Characterization Of Microsatellites Research Articles

Development and characterization of nineteen microsatellite loci for the endangered Mediterranean monk seal Monachus monachus

The Mediterranean monk seal (Monachus monachus) is one of the world’s most endangered marine mammals. Centuries of human persecution along with recent expansion of the fishing industry and intensive shoreline development, have isolated Mediterranean monk seals into small populations in the eastern Mediterranean Basin and eastern North Atlantic. We developed 19 microsatellite loci from the species’ genome and investigated genetic patterns within the two existing populations from the North Atlantic (Cabo Blanco and Madeira; N = 50). Microsatellite characterization was conducted on the Cabo Blanco population before the massive die-off that happened in 1997 (N = 30). Four loci deviated from the Hardy–Weinberg equilibrium and six loci showed an excess of homozygotes, suggesting that both nonequilibrium conditions (small-size, bottlenecked population subject to inbreeding) and null alleles are at stake. We could not detect any linkage disequilibrium among locus pairs. The genetic diversity observed in the population was low (mean number of alleles per locus = 2.68) but higher than reported in previous studies using cross-species amplification for the same population. Observed levels of genetic diversity in the post-crash population (N = 16) were slightly lower than in the precrash population (AR = 2.14 vs. 2.39), but the loss of 10 rare alleles in the post-crash population likely reflects the deleterious impact of the 1997 die-off. The Madeiran individuals (N = 4) were fully homozygous (AR = 1.11). Although they were genetically differentiated from Cabo Blanco, allelic distribution showed that Madeira is a genetically depauperate subpopulation of Cabo Blanco, suggesting that translocations from the latter may be considered beneficial. The newly developed microsatellites shall prove useful in managing populations at the individual level, and will be used to further investigate on the genetic structure and historical demography of past and present populations of Mediterranean monk seals.

Development and characterization of microsatellites from the sweetpotato weevil, Cylas formicarius elegantulus

AbstractTo conduct population genetics analyses on sweetpotato weevils (Cylas formicarius elegantulus) collected from three populations in the United States, microsatellite loci were developed from sweetpotato weevil transcriptome sequences obtained from a publicly available database. Nineteen of 27 microsatellite loci tested were usable for population analyses. Sweetpotato weevil individuals from Georgia (N = 17), Hawaii (N = 16) and South Carolina (N = 12) were analysed. Here, we present microsatellite primer sequences for 19 loci and population genetics statistics, including diversity, population differences and relatedness. Thirty‐nine alleles were detected in the 45 individuals, and four private alleles were observed in individuals from the Hawaii and South Carolina populations. Observed heterozygosities ranged from 0.00 to 1.00, and expected heterozygosities ranged from 0.00 to 0.44. The mean Shannon's information index ranged from 0.30 to 0.49 for the three populations. Pairwise differences among populations (FST estimates) from individuals ranged from a low of 0.022 between Georgia and Hawaii to a high of 0.036 between Georgia and South Carolina, and cluster analysis (PCA and Structure) indicated two populations independent of geographical location.

Genome-wide microsatellite characteristics of five human Plasmodium species, focusing on Plasmodium malariae and P. ovale curtisi.

Microsatellites can be utilized to explore genotypes, population structure, and other genomic features of eukaryotes. Systematic characterization of microsatellites has not been a focus for several species of Plasmodium, including P. malariae and P. ovale, as the majority of malaria elimination programs are focused on P. falciparum and to a lesser extent P. vivax. Here, five human malaria species (P. falciparum, P. vivax, P. malariae, P. ovale curtisi, and P. knowlesi) were investigated with the aim of conducting in-depth categorization of microsatellites for P. malariae and P. ovale curtisi. Investigation of reference genomes for microsatellites with unit motifs of 1–10 base pairs indicates high diversity among the five Plasmodium species. Plasmodium malariae, with the largest genome size, displays the second highest microsatellite density (1421 No./Mbp; 5% coverage) next to P. falciparum (3634 No./Mbp; 12% coverage). The lowest microsatellite density was observed in P. vivax (773 No./Mbp; 2% coverage). A, AT, and AAT are the most commonly repeated motifs in the Plasmodium species. For P. malariae and P. ovale curtisi, microsatellite-related sequences are observed in approximately 18–29% of coding sequences (CDS). Lysine, asparagine, and glutamic acids are most frequently coded by microsatellite-related CDS. The majority of these CDS could be related to the gene ontology terms “cell parts,” “binding,” “developmental processes,” and “metabolic processes.” The present study provides a comprehensive overview of microsatellite distribution and can assist in the planning and development of potentially useful genetic tools for further investigation of P. malariae and P. ovale curtisi epidemiology.

