Presence Of Simple Sequence Repeats Research Articles

The complete chloroplast genome of Erodium stephanianum (Geraniaceae)

Erodium stephanianum (Erodium stephanianum Willd. Sp. Pl. 1800) has not had its complete chloroplast genome reported, which limits our understanding of its genetics and evolution. In this study, we assembled and annotated its chloroplast genome, revealing a quadripartite structure with 76 protein-coding genes. Repeat analysis indicated the presence of simple sequence repeats. Phylogenetic analysis confirmed E. stephanianum's placement within the genus Erodium of the Geraniaceae family. These findings offer valuable genomic resources for comparative studies in Erodium and Geraniaceae, aiding genetic diversity and phylogenetic analyses.

Comparative genomics reveals the presence of simple sequence repeats in genes related to virulence in plant pathogenic Pythium ultimum and Pythium vexans

In this study, we evaluated the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) in the complete genome and transcriptomic sequences of the plant pathogenic species of Pythium to acquire a better knowledge of their genome structure and evolution. Among the species, P. ultimum had the highest RA and RD of SSRs in the genomic sequences, whereas P. vexans had the highest RA and RD in the transcriptomic sequences. The genomic and transcriptomic sequences of P. aphanidermatum showed the lowest RA and RD of SSRs. Trinucleotide SSRs were the most prevalent class in both genomic and transcriptomic sequences, while dinucleotide SSRs were the least prevalent. The G + C content of the transcriptomic sequences was found to be positively correlated with the number (r = 0.601) and RA (r = 0.710) of SSRs. A motif conservation study revealed the highest number of unique motifs in P. vexans (9.9%). Overall, a low conservation of motifs was observed among the species (25.9%). A gene enrichment study revealed P. vexans and P. ultimum carry SSRs in their genes that are directly connected to virulence, whereas the remaining two species, P. aphanidermatum and P. arrhenomanes, harbour SSRs in genes involved in transcription, translation, and ATP binding. In an effort to enhance the genomic resources, a total of 11,002 primers from the transcribed regions were designed for the pathogenic Pythium species. Furthermore, the unique motifs identified in this work could be employed as molecular probes for species identification.

Deciphering the SSR incidences across viral members of Coronaviridae family

Presence of Simple Sequence Repeats (SSRs), both in genic and intergenic regions, have been widely studied in eukaryotes, prokaryotes, and viruses. In the current study, we undertook a survey to analyze the frequency and distribution of microsatellites or SSRs in multiple genomes of Coronaviridae members. We successfully identified 919 SSRs with length ≥12 bp across 55 reference genomes majority of which (838 SSRs) were found abundant in genic regions. The in-silico analysis further identified the preferential abundance of hexameric SSRs than any other size-based motif class. Our analysis shows that the genome size and GC content of the genome had a weak influence on SSR frequency and density. However, we find a positive correlation of SSRs GC content with genomic GC content. We also report relatively low abundances of all theoretically possible 501 repeat motif classes in all the genomes of Coronaviridae. The majority of SSRs were AT-rich. Overall, we see an underrepresentation of SSRs across the genomes of Coronaviridae. Besides, our integrative study highlights the presence of SSRs in ORF1ab (nsp3, nsp4, nsp5A_3CLpro and nsp5B_3CLpro, nsp6, nsp10, nsp12, nsp13, & nsp15 domains), S, ORF3a, ORF7a, N & 3′ UTR regions of SARS-CoV-2 and harbours multiple mutations (3′UTR and ORF1ab SSRs serving as major mutational hotspots). This indicates the genic SSRs are under selection pressure against mutations that might alter the reading frame and at the same time responsible for rapid protein evolution. Our preliminary results indicate the significance of the limited repertoire of SSRs in the genomes of Coronaviridae.

Meningococcal core and accessory phasomes vary by clonal complex.

