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Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.).

ABSTRACT: 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.

SUBMITTER: Soto-Cerda BJ

PROVIDER: S-EPMC3881565 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundQuantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L).ResultsSpecific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes.ConclusionsThe use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.

Project description:BackgroundFlaxseed oil is characterized by high content of essential polyunsaturated fatty acids (PUFA) promoted as a human dietary supplement protecting against atherosclerosis. The disadvantage of the high PUFA content in flax oil is high susceptibility to oxidation, which can result in carcinogenic compound formation. Linola flax cultivar is characterized by high linoleic acid content in comparison to traditional flax cultivars rich in linolenic acid. The changes in fatty acid proportions increase oxidative stability of Linola oil and broaden its use as an edible oil for cooking. However one of investigated transgenic lines has high ALA content making it suitable as omega-3 source. Protection of PUFA oxidation is a critical factor in oil quality. The aim of this study was to investigate the impact of phenylpropanoid contents on the oil properties important during the whole technological process from seed storage to grinding and oil pressing, which may influence health benefits as well as shelf-life, and to establish guidelines for the selection of new cultivars.MethodsThe composition of oils was determined by chromatographic (GS-FID and LC-PDA-MS) methods. Antioxidant properties of secondary metabolites were analyzed by DPPH method. The stability of oils was investigated: a) during regular storage by measuring acid value peroxide value p-anisidine value malondialdehyde, conjugated dienes and trienes; b) by using accelerated rancidity tests by TBARS reaction; c) by thermoanalytical - differential scanning calorimetry (DSC).ResultsIn one approach, in order to increase oil stability, exogenous substances added are mainly lipid soluble antioxidants from the isoprenoid pathway, such as tocopherol and carotene. The other approach is based on transgenic plant generation that accumulates water soluble compounds. Increased accumulation of phenolic compounds in flax seeds was achieved by three different strategies that modify genes coding for enzymes from the phenylpropanoid pathway. The three types of transgenic flax had different phenylpropanoid profiles detected in oil, highly increasing its stability.ConclusionsWe found that hydrophilic phenylpropanoids more than lipophilic isoprenoid compounds determine oil stability however they can work synergistically. Among phenolics the caffeic acid was most effective in increasing oil stability.

Project description:Coexistence allows growers and consumers the choice of producing or purchasing conventional or organic crops with known standards for adventitious presence of genetically engineered (GE) seed. Flax (Linum usitatissimum L.) is multipurpose oilseed crop in which product diversity and utility could be enhanced for industrial, nutraceutical and pharmaceutical markets through genetic engineering. If GE flax were released commercially, pollen-mediated gene flow will determine in part whether GE flax could coexist without compromising other markets. As a part of pre-commercialization risk assessment, we quantified pollen-mediated gene flow between two cultivars of flax. Field experiments were conducted at four locations during 2006 and 2007 in western Canada using a concentric donor (20 × 20 m) receptor (120 × 120 m) design. Gene flow was detected through the xenia effect of dominant alleles of high ?-linolenic acid (ALA; 18:3(cis?9,12,15)) to the low ALA trait. Seeds were harvested from the pollen recipient plots up to a distance of 50 m in eight directions from the pollen donor. High ALA seeds were identified using a thiobarbituric acid test and served as a marker for gene flow. Binomial distribution and power analysis were used to predict the minimum number of seeds statistically required to detect the frequency of gene flow at specific ? (confidence interval) and power (1-?) values. As a result of the low frequency of gene flow, approximately 4 million seeds were screened to derive accurate quantification. Frequency of gene flow was highest near the source: averaging 0.0185 at 0.1 m but declined rapidly with distance, 0.0013 and 0.00003 at 3 and 35 m, respectively. Gene flow was reduced to 50% (O??) and 90% (O??) between 0.85 to 2.64 m, and 5.68 to 17.56 m, respectively. No gene flow was detected at any site or year > 35 m distance from the pollen source, suggesting that frequency of gene flow was ? 0.00003 (P = 0.95). Although it is not possible to eliminate all adventitious presence caused by pollen-mediated gene flow, through harvest blending and the use of buffer zones between GE and conventional flax fields, it could be minimized. Managing other sources of adventitious presence including seed mixing and volunteer populations may be more problematic.

