Dataset Information

Population structure and genetic diversity in red clover (Trifolium pratense L.) germplasm.

ABSTRACT: Red clover (Trifolium pratense L.) is a highly adaptable forage crop for temperate livestock agriculture. Genetic variation can be identified, via molecular techniques, and used to assess diversity among populations that may otherwise be indistinguishable. Here we have used genotyping by sequencing (GBS) to determine the genetic variation and population structure in red clover natural populations from Europe and Asia, and varieties or synthetic populations. Cluster analysis differentiated the collection into four large regional groups: Asia, Iberia, UK, and Central Europe. The five varieties clustered with the geographical area from which they were derived. Two methods (BayeScan and Sam?ada) were used to search for outlier loci indicating signatures of selection. A total of 60 loci were identified by both methods, but no specific genomic region was highlighted. The rate of decay in linkage disequilibrium was fast, and no significant evidence of any bottlenecks was found. Phenotypic analysis showed that a more prostrate and spreading growth habit was predominantly found among populations from Iberia and the UK. A genome wide association study identified a single nucleotide polymorphism (SNP) located in a homologue of the VEG2 gene from pea, associated with flowering time. The identification of genetic variation within the natural populations is likely to be useful for enhancing the breeding of red clover in the future.

SUBMITTER: Jones C

PROVIDER: S-EPMC7239897 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Population structure and genetic diversity in red clover (Trifolium pratense L.) germplasm.

Jones Charlotte C De Vega Jose J Lloyd David D Hegarty Matthew M Ayling Sarah S Powell Wayne W Skøt Leif L

Scientific reports 20200520 1

Red clover (Trifolium pratense L.) is a highly adaptable forage crop for temperate livestock agriculture. Genetic variation can be identified, via molecular techniques, and used to assess diversity among populations that may otherwise be indistinguishable. Here we have used genotyping by sequencing (GBS) to determine the genetic variation and population structure in red clover natural populations from Europe and Asia, and varieties or synthetic populations. Cluster analysis differentiated the co ...[more]

PMID: 32433569

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Project description:Red clover (Trifolium pratense) is one of the most important fodder crops worldwide. The knowledge of genetic diversity among red clover populations, however, is under development. This study provides insights into its genetic diversity, using single nucleotide polymorphism (SNP) markers to define population structure in wild and cultivated red clover. Twenty-nine accessions representing the genetic resources available at NordGen (the Nordic gene bank) and Lantmännen (a Swedish agricultural company with a red clover breeding program) were used for this study. Genotyping was performed via SeqSNP, a targeted genotype by sequencing method that offers the capability to target specific SNP loci and enables de novo discovery of new SNPs. The SNPs were identified through a SNP mining approach based on coding sequences of red clover genes known for their involvement in development and stress responses. After filtering the genotypic data using various criteria, 623 bi-allelic SNPs, including 327 originally targeted and 296 de novo discovered SNPs were used for population genetics analyses. Seventy-one of the SNP loci were under selection considering both Hardy-Weinberg equilibrium and pairwise FST distributions. The average observed heterozygosity (H O ), within population diversity (H S ) and overall diversity (H T ) were 0.22, 0.21 and 0.22, respectively. The tetraploids had higher average H O (0.35) than diploids (0.21). The analysis of molecular variance (AMOVA) showed low but significant variation among accessions (5.4%; P < 0.001), and among diploids and tetraploids (1.08%; P = 0.02). This study revealed a low mean inbreeding coefficient (FIS = -0.04) exhibiting the strict outcrossing nature of red clover. As per cluster, principal coordinate and discriminant analyses, most wild populations were grouped together and were clearly differentiated from the cultivated types. The cultivated types of red clover had a similar level of genetic diversity, suggesting that modern red clover breeding programs did not negatively affect genetic diversity or population structure. Hence, the breeding material used by Lantmännen represents the major genetic resources in Scandinavia. This knowledge of how different types of red clover accessions relate to each other and the level of outcrossing and heterozygosity will be useful for future red clover breeding.

