Simple Sequence Repeat Patterns Research Articles

Investigating the patterns of simple sequence repeats (SSRs) inferred from the whole genome of a critically endangered species Cassine koordersii kosterm

Cassine koordersii Kosterm. (Celastraceae) is a critically endangered species indigenous to Jember, East Java. Programs for genetic conservation and plant breeding have recently implemented next-generation sequencing (NGS) techniques based on genomic data. This research aims to explore and distinguish between perfect and imperfect SSR patterns in the assembled genome. The Abyss assembler produced 3,060,362 scaffolds with 35.63 % GC content for the assembled genome. The investigation and identification of SSRs using the Krait tool found 139,236 and 582,360 sequences for including perfect and imperfect SSRs, respectively. There were six motif repeats of perfect and imperfect SSRs consisting of 73,175 and 202,438 sequences of mononucleotide (the most motif was A); 17,179 and 65,705 sequences of dinucleotide (the most motif was AT); 5,175 and 51,948 sequences of trinucleotide (the most motif was AAT); 3,824 and 14,010 sequences of tetranucleotide (the most motif was AAAT); 659 and 3,082 sequences of pentanucleotide (the most motif is AAAAT); 118 and 757 sequences of hexanucleotide (the most motif is AAAAAT). The depicted perfect and imperfect SSRs markers can be employed in future genetic studies of Cassine and related genera for either recommendation effort or improvement in conservation genetic concerns.

In silico approach to identify microsatellite candidate biomarkers to differentiate the biovar of Corynebacterium pseudotuberculosis genomes.

Corynebacterium pseudotuberculosis is the causative bacterial agent of the zoonotic disease known as caseous lymphadenitis, and it presents several mechanisms of response to host defenses, including the presence of virulence factors (VFs). The genomes of these bacteria have several polymorphic markers known as microsatellites, or simple sequence repeats (SSRs), that can be used to characterize the genome, to study possible polymorphisms existing among strains, and to verify the effects of such polymorphic markers in coding regions and regions associated with VFs. In this study, several SSRs were identified within coding regions throughout the 54 genomes of this species, revealing possible polymorphisms associated with coding regions that could be used as strain-specific or serotype-specific identifiers of C. pseudotuberculosis. The similarities associated with SSRs amongst the different serum variants of C. pseudotuberculosis, biovars equi and ovis, were also evaluated, and it was possible to identify SSRs located in coding regions responsible for a VF enrolled in pathogenesis known to mediate bacterial adherence (SpaH-type pili virulence factor). Phylogenetic analyses revealed that strains sharing SSR patterns, including the possible polymorphisms identified in the same position of gene-coding regions, were displayed by strains with a common ancestor, corroborating with the Genome Tree Report of the NCBI. Statistical analysis showed that the microsatellite groups belonging to equi and ovis biovars have a significance of 0.006 (p-value) in similarity, thus indicating them as good biomarker candidates for C. pseudotuberculosis.

Microsatellite Signature of Reference Genome Sequence of SARS-CoV-2 and 32 Species of Coronaviridae Family

Background: Simple sequence repeats (SSRs) are 1 - 6 bp repeat motif sequences present across both prokaryotic and eukaryotic genomes with various clinical implications besides being tools for conservation and evolutionary studies. Objectives: Analysis of 33 Coronavirus genomes, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for incidence, distribution, and complexity of SSRs patterns to understand their role in host divergence and evolution. Methods: Full-length genome sequences were extracted from National Center for Biotechnology Information (NCBI). Extraction of microsatellites was done using imperfect microsatellite extractor (IMEx) in “Advanced Mode”. Sequences were aligned with MAFFT v6.861b and the maximum likelihood tree was inferred using RAxML v8.1.20 of the GTR + GAMMA+I model with default specifications. Results: A total of 3,442 SSRs and 136 complex sequence repeats (cSSRs) were extracted from the studied 33 genomes. SSR incidence ranged from 82 (CV09) to 144 (CV60). cSSR incidence ranged from 1 (CV42, CV43, CV53) to 11 (CV32). CV61 (SARS-CoV-2) had 107 SSRs and 6 SSRs. Di-nucleotide motifs were the most prevalent followed by tri- and mono-nucleotide motifs. TG/GT was the most represented di-nucleotide motif, followed by CA/AC. In tri-nucleotide SSRs, ACA/TGT was the most represented motif followed by CAA/GTT, whereas in mono-nucleotide SSRs, T was the most observed nucleotide, followed by A. About 94% of SSRs were localized to the coding region. Twenty species, including CV61 (SARS-CoV-2), exhibit mono-nucleotide repeats exclusively in the A/T region, which were clustered in phylogenetic analysis. The sequence similarity of the genomes was assessed through heat map analysis and revealed similar sequences are expectedly placed in proximity on the phylogenetic tree. Conclusions: Mono-nucleotide exclusivity to A/T region and SSR genome signature can be a possible basis for predicting the evolution of viruses in terms of host range.

