scholarly journals A multispecies amplicon sequencing approach for genetic diversity assessment in grassland plant species

2021 ◽  
Author(s):  
Miguel Loera-Sanchez ◽  
Bruno Studer ◽  
Roland Koelliker
Keyword(s):  
Genetic Diversity ◽  
Plant Species ◽  
High Throughput ◽  
Large Scale ◽  
Trifolium Pratense ◽  
Amplicon Sequencing ◽  
Pathogen Resistance ◽  
Production Plant ◽  
Diversity Assessment ◽  
Grassland Plants

Grasslands are widespread and economically relevant ecosystems at the basis of sustainable roughage production. Plant genetic diversity (PGD; i.e., within-species diversity) is related to many beneficial effects to the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large-scale assessment. However, the continuous advancement of high-throughput DNA sequencing approaches have improved the prospects of broad, multispecies PGD monitoring. Among them, amplicon sequencing stands out as a robust and cost-effective method. Here we report a set of twelve multispecies primer pairs that can be used for high-throughput PGD assessment in multiple grassland plant species. The loci targeted by the amplicons were selected and tested in two phases: a "discovery phase" based on a sequence capture assay (611 target nuclear loci assessed in 16 grassland plant species), which resulted in the selection of eleven loci; and a "validation phase", in which the selected loci were targeted and sequenced using twelve multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The resulting multispecies amplicons had overall nucleotide diversities per species ranging from 5.19 × 10-3 to 1.29 × 10-2, which is in the range of flowering-related genes but slightly lower than pathogen resistance genes. We conclude that the methodology, the DNA sequence resources, and the amplicon-specific primer pairs reported in this study provide the basis for large-scale, multispecies PGD monitoring in grassland plants.

scholarly journals DNA-Based Assessment of Genetic Diversity in Grassland Plant Species: Challenges, Approaches, and Applications

Agronomy ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 881
Author(s):  
Miguel Loera-Sánchez ◽  
Bruno Studer ◽  
Roland Kölliker
Keyword(s):  
Genetic Diversity ◽  
Plant Species ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Model Organisms ◽  
Special Focus ◽  
Reduced Representation ◽  
Forage Plants ◽  
Single Method

Grasslands are wide-spread, multi-species ecosystems that provide many valuable services. Plant genetic diversity (i.e., the diversity within species) is closely linked to ecosystem functioning in grasslands and constitutes an important reservoir of genetic resources that can be used to breed improved cultivars of forage grass and legume species. Assessing genetic diversity in grassland plant species is demanding due to the large number of different species and the level of resolution needed. However, recent methodological advances could help in tackling this challenge at a larger scale. In this review, we outline the methods that can be used to measure genetic diversity in plants, highlighting their strengths and limitations for genetic diversity assessments of grassland plant species, with a special focus on forage plants. Such methods can be categorized into DNA fragment, hybridization array, and high-throughput sequencing (HTS) methods, and they differ in terms of resolution, throughput, and multiplexing potential. Special attention is given to HTS approaches (i.e., plastid genome skimming, whole genome re-sequencing, reduced representation libraries, sequence capture, and amplicon sequencing), because they enable unprecedented large-scale assessments of genetic diversity in non-model organisms with complex genomes, such as forage grasses and legumes. As no single method may be suited for all kinds of purposes, we also provide practical perspectives for genetic diversity analyses in forage breeding and genetic resource conservation efforts.

scholarly journals MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction

2019 ◽  
Author(s):  
Bryden Fields ◽  
Sara Moeskjær ◽  
Ville-Petri Friman ◽  
Stig U. Andersen ◽  
J. Peter W. Young
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Error Correction ◽  
High Throughput ◽  
Environmental Dna ◽  
Amplicon Sequencing ◽  
Efficient Elimination

AbstractBackgroundSequencing and PCR errors are a major challenge when characterising genetic diversity using high-throughput amplicon sequencing (HTAS).ResultsWe have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming DADA2 and UNOISE3, and the ability to confidently recognise genuine alleles that are present at low abundance or resemble chimeras.ConclusionsThe method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.

scholarly journals A comparative genetic diversity assessment of industrial and household Brazilian cassava varieties using SSR markers

