Trinucleotide Microsatellite Markers Research Articles

Construction of first genetic linkage map based on microsatellite markers and characterization of di- and tri-nucleotide microsatellite markers for Crassostrea hongkongesis

Hong Kong oyster Crassostrea hongkongensis is an important aquaculture species in the coastal areas of southern China. Because of its high demand and reasonable price, the production of Cohnella hongkongensis has increased drastically. However, the aquaculture of C. hongkongensis has also encountered some problems, such as seasonal mass mortality and reduction of availability and quality of wild seeds. Genetic linkage map is an effective tool for analyzing economically important traits. In this study, a consensus genetic map of C. hongkongensis was constructed by using microsatellite markers of F1 family. The consensus linkage map contained 104 loci, with a span of 653.9 cM and an average resolution of 6.9 cM. The estimated coverage of consensus linkage map was 85%. We identified 10 linkage groups, which were consistent with the haploid chromosome number of the species. The linkage map of male C. hongkongensis comprised 57 markers with a span of 467.6 cM, while the linkage map of female C. hongkongensis comprised 72 markers with a span of 570.9 cM. The average recombination ratio between males and females was 1:1.2. This map is vital for future QTL mapping framework of C. hongkongensis to perform marker assisted selection. Moreover, compared with di-nucleotide microsatellite markers, tri-nucleotide microsatellite markers have significantly stronger correlation with functional genes and cross-amplification success. We also found that the application potential of tri-nucleotide microsatellite markers in molecular-marker assisted selection gene mapping, and comparative linkage mapping was greater than that of di-nucleotide markers.

Development of tri-nucleotide microsatellite markers from Crassostrea hongkongensis using enriched genomic libraries and cross-species amplification in two closely related species

An enrichment protocol was used to isolate and characterize tri-nucleotide microsatellite markers in Crassostrea hongkongensis, an economically important and extensively cultured oyster species along the southern coastal areas of China. Thirty tri-nucleotide microsatellite loci were assessed in 32 individuals from a wild population. The number of alleles ranged from 2 to 12, averaging 7.63 alleles per locus. The observed and expected heterozygosity values ranged from 0.031 to 0.844 (averaging 0.579) and 0.062 to 0.881 (averaging 0.665), respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction. BLAST analysis showed that the tri-nucleotide microsatellite markers randomly developed from the genome of C.hongkongensis have a higher correlation with functional genes than di-nucleotide markers. Cross-amplification in C. ariakensis and C. gigas was performed and 21 loci showed reasonable polymorphism between C. hongkongensis and C. ariakensis, while 15 loci showed reasonable polymorphism between C. hongkongensis and C. gigas. The tri-nucleotide microsatellite markers identified in this study have greater potential for future use in gene mapping, molecular-marker assisted selection and comparative linkage mapping than previously identified di-nucleotide markers. Additionally, we found two microsatellite loci that can effectively identify the purebred and hybrid offspring of these three oyster species.

Assessment of the genetic relationship between the recently established benthic population and the adjacent floating populations of Sargassum horneri (Phaeophyceae) in Dalian of China by newly developed trinucleotide microsatellite markers

Sargassum horneri is a conspicuous canopy-forming brown seaweed growing on solid substrates or floating on sea surface. A recently established benthic population was discovered in a bay in Dalian, China, in 2016 and suggested to be derived from the earlier floating population. Since 2016, floating populations have kept appearing in the adjacent area. The genetic connectivity between this recently established benthic population and the adjacent floating populations remains unclear. In this study, 10 novel informative trinucleotide microsatellite markers were developed through the high throughput sequencing technology, and employed to analyze the genetic structure of the benthic population (DS16 and DS18) and three adjacent floating populations (DL16, BL16, and DL18) sampled in 2016 and 2018, with one floating population collected from Shidao in 2016 (SD16) as reference. Significant genetic differentiation was found to exist between the benthic and floating populations, and among floating populations from different years. Almost no membership of floating populations was detected in the benthic population and vice versa by the Bayesian model–based structure analysis, indicating very limited genetic connectivity between them. Significant excess of heterozygosity, significant multilocus linkage disequilibrium, high frequency of repeated genotypes, and putative clones were detected in all floating populations, indicating that asexual reproduction is likely dominant in them. Inbreeding and founder effect were revealed to be present in the benthic population. Further studies will focus on tracking the dynamics of genetic structure in the recently established benthic population and investigating the exact reproductive means and patterns of floating populations.

