Finding the perfect pairs: A matchmaking of plant markers and primers for multi-marker eDNA metabarcoding.

As the scope of plant eDNA metabarcoding diversifies, so do the primers, markers and methods. A wealth of primers exists today, but their comparative evaluation is lacking behind. Similarly, multi-marker approaches are recommended but debates persist regarding barcode complementarity and optimal combinations. After a literature compilation of used primers, we compared in silico 102 primer pairs based on amplicon size, coverage and specificity, followed by an experimental evaluation of 15 primer pairs on a mock community sample covering 268 plant species and genera, and about 100 families. The analysis was done for the four most common plant metabarcoding markers, rbcL, trnL, ITS1 and ITS2 and their complementarity was assessed based on retrieved species. By focusing on existing primers, we identify common designs, promote alternatives and enhance prior-supported primers for immediate applications. The ITS2 was the best-performing marker for flowering vascular plants and was congruent to ITS1. However, the combined taxonomic breadth of ITS2 and rbcL surpassed any other combination, highlighting their high complementarity across Streptophyta. Overall, our study underscores the significance of comprehensive primer and barcode evaluations tailored to metabarcoding applications.

Co-Infection of Tomato Brown Rugose Fruit Virus and Pepino Mosaic Virus in Grocery Tomatoes in South Florida: Prevalence and Genomic Diversity.

Tomato brown rugose fruit virus (ToBRFV) is an economically important seed and mechanically transmitted pathogen of significant importance to tomato production around the globe. Synergistic interaction with pepino mosaic virus (PepMV), another seed and mechanically transmitted virus, and long-distance dissemination of these two viruses via contaminated tomato fruits through global marketing were previously suggested. In 2019, we detected both viruses in several grocery store-purchased tomatoes in South Florida, USA. In this study, to identify potential sources of inoculum, co-infection status, prevalence, and genomic diversity of these viruses, we surveyed symptomatic and asymptomatic imported tomatoes sold in ten different groceries in four cities in South Florida. According to the product labels, all collected tomatoes originated from Canada, Mexico, or repacking houses in the United States. With high prevalence levels, 86.5% of the collected samples were infected with ToBRFV, 90% with PepMV alone, and 73% were mixed-infected. The phylogenetic study showed no significant correlations between ToBRFV genomic diversity and the tomato label origin. Phylogenetic analysis of PepMV isolates revealed the prevalence of the PepMV strains, Chilean (CH2) and recombinant (US2). The results of this study highlight the continual presence of PepMV and ToBRFV in imported tomatoes in Florida grocery stores.

Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) for Gene Expression Analyses.

The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is considered to be the gold standard for gene expression research. However, for this claim to be valid, RT-qPCR studies must test and optimize the quality of its RNA templates and assays. This chapter describes the experimental procedures required to generate reliable and reproducible gene expression results using RT-qPCR.

Newly-designed primer pairs for the detection of type 2 porcine reproductive and respiratory syndrome virus genes.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, caused by PRRS virus (PRRSV), that critically affects the swine industry. While the detection of PRRSV genes plays a key role in PRRS control, the PRRSV genome is known to undergo frequent mutation. Nevertheless, primer pairs widely used for the detection of PRRSV genes were designed between 1995 and 2010. The reliability of these primer pairs for the detection of currently circulating PRRSVs is therefore questionable. Here, we investigated the sensitivity of the previously reported primer pairs to detect PRRSV genes that have been recently isolated or detected in Japan. In addition, based on nucleotide sequences from the recent Japanese PRRSVs, we designed four new primer pairs for the detection of PRRSV genes. The sensitivity and specificity of the new primer pairs were evaluated by quantitative reverse transcription PCR using RNA extracted from PRRSV isolates, swine serum, and oral fluid specimens collected from PRRS-affected pigs, and swine sera collected from a PRRSV-free pig farm in Japan. One of novel primer pairs used in our study exhibited greater sensitivity than the previously reported primer pairs, and is thus more reliable for the detection of PRRSV genes.

The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure.

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

Tubulin-Based DNA Barcode: Principle and Applications to Complex Food Matrices.