Bragantia ◽  
2011 ◽  
Vol 70 (4) ◽  
pp. 745-752 ◽  
Author(s):  
Marcos Vinicius Bohrer Monteiro Siqueira ◽  
Aline Borges ◽  
Teresa Losada Valle ◽  
Elizabeth Ann Veasey
Keyword(s):  
Genetic Diversity ◽  
Genetic Variability ◽  
Large Scale ◽  
Gene Diversity ◽  
Household Consumption ◽  
Manihot Esculenta Crantz ◽  
Diversity Assessment ◽  
Source Of Information ◽  
Selection Of ◽  
Cassava Varieties

This study was carried out in order to assess the genetic diversity of 20 cassava (Manihot esculenta Crantz) genotypes with high phenotypic performance using microsatellite markers. Two groups were considered for this study: eleven varieties for industrial uses and nine for household consumption. By using nine microsatellite primers, high polymorphism was identified in all the loci analyzed, with values reaching 100%. On average, 3.4 alleles per locus were found, with 0.371 the value estimated for the observed heterozygosity and 0.555 for gene diversity for the entire set of varieties. The genetic variability found in both varieties, cultivated on a large-scale in the South Center region of Brazil, is wide enough to allow the choice of divergent parental genotypes to be used in crosses to obtain new recombinant genotypes. Furthermore, the analyses indicated a high genetic variability within the two groups (I: varieties for industrial uses; II: varieties for household consumption). However, varieties for household consumption attain higher genetic variability, probably due to high priority placed on selection of different sensorial traits. In the cluster analysis, a tendency for separation of varieties for industrial use and household consumption was verified. Our results represent an important source of information to the cassava breeding program in Brazil.

scholarly journals Deciphering the Genetic Diversity of Landraces With High-Throughput SNP Genotyping of DNA Bulks: Methodology and Application to the Maize 50k Array

2021 ◽  
Vol 11 ◽  
Author(s):  
Mariangela Arca ◽  
Tristan Mary-Huard ◽  
Brigitte Gouesnard ◽  
Aurélie Bérard ◽  
Cyril Bauland ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
High Throughput ◽  
Large Scale ◽  
Allelic Frequency ◽  
Laboratory Equipment ◽  
Fluorescent Intensity ◽  
Allelic Frequencies ◽  
Wide Range ◽  
Young Leaves ◽  
Scale Characterization

Genebanks harbor original landraces carrying many original favorable alleles for mitigating biotic and abiotic stresses. Their genetic diversity remains, however, poorly characterized due to their large within genetic diversity. We developed a high-throughput, cheap and labor saving DNA bulk approach based on single-nucleotide polymorphism (SNP) Illumina Infinium HD array to genotype landraces. Samples were gathered for each landrace by mixing equal weights from young leaves, from which DNA was extracted. We then estimated allelic frequencies in each DNA bulk based on fluorescent intensity ratio (FIR) between two alleles at each SNP using a two step-approach. We first tested either whether the DNA bulk was monomorphic or polymorphic according to the two FIR distributions of individuals homozygous for allele A or B, respectively. If the DNA bulk was polymorphic, we estimated its allelic frequency by using a predictive equation calibrated on FIR from DNA bulks with known allelic frequencies. Our approach: (i) gives accurate allelic frequency estimations that are highly reproducible across laboratories, (ii) protects against false detection of allele fixation within landraces. We estimated allelic frequencies of 23,412 SNPs in 156 landraces representing American and European maize diversity. Modified Roger’s genetic Distance between 156 landraces estimated from 23,412 SNPs and 17 simple sequence repeats using the same DNA bulks were highly correlated, suggesting that the ascertainment bias is low. Our approach is affordable, easy to implement and does not require specific bioinformatics support and laboratory equipment, and therefore should be highly relevant for large-scale characterization of genebanks for a wide range of species.

scholarly journals Deciphering the genetic diversity of landraces with high-throughput SNP genotyping of DNA bulks: methodology and application to the maize 50k array

2020 ◽  
Author(s):  
Mariangela Arca ◽  
Tristan Mary-Huard ◽  
Brigitte Gouesnard ◽  
Aurélie Bérard ◽  
Cyril Bauland ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
High Throughput ◽  
Large Scale ◽  
Allelic Frequency ◽  
Laboratory Equipment ◽  
Fluorescent Intensity ◽  
Allelic Frequencies ◽  
Wide Range ◽  
Young Leaves ◽  
Scale Characterization