Project description:Cultivated flax (Linum usitatissimum L.) is widely used for production of textile, food, chemical and pharmaceutical products. However, various stresses decrease flax production. Search for genes, which are involved in stress response, is necessary for breeding of adaptive cultivars. Imbalanced concentration of nutrient elements in soil decrease flax yields and also results in heritable changes in some flax lines. The appearance of Linum Insertion Sequence 1 (LIS-1) is the most studied modification. However, LIS-1 function is still unclear.High-throughput sequencing of transcriptome of flax plants grown under normal (N), phosphate deficient (P), and nutrient excess (NPK) conditions was carried out using Illumina platform. The assembly of transcriptome was performed, and a total of 34924, 33797, and 33698 unique transcripts for N, P, and NPK sequencing libraries were identified, respectively. We have not revealed any LIS-1 derived mRNA in our sequencing data. The analysis of high-throughput sequencing data allowed us to identify genes with potentially differential expression under imbalanced nutrition. For further investigation with qPCR, 15 genes were chosen and their expression levels were evaluated in the extended sampling of 31 flax plants. Significant expression alterations were revealed for genes encoding WRKY and JAZ protein families under P and NPK conditions. Moreover, the alterations of WRKY family genes differed depending on LIS-1 presence in flax plant genome. Besides, we revealed slight and LIS-1 independent mRNA level changes of KRP2 and ING1 genes, which are adjacent to LIS-1, under nutrition stress.Differentially expressed genes were identified in flax plants, which were grown under phosphate deficiency and excess nutrition, on the basis of high-throughput sequencing and qPCR data. We showed that WRKY and JAS gene families participate in flax response to imbalanced nutrient content in soil. Besides, we have not identified any mRNA, which could be derived from LIS-1, in our transcriptome sequencing data. Expression of LIS-1 flanking genes, ING1 and KRP2, was suggested not to be nutrient stress-induced. Obtained results provide new insights into edaphic stress response in flax and the role of LIS-1 in these process.

Project description:BackgroundA sustainable breeding program requires a minimum level of germplasm diversity to provide varied options for the selection of new breeding lines. To maximize genetic gain of the North Dakota State University (NDSU) flax breeding program, we aimed to increase the genetic diversity of its parental stocks by incorporating diverse genotypes. For this purpose, we analyzed the genetic diversity, linkage disequilibrium, and population sub-structure of 350 globally-distributed flax genotypes with 6200 SNP markers.ResultsAll the genotypes tested clustered into seven sub-populations (P1 to P7) based on the admixture model and the output of neighbor-joining (NJ) tree analysis and principal coordinate analysis were in line with that of structure analysis. The largest sub-population separation arose from a cluster of NDSU/American genotypes with Turkish and Asian genotypes. All sub-populations showed moderate genetic diversity (average H = 0.22 and I = 0.34). The pairwise Fst comparison revealed a great degree of divergence (Fst > 0.25) between most of the combinations. A whole collection mantel test showed significant positive correlation (r = 0.30 and p < 0.01) between genetic and geographic distances, whereas it was non-significant for all sub-populations except P4 and P5 (r = 0.251, 0.349 respectively and p < 0.05). In the entire collection, the mean linkage disequilibrium was 0.03 and it decayed to its half maximum within < 21 kb distance.ConclusionsTo maximize genetic gain, hybridization between NDSU stock (P5) and Asian individuals (P6) are potentially the best option as genetic differentiation between them is highest (Fst > 0.50). In contrast, low genetic differentiation between P5 and P2 may enhance the accumulation of favorable alleles for oil and fiber upon crossing to develop dual purpose varieties. As each sub-population consists of many genotypes, a Neighbor-Joining tree and kinship matrix assist to identify distantly related genotypes. These results also inform genotyping decisions for future association mapping studies to ensure the identification of a sufficient number of molecular markers to tag all linkage blocks.

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Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.).

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