Development and Characterization of Genic Microsatellites for the Ornamental Plant Green and Gold (Chrysogonum virginianum)

The genus Chrysogonum is native to the eastern United States. Three entities have been recognized—either as three varieties of Chrysogonum virginianum or as two species, one of them with two varieties. The current study suggests that a fourth entity should be recognized. Several forms of the complex are in commercial trade as ornamentals. As very limited molecular information on Chrysogonum is available, we developed a set of genic simple sequence repeat markers (eSSRs) from de novo transcriptome sequencing. We tested a set of 17 eSSRs on a collection of C. virginianum genomic DNA samples from the three botanical varieties, and a new putative type observed in Tennessee, dubbed “Ocoee-type” for its geographic origin. The polymerase chain reaction and capillary electrophoresis analyses with downstream population genetics tools verified the usefulness of the eSSRs. By applying this approach, we showed recognizable variation within Chrysogonum, although it did not correspond exactly to previous infraspecific classifications. Finally, as demonstrated for the commercial cultivar Pierre included in the study, the eSSRs can be used for enhancing the future breeding or hybridization efforts of this ornamental plant.

Microsatellite characterization and marker development for the fungus Penicillium digitatum, causal agent of green mold of citrus.

Penicillium digitatum is one of the most important postharvest pathogens of citrus on a global scale causing significant annual losses due to fruit rot. However, little is known about the diversity of P. digitatum populations. The genome of P. digitatum has been sequenced, providing an opportunity to determine the microsatellite distribution within P. digitatum to develop markers that could be valuable tools for studying the population biology of this pathogen. In the analyses, a total of 3,134 microsatellite loci were detected; 66.73%, 23.23%, 8.23%, 1.24%, 0.16%, and 0.77% were detected as mono‐, di‐, tri‐, tetra‐, penta‐, and hexanucleotide repeats, respectively. As consistent with other ascomycete fungi, the genome size of P. digitatum does not seem to correlate with the density of microsatellite loci. However, significantly longer motifs of mono‐, di‐, and tetranucleotide repeats were identified in P. digitatum compared to 10 other published ascomycete species with repeats of over 800, 300, and 900 motifs found, respectively. One isolate from southern California and five additional isolates from other countries (“global isolates”) were used to initially screen microsatellite markers developed in this study. Twelve additional isolates, referred to as the “local isolates,” were also collected from citrus at the University of California Riverside agricultural experiment station and were subsequently used to screen the primers that sequenced well and were polymorphic based on the global isolates. Thirty‐six primers were screened, and nine trinucleotide loci and one hexanucleotide locus were chosen as robust markers. These loci yielded two to seven alleles and will be useful to study population genetic structure of P. digitatum populations.

Genetic monitoring of natural sturgeon spawning in the lower Volga for the period 2017-2018

Genetic monitoring of natural sturgeon spawning in the lower Volga for the period 2017-2018

Isolation and Characterization of Polymorphic Microsatellites from Draft Genome Data for Large Yellow Croaker, Pseudosciaena Crocea

In this study, we isolated thirty-eight microsatellite loci from the draft genome of large yellow croaker, Pseudosciaena crocea. These loci were tested on 34 individuals from Zhangzhou prefecture city in Fujian. Most loci were highly polymorphic. A total of 214 alleles were detected with an average of 5.63 alleles per locus. The observed and expected heterozygosity ranged from 0.454 to 0.909 and from 0.503 to 0.918, with an average of 0.635 and 0.744, respectively. The PIC values of these loci ranged from 0.372 to 0.878, with an average of 0.712. Eight loci significantly deviated from Hardy-Weinberg equilibrium (HWE). Significant linkage disequilibrium (LD) was not found in these 38 loci. The newly identified polymorphic markers will contribute to the study of genetic diversity, population structure, and conservation of P. crocea.

Identification and characterization of microsatellites in Aconitum reclinatum (Ranunculaceae), a rare species endemic to North America.

Premise of the Study Aconitum reclinatum is the only representative species of Aconitum subg. Lycoctonum in North America, with restricted ranges and endangered populations. Polymorphic microsatellite markers were developed for A. reclinatum for further investigation of genetic diversity and population structure.Methods and ResultsUsing Illumina HiSeq technology, we sequenced a genomic library for identification of simple sequence repeat markers. A total of 12 polymorphic primer pairs were developed and tested on 66 individuals from four populations in North America. The number of alleles ranged from one to seven per locus with an average of 3.48. Levels of observed and expected heterozygosity varied from 0 to 1.000 and 0 to 0.736, respectively, at population level. Three primer pairs were successfully amplified in three of four closely related species.ConclusionsThe microsatellites isolated in this study will be useful in further research on the genetic diversity and conservation genetics of A. reclinatum populations in North America.