Neisseria meningitidis is a Gram-negative human commensal pathogen, with extensive phenotypic plasticity afforded by phase-variable (PV) gene expression. Phase variation is a stochastic switch in gene expression from an ON to an OFF state, mediated by localized hypermutation of simple sequence repeats (SSRs). Circulating N. meningitidis clones vary in propensity to cause disease, with some clonal complexes (ccs) classified as hypervirulent and others as carriage-associated. We examined the PV gene repertoires, or phasome, of these lineages in order to determine whether phase variation contributes to disease propensity. We analysed 3328 genomes representative of nine circulating meningococcal ccs with PhasomeIt, a tool that identifies PV genes by the presence of SSRs and homologous gene clusters. The presence, absence and functions of all identified PV gene clusters were confirmed by annotation or blast searches within the Neisseria PubMLST database. While no significant differences were detected in the number of PV genes or the core, conserved phasome content between hypervirulent and carriage lineages, individual ccs exhibited major variations in PV gene numbers. Phylogenetic clusters produced by phasome or core genome analyses were similar, indicating co-evolution of PV genes with the core genome. While conservation of PV clusters is high, with 76 % present in all meningococcal isolates, maintenance of an SSR is variable, ranging from conserved in all isolates to present only in a single cc, indicating differing evolutionary trajectories for each lineage. Diverse functional groups of PV genes were present across the meningococcal lineages; however, the majority directly or indirectly influence bacterial surface antigens and could impact on future vaccine development. Finally, we observe that meningococci have open pan phasomes, indicating ongoing evolution of PV gene content and a significant potential for adaptive changes in this clinically relevant genus.

The Increase of Simple Sequence Repeats during Diversification of Marchantiidae, An Early Land Plant Lineage, Leads to the First Known Expansion of Inverted Repeats in the Evolutionarily-Stable Structure of Liverwort Plastomes.

The chloroplast genomes of liverworts, an early land plant lineage, exhibit stable structure and gene content, however the known resources are very limited. The newly sequenced plastomes of Conocephalum, Riccia and Sphaerocarpos species revealed an increase of simple sequence repeats during the diversification of complex thalloid liverwort lineage. The presence of long TA motifs forced applying the long-read nanopore sequencing method for proper and dependable plastome assembly, since the length of dinucleotide repeats overcome the length of Illumina short reads. The accumulation of SSRs (simple sequence repeats) enabled the expansion of inverted repeats by the incorporation of rps12 and rps7 genes, which were part of large single copy (LSC) regions in the previously sequenced plastomes. The expansion of inverted repeat (IR) at the genus level is reported for the first time for non-flowering plants. Moreover, comparative analyses with remaining liverwort lineages revealed that the presence of SSR in plastomes is specific for simple thalloid species. Phylogenomic analysis resulted in trees confirming monophyly of Marchantiidae and partially congruent with previous studies, due to dataset-dependent results of Dumortiera-Reboulia relationships. Despite the lower evolutionary rate of Marchantiales plastomes, significant barcoding gap was detected, even for recently divergent holarctic Conocephalum species. The sliding window analyses revealed the presence of 18 optimal (500 bp long) barcodes that enable the molecular identification of all studied species.

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

AbstractShotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.

Phasome analysis of pathogenic and commensal Neisseria species expands the known repertoire of phase variable genes, and highlights common adaptive strategies.

Pathogenic Neisseria are responsible for significantly higher levels of morbidity and mortality than their commensal relatives despite having similar genetic contents. Neisseria possess a disparate arsenal of surface determinants that facilitate host colonisation and evasion of the immune response during persistent carriage. Adaptation to rapid changes in these hostile host environments is enabled by phase variation (PV) involving high frequency, stochastic switches in expression of surface determinants. In this study, we analysed 89 complete and 79 partial genomes, from the NCBI and Neisseria PubMLST databases, representative of multiple pathogenic and commensal species of Neisseria using PhasomeIt, a new program that identifies putatively phase-variable genes and homology groups by the presence of simple sequence repeats (SSR). We detected a repertoire of 884 putative PV loci with maxima of 54 and 47 per genome in gonococcal and meningococcal isolates, respectively. Most commensal species encoded a lower number of PV genes (between 5 and 30) except N. lactamica wherein the potential for PV (36–82 loci) was higher, implying that PV is an adaptive mechanism for persistence in this species. We also characterised the repeat types and numbers in both pathogenic and commensal species. Conservation of SSR-mediated PV was frequently observed in outer membrane proteins or modifiers of outer membrane determinants. Intermittent and weak selection for evolution of SSR-mediated PV was suggested by poor conservation of tracts with novel PV genes often occurring in only one isolate. Finally, we describe core phasomes—the conserved repertoires of phase-variable genes—for each species that identify overlapping but distinctive adaptive strategies for the pathogenic and commensal members of the Neisseria genus.

