Development Of Species-specific Markers Research Articles

Distribution and conservation of simple sequence repeats in plant pathogenic species of Zymoseptoria and development of genomic resources for its orphaned species.

To better understand the structure and evolution of the genomes of four plant pathogenic species of Zymoseptoria, we analyzed the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) in their whole genome and transcriptome sequences. In this study, SSRs are defined as repeats of more than 12 bases in length. The genome and transcriptome sequences of Zymoseptoria ardabiliae show the highest RA (201.1 and 129.9) and RD (3229.4 and 1928.2) of SSRs, while those of Zymoseptoria pseudotritici show the lowest RA (167.2 and 118.5) and RD (2482.2 and 1687.0). The majority of SSRs in the genomic and transcriptome sequences of species were trinucleotide SSRs, while dinucleotide SSRs were the least common. The most common trinucleotide motifs in the transcriptomic sequences across all species were those that encoded the amino acid arginine. As per our motif conservation study, Zymoseptoria tritici (12.4%) possessed the most unique motifs, while Z. pseudotritici (3.9%) had the fewest. Overall, only 38.1% of the motifs were found to be conserved among the species. Gene enrichment studies reveal that three of the species, Z. ardabiliae, Zymoseptoria brevis, and Z. pseudotritici, have SSRs in their genes related to cellular metabolism, while the remaining Z. tritici harbors SSRs in genes related to DNA synthesis and gene expression. In an effort to improve the genetic resources for the orphan species of pathogenic Zymoseptoria, a total of 73,134 primers were created. The genomic resources developed in this study could help with analyses of genetic relatedness within the population and the development of species-specific markers.

Complete organellar genomes of six Sargassum species and development of species-specific markers

Sargassum is one of the most important brown algal genera that can be used as food and raw material for medicinal purpose, and has various beneficial effects. As the classification of Sargassum species is currently based on their morphological characteristics, organellar genome sequences of Sargassum would provide important information for accurate identification of species and developing species-specific markers. We sequenced the complete organellar genomes of six Sargassum species, including the first complete chloroplast genome sequences of S. fulvellum, S. serratifolium, S. macrocarpum, and S. siliquastrum, and the first complete mitochondrial genome sequences of S. fulvellum, S. serratifolium, and S. macrocarpum. The chloroplast genomes of the 6 Sargassum species contained 139 protein-coding genes (PCGs), and the mitochondrial genomes possessed 37 PCGs. A comparative study was performed between the newly sequenced organellar genomes and 44 other species belonging to class Phaeophyceae. Phylogenetic relationships using PCGs shared by Phaeophyceae species were constructed with IQ-TREE 2 using the maximum likelihood method. In addition, we developed real-time PCR markers based on SNPs to distinguish the 6 Sargassum species. Our results provide useful information for establishing phylogenetic relationships between brown algae.

Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

Tilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

Development of specific markers for the detection of Tolypella canadensis eDNA in water samples

Assessing the biodiversity of an ecosystem plays a major role in ecosystem management. However, proper determination on species-level is often tricky when morphological features are scarce and especially rare species require huge sampling efforts to be detected in the aquatic realm. As an alternative to conventional methods, environmental samples can be examined via the eDNA method, allowing for large-scale integration as well as taxa resolution independent from expression of morphological characters. However, to apply this technique genetic markers that are specific to a species or at least a genus are required. Such markers until now have been successfully developed only for a few well studied taxonomic groups like, e.g., fishes and amphibians, but are still missing for others, especially plants and algae (e.g. Bista et al. 2017). This project focusses on the development of species-specific markers for the macrophytic green algae Tolypella canadensis (Characeae, Charophyta), a rare alga preferring deep water and known so far mainly from remote places. Tolypella canadensis is a circumpolar species and prefers oligotrophic lakes, where it grows in depths up to 13 m (Langangen 2002; Romanov and Kopyrina 2016). In addition, proper determination of Tolypella-species is a field of a few specialists, further complicating monitoring or even detection of this rare species. The design of the species-specific primers was based on reference nucleotide sequences of the chloroplast genes rbcL, psbC and atpB and of the ribosomal internal transcribed spacer regions ITS1 and ITS2, obtained from GenBank (Perez et al. 2017). To determine the specificity of the newly designed primers, DNA isolates obtained from T. canadensis specimens collected from the Torneträsk (Sweden, 2018) and other charophyte species were prepared in different proportions. The sensitivity of the primers was experimentally assayed by using serial dilutions of T. canadensis DNA. Additionally, a mock test comprised of a sample with the DNA of several charophyte species was conducted and finally, the markers were tested on environmental samples from the Torneträsk. Tolypella canadensis-specific primers of the ITS2 region yielded positive PCR amplifications of one single band when T. canadensis was present in a sample. Cross-amplification was not found during the mock test; other charophyte species did not yield positive amplification. The eDNA samples from the Torneträsk validated the performance of the ITS2 marker. The T. canadensis-specific marker designed in this project was proven to be sensitive and accurate. It could be recommended as a useful tool to detect the presence of T. canadensis DNA, even at low concentration and in complex samples containing other charophyte species.

