Showing papers on "Microsatellite published in 2016"

Characterization of genetic sequence variation of 58 STR loci in four major population groups.

Abstract: Massively parallel sequencing (MPS) can identify sequence variation within short tandem repeat (STR) alleles as well as their nominal allele lengths that traditionally have been obtained by capillary electrophoresis. Using the MiSeq FGx Forensic Genomics System (Illumina), STRait Razor, and in-house excel workbooks, genetic variation was characterized within STR repeat and flanking regions of 27 autosomal, 7 X-chromosome and 24 Y-chromosome STR markers in 777 unrelated individuals from four population groups. Seven hundred and forty six autosomal, 227 X-chromosome, and 324 Y-chromosome STR alleles were identified by sequence compared with 357 autosomal, 107 X-chromosome, and 189 Y-chromosome STR alleles that were identified by length. Within the observed sequence variation, 227 autosomal, 156 X-chromosome, and 112 Y-chromosome novel alleles were identified and described. One hundred and seventy six autosomal, 123 X-chromosome, and 93 Y-chromosome sequence variants resided within STR repeat regions, and 86 autosomal, 39 X-chromosome, and 20 Y-chromosome variants were located in STR flanking regions. Three markers, D18S51, DXS10135, and DYS385a-b had 1, 4, and 1 alleles, respectively, which contained both a novel repeat region variant and a flanking sequence variant in the same nucleotide sequence. There were 50 markers that demonstrated a relative increase in diversity with the variant sequence alleles compared with those of traditional nominal length alleles. These population data illustrate the genetic variation that exists in the commonly used STR markers in the selected population samples and provide allele frequencies for statistical calculations related to STR profiling with MPS data.

Sequence variation of 22 autosomal STR loci detected by next generation sequencing.

Abstract: Sequencing short tandem repeat (STR) loci allows for determination of repeat motif variations within the STR (or entire PCR amplicon) which cannot be ascertained by size-based PCR fragment analysis. Sanger sequencing has been used in research laboratories to further characterize STR loci, but is impractical for routine forensic use due to the laborious nature of the procedure in general and additional steps required to separate heterozygous alleles. Recent advances in library preparation methods enable high-throughput next generation sequencing (NGS) and technological improvements in sequencing chemistries now offer sufficient read lengths to encompass STR alleles. Herein, we present sequencing results from 183 DNA samples, including African American, Caucasian, and Hispanic individuals, at 22 autosomal forensic STR loci using an assay designed for NGS. The resulting dataset has been used to perform population genetic analyses of allelic diversity by length compared to sequence, and exemplifies which loci are likely to achieve the greatest gains in discrimination via sequencing. Within this data set, six loci demonstrate greater than double the number of alleles obtained by sequence compared to the number of alleles obtained by length: D12S391, D2S1338, D21S11, D8S1179, vWA, and D3S1358. As expected, repeat region sequences which had not previously been reported in forensic literature were identified.

The Report of My Death was an Exaggeration: A Review for Researchers Using Microsatellites in the 21st Century

Abstract: Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites. We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers.

Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis

Abstract: Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been difficult and costly. The whole genome sequencing with next-generation sequencing (NGS) technologies provides large amounts of sequence data to develop numerous microsatellite markers at whole genome scale. SSR markers have great advantage in cross-species comparisons and allow investigation of karyotype and genome evolution through highly efficient computation approaches such as in silico PCR. Here we described genome wide development and characterization of SSR markers in the watermelon (Citrullus lanatus) genome, which were then use in comparative analysis with two other important crop species in the Cucurbitaceae family: cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). We further applied these markers in evaluating the genetic diversity and population structure in watermelon germplasm collections. A total of 39,523 microsatellite loci were identified from the watermelon draft genome with an overall density of 111 SSRs/Mbp, and 32,869 SSR primers were designed with suitable flanking sequences. The dinucleotide SSRs were the most common type representing 34.09 % of the total SSR loci and the AT-rich motifs were the most abundant in all nucleotide repeat types. In silico PCR analysis identified 832 and 925 SSR markers with each having a single amplicon in the cucumber and melon draft genome, respectively. Comparative analysis with these cross-species SSR markers revealed complicated mosaic patterns of syntenic blocks among the genomes of three species. In addition, genetic diversity analysis of 134 watermelon accessions with 32 highly informative SSR loci placed these lines into two groups with all accessions of C.lanatus var. citorides and three accessions of C. colocynthis clustered in one group and all accessions of C. lanatus var. lanatus and the remaining accessions of C. colocynthis clustered in another group. Furthermore, structure analysis was consistent with the dendrogram indicating the 134 watermelon accessions were classified into two populations. The large number of genome wide SSR markers developed herein from the watermelon genome provides a valuable resource for genetic map construction, QTL exploration, map-based gene cloning and marker-assisted selection in watermelon which has a very narrow genetic base and extremely low polymorphism among cultivated lines. Furthermore, the cross-species transferable SSR markers identified herein should also have practical uses in many applications in species of Cucurbitaceae family whose whole genome sequences are not yet available.