ABSTRACTGenebanks harbor original landraces carrying many original favorable alleles for mitigating biotic and abiotic stresses. Their genetic diversity remains however poorly characterized due to their large within genetic diversity. We developed a high-throughput, cheap and labor saving DNA bulk approach based on SNP Illumina Infinium HD array to genotype landraces. Samples were gathered for each landrace by mixing equal weights from young leaves, from which DNA was extracted. We then estimated allelic frequencies in each DNA bulk based on fluorescent intensity ratio (FIR) between two alleles at each SNP using a two step-approach. We first tested either whether the DNA bulk was monomorphic or polymorphic according to the two FIR distributions of individuals homozygous for allele A or B, respectively. If the DNA bulk was polymorphic, we estimated its allelic frequency by using a predictive equation calibrated on FIR from DNA bulks with known allelic frequencies. Our approach: (i) gives accurate allelic frequency estimations that are highly reproducible across laboratories, (ii) protects against false detection of allele fixation within landraces. We estimated allelic frequencies of 23,412 SNPs in 156 landraces representing American and European maize diversity. Modified Roger’s genetic Distance between 156 landraces estimated from 23,412 SNPs and 17 SSRs using the same DNA bulks were highly correlated, suggesting that the ascertainment bias is low. Our approach is affordable, easy to implement and does not require specific bioinformatics support and laboratory equipment, and therefore should be highly relevant for large-scale characterization of genebanks for a wide range of species.

scholarly journals MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction

2020 ◽  
Author(s):  
Bryden Fields ◽  
Sara Moeskjær ◽  
Ville-Petri Friman ◽  
Stig U. Andersen ◽  
J. Peter W. Young
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Error Correction ◽  
High Throughput ◽  
Environmental Dna ◽  
Amplicon Sequencing ◽  
Efficient Elimination

Abstract Background Sequencing and PCR errors are a major challenge when characterising genetic diversity using high-throughput amplicon sequencing (HTAS). Results We have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming DADA2 and UNOISE3, and the ability to confidently recognise genuine alleles that are present at low abundance or resemble chimeras. Conclusions The method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.

scholarly journals High throughput amplicon sequencing to assess within- and between-host genetic diversity in plant viruses

2017 ◽  
Author(s):  
Sylvain Piry ◽  
Catherine Wipf-Scheibel ◽  
Jean-François Martin ◽  
Maxime Galan ◽  
Karine Berthier
Keyword(s):  
Genetic Diversity ◽  
High Throughput ◽  
Variant Calling ◽  
Amplicon Sequencing ◽  
Plant Viruses ◽  
Sequencing Error ◽  
List Type ◽  
Data Generating Process ◽  
Host Genetic ◽  
Robust Variant

AbstractMolecular epidemiology approaches at the landscape scale require to study the genetic diversity of viral populations from numerous hosts and to characterize mixed infections. In such a context, high-throughput amplicon sequencing (HTAS) techniques create interesting opportunities as they allow identifying distinct variants within a same host while simultaneously genotyping a high number of samples. Validating variants produced by HTAS may, however, remain difficult due to biases occurring at different steps of the data-generating process (e.g. environmental contaminations and sequencing error). Here, we focused on Endive necrotic mosaic virus (ENMV), a member of family Potyviridae, genus Potyvirus to develop an HTAS approach and to characterize the genetic diversity at the intra- and inter-host levels from 430 samples collected over an area of 1660 km2 located in south-eastern France. We demonstrated how it is possible, by incorporating various controls in the experimental design and by performing independent sample replicates, to estimate potential biases in HTAS results and to implement an automated and robust variant calling procedure.HighlightsHigh-throughput amplicon sequencing to assess plant virus genetic diversityEstimating bias in high throughput amplicon sequencing resultsAutomated variant calling procedure for robust high throughput amplicon sequencing

scholarly journals Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period

2021 ◽  
Vol 12 ◽  
Author(s):  
Suhyun Kim ◽  
Md. Rashedul Islam ◽  
Ilnam Kang ◽  
Jang-Cheon Cho
Keyword(s):  
16S Rrna ◽  
High Throughput ◽  
Large Scale ◽  
Phylogenetic Analyses ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Taxonomic Groups ◽  
Bacterial Groups

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

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