The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal species. Similarly, many plant varieties can also be barcoded by TBP. The method is based on a simple cell biology concept that finds a conveniently exploitable molecular basis. It does not depend on DNA sequencing as the most classically established DNA barcode strategies. Successful applications, diversified for the different target sequences or experimental purposes, have been reported in many different plant species and, of late, a new a version applicable to animal species, including fishes, has been developed. Also, the TBP method is currently used for the genetic authentication of plant material and derived food products. Due to the use of a couple of universal primer pairs, specific for plant and animal organisms, respectively, it is effective in metabarcoding a complex matrix allowing an easy and rapid recognition of the different species present in a mixture. A simple, dedicated database made up by the genomic profile of reference materials is also part of the analytical procedure. Here we will provide some example of the TBP application and will discuss its features and uses in comparison with the DNA sequencing-based methods.

Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities.

Metabarcoding combines DNA barcoding with high-throughput sequencing, often using one genetic marker to understand complex and taxonomically diverse samples. However, species-level identification depends heavily on the choice of marker and the selected primer pair, often with a trade-off between successful species amplification and taxonomic resolution. We present a versatile metabarcoding protocol for biomonitoring that involves the use of two barcode markers (COI and 18S) and four primer pairs in a single high-throughput sequencing run, via sample multiplexing. We validate the protocol using a series of 24 mock zooplanktonic communities incorporating various levels of genetic variation. With the use of a single marker and single primer pair, the highest species recovery was 77%. With all three COI fragments, we detected 62%-83% of species across the mock communities, while the use of the 18S fragment alone resulted in the detection of 73%-75% of species. The species detection level was significantly improved to 89%-93% when both markers were used. Furthermore, multiplexing did not have a negative impact on the proportion of reads assigned to each species and the total number of species detected was similar to when markers were sequenced alone. Overall, our metabarcoding approach utilizing two barcode markers and multiple primer pairs per barcode improved species detection rates over a single marker/primer pair by 14% to 35%, making it an attractive and relatively cost-effective method for biomonitoring natural zooplankton communities. We strongly recommend combining evolutionary independent markers and, when necessary, multiple primer pairs per marker to increase species detection (i.e., reduce false negatives) in metabarcoding studies.

Genome Wide SSR High Density Genetic Map Construction from an Interspecific Cross of Gossypium hirsutum × Gossypium tomentosum.

A high density genetic map was constructed using F2 population derived from an interspecific cross of G. hirsutum × G. tomentosum. The map consisted of 3093 marker loci distributed across all the 26 chromosomes and covered 4365.3 cM of cotton genome with an average inter-marker distance of 1.48 cM. The maximum length of chromosome was 218.38 cM and the minimum was 122.09 cM with an average length of 167.90 cM. A sub-genome covers more genetic distance (2189.01 cM) with an average inter loci distance of 1.53 cM than D sub-genome which covers a length of 2176.29 cM with an average distance of 1.43 cM. There were 716 distorted loci in the map accounting for 23.14% and most distorted loci were distributed on D sub-genome (25.06%), which were more than on A sub-genome (21.23%). In our map 49 segregation hotspots (SDR) were distributed across the genome with more on D sub-genome as compared to A genome. Two post-polyploidization reciprocal translocations of "A2/A3 and A4/A5" were suggested by seven pairs of duplicate loci. The map constructed through these studies is one of the three densest genetic maps in cotton however; this is the first dense genome wide SSR interspecific genetic map between G. hirsutum and G. tomentosum.

Development and characterization of 25 EST-SSR markers in Pinus sylvestris var. mongolica (Pinaceae).

PREMISE OF THE STUDY: A set of novel expressed sequence tag (EST) microsatellite markers was developed in Pinus sylvestris var. mongolica to promote further genetic studies in this species. • METHODS AND RESULTS: One hundred seventy-five EST-simple sequence repeat (SSR) primers were designed and synthesized for 31,653 isotigs based on P. tabuliformis EST sequences. The primer pairs were used to identify 25 polymorphic loci in 48 individuals. The number of alleles ranged from two to eight with observed and expected heterozygosity values of 0.0435 to 0.8125 and 0.0430 to 0.7820, respectively. • CONCLUSIONS: These new polymorphic EST-SSR markers will be useful for assessing genetic diversity, molecular breeding and genetic improvement, and conservation of P. sylvestris var. mongolica.

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.