Portuguese Pinus nigra J.F. Arnold populations: genetic diversity, structure and relationships inferred by SSR markers

Pinus nigra J.F. Arnold has an ecological and economic interest in Europe, but many of the planted populations have an unknown origin and infraspecific taxonomy. Six Portuguese P. nigra populations characterised with microsatellites revealed high intra-population diversity structured into two clusters with low differentiation that might suggest two provenances or infraspecific taxa. Despite compared with foreign samples from different subspecies, we were not able to infer about the origin or infraspecific taxonomy of the Portuguese populations based on the pooled microsatellite data . Many of the European Pinus nigra J.F. Arnold forests were planted with the material of unknown origin. Efforts have been made to determine their provenances and infraspecific taxonomy regarding their relevance for defining strategies of genetic resources conservation, germplasm use, forest management and genetic improvement. The Portuguese P. nigra populations are allochthonous, and their provenance and infraspecific taxonomy are unknown. With this work, we intended to characterise the Portuguese P. nigra populations regarding its genetic diversity, structure and relationships using simple sequence repeat (SSR) markers and/or to infer about its origin and infraspecific taxonomy by comparing the SSR patterns of foreign P. nigra samples of known taxonomic classification. A total of 224 Portuguese P. nigra individuals from six populations were characterised using 14 SSR markers developed specifically to different Pinus sp., including P. nigra, by other authors. Thirteen SSR loci were selected and showed 100% of polymorphism among individuals. The genetic diversity was higher within (95%) rather than among (5%) populations. The Portuguese individuals were structured into two main clusters (K = 2) with low genetic differentiation (FST = 0.04), and the foreign samples were genetically distant from the Portuguese individuals. The six Portuguese P. nigra populations revealed high genetic diversity and seemed to be structured into two main clusters with low differentiation suggesting two provenances or infraspecific taxa. Nonetheless, the comparative analyses with foreign samples did not allow a clear inference about its origin and/or infraspecific taxonomy. Additional foreign samples and/or molecular markers will be tested to pursue these goals.

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium (TM2G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium (SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium (MSN), embryo maturation medium (CMM), and shoot elongation medium (CEM), respectively. And the shoots were then rooted on Murashige and Skoog (1962) medium (MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat (SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patterns. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.

Identifying significant associations of orthologous simple sequence repeats with gene ontologies.

Simple Sequence Repeats (SSRs), also known as microsatellites, regulate gene functions. SSR mutations in a disease gene may cause various genetic disorders. To identify putative functional SSRs, a web-based system, Gene Ontology SSR Hierarchy (GOSH), was developed to facilitate discovery of significant associations between SSRs and Gene Ontology (GO) terms. Using the GO hierarchy term structure, GOSH assists users with selecting functional or biological gene subsets. Significant SSR patterns are retrieved and identified via comprehensive overrepresentation analysis within a target gene subset and by comparing results with orthologous genes. Pattern relationships between different biological subsets or supersets can be observed by using the GO hierarchy structure directly. GOSH also supports GO searching through identified significant SSR patterns and all GO terms possessing such patterns are listed for consultation. GOSH is the first comprehensive and efficient online mining tool for discovering significant orthologous SSR patterns in GO terms and is available at http://gosh.cs.ntou.edu.tw/.

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species.

