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 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 Application of propylene glycol in DNA-based studies of invertebrates

2021 ◽  
Vol 5 ◽  
Author(s):  
Alexander M. Weigand ◽  
Noah Desquiotz ◽  
Hannah Weigand ◽  
Nikolaus Szucsich
Keyword(s):  
Propylene Glycol ◽  
High Throughput Sequencing ◽  
Amplicon Sequencing ◽  
Morphological Characters ◽  
Storage Conditions ◽  
Steady Increase ◽  
Reduced Representation ◽  
Study Results ◽  
Integrative Study ◽  
Limited Application

High-throughput sequencing (HTS) studies on invertebrates commonly use ethanol as the main sample fixative (upon collection) and preservative (for storage and curation). However, alternative agents exists, which should not be automatically neglected when studies are newly designed. This review provides an overview of the application of propylene glycol (PG) in DNA-based studies of invertebrates, thus to stimulate an evidence-based discussion. The use of PG in DNA-based studies of invertebrates is still limited (n = 79), but a steady increase has been visible since 2011. Most studies used PG as a fixative for passive trapping (73%) and performed Sanger sequencing (66%; e.g. DNA barcoding). More recently, HTS setups joined the field (11%). Terrestrial Coleoptera (30%) and Diptera (20%) were the most studied groups. Very often, information on the grade of PG used (75%) or storage conditions (duration, temperature) were lacking. This rendered direct comparisons of study results difficult, and highlight the need for further systematic studies on these subjects. When compared to absolute ethanol, PG can be more widely and cheaply acquired (e.g. as an antifreeze, 13% of studies). It also enables longer trapping intervals, being especially relevant at remote or hard-to-reach places. Shipping of PG-conserved samples is regarded as risk-free and is authorised, pinpointing its potential for larger trapping programs or citizen science projects. Its property to retain flexibility of morphological characters as well as to lead to a reduced shrinkage effect was especially appraised by integrative study designs. Finally, the so far limited application of PG in the context of HTS showed promising results for short read amplicon sequencing and reduced representation methods. Knowledge of the influence of PG fixation and storage for long(er) read HTS setups is currently unavailable. Given our review results and taking difficulties of direct methodological comparisons into account, future DNA-based studies of invertebrates should on a case-by-case basis critically scrutinise if the application of PG in their anticipated study design can be of benefit.

scholarly journals Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

2019 ◽  
Author(s):  
Satsuki Tsuji ◽  
Atsushi Maruyama ◽  
Masaki Miya ◽  
Masayuki Ushio ◽  
Hirotoshi Sato ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Water Sample ◽  
Sanger Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Environmental Dna ◽  
Intraspecific Diversity ◽  
Survey Method ◽  
Intraspecific Genetic Diversity

AbstractEnvironmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, UNOISE3 and DADA2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (UNOISE3) and 41 (DADA2) haplotypes were detected by eDNA analysis. When DADA2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. This study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods, suggesting that it could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

scholarly journals Identification, evaluation, and application of the genomic-SSR loci in ramie

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Ming-Bao Luan ◽  
Ze-Mao Yang ◽  
Juan-Juan Zhu ◽  
Xin Deng ◽  
Chen-Chen Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
High Throughput Sequencing ◽  
Trinucleotide Repeat ◽  
Genetic Relationships ◽  
Poor Correlation ◽  
Similarity Coefficients ◽  
Reduced Representation ◽  
Ssr Primers ◽  
Ssr Loci ◽  
Trait Locus