Development and characterization of simple sequence repeat (SSR) markers based on a full-length cDNA library of Napier Grass (Pennisetum purpureum Schum)

Napier grass (Pennisetum purpureum Schum.) is an economically important species used as a fodder crop, reflecting its high biomass, perennial nature and pest resistance. In order to understand the genomic information and development of this species based on SSR markers, the transcriptome for Napier grass was obtained using next-generation sequencing. A total of 117,076 sequence contigs were obtained, with lengths of 201–10,652 bp. Among these contigs, 44,313 (37.85%) sequences were annotated against public protein databases (E-value <10−5). In addition, the contigs generated from the transcript sequencing were also analysed for the presence of simple sequence repeats (SSRs). A total of 12,744 SSR motifs were identified with in these contigs and corresponding primer pairs were designed. Empirical validation of a cohort of 200 SSRs was performed, with 34% being polymorphic for 20 Napier grass accessions. The polymorphic index content values for each primer ranged from 0.105 to 0.749, with an average of 0.409. The development of genetic, genomic resources and EST-SSRs for Napier grass will contribute to novel gene discovery and the marker-assisted selective breeding of this species.

A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5′ flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5′UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8–21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5′-UTR significantly enhances the level of gene expression. We termed this phenomenon as “microsatellite mediated enhancement” (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids.

Microsatellite resources of Eucalyptus: current status and future perspectives.

Eucalyptus is the premier paper pulp, short rotation plantation species grown all over the world. Genetic improvement programs integrating molecular marker tools are in progress in many parts of the globe to increase the productivity. Whole genome sequence and expressed sequence tags (ESTs) of the eucalypts paved way for introduction of molecular genetics and breeding in this genus. Different molecular characterization approaches have been used simultaneously in eucalypts, however, microsatellites or simple sequence repeats (SSRs) with their prolific characteristics could occupy a special niche in Eucalyptus genetic improvement. Further, highly informative SSRs were used for the clonal identity, genetic fidelity and in certification of breeder’s rights. Eucalyptus genetic linkage maps generated with microsatellite loci were used successfully to identify quantitative trait loci (QTLs) for various economically important traits. Progressively more numbers of microsatellites are being linked to genes associated with adaptive and functional variations, therefore making their utility broader in genetic applications. Availability of common SSR markers across the species provides an opportunity to validate the expression of QTLs across variable genetic backgrounds and accurately compare the position of QTLs in other species. Recent evidences suggest that the presence of SSRs in micro RNAs of plant species play a role in the quantitative trait expression. Similar studies in eucalypts may provide new insights into the genetic architecture of transcript-level variations and post transcriptional gene regulation. This review on eucalypts microsatellites, highlights the availability and characteristics of genomic and eSSRs and their potential in genetic analysis of natural and breeding populations and also discusses the future prospects in population genetics and marker assisted selection.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0073-3) contains supplementary material, which is available to authorized users.

Gene Discovery and Molecular Marker Development, Based on High-Throughput Transcript Sequencing of Paspalum dilatatum Poir

Background Paspalum dilatatum Poir. (common name dallisgrass) is a native grass species of South America, with special relevance to dairy and red meat production. P. dilatatum exhibits higher forage quality than other C4 forage grasses and is tolerant to frost and water stress. This species is predominantly cultivated in an apomictic monoculture, with an inherent high risk that biotic and abiotic stresses could potentially devastate productivity. Therefore, advanced breeding strategies that characterise and use available genetic diversity, or assess germplasm collections effectively are required to deliver advanced cultivars for production systems. However, there are limited genomic resources available for this forage grass species.ResultsTranscriptome sequencing using second-generation sequencing platforms has been employed using pooled RNA from different tissues (stems, roots, leaves and inflorescences) at the final reproductive stage of P. dilatatum cultivar Primo. A total of 324,695 sequence reads were obtained, corresponding to c. 102 Mbp. The sequences were assembled, generating 20,169 contigs of a combined length of 9,336,138 nucleotides. The contigs were BLAST analysed against the fully sequenced grass species of Oryza sativa subsp. japonica, Brachypodium distachyon, the closely related Sorghum bicolor and foxtail millet (Setaria italica) genomes as well as against the UniRef 90 protein database allowing a comprehensive gene ontology analysis to be performed. The contigs generated from the transcript sequencing were also analysed for the presence of simple sequence repeats (SSRs). A total of 2,339 SSR motifs were identified within 1,989 contigs and corresponding primer pairs were designed. Empirical validation of a cohort of 96 SSRs was performed, with 34% being polymorphic between sexual and apomictic biotypes.ConclusionsThe development of genetic and genomic resources for P. dilatatum will contribute to gene discovery and expression studies. Association of gene function with agronomic traits will significantly enable molecular breeding and advance germplasm enhancement.