Mining and characterization of microsatellites from a genome of Venturia carpophila

A total of 4021 microsatellites were mined from a genome of Venturia carpophila, and 192 were selected to screen 39 isolates of the fungus collected from peach and nectarine in the southeastern USA. Of the 192 selected, 32 primers consistently and reliably produced polymorphic amplicons. Subsequently, the genotyping data from these 32 primers were used for preliminary analysis of the genetic diversity among the 39 isolates. The number of alleles identified ranged from 2 to 9, and the polymorphic information content from 0.097 to 0.792. Over all isolates, Shannon’s information index was 0.914, indicating genetic diversity. Stoddart and Taylor’s index of diversity and Simpson’s index also indicated high diversity (32.4 and 0.969, respectively). Evenness within the sample was high (0.955), but there was strong evidence for haploid linkage disequilibrium (3.799, P = 0.001). Observations on diversity were supported by analysis of genetic distance, which showed little affinity for clustering based on isolate source population, location, or host. The microsatellites developed in this study should be useful in future research of the population genetic structure and dynamics of V. carpophila and evaluating the risks posed by the ability of the pathogen to adapt on peach and possibly other stone fruit hosts in the USA and elsewhere in the world.

Genome-wide characterization of microsatellites and genetic diversity assessment of spinach in the Chinese germplasm collection.

Spinach is a nutritional leafy green vegetable, and it also serves as a model species for studying sex chromosome evolution. Genetic marker development and genome structure analysis are important in breeding practice and theoretical evolution studies of spinach. In this study, the frequency and distribution of different microsatellites in the recently released draft spinach genome were characterized. A total of 261,002 perfect microsatellites were identified (estimated frequency: ~262.1 loci/Mbp). The most abundant microsatellites were tetranucleotide and trinucleotide, accounting for 33.2% and 27.7% of the total number of microsatellites, respectively. A total of 105 primer pairs were designed and screened, and 34 were polymorphic among the detected spinach cultivars. Combined with seven primer sets developed previously, 41 primer pairs were used to investigate genetic diversity among 43 spinach cultivars in China. The average polymorphism information content value of the 41 markers was 0.43, representing an intermediate level. The spinach cultivars had a low genetic diversity, and no detectable common factors were shared by each group in the UPGMA dendrogram. This study’s findings facilitate further investigations on the organization of the microsatellites in spinach genome and provide clues for future breeding applications of spinach in China.

Genome-wide mapping and characterization of microsatellites in the swamp eel genome

We described genome-wide screening and characterization of microsatellites in the swamp eel genome. A total of 99,293 microsatellite loci were identified in the genome with an overall density of 179 microsatellites per megabase of genomic sequences. The dinucleotide microsatellites were the most abundant type representing 71% of the total microsatellite loci and the AC-rich motifs were the most recurrent in all repeat types. Microsatellite frequency decreased as numbers of repeat units increased, which was more obvious in long than short microsatellite motifs. Most of microsatellites were located in non-coding regions, whereas only approximately 1% of the microsatellites were detected in coding regions. Trinucleotide repeats were most abundant microsatellites in the coding regions, which represented amino acid repeats in proteins. There was a chromosome-biased distribution of microsatellites in non-coding regions, with the highest density of 203.95/Mb on chromosome 8 and the least on chromosome 7 (164.06/Mb). The most abundant dinucleotides (AC)n was mainly located on chromosome 8. Notably, genomic mapping showed that there was a chromosome-biased association of genomic distributions between microsatellites and transposon elements. Thus, the novel dataset of microsatellites in swamp eel provides a valuable resource for further studies on QTL-based selection breeding, genetic resource conservation and evolutionary genetics.

Characterization of Microsatellites in Pseudogymnoascus destructans for White-nose Syndrome Genetic Analysis.

Despite only emerging in the past decade, white-nose syndrome has become among the most devastating wildlife diseases known. The pathogenic fungus Pseudogymnoascus destructans infects hibernating bats and typically leads to high rates of mortality at hibernacula during winter in North America. We developed a set of genetic markers to better differentiate P. destructans isolates. We designed and successfully characterized these 23 microsatellite markers of P. destructans for use in disease ecology and epidemiology research. We validated these loci with DNA extracted from a collection of P. destructans isolates from the US and Canada, as well as from Europe (the likely introduction source based on currently available data). Genetic diversity calculated for each locus and for the multilocus panel as a whole indicates sufficient allelic diversity to differentiate among and between samples from both Europe and North America. Indices of genetic diversity indicate a loss of allelic diversity that is consistent with the recent introduction and rapid spread of an emerging pathogen.

