Dinucleotide Motifs Research Articles

Non-random distribution of microsatellite motifs and (TTAGGG)n repeats in the monkey frog Pithecopus rusticus (Anura, Phyllomedusidae) karyotype.

The monkey frog, Pithecopus rusticus (Anura, Phyllomedusidae) is endemic to the grasslands of the Araucarias Plateau, southern Brazil. This species is known only from a small population found at the type locality. Here, we analyzed for the first time the chromosomal organization of the repetitive sequences, including seven microsatellite repeats and telomeric sequences (TTAGGG)n in the karyotype of the species by Fluorescence in situ Hybridization. The dinucleotide motifs had a pattern of distribution clearly distinct from those of the tri- and tetranucleotides. The dinucleotide motifs are abundant and widely distributed in the chromosomes, located primarily in the subterminal regions. The tri- and tetranucleotides, by contrast, tend to be clustered, with signals being observed together in the secondary constriction of the homologs of pair 9, which are associated with the nucleolus organizer region. As expected, the (TTAGGG)n probe was hybridized in all the telomeres, with hybridization signals being detected in the interstitial regions of some chromosome pairs. We demonstrated the variation in the abundance and distribution of the different microsatellite motifs and revealed their non-random distribution in the karyotype of P. rusticus. These data contribute to understand the role of repetitive sequences in the karyotype diversification and evolution of this taxon.

Rapid development of novel microsatellite markers from Mauremys reevesii (Testudines: Geoemydidae) using next-generation DNA sequencing technology

Mauremys reevesii (Gray, 1831), which belongs to Mauremys of Geoemydidae (Testudines), distributed in China, as well as Japan and Korea. Previous studies have developed several polymorphic microsatellite loci, but most of them were dinucleotide motifs. Here, we developed 15 polynucleotide-repeat microsatellite loci (including di-, tri, tetra-and pentanucleotide motifs) for M. reevesii through Restriction-site Associated DNA tags sequencing (RAD-seq). A total of 987 microsatellite loci with flanking sequences were suitable for setting primers for polymerase chain reactions (PCR). To verify the identified SSRs, 40 primer pairs were selected for PCR detection. In total, 32 primer sets produced strong PCR products matching their expected sizes, in which species amplification tests showed that 15 were polymorphic. And the number of alleles per locus ranged from 3 to 16. The observed and expected heterozygosity per locus varied from 0.3784 to 1.000 and from 0.3995 to 0.9700, respectively. The methodology of microsatellite isolation constructed in this study is not only cost-effective and time-saving in comparison to traditional approaches, but also can be served as useful tools which benefit population genetics studies and conservation management of M. reevesii.

Development of EST-SSR markers and their application in an analysis of the genetic diversity of the endangered species Magnolia sinostellata.

Magnolia sinostellata is an endemic species of Magnoliaceae that is narrowly distributed in the south of Zhejiang Province, China. To explore the genetic diversity and population structure of this endangered species, this study developed sequence tag-simple sequence repeat (EST-SSR) markers based on transcriptome data of M. sinostellata. In total, 25472 SSRs were identified among 110644 unique assembled sequences with a total of 90.83Mb and an average frequency of 23.02%. The mononucleotide (33.53%) and dinucleotide (42.08%) motifs appeared to be the most abundant. In total, 150 potential loci were randomly selected to validate the quality of the developed SSR markers; an effective PCR rate of 32.00% and a polymorphism rate of 15.33% were obtained for these loci. After performing sequencing and cloning for validation, 23 pairs of SSR primers were retained and used to characterize the genetic diversity and population structure of M. sinostellata. Overall, 204 alleles were amplified. The results of Shannon's information index (I), heterozygosity (Ho), heterozygosity (He) and Nei's expected heterozygosity (H) indicated rich genetic diversity in M. sinostellata. However, the high inbreeding coefficient and differential coefficient suggest that serious genetic drift occurred within populations, and genetic differentiation is apparent among the populations. Consequently, although M. sinostellata has high genetic diversity among populations, it is still in a serious and dangerous condition. Habitat destruction caused by human activities is the main threat to this species, and enhancing the species abundance by adopting some conservation measures should be favourable for saving the species.