Genome-wide SSR-based association mapping for fiber quality in nation-wide upland cotton inbreed cultivars in China.

Abstract: Since upland cotton was introduced into China during the 1920s–1950s, hundreds of inbreed cultivars have been developed. To explore the molecular diversity, population structure and elite alleles, 503 inbred cultivars developed in China and some foreign cultivars from the United States and the Soviet Union were collected and analyzed by 494 genome-wide SSRs (Simple Sequence Repeats). Four hundred and ninety-four pairs of SSRs with high polymorphism and uniform distribution on 26 chromosomes were used to scan polymorphisms in 503 nation-wide upland cottons. The programming language R was used to make boxplots for the phenotypic traits in different environments. Molecular marker data and 6 fiber quality traits were analyzed by the method of MLM (mixed linear model) (P + G + Q + K) in the TASSEL software package on the basis of the population structure and linkage disequilibrium analysis. The loci of elite allelic variation and typical materials carrying elite alleles were identified based on phenotypic effect values. A total of 179 markers were polymorphic and generated 426 allele loci; the population based on molecular diversity was classified into seven subpopulations corresponding to pedigree origin, ecological and geographical distribution. The attenuation distance of linkage disequilibrium dropped significantly up to 0–5 cM. Association mapping for fiber quality showed that 216 marker loci were associated with fiber quality traits (P < 0.05) explaining 0.58 % ~ 5.12 % of the phenotypic variation, with an average of 2.70 %. Thirteen marker loci were coincident with other studies, and three were detected for the same trait. Seven quantitative trait loci were related to known genes in fiber development. Based on phenotypic effects, 48 typical materials that contained the elite allele loci related to fiber quality traits were identified and are widely used in practical breeding. The molecular diversity and population structure of 503 nation-wide upland cottons in China were evaluated by 494 genome-wide SSRs, and association mapping for fiber quality revealed known and novel elite alleles. The molecular diversity provides a guide for parental mating in cotton breeding, and the association mapping results will aid in the fine-mapping genes related to fiber quality traits and facilitate further studies on candidate genes.

A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding.

Abstract: This study examines the potential of next-generation sequencing based ‘genotyping-by-sequencing’ (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data.

Population-Scale Sequencing Data Enable Precise Estimates of Y-STR Mutation Rates

Abstract: Short tandem repeats (STRs) are mutation-prone loci that span nearly 1% of the human genome. Previous studies have estimated the mutation rates of highly polymorphic STRs by using capillary electrophoresis and pedigree-based designs. Although this work has provided insights into the mutational dynamics of highly mutable STRs, the mutation rates of most others remain unknown. Here, we harnessed whole-genome sequencing data to estimate the mutation rates of Y chromosome STRs (Y-STRs) with 2–6 bp repeat units that are accessible to Illumina sequencing. We genotyped 4,500 Y-STRs by using data from the 1000 Genomes Project and the Simons Genome Diversity Project. Next, we developed MUTEA, an algorithm that infers STR mutation rates from population-scale data by using a high-resolution SNP-based phylogeny. After extensive intrinsic and extrinsic validations, we harnessed MUTEA to derive mutation-rate estimates for 702 polymorphic STRs by tracing each locus over 222,000 meioses, resulting in the largest collection of Y-STR mutation rates to date. Using our estimates, we identified determinants of STR mutation rates and built a model to predict rates for STRs across the genome. These predictions indicate that the load of de novo STR mutations is at least 75 mutations per generation, rivaling the load of all other known variant types. Finally, we identified Y-STRs with potential applications in forensics and genetic genealogy, assessed the ability to differentiate between the Y chromosomes of father-son pairs, and imputed Y-STR genotypes.

