthesis of oxylipin hormones as regulators of development and defense. Knockout mutants of Arabidopsis, deficient in various key components of the chloroplast photoprotection system, consistently produced greater concentrations of the hormone jasmonic acid or its precursor 12-oxo-phytodienoic acid (OPDA), both members of the oxylipin messenger family. Characterized plants include several mutants deficient in PsbS (an intrinsic chlorophyll-binding protein of photosystem II) or pigments (zeaxanthin and/or lutein) required for photoprotective thermal dissipation of excess excitation energy in the chloroplast and a mutant deficient in reactive oxygen detoxification via the antioxidant vitamin E (tocopherol). Evidence is also presented that certain plant defenses against herbivores or pathogens are elevated for these mutants. This evidence furthermore indicates that wild-type Arabidopsis plants possess less than maximal defenses against herbivores or pathogens, and suggest that plant lines with superior defenses against abiotic stress may have lower biotic defenses. The implications of this apparent trade-off between abiotic and biotic plant defenses for plant ecology as well as for plant breeding/engineering are explored, and the need for research further addressing this important issue is highlighted.
Ramadugu C., Pfeil B. E., Manjunath K. L., Lee R. F., Maureira-Butler I. J. and Roose M. L. (2013). A six nuclear gene phylogeny of Citrus (Rutaceae) taking into account hybridization and lineage sorting. PLoS One. 8; 7; e68410.
Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4–12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species.
Methodology and Principal Findings
(1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test.
We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches–gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus.
Soto-Cerda B, Westermeyer F, Iñiguez-Luy F, Muñoz G, Montenegro A, Cloutier S. (2013). Assessing the agronomic potential of linseed genotypes by multivariate analyses and association mapping of agronomic traits. Euphytica. 196; 1; 35-49.
High prices of fish oil make linseed attractive for aquaculture and animal feed. To ensure a constant supply of linseed, the development of stable cultivars is of strategic importance. In this study, 35 linseed genotypes were evaluated in five Chilean environments (E) from 2009 to 2012. The additive main effect and multiplicative interaction analysis (AMMI), genotype (G) plus genotype by environment (GE) interaction (GGE) biplot analysis and three stability parameters were tested with the aim of identifying adapted genotypes for the development of linseed cultivars. An association mapping (AM) analysis was also conducted for four agronomic traits and the stability of the associated markers was evaluated using the QQE (QTL main effect and QTL by environment interaction) approach. Combined analysis of variance for yield, seeds per boll (SPB), plant height (PH) and days to flowering (DTF) were significant for G, E and GE (P < 0.001). The combined stability analysis identified some Canadian, Argentinean and Chilean accessions to be the best adapted and highest yielding genotypes. Coancestry analysis indicated that crossing Canadian and Chilean genotypes could maximize transgressive segregation for yield. Significant associations for DTF, PH and SPB explained up to 59 % of the phenotypic variation for these traits. The QQE and AM analyses were consistent in identifying marker LGM27B as the most stable and significant across all environments with the largest effect in reducing DTF. The combined application of the stability, AM and QQE analyses could accelerate the development of marketable linseed cultivars adapted to Southern Chile.
Clarke WE, Parkin IA, Gajardo HA, Gerhardt DJ, Higgins E, Sidebottom C, Sharpe AG, Snowdon RJ, Federico ML, Iniguez-Luy FL. (2013). Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L. PLoS One. 8; 12; e81992.
Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.
Ogura T, Hernández A, Aizawa T, Ogihara J, Sunairi M, Alcaino J, Salvo-Garrido H,Maureira-Butler I. (2013). 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. PLoS One. 8; 11; e80369.
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.
Dechaine JM, Brock MT, Iniguez-Luy FL, Weinig C. (2013). Quantitative trait loci × environment interactions for plant morphology vary over ontogeny in Brassica rapa. New Phytologist. 201; 2; 657-669.