First draft genome sequencing of fennel (Foeniculum vulgare Mill.): identification of simple sequence repeats and their application in marker-assisted breeding

The development of F1 hybrid varieties benefits from the synergistic effect of conventional and molecular marker-assisted breeding schemes. A sequencing run was carried out in Foeniculum vulgare (2n = 2x = 22) to develop the first genome draft and to identify microsatellites suitable for implementing multilocus SSR marker assays. A preliminary cytometric analysis allowed us to estimate the genome size (2C = 2.64–2.86 pg), equal to about 1.34 Mbp for 1C genome, and to calculate the sequencing coverage (53×). The genome draft assembly into 300,408 scaffolds and its bioinformatic analysis enabled the annotation of coding and non-coding regions across the genome, including 103,306 SSR elements. A total of 100 microsatellites were randomly chosen among those with dinucleotide and trinucleotide repeat motifs and with a repeat motif length ≥ 25 times and were preliminarily tested. Of these, 27 SSR markers, classified as suitable for genetic diversity analyses, were efficiently organized in five PCR multiplex assays and validated using a core collection of 100 fennel individuals potentially useful for the development of inbred lines and F1 hybrids. All SSR loci were found to be polymorphic, scoring an observed number of marker alleles Na = 207 and an average polymorphism information content PIC = 0.69. The SSR data were used to calculate (i) the degree of homozygosity for the individual inbred lines (0.35 < Ho < 0.96), to eventually plan additional selfing or sibling cycles, and (ii) the degree of genetic similarity for all possible pair-wise comparisons between parental inbred lines (GS = 0.55–0.77), to identify the most divergent combinations for the constitution of experimental F1 hybrids. The integration of genotypic and phenotypic data was useful for implementing guidelines for precision hybrid breeding schemes in fennel.

Chitosan-based Nanoparticles in Mucosal Vaccine Delivery.

Most infectious diseases are caused by pathogenic infiltrations from the mucosal tract. Nowadays, the use of vaccines has been widely investigated for the prevention of different infectious diseases, infertility, immune disorders, malignancies, and allergies. Broad-spectrum adjuvant substances have been studied for immune system stimulation with a greater efficiency against specific antigens. Various adjuvants have been developed such as inorganic, oil-based, and emulsion adjuvants, bacterial products and their derivatives, cytokines, cytosine-guanine dinucleotide (CpG) motifs, and particulate systems. Mucosal vaccine delivery is an alternative route to induce both humoral and cellular immune responses. Applying nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery, and consequently, more specific release of the agent of interest. Chitosan nanoparticles have immunological activity and mucoadhesive properties. They have been used as a mucosal vaccine delivery system for many antigens. This review provides an overview of the recent advances in chitosan nanoparticles as a novel mucosal vaccine delivery system.

Dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids

Lipid nanoparticles (LNPs) are playing a leading role in enabling clinical applications of gene therapies based on DNA or RNA polymers. One factor impeding clinical acceptance of LNP therapeutics is that LNP formulations of nucleic acid polymers can be immunostimulatory, necessitating co-administration of potent corticosteroid immunosuppressive agents. Here, we describe the development of hydrophobic prodrugs of a potent corticosteroid, dexamethasone, that can be readily incorporated into LNP systems. We show that the presence of the dexamethasone prodrug LD003 effectively suppresses production of cytokines such as KC-GRO, TNFα, IL-1β and IL-6 following intravenous administration of LNP loaded with immune stimulatory oligodeoxynucleotides containing cytosine-guanine dinucleotide motifs. Remarkably, LD003 dose levels corresponding to 0.5 mg/kg dexamethasone achieve a greater immunosuppressive effect than doses of 20 mg/kg of free dexamethasone. Similar immunosuppressive effects are observed for subcutaneously administered LNP-siRNA. Further, the incorporation of low levels of LD003 in LNP containing unmodified mRNA or plasmid DNA significantly reduced pro-inflammatory cytokine levels following intravenous administration. Our results suggest that incorporation of hydrophobic prodrugs such as LD003 into LNP systems could provide a convenient method for avoiding the immunostimulatory consequences of systemic administration of genetic drug formulations.