Microsatellite Identification in Perennial Ryegrass using Next‐Generation Sequencing

Microsatellite markers are potentially valuable molecular genetic markers for conservation ecology, paternity testing, pedigree reconstruction, population genetics, and linkage mapping. Traditional methods for the development of microsatellite markers can be time‐consuming, laborious, and expensive. Next‐generation sequencing (NGS) is a more recent and promising approach to microsatellite marker development. Perennial ryegrass (Lolium perenne L.) is an important turfgrass species with a limited set of publicly available microsatellite markers. Here we conducted Illumina NGS, as well as genotyping of a Lolium test population, to identify and characterize perennial ryegrass microsatellite markers. Sequencing and assembly results returned a microsatellite marker database containing 10,830 perfect di‐ and 42,718 perfect trinucleotide microsatellites with repeat unit lengths equal to or greater than six and four repeat units, respectively, as well as sufficient flanking sequence to enable polymerase chain reaction (PCR) primer design. Genotyping results from a subset of 172 di‐ and trinucleotide microsatellite markers indicated that 38.7% of these markers were polymorphic in perennial ryegrass, and 21.5% were transferable to and polymorphic in annual ryegrass (Lolium multiflorum Lam.). The thousands of expected polymorphic markers reported herein provide a significant new resource for studying Lolium genetic diversity, parentage analysis, linkage analysis, and germplasm in turf and forage breeding programs.

Development of novel tetra- and trinucleotide microsatellite markers for giant grouper Epinephelus lanceolatus using 454 pyrosequencing.

Giant grouper (Epinephelus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable. This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and trinucleotide microsatellite markers in giant grouper from Sabah, Malaysia. In total, of 62,763 sequences containing simple sequence repeats (SSRs) were obtained, and 78 SSR loci were selected to possibly contain tetra- and trinucleotide repeats. Of these loci, 16 had tetra- and 8 had trinucleotide repeats, all of which exhibited polymorphisms within easily genotyped regions. A total of 143 alleles were identified with an average of 5.94 alleles per locus, with mean observed and expected heterozygosities of 0.648 and 0.620, respectively. Among of them, 15 microsatellite markers were identified without null alleles and with Hardy-Weinberg equilibrium. These alleles showed a combined non-exclusion probability of 0.01138. The probability of individual identification (PID) value combined with in descending order 12 microsatellite markers was 0.00008, which strongly suggests that the use of the microsatellite markers developed in this study in various combinations would result in a high resolution method for parentage analysis and individual identification. These markers could be used to establish a broodstock management program for giant grouper and to provide a foundation for genetic studies such as population structure, parentage analysis, and kinship selection.

Forensic Characterization of Four New Bovine Tri-nucleotide Microsatellite Markers in Korean Cattle (Hanwoo)

We identified four new bovine tri-nucleotide microsatellite loci and analyzed their sequence structures and genetic parameters in 105 randomly selected Korean cattle (Hanwoo). Allele numbers of the loci B17S0808, B15S6253, B8S7996, and B17S4998 were 10, 11, 12, and 29, respectively. These alleles contained a simple or compound repeat sequences with some variations. Allele distributions of all these loci were in Hardy-Weinberg equilibrium (P > 0.05). Observed heterozygosity and expected heterozygosity ranged from 0.54 (B15S6253) to 0.92 (B17S4998) and from 0.599 (B15S6253) to 0.968 (B17S4998), respectively, and two measures of heterozygosity at each locus were highly correlated. Polymorphism information content (PIC) for these 4 loci ranged from 0.551 (B15S6253) to 0.932 (B17S4998), which means that all these loci are highly informative (PIC > 0.5). Other genetic parameters, power of discrimination (PD) and probability of exclusion (PE) ranged from 0.783 (B15S6253) to 0.984 (B17S4998) and from 0.210 (B15S6253) to 0.782 (B17S4998), respectively. Their combined PD and PE values were 0.9999968 and 0.98005176, respectively. Capillary electrophoresis revealed that average peak height ratio for a stutter was 13.89% at B17S0808, 26.67% at B15S6253, 9.09% at B8S7996, and 43.75% at B17S4998. Although the degree of genetic variability of the locus B15S6253 was relatively low among these four microsatellite markers, their favorable parameters and low peak height ratios for stutters indicate that these four new tri-nucleotide microsatellite loci could be useful multiplex PCR markers for the forensic and population genetic studies in cattle including Korean native breed.

Characterization of 12 polymorphic microsatellite markers for a facultatively eusocial sweat bee (Megalopta genalis)

We developed a library of twelve polymorphic di- and tri-nucleotide microsatellite markers for Megalopta genalis, a facultatively eusocial sweat bee. We tested each locus in a panel of 23 unrelated females and found 7-20 alleles per locus. Observed and expected heterozygosities ranged from 0.65 to 0.96 and from 0.69 to 0.95 respectively. None of the loci deviated from Hardy-Weinberg equilibrium proportions or was found to be in gametic disequilibrium.