Characterization of RFLP probe sequences for gene discovery and SSR development in Sorghum bicolor (L.) Moench.

In this study, we collected and analyzed DNA sequence data for 789 previously mapped RFLP probes from Sorghum bicolor (L.) Moench. DNA sequences, comprising 894 non-redundant contigs and end sequences, were searched against three GenBank databases, nucleotide (nt), protein (nr) and EST (dbEST), using BLAST algorithms. Matching ESTs were also searched against nt and nr. Translated DNA sequences were then searched against the conserved domain database (CDD) to determine if functional domains/motifs were congruent with the proteins identified in previous searches. More than half (500/894 or 56%) of the query sequences had significant matches in at least one of the GenBank searches. Overall, proteins identified for 148 sequences (17%) were consistent among all searches, of which 66 sequences (7%) contained congruent coding domains. The RFLP probe sequences were also evaluated for the presence of simple sequence repeats (SSRs) and 60 SSRs were developed and assayed in an array of sorghum germplasm comprising inbreds, landraces and wild relatives. Overall, these SSR loci had lower levels of polymorphism ( D = 0.46, averaged over 51 polymorphic loci) compared with sorghum SSRs that were isolated by library hybridization screens ( D = 0.69, averaged over 38 polymorphic loci). This result was probably due to the relatively small proportion of di-nucleotide repeat-containing markers (42% of the total SSR loci) obtained from the DNA sequence data. These di-nucleotide markers also contained shorter repeat motifs than those isolated from genomic libraries. Based on BLAST results, 24 SSRs (40%) were located within, or near, previously annotated or hypothetical genes. We determined the location of 19 of these SSRs relative to putative coding regions. In general, SSRs located in coding regions were less polymorphic ( D = 0.07, averaged over three loci) than those from gene flanking regions, UTRs and introns ( D = 0.49, averaged over 16 loci). The sequence information and SSR loci generated through this study will be valuable for application to sorghum genetics and improvement, including gene discovery, marker-assisted selection, diversity and pedigree analyses, comparative mapping and evolutionary genetic studies.

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of 'streamlined’ laboratory techniques and data analysis to Developing Countries are discussed.

Simple Sequence Repeat Markers Developed from Maize Sequences Found in the GENBANK Database: Map Construction

Simple sequence repeats (SSRs) are rapidly becoming an important class of DNA markers that are being widely used to map both plant and animal genomes. SSRs have the advantage of providing a codominant marker system based on polymerase chain reaction (PCR) methodology. Although the presence of SSRs is now well documented in the plant kingdom, a mapped set of primer sequences in maize (Zea mays L.) is not available. Polymorphic primer pairs developed from maize sequences in GENBANK were mapped to 42 loci in maize by means of either a B73 × Mo17, Mo17 × H99, or B73 × G35 recombinant inbred population. All SSR loci were found to be linked to one or more adjacent restriction fragment length polymorphism (RFLP) and/or isozyme loci. Segregation followed a pattern of Mendelian inheritance with one SSR locus deviating from expected ratios at a 1% level of significance. The SSRs were distributed throughout the maize genome with no evidence of clustering. Each SSR marker detected a single locus.

Characterization and genetic mapping of simple repeat sequences in the tomato genome.

Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microstellites was measured by PCR amplification of individual loci of by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited between Lycopersicon esculentum and L. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.

Methodology of generation and characteristics of simple sequence repeat DNA markers in avocado (Persea americana M.)

Generation of Simple Sequence Repeat (SSR) DNA markers was based on the construction of genomic DNA library of avocado (Persea americana M.). The library was screened with the four dinucleotide probes (AG), (AT), (GC) and (CA). Positive clones were sequenced to validate the presence of simple sequence repeats (SSR) and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the simple sequence repeat. Twenty six different pairs of primers which yield a PCR product in the initial screening were synthesized. The SSR A1E11 was found to have eleven alleles while A3F8 has eight alleles. The SSRs in avocado were found to be inherited in a Mendelian fashion.