Abundance and Characterization of Perfect Microsatellites on the Cattle Y Chromosome

ABSTRACTMicrosatellites or simple sequence repeats (SSRs) are found in most organisms and play an important role in genomic organization and function. To characterize the abundance of SSRs (1-6 base-pairs [bp]) on the cattle Y chromsome, the relative frequency and density of perfect or uninterrupted SSRs based on the published Y chromosome sequence were examined. A total of 17,273 perfect SSRs were found, with total length of 324.78 kb, indicating that approximately 0.75% of the cattle Y chromosome sequence (43.30 Mb) comprises perfect SSRs, with an average length of 18.80 bp. The relative frequency and density were 398.92 loci/Mb and 7500.62 bp/Mb, respectively. The proportions of the six classes of perfect SSRs were highly variable on the cattle Y chromosome. Mononucleotide repeats had a total number of 8073 (46.74%) and an average length of 15.45 bp, and were the most abundant SSRs class, while the percentages of di-, tetra-, tri-, penta-, and hexa-nucleotide repeats were 22.86%, 11.98%, 11.58%, 6.65%, and 0.19%, respectively. Different classes of SSRs varied in their repeat number, with the highest being 42 for dinucleotides. Results reveal that repeat categories A, AC, AT, AAC, AGC, GTTT, CTTT, ATTT, and AACTG predominate on the Y chromosome. This study provides insight into the organization of cattle Y chromosome repetitive DNA, as well as information useful for developing more polymorphic cattle Y-chromosome-specific SSRs.

Characterization of Microsatellites in Genome of Pinus squamata, a Critically Endangered Species in the World

Characterization of Microsatellites in Genome of Pinus squamata, a Critically Endangered Species in the World

Genome-wide In Silico Analysis, Characterization and Identification of Microsatellites in Spodoptera littoralis Multiple nucleopolyhedrovirus (SpliMNPV).

In this study, we undertook a survey to analyze the distribution and frequency of microsatellites or Simple Sequence Repeats (SSRs) in Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV) genome (isolate AN–1956). Out of the 55 microsatellite motifs, identified in the SpliMNPV-AN1956 genome using in silico analysis (inclusive of mono-, di-, tri- and hexa-nucleotide repeats), 39 were found to be distributed within coding regions (cSSRs), whereas 16 were observed to lie within intergenic or noncoding regions. Among the 39 motifs located in coding regions, 21 were located in annotated functional genes whilst 18 were identified in unknown functional genes (hypothetical proteins). Among the identified motifs, trinucleotide (80%) repeats were found to be the most abundant followed by dinucleotide (13%), mononucleotide (5%) and hexanucleotide (2%) repeats. The 39 motifs located within coding regions were further validated in vitro by using PCR analysis, while the 21 motifs located within known functional genes (15 genes) were characterized using nucleotide sequencing. A comparison of the sequence analysis data of the 21 sequenced cSSRs with the published sequences is presented. Finally, the developed SSR markers of the 39 motifs were further mapped/localized onto the SpliMNPV-AN1956 genome. In conclusion, the SSR markers specific to SpliMNPV, developed in this study, could be a useful tool for the identification of isolates and analysis of genetic diversity and viral evolutionary status.

Genome-wide characterization of microsatellites in Triticeae species: abundance, distribution and evolution.

Microsatellites are an important constituent of plant genome and distributed across entire genome. In this study, genome-wide analysis of microsatellites in 8 Triticeae species and 9 model plants revealed that microsatellite characteristics were similar among the Triticeae species. Furthermore, genome-wide microsatellite markers were designed in wheat and then used to analyze the evolutionary relationship of wheat and other Triticeae species. Results displayed that Aegilops tauschii was found to be the closest species to Triticum aestivum, followed by Triticum urartu, Triticum turgidum and Aegilops speltoides, while Triticum monococcum, Aegilops sharonensis and Hordeum vulgare showed a relatively lower PCR amplification effectivity. Additionally, a significantly higher PCR amplification effectivity was found in chromosomes at the same subgenome than its homoeologous when these markers were subjected to search against different chromosomes in wheat. After a rigorous screening process, a total of 20,666 markers showed high amplification and polymorphic potential in wheat and its relatives, which were integrated with the public available wheat markers and then anchored to the genome of wheat (CS). This study not only provided the useful resource for SSR markers development in Triticeae species, but also shed light on the evolution of polyploid wheat from the perspective of microsatellites.