Genome survey of pistachio ( Pistacia vera L.) by next generation sequencing: Development of novel SSR markers and genetic diversity in Pistacia species

Abstract: Pistachio (Pistacia vera L.) is one of the most important nut crops in the world. There are about 11 wild species in the genus Pistacia, and they have importance as rootstock seed sources for cultivated P. vera and forest trees. Published information on the pistachio genome is limited. Therefore, a genome survey is necessary to obtain knowledge on the genome structure of pistachio by next generation sequencing. Simple sequence repeat (SSR) markers are useful tools for germplasm characterization, genetic diversity analysis, and genetic linkage mapping, and may help to elucidate genetic relationships among pistachio cultivars and species. To explore the genome structure of pistachio, a genome survey was performed using the Illumina platform at approximately 40× coverage depth in the P. vera cv. Siirt. The K-mer analysis indicated that pistachio has a genome that is about 600 Mb in size and is highly heterozygous. The assembly of 26.77 Gb Illumina data produced 27,069 scaffolds at N50 = 3.4 kb with a total of 513.5 Mb. A total of 59,280 SSR motifs were detected with a frequency of 8.67 kb. A total of 206 SSRs were used to characterize 24 P. vera cultivars and 20 wild Pistacia genotypes (four genotypes from each five wild Pistacia species) belonging to P. atlantica, P. integerrima, P. chinenesis, P. terebinthus, and P. lentiscus genotypes. Overall 135 SSR loci amplified in all 44 cultivars and genotypes, 41 were polymorphic in six Pistacia species. The novel SSR loci developed from cultivated pistachio were highly transferable to wild Pistacia species. The results from a genome survey of pistachio suggest that the genome size of pistachio is about 600 Mb with a high heterozygosity rate. This information will help to design whole genome sequencing strategies for pistachio. The newly developed novel polymorphic SSRs in this study may help germplasm characterization, genetic diversity, and genetic linkage mapping studies in the genus Pistacia.

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Abstract: Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity.

Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

Abstract: Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Abstract: Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Abstract: Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (FST = 0.049) as evidenced by high level of gene flow estimate (Nm = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.

QTL mapping for bacterial wilt resistance in peanut ( Arachis hypogaea L.).

Abstract: Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious, global, disease of peanut (Arachis hypogaea L.), but it is especially destructive in China. Identification of DNA markers linked to the resistance to this disease will help peanut breeders efficiently develop resistant cultivars through molecular breeding. A F2 population, from a cross between disease-resistant and disease-susceptible cultivars, was used to detect quantitative trait loci (QTL) associated with the resistance to this disease in the cultivated peanut. Genome-wide SNPs were identified from restriction-site-associated DNA sequencing tags using next-generation DNA sequencing technology. SNPs linked to disease resistance were determined in two bulks of 30 resistant and 30 susceptible plants along with two parental plants using bulk segregant analysis. Polymorphic SSR and SNP markers were utilized for construction of a linkage map and for performing the QTL analysis, and a moderately dense linkage map was constructed in the F2 population. Two QTL (qBW-1 and qBW-2) detected for resistance to BW disease were located in the linkage groups LG1 and LG10 and account for 21 and 12 % of the bacterial wilt phenotypic variance. To confirm these QTL, the F8 RIL population with 223 plants was utilized for genotyping and phenotyping plants by year and location as compared to the F2 population. The QTL qBW-1 was consistent in the location of LG1 in the F8 population though the QTL qBW-2 could not be clarified due to fewer markers used and mapped in LG10. The QTL qBW-1, including four linked SNP markers and one SSR marker within 14.4-cM interval in the F8, was closely related to a disease resistance gene homolog and was considered as a candidate gene for resistance to BW. QTL identified in this study would be useful to conduct marker-assisted selection and may permit cloning of resistance genes. Our study shows that bulk segregant analysis of genome-wide SNPs is a useful approach to expedite the identification of genetic markers linked to disease resistance traits in the allotetraploidy species peanut.