- Growth in plants occurs via the addition of repeating modules, suggesting that the genetic architecture of similar subunits may vary between earlier- and later-developing modules. These complex environment × ontogeny interactions are not well elucidated, as studies examining quantitative trait loci (QTLs) expression over ontogeny have not included multiple environments.
- Here, we characterized the genetic architecture of vegetative traits and onset of reproduction over ontogeny in recombinant inbred lines of Brassica rapa in the field and glasshouse.
- The magnitude of genetic variation in plasticity of seedling internodes was greater than in those produced later in ontogeny. We correspondingly detected that QTLs for seedling internode length were environment-specific, whereas later in ontogeny the majority of QTLs affected internode lengths in all treatments. The relationship between internode traits and onset of reproduction varied with environment and ontogenetic stage. This relationship was observed only in the glasshouse environment and was largely attributable to one environment-specific QTL.
- Our results provide the first evidence of a QTL × environment × ontogeny interaction, and provide QTL resolution for differences between early- and later-stage plasticity for stem elongation. These results also suggest potential constraints on morphological evolution in early vs later modules as a result of associations with reproductive timing.
Jolivet P., Acevedo F., Boulard C., d’Andréa S., Faure JD., Kohli A., Nesi N., Valot B., Chardot T. (2013). Crop seed oil bodies: from challenges in protein identification to an emerging picture of the oil body proteome. Proteomics. 13; 12-13; 1836-1849.
Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics-mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well-characterized plants, including the well-established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Toward an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) PTM of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops.
Shene, C., Spuler, M. J., Leyton, A., Duarte, C., Acevedo, F. and Rubilar, M. (2013). Pancreatic lipase activity in emulsions containing seed meals: Effect of extrusion. European Journal of Lipid Science and Technology. 115, 2, 217-223.
The effect of extrusion of soybean (Glycine max), yellow lupin (Lupinus luteus), and linseed (Linum usitatissimum) on the activity of porcine pancreatic lipase (PPL) was evaluated through pH-Stat methodology with tributyrin emulsions in which the seed meals [extruded (E) and non-extruded (NE)] were dispersed. The seed meals affected PPL activity in a concentration-dependent fashion; at 2.3 mg/mL the meals reduced PPL activity (11.7–45.0% reduction) while the opposite was true (3.1–28.0% increase) at 6.8 mg/mL. PPL activity in the assays containing E-lupin, E-soybean and E-linseed meals at 2.3 mg/mL was 109.7 and 11.3% higher and 25.5% lower than the activity in the emulsions containing the NE-meals, respectively. The differences between the PPL activity in emulsions containing the extruded and NE-meals could be due to changes in the emulsifying properties of the meals after extrusion; extrusion increased (10.2%) the emulsification capacity of lupin meal and for linseed meal this property was reduced (9.2%). In spite of the low PPL activity at low meal concentrations, the percentage of free fatty acids released after 30 min was similar to the theoretical maximum (66.6%) with two exceptions – NE-soybean meal (59.5 ± 2.7%) and E-linseed meal (57.9 ± 1.2%).
Shene, C., Leyton, A., Rubilar, M., Pinelo, M., Acevedo, F. and Morales, E. (2013). Production of lipids and docosahexasaenoic acid (DHA) by a nativeThraustochytrium strain. European Journal of Lipid Science and Technology. 115; 8; 890-900.
Production of docosahexasaenoic acid (DHA) by a native Labyrinthulomycetes strain, Thraustochytriidae sp. TN5, whose growth characteristics present some differences to related strains, was scaled from shaken flask to a laboratory fermentor. The effect of the growth medium composition and growth conditions for (i) biomass production, (ii) lipid content of the biomass, and (iii) DHA content in the lipids was determined. Taguchi’s design of experiments was used to study the influence of two discrete – carbon and nitrogen sources – and six continuous – concentrations of the carbon and nitrogen sources, yeast extract and artificial seawater, incubation temperature, and time- factors on the three mentioned variables. In the flask experiments the lipidic content in the biomass (25.2% w/w) and DHA concentration (0.48 g/L) were the highest at the following conditions: maltose 20 g/L, sodium glutamate 2.4 g/L, yeast extract 6 g/L, 72 h, 25°C, and artificial seawater 30% v/v; under these conditions biomass concentration was 5.1 g/L. Fed-batch allowed to increase biomass concentration to 14 g/L. The lipidic fraction of Thraustochytriidae sp. TN5 biomass in the repeated batches was found between 16.2 and 34.8% w/w. Under this growth condition lipids and DHA productivities were 50 and 23 mg/(L h), respectively.