Characterization of perfect microsatellite based on genome-wide and chromosome level in Rhesus monkey (Macaca mulatta)

Microsatellite studies based on chromosomes level would contribute to the biometric correlation analysis of chromosome and microsatellite applications on the specific chromosome. In this study, the total microsatellite length of 1,141,024 loci was 21.8Mb, which covered about 0.74% of the male Rhesus monkey genome. Perfect mononucleotide SSRs were the most abundant, followed by the pattern: perfect di->tetra->tri->penta->hexanucleotide SSRs. The main range of repeat times focused on 12–32 times (mono-), 7–23 times (di-), 5–10 times (tri-), 4–14 times (tetra-), 4–9 times (penta-), 4–8 times (hexa-), respectively. The largest SSRs number was found in chromosome 1 with 94,347 loci, followed by chromosome 3, 2, 7 and 5, and the smallest number was in chromosome 18. The predominant repeat types in male Rhesus monkey genome and chromosome Y were basically A, AC, AG, AAT, AAC, AAAT, AAAC, AAAG, AAACA and AAACAA. SSRs number of all chromosomes was closely positively correlated with chromosome sequence size (r=0.969, p<0.01), and significantly negatively correlated with abundance (r=−0.24, 0.01<p<0.05). The lengths of all chromosomes were significantly negatively correlated with microsatellite density (r=−0.456, 0.01<p<0.05), and relative abundance and density of SSRs in all chromosomes were significantly negatively correlated with SSR GC content (r=−0.939/−0.928, p<0.01). The SSRs GC content on chromosome X (accounting for 16.71%) was found to be the highest in female Rhesus monkey, which might contributed to the DNA methylation of CpG islands for sex chromosome X inactivation and expression regulation. These results and exported tetranucleotide repeat sequences in each chromosome for primer design would facilitate the exploration of microsatellites structural function, composition mode and molecular markers development in Rhesus monkey genome.

Characterization and utilization of microsatellites in the Coffea canephora genome to assess genetic association between wild species in Kenya and cultivated coffee

Coffee is an important beverage crop in the world and has a significant contribution to Kenya’s economy. Here, we analyzed the genome-wide distribution of microsatellites in the Coffea canephora genome. A total of 159,041 SSRs were identified, with an overall density of 308 SSRs per Mb. Tetra-nucleotide repeats are the most abundant, accounting for 32 % of the total SSRs. AT-rich motifs are dominant across all SSR repeat units, while GC-rich motifs were generally rare. A set of 100 SSRs was selected to amplify 96 coffee accessions, including 10 wild accessions collected from Mt. Marsabit (Kenya). Of these SSRs, 33 % generated clear polymorphic bands among all tested accessions, with an average of 3.9 alleles per SSR locus. Wild coffee species from Mt. Marsabit showed a close genetic similarity with cultivated accessions in Kenya, suggesting that the wild species in Mt. Marsabit played an important role in the domestication of cultivated coffee in Kenya. Significantly low pairwise genetic divergence was observed between cultivated and wild accessions in Kenya, suggesting a relatively narrow level of genetic basis among coffee germplasm in Kenya. In addition, cultivated and wild coffee accessions in Kenya show a great divergence from those in other countries. Our results not only provide molecular tools for genetic studies in coffee but are also helpful for conservation and coffee breeding programs in Kenya.

Characterization of genome-wide microsatellites of Saccharina japonica based on a preliminary assembly of Illumina sequencing reads

Microsatellites or simple sequence repeats (SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp (Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N50 of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.

Characterization of microsatellites identified by next-generation sequencing in the Neotropical tree Handroanthus billbergii (Bignoniaceae).

Premise of the study:We developed microsatellite (simple sequence repeat [SSR]) markers in the Neotropical tree Handroanthus billbergii (Bignoniaceae), to be applied in assessment of genetic diversity in this species as a reference for inferring the impact of dry forest fragmentation in Ecuador.Methods and Results:Using next-generation sequencing, we detected a total of 26,893 putative SSRs reported here. Using an ABI 3500xl sequencer, we identified and characterized a set of polymorphic markers in 23 individuals belonging to three populations of H. billbergii.Conclusions:We report a set of 30 useful SSR markers for H. billbergii and a large list of potential microsatellites for developing new markers for this or related species.