Development of Microsatellite Markers for the Nipa Palm Hispid Beetle, Octodonta nipae (Maulik).

The nipa palm hispid beetle, Octodonta nipae (Maulik) (Coleoptera: Chrysomelidae), is an important invasive pest on palm plants in southern China. Based on existing transcriptome data, polymorphism simple sequence repeat (SSR) loci were identified. In total, 1274 SSR loci were identified from 49919 unigenes. The majority of them contained mononucleotide, dinucleotide, and trinucleotide motifs (43.56%, 26.14%, and 28.18%), in which A/T (41.21%) and AT/TA (15.86%) were the most abundant motifs. 104 pairs of the SSR primers produced amplification bands of expected sizes in O. nipae, 80 pairs of SSR primers were tested randomly for polymorphism, 9 loci of them were validated to be polymorphic markers, and the number of alleles ranged from 2 to 3, with an average of 2.56 per locus. The population of Zhangzhou and Fuzhou was analyzed by the 9 loci (On1–On9). These SSR transcriptome data can provide invaluable resource for SSR development, population genetics research, invasion and expansion mechanism, paternity testing, and other research on O. nipae and its related species.

Development of microsatellite genetic markers in Siberian larch (Larix sibirica Ledeb.) based on the de novo whole genome sequencing

This special issue of the journal is devoted to the outstanding population geneticist Yuri Petrovich Altukhov, who paid much attention in his research to the development of molecular genetic markers for population studies. Over the past time markers and methods of their development have undergone significant change. Thanks to modern methods of whole genome sequencing, it has become possible to develop markers of very different types—selectively neutral, as well as functional. Among them, microsatellite markers remain the most informative, convenient, reproducible, relatively inexpensive, and polymorphic. Whole genome sequencing greatly facilitates their discovery and development. This paper is devoted to the development of new microsatellite markers for a very important species of boreal forest—Siberian larch (Larix sibirica Ledeb.). Using a draft assembly of the larch genome, several thousand contigs containing microsatellite loci with di-, tri, tetra-, and pentanucleotide motifs were selected. A total of 59 pairs of PCR primers were tested for loci with dinucleotide motifs as the most variable. From them, 11 pairs were finally selected for 11 loci with dinucleotide repeats, which showed a high level of polymorphism and can be used in various population genetic studies and to identify the origin of wood and plant material. This study was done at the Laboratory of Forest Genomics of the Genome Research and Education Center of the Siberian Federal University with the support of the Department of Forest Genetics and Forest Tree Breeding of the Georg-August University of Gottingen, the Department for Monitoring of Forest Genetic Resources of the Forest Protection Center of the Krasnoyarsk Territory, and the Laboratory of Forest Genetics and Selection of the Sukachev Institute of Forest of the Siberian Branch of the Russian Academy of Sciences within the framework of the project “Genomics of the Key Boreal Forest Conifer Species and Their Major Phytopathogens in the Russian Federation” funded by the Government of the Russian Federation (grant no. 14.Y26.31.0004).

Radiation-enhanced delivery of systemically administered amphiphilic-CpG oligodeoxynucleotide

Along with vaccines and checkpoint blockade, immune adjuvants may have an important role in tumor immunotherapy. Oligodeoxynucleotides containing unmethylated cytidyl guanosyl dinucleotide motifs (CpG ODN) are TLR9 ligands with attractive immunostimulatory properties, but intratumoral administration has been required to induce an effective anti-tumor immune response. Following on recent studies with radiation-targeted delivery of nanoparticles, we examined enhanced tumor-specific delivery of amphiphile-CpG, an albumin-binding analog of CpG ODN, following systemic administration 3days after tumor irradiation. The combination of radiation and CpG displayed superior tumor control over either treatment alone. Intravital imaging of fluorescently labeled amphiphilic-CpG revealed increased accumulation in irradiated tumors along with decreased off-target accumulation in visceral organs. Within 48h after amphiphile-CpG administration, immune activation could be detected by increased Granzyme B and Interferon gamma activity in the tumor as well as in circulating monocytes and activated CD8+ T cells. Using radiotherapy to enhance the targeting of CpG to tumors may help advance this once promising therapy to clinical relevance.