PeakSeeker: a program for interpreting genotypes of mononucleotide repeats

BackgroundMononucleotide repeat microsatellites are abundant, highly polymorphic DNA sequences, having the potential to serve as valuable genetic markers. Use of mononucleotide microsatellites has been limited by their tendency to produce "stutter", confounding signals from insertions and deletions within the mononucleotide tract that occur during PCR, which complicates interpretation of genotypes by masking the true position of alleles. Consequently, microsatellites with larger repeating subunits (dinucleotide and trinucleotide motifs) are used, which produce less stutter but are less genetically heterogeneous and less informative. A method to interpret the genotypes of mononucleotide repeats would permit the widespread use of those highly informative microsatellites in genetic research.FindingsWe have developed an approach to interpret genotypes of mononucleotide repeats using a software program, named PeakSeeker. PeakSeeker interprets experimental electropherograms as the most likely product of signals from individual alleles. Because mononucleotide tracts demonstrate locus-specific patterns of stutter peaks, this approach requires that the genotype pattern from a single allele is defined for each marker, which can be approximated by genotyping single DNA molecules or homozygotes. We have evaluated the program's ability to discriminate various types of homozygous and heterozygous mononucleotide loci using simulated and experimental data.ConclusionMononucleotide tracts offer significant advantages over di- and tri-nucleotide microsatellite markers traditionally employed in genetic research. The PeakSeeker algorithm provides a high-throughput means to type mononucleotide tracts using conventional and widely implemented fragment length polymorphism genotyping. Furthermore, the PeakSeeker algorithm could potentially be adapted to improve, and perhaps to standardize, the analysis of conventional microsatellite genotypes.

Isolation of trinucleotide microsatellite markers for Mystus nemurus

Twelve single locus trinucleotide microsatellite markers were developed to characterize the Asian river catfish, Mystus nemurus, an important food fish in South East Asia. They were obtained by using a rapid method namely the 5' anchored PCR enrichment protocol. The specific primers were designed to flank the repeat sequences and these were subsequently used to characterize 90 unrelated fish from Malaysia. The number of alleles per locus ranged from 2 (MnVj2-281) to 12 (MnBp8-4-43b) while the levels of heterozygosity ranged from 0.0444 (MnVj2-1-19) to 0.7458 (MnVj2-291).

Isolation and characterization of microsatellite loci in the sand fly Phlebotomus papatasi (Diptera: Psychodidae)

Abstract Phlebotomus papatasi is a proven vector of Leishmania major which is one of the causative agents of cutaneous leishmaniasis in the Old World. Although it has a wide geographical range, its population structure is not yet well understood. In an effort to better understand the population dynamics of this vector, we developed a panel of di‐ and trinucleotide microsatellite markers, using a magnetic bead hybridization enrichment protocol. These microsatellite loci showed three to seven alleles with an expected heterozygosity range between 0.702 and 0.876. The level of polymorphisms found in this study suggests that these microsatellite loci can be used for population analysis of P. papatasi.

Characterization of trinucleotide SSR motifs in wheat.

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer "stutter bands" as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat ( Triticum aestivumL.). Four genomic libraries of cultivar 'Chinese Spring' were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) (n), (GGA/CCT) (n), (TAA/ATT) (n), (CAA/GTT) (n), (GGT/CCA) (n), (CAT/GTA) (n), (CGA/GCT) (n), (CTA/GAT) (n), and (CGT/GCA) (n) repeats were estimated to be 5.4x10(4), 3.5x10(4), 3.2x10(4), 1.2x10(4), 6.3x10(3), 4.9x10(3), 4.5x10(3), 4.5x10(3) and 3.6x10(3), i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) (n), 30 (43%) (CTT/GAA) (n), 16 (59%) (CAA/GTT) (n), 3 (27%) (CAT/GTA) (n) and 2 (4%) (GGA/CCT) (n) clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) (n), (CTT/GAA) (n) and (CAA/GTT) (n) microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) (n), four (14.8%) to (CTT/GAA) (n), and two (12.5%) to (CAA/GTT) (n) resulted in polymorphic markers. The results indicated that (TAA/ATT) (n) microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat.

Sequence-Tagged Site Markers in Grapevine and Citrus.

DNA markers based on PCR are becoming increasingly important in a wide range of applications including cultivar identification, phylogenetics and the construction of linkage maps for marker assisted breeding and map-based cloning of genes. We have investigated the utility of the microsatellite sequence-tagged site marker type because of the high level of associated polymorphism and simple genetics. For grapevine cultivar identification the marker type has been very successful. For phylogenetic analysis, studies with Citrus and related genera suggests that microsatellite length variation may be better suited to construct relatedness over small genetic distances rather than large genetic distances. The integration of trinucleotide microsatellite markers into a linkage map of Citrus has demonstrated the utility of this marker type for genetic analysis within wide intergeneric crosses and the potential to act as anchor loci to align linkage maps from different crosses and laboratories.