Project description:Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is used as forage. Leaf senescence is a critical developmental process that negatively affects plant quality and yield. The regulatory mechanism of leaf senescence has been studied, and genes involved in leaf senescence have been cloned and characterized in many plants. However, those works mainly focused on model plants. Information about regulatory pathways and the genes involved in leaf senescence in red clover is very sparse. In this study, to better understand leaf senescence in red clover, transcriptome analysis of mature and senescent leaves was investigated using RNA-Seq. A total of about 35,067 genes were identified, and 481 genes were differentially expressed in mature and senescent leaves. Some identified differentially expressed genes showed similar expression patterns as those involved in leaf senescence in other species, such as Arabidopsis, Medicago truncatula and rice. Differentially expressed genes were confirmed by quantitative real-time PCR (qRT-PCR). Genes involved in signal transduction, transportation and metabolism of plant hormones, transcription factors and plant senescence were upregulated, while the downregulated genes were primarily involved in nutrient cycling, lipid/carbohydrate metabolism, hormone response and other processes. There were 64 differentially expressed transcription factor genes identified by RNA-Seq, including ERF, WRKY, bHLH, MYB and NAC. A total of 90 genes involved in biosynthesis, metabolism and transduction of plant hormones, including abscisic acid, jasmonic acid, cyokinin, brassinosteroid, salicylic acid and ethylene, were identified. Furthermore, 207 genes with direct roles in leaf senescence were demonstrated, such as senescence-associated genes. These genes were associated with senescence in other plants. Transcriptome analysis of mature and senescent leaves in red clover provides a large number of differentially expressed genes. Further analysis and identification of senescence-associated genes can provide new insight into the regulatory mechanisms of leaf development and senescence in legume plant and red clover.

Project description:BackgroundImprovement of freezing tolerance of red clover (Trifolium pratense L.) would increase its persistence under cold climate. In this study, we assessed the freezing tolerance and compared the proteome composition of non-acclimated and cold-acclimated plants of two initial cultivars of red clover: Endure (E-TF0) and Christie (C-TF0) and of populations issued from these cultivars after three (TF3) and four (TF4) cycles of phenotypic recurrent selection for superior freezing tolerance. Through this approach, we wanted to identify proteins that are associated with the improvement of freezing tolerance in red clover.ResultsFreezing tolerance expressed as the lethal temperature for 50 % of the plants (LT50) increased markedly from approximately -2 to -16 °C following cold acclimation. Recurrent selection allowed a significant 2 to 3 °C increase of the LT50 after four cycles of recurrent selection. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to study variations in protein abundance. Principal component analysis based on 2D-DIGE revealed that the largest variability in the protein data set was attributable to the cold acclimation treatment and that the two genetic backgrounds had differential protein composition in the acclimated state only. Vegetative storage proteins (VSP), which are essential nitrogen reserves for plant regrowth, and dehydrins were among the most striking changes in proteome composition of cold acclimated crowns of red clovers. A subset of proteins varied in abundance in response to selection including a dehydrin that increased in abundance in TF3 and TF4 populations as compared to TF0 in the Endure background.ConclusionRecurrent selection performed indoor is an effective approach to improve the freezing tolerance of red clover. Significant improvement of freezing tolerance by recurrent selection was associated with differential accumulation of a small number of cold-regulated proteins that may play an important role in the determination of the level of freezing tolerance.

Project description:Starting with an isoflavone-rich red clover extract (RCE), this study expands on the DESIGNER approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources using countercurrent separation (CCS) methodology. A hydrostatic CCS (also known as centrifugal partition chromatography, CPC) technique was used to enrich and deplete selected bioactive isoflavones of RCE extracts. In order to efficiently prepare large enough DESIGNER extracts from RCE for biological testing including in vivo assays, it was necessary to choose a balance between resolution and a loading capacity of at least 1?g per separation for the selected solvent system (SS). Adding 3?mL of DMSO to the sample containing equal amounts of upper and lower phases of hexanes-ethyl acetate-methanol-water (HEMWat 5.5/4.5/5/5, v/v) allowed 1?g of RCE to be dissolved in the sample without disrupting the chromatographic resolution of the target isoflavones. CPC experiments using other solubility modifiers, acetone and acetonitrile indicated that these modifiers increase solubility significantly, even better than DMSO, but the separation of target compounds was sufficiently disturbed to be unacceptable for producing the desired DESIGNER extracts. The preparation of DESIGNER extracts was achieved with two sequential CPC separations. The first produced a biochanin A enriched fraction (93.60% w/w) with only small amounts of other isoflavones: 2.30% w/w prunetin, 1.17% w/w formononetin, and 0.12% w/w irilone. Gravimetric investigations of this step demonstrated the high efficiency of CCS technology for full and unbiased sample recovery, confirmed experimentally to be 99.80%. A formononetin enriched fraction from this first separation was re-chromatographed on a more polar HEMWat (4/6/4/6, v/v) SS to produce a formononetin enriched DESIGNER fraction of 94.70% w/w purity. The presence of the minor (iso)flavonoids: 3.16% w/w pseudobaptigenin, 0.39% w/w kaempferol, and 0.31% w/w genistein was also monitored in these fractions. Chromatographic fractions, combined fractions, and DESIGNER extracts were analyzed with quantitative 1H NMR (qHNMR) spectroscopy which provided purity information, quantitation, and structural identification of the components.

Dataset Information

Population structure and genetic diversity in red clover (Trifolium pratense L.) germplasm.

Publications

Population structure and genetic diversity in red clover (Trifolium pratense L.) germplasm.

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