Novel 307 polymorphic SSR markers from BAC-end sequences in walnut (Juglans regia L.): Effects of motif types and repeat lengths on polymorphism and genetic diversity

Walnut, Juglans regia L., has been cultivated for its edible nut and timber since ancient times. It is one of the most valuable and widely cultivated horticultural commodities in the world. Simple sequence repeats (SSRs) have a wide range of applications in plants, and there is still a necessity to develop novel SSR markers for each plant species. Bacterial artificial chromosome-end sequences (BES) are good sources to generate novel SSR markers. In pursuit of this objective, a total of 558 BES-SSR primer pairs were designed for J. regia. Of them, 507 (91%) had amplifications, and 307 were polymorphic when tested in eight walnut cultivars. A total of 1097 alleles were generated from 307 polymorphic SSR loci, ranging from two to eleven, with an average of 3.6 per locus. Polymorphism information contents (PIC) varied from 0.11 to 0.88 with an average of 0.46. Di-nucleotide motifs had higher polymorphism and genetic diversity values than the other motif types. The rate of polymorphism increased with the repeat length, therefore, class I SSRs are useful source of polymorphic DNA markers in walnut. Novel SSR markers developed in this study may possess potential applications such as fingerprinting, marker-assisted breeding, germplasm characterization and genetic linkage mapping in walnut and other species of Juglans.

A Genome-Wide Survey of the Microsatellite Content of the Globe Artichoke Genome and the Development of a Web-Based Database.

The recently acquired genome sequence of globe artichoke (Cynara cardunculus var. scolymus) has been used to catalog the genome’s content of simple sequence repeat (SSR) markers. More than 177,000 perfect SSRs were revealed, equivalent to an overall density across the genome of 244.5 SSRs/Mbp, but some 224,000 imperfect SSRs were also identified. About 21% of these SSRs were complex (two stretches of repeats separated by <100 nt). Some 73% of the SSRs were composed of dinucleotide motifs. The SSRs were categorized for the numbers of repeats present, their overall length and were allocated to their linkage group. A total of 4,761 perfect and 6,583 imperfect SSRs were present in 3,781 genes (14.11% of the total), corresponding to an overall density across the gene space of 32,5 and 44,9 SSRs/Mbp for perfect and imperfect motifs, respectively. A putative function has been assigned, using the gene ontology approach, to the set of genes harboring at least one SSR. The same search parameters were applied to reveal the SSR content of 14 other plant species for which genome sequence is available. Certain species-specific SSR motifs were identified, along with a hexa-nucleotide motif shared only with the other two Compositae species (sunflower (Helianthus annuus) and horseweed (Conyza canadensis)) included in the study. Finally, a database, called “Cynara cardunculus MicroSatellite DataBase” (CyMSatDB) was developed to provide a searchable interface to the SSR data. CyMSatDB facilitates the retrieval of SSR markers, as well as suggested forward and reverse primers, on the basis of genomic location, genomic vs genic context, perfect vs imperfect repeat, motif type, motif sequence and repeat number. The SSR markers were validated via an in silico based PCR analysis adopting two available assembled transcriptomes, derived from contrasting globe artichoke accessions, as templates.