Genetic Diversity in A Core Subset of Wild Barley Germplasm

Wild barley [Hordeum vulgare ssp. spontaneum (C. Koch) Thell.] is a part of the primary gene pool with valuable sources of beneficial genes for barley improvement. This study attempted to develop a core subset of 269 accessions representing 16 countries from the Plant Gene Resources of Canada (PGRC) collection of 3,782 accessions, and to characterize them using barley simple sequence repeat (SSR) markers. Twenty-five informative primer pairs were applied to screen all samples and 359 alleles were detected over seven barley chromosomes. Analyses of the SSR data showed the effectiveness of the stratified sampling applied in capturing country-wise SSR variation. The frequencies of polymorphic alleles ranged from 0.004 to 0.708 and averaged 0.072. More than 24% or 7% SSR variation resided among accessions of 16 countries or two regions, respectively. Accessions from Israel and Jordan were genetically most diverse, while accessions from Lebanon and Greece were most differentiated. Four and five optimal clusters of accessions were obtained using STRUCTURE and BAPS programs and partitioned 16.3% and 20.3% SSR variations, respectively. The five optimal clusters varied in size from 15 to 104 and two clusters had only country-specific accessions. A genetic separation was detected between the accessions east and west of the Zagros Mountains only at the country, not the individual, level. These SSR patterns enhance our understanding of the wild barley gene pool, and are significant for conserving wild barley germplasm and exploring new sources of useful genes for barley improvement.

Distribution and localization of microsatellites in the Perigord black truffle genome and identification of new molecular markers

The level of genetic diversity and genetic structure in the Perigord black truffle (Tuber melanosporum Vittad.) has been debated for several years, mainly due to the lack of appropriate genetic markers. Microsatellites or simple sequence repeats (SSRs) are important for the genome organisation, phenotypic diversity and are one of the most popular molecular markers. In this study, we surveyed the T. melanosporum genome (1) to characterise its SSR pattern; (2) to compare it with SSR patterns found in 48 other fungal and three oomycetes genomes and (3) to identify new polymorphic SSR markers for population genetics. The T. melanosporum genome is rich in SSRs with 22,425 SSRs with mono-nucleotides being the most frequent motifs. SSRs were found in all genomic regions although they are more frequent in non-coding regions (introns and intergenic regions). Sixty out of 135 PCR-amplified mono-, di-, tri-, tetra, penta, and hexa-nucleotides were polymorphic (44%) within black truffle populations and 27 were randomly selected and analysed on 139 T. melanosporum isolates from France, Italy and Spain. The number of alleles varied from 2 to 18 and the expected heterozygosity from 0.124 to 0.815. One hundred and thirty-two different multilocus genotypes out of the 139 T. melanosporum isolates were identified and the genotypic diversity was high (0.999). Polymorphic SSRs were found in UTR regulatory regions of fruiting bodies and ectomycorrhiza regulated genes, suggesting that they may play a role in phenotypic variation. In conclusion, SSRs developed in this study were highly polymorphic and our results showed that T. melanosporum is a species with an important genetic diversity, which is in agreement with its recently uncovered heterothallic mating system.

Comparative genomic analysis of simple sequence repeats in three Plasmodium species

Simple sequence repeats (SSRs) are known to be responsible for genetic complexities and play major roles in gene and genome evolution. To this respect, malaria parasites are known to have rapidly evolving and complex genomes with complicated and differential pathogenic behaviors. Hence, by studying the whole genome comparative SSRs patterns, one can understand genomic complexities and differential evolutionary patterns of these species. We herein utilized the whole genome sequence information of three Plasmodium species, Plasmodium falciparum, Plasmodium vivax, and Plasmodium knowlesi, to comparatively analyze genome-wide distribution of SSRs. The study revealed that despite having the smallest genome size, P. falciparum bears the highest SSR content among the three Plasmodium species. Furthermore, distribution patterns of different SSRs types (e.g., mono, di, tri, tetra, penta, and hexa) in term of relative abundance and relative density provide evidences for greater accumulation of di-repeats and marked decrease of mono-repeats in P. falciparum in comparison to other two species. Overall, the types and distribution of SSRs in P. falciparum genome was found to be different than that of P. vivax and P. knowlesi. The latter two species have quite similar SSR organizations in many aspects of the data. The results were discussed in terms of comparative SSR patterns among the three Plasmodium species, uniqueness of P. falciparum in SSR organization and general pattern of evolution of SSRs in Plasmodium.