To provide a theoretical and practical foundation for ramie genetic analysis, simple sequence repeats (SSRs) were identified in the ramie genome and employed in this study. From the 115 369 sequences of a specific-locus amplified fragment library, a type of reduced representation library obtained by high-throughput sequencing, we identified 4774 sequences containing 5064 SSR motifs. SSRs of ramie included repeat motifs with lengths of 1 to 6 nucleotides, and the abundance of each motif type varied greatly. We found that mononucleotide, dinucleotide, and trinucleotide repeat motifs were the most prevalent (95.91%). A total of 98 distinct motif types were detected in the genomic-SSRs of ramie. Of them, The A/T mononucleotide motif was the most abundant, accounting for 41.45% of motifs, followed by AT/TA, accounting for 20.30%. The number of alleles per locus in 31 polymorphic microsatellite loci ranged from 2 to 7, and observed and expected heterozygosities ranged from 0.04 to 1.00 and 0.04 to 0.83, respectively. Furthermore, molecular identity cards (IDs) of the germplasms were constructed employing the ID Analysis 3.0 software. In the current study, the 26 germplasms of ramie can be distinguished by a combination of five SSR primers including Ibg5-5, Ibg3-210, Ibg1-11, Ibg6-468, and Ibg6-481. The allele polymorphisms produced by all SSR primers were used to analyze genetic relationships among the germplasms. The similarity coefficients ranged from 0.41 to 0.88. We found that these 26 germplasms were clustered into five categories using UPGMA, with poor correlation between germplasm and geographical distribution. Our study is the first large-scale SSR identification from ramie genomic sequences. We have further studied the SSR distribution pattern in the ramie genome, and proposed that it is possible to develop SSR loci from genomic data for population genetics studies, linkage mapping, quantitative trait locus mapping, cultivar fingerprinting, and as genetic diversity studies.

scholarly journals Leveraging targeted sequencing for non-model species: a step-by-step guide to obtain a reduced SNP set and a pipeline to automate data processing in the Antarctic Midge, Belgica antarctica

2019 ◽  
Author(s):  
Vitor A. C. Pavinato ◽  
Saranga Wijeratne ◽  
Drew Spacht ◽  
David L. Denlinger ◽  
Tea Meulia ◽  
...  
Keyword(s):  
Rapid Development ◽  
Molecular Ecology ◽  
Amplicon Sequencing ◽  
Model Organisms ◽  
Reduced Representation ◽  
Bioinformatic Tools ◽  
Belgica Antarctica ◽  
Genomic Regions ◽  
Low Coverage ◽  
The Antarctic

AbstractThe sequencing of whole or partial (e.g. reduced representation) genomes are commonly employed in molecular ecology and conservation genetics studies. However, due to sequencing costs, a trade-off between the number of samples and genome coverage can hinder research for non-model organisms. Furthermore, the processing of raw sequences requires familiarity with coding and bioinformatic tools that are not always available. Here, we present a guide for isolating a set of short, SNP-containing genomic regions for use with targeted amplicon sequencing protocols. We also present a python pipeline--PypeAmplicon-- that facilitates processing of reads to individual genotypes. We demonstrate the applicability of our method by generating an informative set of amplicons for genotyping of the Antarctic midge, Belgica antarctica, an endemic dipteran species of the Antarctic Peninsula. Our pipeline analyzed raw sequences produced by a combination of high-multiplexed PCR and next-generation sequencing. A total of 38 out of 47 (81%) amplicons designed by our panel were recovered, allowing successful genotyping of 42 out of 55 (76%) targeted SNPs. The sequencing of ∼150 bp around the targeted SNPs also uncovered 80 new SNPs, which complemented our analyses. By comparing overall patterns of genetic diversity and population structure of amplicon data with the low-coverage, whole-genome re-sequencing (lcWGR) data used to isolate the informative amplicons, we were able to demonstrate that amplicon sequencing produces information and results similar to that of lcWGR. Our methods will benefit other research programs where rapid development of population genetic data is needed but yet prevented due to high expense and a lack of bioinformatic experience.

scholarly journals Estimating intraspecific genetic diversity from community DNA metabarcoding data

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4644 ◽  
Author(s):  
Vasco Elbrecht ◽  
Ecaterina Edith Vamos ◽  
Dirk Steinke ◽  
Florian Leese
Keyword(s):  
Genetic Diversity ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Intraspecific Diversity ◽  
Great Promise ◽  
Mock Community ◽  
Dna Metabarcoding ◽  
Intraspecific Genetic Diversity ◽  
Primer Sets ◽  
Haplotype Information

BackgroundDNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high-throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTUs), losing information on intraspecific diversity in the process. While Cytochrome c oxidase subunit I (COI) haplotype information is limited in resolving intraspecific diversity it is nevertheless often useful e.g. in a phylogeographic context, helping to formulate hypotheses on taxon distribution and dispersal.MethodsThis study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotype information from freshwater macroinvertebrate metabarcoding datasets. This novel approach was added to the R package “JAMP” and can be applied to COI amplicon datasets. We tested our haplotyping method by sequencing (i) a single-species mock community composed of 31 individuals with 15 different haplotypes spanning three orders of magnitude in biomass and (ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates.ResultsWe detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177–200 OTUs, each containing an average of 2.40–3.30 haplotypes per OTU. The derived intraspecific diversity data showed population structures that were consistent between replicates and similar between primer pairs but resolution depended on the primer length. A closer look at abundant taxa in the dataset revealed various population genetic patterns, e.g. the stoneflyTaeniopteryx nebulosaand the caddisflyHydropsyche pellucidulashowed a distinct north–south cline with respect to haplotype distribution, while the beetleOulimnius tuberculatusand the isopodAsellus aquaticusdisplayed no clear population pattern but differed in genetic diversity.DiscussionWe developed a strategy to infer intraspecific genetic diversity from bulk invertebrate metabarcoding data. It needs to be stressed that at this point this metabarcoding-informed haplotyping is not capable of capturing the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding datasets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about species diversity but also underlying genetic diversity.

scholarly journals Traditional ecological knowledge among Sami reindeer herders in northern Sweden about vascular plants grazed by reindeer

Rangifer ◽  
2013 ◽  
Vol 32 (1) ◽  
pp. 1-17 ◽  
Author(s):  
Berit Inga ◽  
Öje Danell
Keyword(s):  
Plant Species ◽  
Vascular Plants ◽  
Large Scale ◽  
Species Level ◽  
Food Plants ◽  
Ecological Knowledge ◽  
Individual Plant ◽  
Detailed Knowledge ◽  
Forage Plants ◽  
Plant Groups

Traditional knowledge about how reindeer utilize forage resources was expected to be crucial to reindeer herders. Seventeen Sami reindeer herders in four reindeer herding communities in Sweden (“samebyar” in Swedish) were interviewed about plants species considered to be important reindeer food plants in scientific literature. Among 40 plant species, which the informants were asked to identify and indicate whether and when they were grazed by reindeer, they identified a total of 21 plant taxa and five plant groups. They especially recognised species that were used as human food by the Sami themselves, but certain specific forage plants were also identified. Detailed knowledge of vascular plants at the species level was surprisingly general, which may indicate that knowledge of pasture resources in a detailed species level is not of vital importance. This fact is in sharp contradiction to the detailed knowledge that Sami people express for example about reindeer (as an animal) or snow (as physical element). The plausible explanation is that observations of individual plant species are unnecessarily detailed information in large-scale reindeer pastoralism, because the animals graze freely under loose herding and border surveillance.

scholarly journals AlnC: An extensive database of long non-coding RNAs in angiosperms

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0247215
Author(s):  
Ajeet Singh ◽  
A. T. Vivek ◽  
Shailesh Kumar
Keyword(s):  
Plant Species ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Significant Rise ◽  
Open Reading Frames ◽  
Sequencing Data ◽  
High Throughput Sequencing Data ◽  
Non Coding Rnas ◽  
Public Repositories ◽  
User Friendly

Long non-coding RNAs (lncRNAs) are defined as transcripts of greater than 200 nucleotides that play a crucial role in various cellular processes such as the development, differentiation and gene regulation across all eukaryotes, including plant cells. Since the last decade, there has been a significant rise in our understanding of lncRNA molecular functions in plants, resulting in an exponential increase in lncRNA transcripts, while these went unannounced from the major Angiosperm plant species despite the availability of large-scale high throughput sequencing data in public repositories. We, therefore, developed a user-friendly, open-access web interface, AlnC (Angiosperm lncRNA Catalogue) for the exploration of lncRNAs in diverse Angiosperm plant species using recent 1000 plant (1KP) trancriptomes data. The current version of AlnC offers 10,855,598 annotated lncRNA transcripts across 682 Angiosperm plant species encompassing 809 tissues. To improve the user interface, we added features for browsing, searching, and downloading lncRNA data, interactive graphs, and an online BLAST service. Additionally, each lncRNA record is annotated with possible small open reading frames (sORFs) to facilitate the study of peptides encoded within lncRNAs. With this user-friendly interface, we anticipate that AlnC will provide a rich source of lncRNAs for small-and large-scale studies in a variety of flowering plants, as well as aid in the improvement of key characteristics in relevance to their economic importance. Database URL: http://www.nipgr.ac.in/AlnC

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