Increased expression of interleukin-6 (IL-6) gene transcript in relation to IL-6 promoter hypomethylation in gingival tissue from patients with chronic periodontitis

ObjectiveDNA methylation of the cytokine genes may play a role in the pathogenesis of periodontitis. The aim of this study is to evaluate whether the alteration of interleukin-6 (IL-6) gene promoter methylation in the gingival tissue (GT) and peripheral blood (PB) is unique to chronic periodontitis (CP). DesignDNA isolated from the GT and PB of 25 patients with (CP) and 20 healthy controls (H) was modified with sodium bisulfite and analyzed for IL-6 promoter methylation with direct sequencing. The levels of IL-6 mRNA and serum IL-6 protein were evaluated by a quantitative reverse transcription polymerase chain reaction and an enzyme-linked immunosorbent assay. ResultsThe CP group showed that the overall methylation rates of IL-6 promoter that contained 19 cytosine-guanine dinucleotide (CpG) motifs were significantly decreased in GT in comparison to PB (p<0.001), which was significantly negatively correlated with the probing depth (p=0.003). The GT and PB of the H group displayed similar overall methylation rates. No significant difference was observed in the methylation rates at each CpG in GT in comparison to the PB in both groups. The levels of IL-6 mRNA in the GT and PB and serum IL-6 of the two groups were comparable. The ratio of IL-6 mRNA in the GT relative to the PB was significantly higher in the CP group than in the H group (p=0.03). ConclusionThe increased expression of IL-6 gene transcription may be related to IL-6 promoter hypomethylation in the GT from CP patients.

Development of new SSR markers for homoeologous WFZP gene loci based on the study of the structure and location of microsatellites in gene-rich regions of chromosomes 2AS, 2BS, and 2DS in bread wheat

Microsatellites, or simple sequence repeats, are widely distributed in eukaryotic genomes, including plant genomes. The peculiarities of the structure and location of the microsatellite loci determine their potential as molecular genetic markers and can influence the assumed function of microsatellites in important biological processes. The identification and study of the distribution of microsatellite loci in gene-rich genome regions of the bread wheat and the development (based on them) of new microsatellite markers are of practical interest and are important for the study of the organization of the bread wheat genome. The sequences of BAC clones that contain the homoeologous WFZP genes of the bread wheat (Triticum aestivum L.) controlling the development of the ear were the basis for the identification and localization of microsatellite loci in gene-rich regions of the 2AS, 2BS, and 2DS chromosomes. Diand trinucleotide microsatellite repeats are the most widespread in the studied sequences. The AG and GA/TC motifs prevail among the dinucleotide motifs; the dinucleotide repeats are found in noncoding gene regions, mobile elements, and nonannotated DNA sequences. Most of the trinucleotide repeats are associated with mobile genetic elements. It was found that homoeologous microsatellite loci are located either in the genes or in the nonannotated DNA sequences. The comparison of the structure of homoeologous loci demonstrated that the divergence in them is associated both with a change in the number of repeats and with nucleotide substitutions. The new microsatellite markers, which are colocalized in the genetic maps with the WFZP-A-B-D genes and can be used for marking these genes in molecular genetic studies and in breeding controlled by markers, were developed.

Vaccination with the Secreted Glycoprotein G of Herpes Simplex Virus 2 Induces Protective Immunity after Genital Infection.

Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in sensory ganglia. After primary infection HSV-2 may reactivate causing recurrent genital ulcerations. HSV-2 infection is prevalent, and globally more than 400 million individuals are infected. As clinical trials have failed to show protection against HSV-2 infection, new vaccine candidates are warranted. The secreted glycoprotein G (sgG-2) of HSV-2 was evaluated as a prophylactic vaccine in mice using two different immunization and adjuvant protocols. The protocol with three intramuscular immunizations combining sgG-2 with cytosine-phosphate-guanine dinucleotide (CpG) motifs and alum induced almost complete protection from genital and systemic disease after intra-vaginal challenge with HSV-2. Robust immunoglobulin G (IgG) antibody titers were detected with no neutralization activity. Purified splenic CD4+ T cells proliferated and produced interferon-γ (IFN-γ) when re-stimulated with the antigen in vitro. sgG-2 + adjuvant intra-muscularly immunized mice showed a significant reduction of infectious HSV-2 and increased IFN-γ levels in vaginal washes. The HSV-2 DNA copy numbers were significantly reduced in dorsal root ganglia, spinal cord, and in serum at day six or day 21 post challenge. We show that a sgG-2 based vaccine is highly effective and can be considered as a novel candidate in the development of a prophylactic vaccine against HSV-2 infection.