Development and characterization of 18 microsatellite loci from the endemic elongate loach (Leptobotia elongata)

18 novel microsatellite loci were separated by the method of triplex affinity capture for Leptobotia elongate. All these 18 loci were polymorphic in L. elongate and the number of alleles per locus ranged from 5 to 14. The observed and expected heterozygosity were 0.259–0.966 and 0.486–0.918, respectively. Deviation from Hardy–Weinberg equilibrium was indicated at four loci. The significant pairwise linkage disequilibrium was found in one pair of loci. This is the first development of species-specific markers for L. elongate. These microsatellite loci would provide new opportunities for a wide range of population genetic and taxonomic studies of L. elongate.

Isolation and characterization of species-specific microsatellite loci in African penguin (Spheniscus demersus)

Eight microsatellite markers were developed via pyrosequencing of a microsatellite-enriched library for the African Penguin (Spheniscus demersus). These microsatellite loci displayed 2–6 alleles with expected heterozygosity values ranging between 0.316 and 0.782 and observed heterozygosity between 0.381 and 0.84. These loci may be suitable for assessing patterns of genetic variability in African penguin. This is the first development of species-specific markers for the African penguin.

Isolation and characterization of species-specific microsatellite loci in red-billed oxpeckers (Buphagus erythrorhynchus)

Ten microsatellite markers were developed via pyrosequencing for the red-billed oxpecker (Buphagus erythrorhynchus). These microsatellite loci displayed 3–15 alleles with expected heterozygosity values ranging between 0.580 and 0.907 and observed heterozygosity between 0.509 and 0.870. The high level of polymorphisms observed in the microsatellite markers supports future investigations to improve our knowledge of the genetic structure and relatedness of these birds. This is the first development of species specific markers for the red-billed oxpecker.

Development of DNA Barcode and Species-Specific Markers forHelopeltis antoniiSignoret andPachypeltis maesarum(Kirkaldy) (Heteroptera: Miridae), Pests of Cashew in India

Some species of mirids are major pests of cashew damaging tender shoots, inflorescence, immature nuts, and apples at various stages of development. In India, crop loss is often more than 40% in cashew alone, and the genus Helopeltis closely resembles the genus Pachypeltis. Using molecular biology tools, morphological identification is further strengthened by DNA barcoding (using COX-1) and development of species-specific markers, which helps a non-specialist to identify a species. The molecular identification of Helopeltis and Pachypeltis spp. has been corroborated with morphological identification. The investigation results help in quick, accurate, and timely identification of Helopeltis antonii and Pachypeltis maesarum, a critical factor in understanding the epidemiology of the crop losses in cashew, their management and also in quarantine.

Development and characterization of ten polymorphic microsatellites isolated from the scallop Argopecten purpuratus

The scallop, Argopecten purpuratus, is a bivalve, Pectinidae distributed along the Pacific coasts of Peru and northern Chile, between Paita (5◦S, 81◦W) and Valparaiso (33◦S, 71◦W), (Bore and Martinez 1980; Wolf and Mendo 2000; Winkler et al. 2001). The three major natural populations in A. purpuratus along the Chilean coast are located in the bays of Mejillones (23◦54′S), La Rinconada (23◦28′S) and Tongoy (30◦14′S) (Avendano et al. 2007, 2008; Avendano and Cantillanez 2008). There is a need to recognize various characteristics and properties of different populations of A. purpuratus. This will help in commercial exploitation of this species and also enable the avoidance of problems due to inbreeding. The development of species specific markers is essential to achieve these objectives (Freiwald et al. 2009; Arias et al. 2010). Microsatellite DNA loci have become important sources of genetic information for a variety of purposes (Goldstein and Schlotterer 1999; Perera et al. 2000; Webster and Reichart 2004). To this end, we developed 10 autosomal microsatellite markers for this species. To evaluate the usefulness and variability of each marker, 43 individuals were characterized along the Chilean coast. Seventeen loci were originally isolated. Two failed to amplify using labelled primers. Of the remaining 15, five are considered unsuitable due to apparently high incidence of null alleles,

Molecular Identification and Phylogeny ofBactroceraSpecies (Diptera: Tephritidae)

Fruit flies that belong to the genus Bactrocera (Diptera: Tephritidae) are major invasive pests of agricultural crops in Asia and Australia. Increased transboundary movement of agricultural produce has resulted in the chance introduction of many invasive species that include Bactrocera mainly as immature stages. Therefore quick and accurate species diagnosis is important at the port of entry, where morphological identification has a limited role, as it requires the presence of adult specimens and the availability of a specialist. Unfortunately when only immature stages are present, a lacunae in their taxonomy impedes accurate species diagnosis. At this juncture, molecular species diagnostics based on COX-I have become handy, because diagnosis is not limited by developmental stages. Yet another method of quick and accurate species diagnosis for Bactrocera spp. is based on the development of species-specific markers. This study evaluated the utility of COX-I for the quick and accurate species diagnosis of eggs, larvae, pupae and adults of B. zonata Saunders, B. tau Walker, and B. dorsalis Hendel. Furthermore the utility of species-specific markers in differentiating B. zonata (500bp) and B. tau (220bp) was shown. Phylogenetic relationships among five subgenera, viz., Austrodacus, Bactrocera, Daculus, Notodacus and Zeugodacus have been resolved employing the 5′ region of COX-I (1490-2198); where COX-I sequences for B. dorsalis Hendel, B. tau Walker, B. correcta Bezzi and B. zonata Saunders from India were compared with other NCBI-GenBank accessions. Phylogenetic analysis employing Maximum Parsimony (MP) and Bayesian phylogenetic approach (BP) showed that the subgenus Bactrocera is monophyletic.