Acevedo, F., Rubilar, M., Scheuermann, E., Cancino, B., Uquiche, E., Garcés, M., Inostroza, K., Shene, C. (2013). Spent coffee grounds as a renewable source of bioactive compounds. Journal of Biobased Materials and Bioenergy. 7; 1; 420-428.
Spent coffee grounds (SCG) are the main residues from the soluble coffee industry. The objective of this work was to evaluate different extraction methods of oil and diterpenes (kahweol and cafestol) from SCG, such as solid–liquid extraction, supercritical extraction or direct saponification, as well as to determine the phenolic composition and antioxidant capacities (by radical scavenging activity, inhibition of linoleic acid oxidation and β-carotene bleaching method) of SCG and defatted SCG. Among assayed oil extraction methods, the Soxhlet extraction process recovered the highest oil amounts (26.4%). The free fatty acid profile showed a high content of polyunsaturated fatty acids (50%) for the lipid fraction extracted from SCG by each method used, mainly linoleic and palmitic acids (about 45 and 30%, respectively). SCG was found to be an important source of polyphenolic compounds, showing 255 and 273 mg GAE/g dm in ethanolic extracts from SCG and defatted SCG, respectively, and was active as an antioxidant. The method that reached the highest level of diterpenes (214 mg/100 g SCG for kahweol and 466 mg/100 g SCG for cafestol) was direct saponification compared with the saponification of oil extracted by solid–liquid extraction or supercritical extraction. Finally, the presence of bioactive compounds of SCG identified in this study could be of great interest for the food, cosmetic and pharmaceutical industries, giving added value to a residue generated from instant coffee industrial processing, readily available in large quantities.
Soto-Cerda BJ, Duguid S, Booker H, Rowland G, Diederichsen A, Cloutier S. (2013). Genomic Regions Underlying Agronomic Traits in Linseed (Linum usitatissimum L.) as Revealed by Association Mapping. Journal of Integrative Plant Biology. 56; 1; 75-87.
The extreme climate of the Canadian Prairies poses a major challenge to improve yield. Although it is possible to breed for yield per se, focusing on yield-related traits could be advantageous because of their simpler genetic architecture. The Canadian flax core collection of 390 accessions was genotyped with 464 simple sequence repeat markers, and phenotypic data for nine agronomic traits including yield, bolls per area, 1,000 seed weight, seeds per boll, start of flowering, end of flowering, plant height, plant branching, and lodging collected from up to eight environments was used for association mapping. Based on a mixed model (principal component analysis (PCA) + kinship matrix (K)), 12 significant marker-trait associations for six agronomic traits were identified. Most of the associations were stable across environments as revealed by multivariate analyses. Statistical simulation for five markers associated with 1000 seed weight indicated that the favorable alleles have additive effects. None of the modern cultivars carried the five favorable alleles and the maximum number of four observed in any accessions was mostly in breeding lines. Our results confirmed the complex genetic architecture of yield-related traits and the inherent difficulties associated with their identification while illustrating the potential for improvement through marker-assisted selection.
Burgos-Díaz, C., Martín-Venegas, R., Martinez, V., Storniolo C.E., Teruel, J.A., Aranda, F.J., Ortiz, A., Manresa, A., Ferrer, R., Marques, A.M. (2013). In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced bySphingobacterium detergens. International Journal of Pharmaceutics 453; 2; 433-440.
The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.