Distribution of CpG Motifs in Upstream Gene Domains in a Reef Coral and Sea Anemone: Implications for Epigenetics in Cnidarians.

Coral reefs are under assault from stressors including global warming, ocean acidification, and urbanization. Knowing how these factors impact the future fate of reefs requires delineating stress responses across ecological, organismal and cellular scales. Recent advances in coral reef biology have integrated molecular processes with ecological fitness and have identified putative suites of temperature acclimation genes in a Scleractinian coral Acropora hyacinthus. We wondered what unique characteristics of these genes determined their coordinate expression in response to temperature acclimation, and whether or not other corals and cnidarians would likewise possess these features. Here, we focus on cytosine methylation as an epigenetic DNA modification that is responsive to environmental stressors. We identify common conserved patterns of cytosine-guanosine dinucleotide (CpG) motif frequencies in upstream promoter domains of different functional gene groups in two cnidarian genomes: a coral (Acropora digitifera) and an anemone (Nematostella vectensis). Our analyses show that CpG motif frequencies are prominent in the promoter domains of functional genes associated with environmental adaptation, particularly those identified in A. hyacinthus. Densities of CpG sites in upstream promoter domains near the transcriptional start site (TSS) are 1.38x higher than genomic background levels upstream of -2000 bp from the TSS. The increase in CpG usage suggests selection to allow for DNA methylation events to occur more frequently within 1 kb of the TSS. In addition, observed shifts in CpG densities among functional groups of genes suggests a potential role for epigenetic DNA methylation within promoter domains to impact functional gene expression responses in A. digitifera and N. vectensis. Identifying promoter epigenetic sequence motifs among genes within specific functional groups establishes an approach to describe integrated cellular responses to environmental stress in reef corals and potential roles of epigenetics on survival and fitness in the face of global climate change.

Genome Survey Sequencing for the Characterization of the Genetic Background of Rosa roxburghii Tratt and Leaf Ascorbate Metabolism Genes.

Rosa roxburghii Tratt is an important commercial horticultural crop in China that is recognized for its nutritional and medicinal values. In spite of the economic significance, genomic information on this rose species is currently unavailable. In the present research, a genome survey of R. roxburghii was carried out using next-generation sequencing (NGS) technologies. Total 30.29 Gb sequence data was obtained by HiSeq 2500 sequencing and an estimated genome size of R. roxburghii was 480.97 Mb, in which the guanine plus cytosine (GC) content was calculated to be 38.63%. All of these reads were technically assembled and a total of 627,554 contigs with a N50 length of 1.484 kb and furthermore 335,902 scaffolds with a total length of 409.36 Mb were obtained. Transposable elements (TE) sequence of 90.84 Mb which comprised 29.20% of the genome, and 167,859 simple sequence repeats (SSRs) were identified from the scaffolds. Among these, the mono-(66.30%), di-(25.67%), and tri-(6.64%) nucleotide repeats contributed to nearly 99% of the SSRs, and sequence motifs AG/CT (28.81%) and GAA/TTC (14.76%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. Genome analysis predicted a total of 22,721 genes which have an average length of 2311.52 bp, an average exon length of 228.15 bp, and average intron length of 401.18 bp. Eleven genes putatively involved in ascorbate metabolism were identified and its expression in R. roxburghii leaves was validated by quantitative real-time PCR (qRT-PCR). This is the first report of genome-wide characterization of this rose species.

Development and validation of novel fiber relevant dbEST–SSR markers and their utility in revealing genetic diversity in diploid cotton (Gossypium herbaceum and G. arboreum)