The second highest chromosome count among vertebrates is observed in cultured sturgeon and is associated with genome plasticity

Abstract: One of the five basal actinopterygian lineages, the Chondrostei, including sturgeon, shovelnose, and paddlefish (Order Acipenseriformes) show extraordinary ploidy diversity associated with three rounds of lineage-specific whole-genome duplication, resulting in three levels of ploidy in sturgeon. Recently, incidence of spontaneous polyploidization has been reported among cultured sturgeon and it could have serious negative implications for the economics of sturgeon farming. We report the occurrence of seven spontaneous heptaploid (7n) Siberian sturgeon Acipenser baerii, which is a functional tetraploid species (4n) with ~245 chromosomes. Our aims were to assess ploidy level and chromosome number of the analysed specimens and to identify the possible mechanism that underlies the occurrence of spontaneous additional chromosome sets in their genome. Among 150 specimens resulting from the mating of a tetraploid (4n) A. baerii (~245 chromosomes) dam with a hexaploid (6n) A. baerii (~368 chromosomes) sire, 143 displayed a relative DNA content that corresponds to pentaploidy (5n) with an absolute DNA content of 8.98 ± 0.03 pg DNA per nucleus and nuclear area of 35.3 ± 4.3 μm2 and seven specimens exhibited a relative DNA content that corresponds to heptaploidy (7n), with an absolute DNA content of 15.02 ± 0.04 pg DNA per nucleus and nuclear area of 48.4 ± 5.1 μm2. Chromosome analyses confirmed a modal number of ~437 chromosomes in these heptaploid (7n) individuals. DNA genotyping of eight microsatellite loci followed by parental assignment confirmed spontaneous duplication of the maternal chromosome sets via retention of the second polar body in meiosis II as the mechanism for the formation of this unusual chromosome number and ploidy level in a functional tetraploid A. baerii. We report the second highest chromosome count among vertebrates in cultured sturgeon (~437) after the schizothoracine cyprinid Ptychobarbus dipogon with ~446 chromosomes. The finding also represents the highest documented chromosome count in Acipenseriformes, and the first report of a functional heptaploid (7n) genome composition in sturgeon. To our knowledge, this study provides the first clear evidence of a maternal origin for spontaneous polyploidization in cultured A. baerii. To date, all available data indicate that spontaneous polyploidization occurs frequently among cultured sturgeons.

The population history of Garra orientalis (Teleostei: Cyprinidae) using mitochondrial DNA and microsatellite data with approximate Bayesian computation

Abstract: The South China landmass has been characterized by a complex geological history, including mountain lifting, climate changes, and river capture/reversal events. To determine how this complexity has influenced the landmass’s phylogeography, our study examined the phylogeography of Garra orientalis, a cyprinid widely distributed in South China, using sequences from the mitochondrial DNA control region and cytochrome b gene (1887 bp) and polymorphisms of thirteen microsatellite loci. In total, 157 specimens were collected from eight populations. All 88 mtDNA haplotypes were identified as belonging to three major lineages, and these lineages were almost allopatric in their distributions. The results of a statistical dispersal-vicariance analysis suggested that the ancestral populations of G. orientalis were distributed south of the Yunkai Mountains, including on Hainan Island. The mtDNA data revealed a strong relationship between phylogeny and geography. In the microsatellite analysis, a total of 339 alleles with an average of 26 alleles per locus were observed across thirteen microsatellite loci. A clustering algorithm for microsatellite data revealed an admixture-like genetic structure. Although the mtDNA and microsatellite data sets displayed a discordant population structure, the results of an approximate Bayesian computation approach showed that these two markers revealed congruent historical signals. The population history of G. orientalis reflects vicariance events and dispersal related to the complex geological history of South China. Our results (i) found that the discordances between mtDNA and microsatellite markers were accounted for by admixtures; (ii) showed that the Wuzhishan and Yinggeling mountain ranges and Qiongzhou Strait were important barriers limiting gene exchange between populations on both sides; (iii) indicated that during glaciation and inter-glacial periods, the strait and continental shelves were exposed and sank, which contributed with the dispersion and differentiation of populations; and (iv) displayed that the admixtures between lineages took place in coastal populations and then colonized the tributaries of the Pearl River.