Efficient algorithms for identifying orthologous simple sequence repeats of disease genes

Dynamic mutations of simple sequence repeats (SSRs) have been demonstrated to affect normal gene function and cause different genetic disorders. Several conserved and even partial functional SSR patterns are discovered in inherited orthologous disease genes. To explore a wide range of SSRs in genetic diseases, a comprehensive system focusing on identifying orthologous SSRs of disease genes through a comparative genomics mechanism is constructed and accomplished by adopting online Mendelian inheritance in man (OMIM) and NCBI HomoloGene databases as the fundamental resources of human genetic diseases and homologous gene information. In addition, an efficient and effective algorithm for searching SSR patterns is also developed for providing annotated SSR information among various model species. By integrating these data resources and mining technologies, biologists and doctors can systematically retrieve novel and important conserved SSR information among orthologous disease genes. The proposed system, Orthologous SSR for Disease Genes (OSDG), is the first comprehensive framework for identifying orthologous SSRs as potential causative factors of genetic disorders and is freely available at http://osdg.cs.ntou.edu.tw/.

Genetic variation of white clover (Trifolium repens L.) collections from China detected by morphological traits, RAPD and SSR.

White clover is an important legume naturalized in China. But genetic variation of Chinese local white clover germplasm has not been reported. There is no information for parents’ selection and genetic resource conservation. The objective of this study was to investigate and characterize genetic variation of ten local white clover germplasms collected from different geographical regions of China. Morphological data analysis of white clover populations was coupled with molecular markers of random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) to investigate the genetic relationships among white clover germplasms of China, and four commercial cultivars were included for a comparison. The results revealed that the populations showed diverse morphological traits, RAPD and SSR patterns. Despite the certain similarities among the dendrograms of particular populations, the overall correlations among the distance matrices appeared to be rather low. In the present study, cluster analysis only slightly reflected the geographical origins of populations, and the dendrogram employing SSR data showed the closest agreement with collections’ geographical origins. The analysis of molecular variance (AMOVA) analysis based on both RAPD and SSR markers revealed higher levels of intra-population variation than inter-population variation. Neither RAPD nor SSR based grouping showed obvious agreement with variation of leaf sizes and other morphological traits. Our results suggest that the combination of morphological traits and SSR analysis could be a useful method for assessing genetic variation in white clover, and could be helpful for breeders to plan crosses for positive traits.

An online conserved SSR discovery through cross-species comparison

Simple sequence repeats (SSRs) play important roles in gene regulation and genome evolution. Although there exist several online resources for SSR mining, most of them only extract general SSR patterns without providing functional information. Here, an online search tool, CG-SSR (Comparative Genomics SSR discovery), has been developed for discovering potential functional SSRs from vertebrate genomes through cross-species comparison. In addition to revealing SSR candidates in conserved regions among various species, it also combines accurate coordinate and functional genomics information. CG-SSR is the first comprehensive and efficient online tool for conserved SSR discovery.

Use of Microsatellite DNA to Distinguish Malting and Nonmalting Barley Cultivars

Efficient and reliable approaches for distinguishing barley cultivars are urgently needed for the malting, brewing, and feed grain industries. The potential of microsatellites, also known as simple sequence repeats (SSRs), to uniquely identify barley cultivars was evaluated on a sample of closely related, visually indistinguishable, white aleurone six-rowed malting and feed (general purpose) barley cultivars grown in Canada. A total of 38 SSRs were tested against the DNA extracted from leaf tissue of 11 barley cultivars. Seventeen SSRs showed allelic polymorphisms among the 11 cultivars, providing a total of 40 different alleles. By combining data for five SSR loci, all 11 cultivars could be distinguished. DNA extracted from single seeds gave allelic SSR patterns similar to leaf DNA and could be used for a rapid system of cultivar identification.