Compared to tetraploid, diploid cotton especially Gossypium herbaceum are poor in genomic resources including molecular markers. Expressed sequence tags (ESTs) in public databases are valuable resources and offer opportunity for identification of simple sequence repeat (SSR) markers. ESTs data base of cotton were employed for the development of novel EST–SSR markers. Sequence assembly and clustering of 1,31,464 dbESTs (G. hirsutum, 92,570 and G. arboreum, 38,894) resulted in 28,718 unigenes (17,019 singletons and 11,699 contigs) having 1,760 SSRs. On an average, the unigenes contained 1 SSR/16.3kb of unigene. The AG class was the most common among the dinucleotide motifs (45%), whereas the AAG class (25%) was predominant among the trinucleotide motif. A total of 378 novel primers pairs were developed from the ESTs of cotton fiber cells and ovules. A set of 60 primers associated with fiber development were selected and validated on a panel of 23 G. herbaceum and nine G. arboreum genotypes. Among 60, 44 primers d amplified 71 loci; of which 54 (76%) were polymorphic. The polymorphic information content (PIC) ranged from 0.11–0.74. The class II repeat SSR motifs were more polymorphic than class I repeat SSR markers. Nei’s dissimilarity coefficients based on SSR markers among the all pair-wise combinations of genotypes ranged from 0.02 to 0.49 with a mean of 0.24. The UPGMA clustering grouped 32 genotypes in 2 clusters at cut off value of 0.23. The EST–SSR markers developed in our study would be best assets for characterization of germplasm, population structure analysis and QTL mapping in diploid cotton.

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

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.

Analysis of EST-SSRs in silver birch (Betula pendula Roth.)

Simple sequence repeats (SSRs) defined as sequence repeat units between 1 and 6 bp occur abundantly in both coding and non-coding regions in eukaryotic genomes and these repeats can affect gene expression. In this study, ESTs (expressed sequence tags) of Betula pendula (silver birch) were analyzed for in silico mining of EST-SSRs, protein annotation, open reading frames (ORFs), designing primers, and identifying codon repetitions. In B. pendula, the frequency of ESTs containing SSRs was 7.8 % with an average of 1SSR/4. 78 kb of EST sequences. A total of 188 SSRs was identified by using MISA software and di-nucleotide SSR motifs (65.9 %) were found to be the most abundant type of repeat motif followed by tri- (27.1 %), tetra- (4.8 %), and penta- (2.2 %) motifs. Based on ORF analysis, 175 of 178 sequences were predicted as ORFs and the most frequent SSRs were detected in 5′ UTR (58.43 %), followed by in ORF (31.46 %) and in 3′ UTR (8.43 %). 102 of 178 ESTs were annotated as ribosomal protein, transport protein, membrane protein, carrier protein, binding protein, and transferase protein. For a total of 102 SSRs (57.3 %) with significant matches, a set of 102 primers (100 %) with forward and reverse strands was designed by using Primer3 software. Serine (Ser, 19.6 %) was predominant in putative encoded amino acids and most of amino acids showed nonpolar (35.3 %) nature. Our data provide resources for B. pendula and can be useful for in silico comparative analyses of Betulaceae species, including SSR mining.

Development of microsatellite markers for Hyacinth macaw (Anodorhynchus hyacinthinus) and their cross-amplification in other parrot species.

BackgroundHyacinth macaw Anodorhynchus hyacinthinus is the largest parrot of the world and is considered vulnerable to extinction due to its habitat loss and illegal trade associated to the international pet market demand. Genetic studies on this species are still incipient to generate a consistent characterization of the population dynamics and to develop appropriate conservation strategies. In this sense, microsatellite markers may support the detection of a population genetic structure for this bird species. However, at this time, none Hyacinth macaw species-specific primers for microsatellite loci have been so far established. This study aimed to develop and characterize polymorphic microsatellite markers for A. hyacinthinus and to check for their cross-amplification in other parrot species.FindingsSequences containing repeated dinucleotide motifs were prospected and optimized from a genomic library that was enriched for microsatellites using magnetic beads. The analyses of 43–57 samples from wild individuals of three distinct Brazilian subpopulations led to the characterization of five polymorphic microsatellite loci. Allele richness per locus ranged from two to 12. Three loci exhibited observed heterozygosity values higher than 50 %, but the overall average value among all loci was close to 45 %. In addition, successful primer cross-amplification was verified in seven other investigated species of Neotropical parrots.ConclusionsThe newly developed markers have shown to be potentially useful for in situ and ex situ population studies to support future conservation actions of Hyacinth macaw and other parrots.