Genetics and Molecular Mapping of Black Rot Resistance Locus Xca1bc on Chromosome B-7 in Ethiopian Mustard (Brassica carinata A. Braun)

Abstract: Black rot caused by Xanthomonas campestris pv. campestris (Pam.) Dowson is the most destructive disease of cauliflower causing huge loss to the farmers throughout the world. Since there are limited sources of resistance to black rot in B. oleracea (C genome Brassica), exploration of A and B genomes of Brassica was planned as these were thought to be potential reservoirs of black rot resistance gene(s). In our search for new gene(s) for black rot resistance, F2 mapping population was developed in Brassica carinata (BBCC) by crossing NPC-17, a susceptible genotype with NPC-9, a resistant genotype. Out of 364 Intron length polymorphic markers and microsatellite primers used in this study, 41 distinguished the parental lines. However, resistant and susceptible bulks could be distinguished by three markers At1g70610, SSR Na14-G02 and At1g71865 which were used for genotyping of F2 mapping population. These markers were placed along the resistance gene, according to order, covering a distance of 36.30 cM. Intron length polymorphic markers At1g70610 and At1g71865 were found to be linked to black rot resistance locus (Xca1bc) at 6.2 and 12.8 cM distance, respectively. This is the first report of identification of markers linked to Xca1bc locus in Brassica carinata on B-7 linkage group. Intron length polymorphic markers provided a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness for marker assisted alien gene introgression into cauliflower.

De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing

Abstract: Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.

Development and Characterization of Genic SSR Markers from Indian Mulberry Transcriptome and Their Transferability to Related Species of Moraceae.

Abstract: Improving mulberry leaf production with enhanced leaf quality holds the key to sustain the ever increasing demand for silk. Adoption of modern genomic approaches for crop improvement is severely constrained by the lack of sufficient molecular markers in mulberry. Here, we report development and validation of 206 EST derived SSR markers using transcriptome data generated from leaf tissue of a drought tolerant mulberry genotype, Dudia white. Analysis of transcriptome data containing 10169 EST sequences, revealed 1469 sequences with microsatellite repeat motifs. We designed a total of 264 primers to the most appropriate repeat regions, of which 206 were locus specific. These markers were validated with 25 diverse mulberry accessions and their transferability to closely related species belonging to family Moraceae was examined. Of these markers, 189 revealed polymorphism with up to 8 allelic forms across mulberry species, genotypes and varieties with a mean of 3.5 alleles per locus. The markers also revealed higher polymorphic information content of 0.824 among the accessions. These markers effectively segregated the species and genotypes and hence, can be used for both diversity analysis and in breeding applications. Around 40% of these markers were transferable to other closely related species. Along with the other genic and genomic markers, we report a set of over 750 co-dominant markers. Using these markers we constructed the first genetic linkage map of mulberry exclusively with co-dominant markers.

Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

Abstract: High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data.

Microsatellite landscape evolutionary dynamics across 450 million years of vertebrate genome evolution

Abstract: The evolutionary dynamics of simple sequence repeats (SSRs or microsatellites) across the vertebrate tree of life remain largely undocumented and poorly understood. In this study, we analyzed patterns of genomic microsatellite abundance and evolution across 71 vertebrate genomes. The highest abundances of microsatellites exist in the genomes of ray-finned fishes, squamate reptiles, and mammals, while crocodilian, turtle, and avian genomes exhibit reduced microsatellite landscapes. We used comparative methods to infer evolutionary rates of change in microsatellite abundance across vertebrates and to highlight particular lineages that have experienced unusually high or low rates of change in genomic microsatellite abundance. Overall, most variation in microsatellite content, abundance, and evolutionary rate is observed among major lineages of reptiles, yet we found that several deeply divergent clades (i.e., squamate reptiles and mammals) contained relatively similar genomic microsatellite compositions. Arc...

Low genetic variation and high differentiation across sky island populations of Lupinus alopecuroides (Fabaceae) in the northern Andes

Abstract: The tropical alpine flora in the northern Andes has caught the attention of evolutionary biologists and conservationists because of the extent of its diversity and its vulnerability. Although population genetics studies are essential to understand how diversity arises and how it can be maintained, plant populations occurring above 4100 m a.s.l. in the so-called super-paramo have rarely been studied at the molecular level. Here, we use 11 microsatellite DNA markers to examine genetic structure in populations of Lupinus alopecuroides, a long-lived semelparous giant rosette known from only 10 geographically isolated populations. Each population is located on a different mountain top, of which three are in Colombia and seven in Ecuador. We analysed 220 individuals from all the ten known populations. We find low genetic variation in all but one of the populations. Four populations are completely monomorphic, and another five show only one polymorphic locus each. On the other hand, we find extremely high genetic differentiation between populations. We discuss the mechanisms that might cause this pattern, and we suggest that it is related to founder effects, lack of gene flow, and autogamy. The genetic relationships among the populations, and the lack of correlation between the genetic and geographic distances also point to the importance of founder effects and colonization history in driving differentiation among the populations.

Microsatellite (ssr) amplification by pcr usually led to polymorphic bands: evidence which shows replication slippage occurs in extend or nascent dna strands

Abstract: Microsatellites or simple sequence repeats (SSRs) are very effective molecular markers in population genetics, genome mapping, taxonomic study and other large-scale studies. Variation in number of tandem repeats within microsatellite refers to simple sequence length polymorphism (SSLP); but there are a few studies that are showed SSRs replication slippage may be occurred during in vitro amplification which are produced 'stutter products' differing in length from the main products. The purpose of this study is introducing a reliable method to realize SSRs replication slippage. At first, three unique primers designed to amplify SSRs loci in the great gerbil (Rhombomys opimus) by PCR. Crush and soak method used to isolate interesting DNA bands from polyacrylamide gel. PCR products analyzed using by sequencing methods. Our study has been shown that Taq DNA polymerase slipped during microsatellite in vitro amplification which led to insertion or deletion of repeats in sense or antisense DNA strands. It is produced amplified fragments with various lengths in gel electrophoresis showed as 'stutter bands'. Thus, in population studies by SSRs markers recommend that replication slippage effects and stutter bands have been considered.

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

Abstract: 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.

Development and validation of EST-derived SSR markers and diversity analysis in cluster bean (Cyamopsis tetragonoloba)

Abstract: Cluster bean/guar (Cyamopsis tetragonoloba), has an important place in industry because of its seeds, which contain galactomannan (guar gum) rich endosperm. Guar gum, an important ingredient of many products, is purely an export oriented commodity. Development of molecular markers for this crop is essential to accelerate breeding for guar gum content in seeds. A total of 100 novel primers pairs were developed from 16,476 expressed sequence tags (ESTs) sequence of cluster bean. A total of 50 primers pairs with function annotation of gum synthesis were selected and validated on a panel of 32 genotypes. Among the 50 primers 39 primers were amplified with a total of 45 loci. The polymorphic information content (PIC) ranged from 0.00 to 0.42 with an average of 0.13. With low polymorphic simple sequence repeats (SSRs) and narrow genetic base, most of the genotypes scattered into three clusters regardless of their geographical origin. Present study showed the existence of very low genetic diversity in cluster bean. The results indicated that there is need to explore SSR markers from whole genome or alternative marker systems like SNP (single nucleotide polymorphism) markers, for effective implication of markers in cluster bean breeding.

popSTR: population-scale detection of STR variants

Abstract: Motivation Microsatellites, also known as short tandem repeats (STRs), are tracts of repetitive DNA sequences containing motifs ranging from two to six bases. Microsatellites are one of the most abundant type of variation in the human genome, after single nucleotide polymorphisms (SNPs) and Indels. Microsatellite analysis has a wide range of applications, including medical genetics, forensics and construction of genetic genealogy. However, microsatellite variations are rarely considered in whole-genome sequencing studies, in large due to a lack of tools capable of analyzing them. Results Here we present a microsatellite genotyper, optimized for Illumina WGS data, which is both faster and more accurate than other methods previously presented. There are two main ingredients to our improvements. First we reduce the amount of sequencing data necessary for creating microsatellite profiles by using previously aligned sequencing data. Second, we use population information to train microsatellite and individual specific error profiles. By comparing our genotyping results to genotypes generated by capillary electrophoresis we show that our error rates are 50% lower than those of lobSTR, another program specifically developed to determine microsatellite genotypes. Availability and Implementation Source code is available on Github: https://github.com/DecodeGenetics/popSTR. Contact snaedis.kristmundsdottir@decode.is or bjarni.halldorsson@decode.is.

A New Resource for the Development of SSR Markers: Millions of Loci from a Thousand Plant Transcriptomes

Abstract: Premise of the study: The One Thousand Plant Transcriptomes Project (1KP, 1000+ assembled plant transcriptomes) provides an enormous resource for developing microsatellite loci across the plant tree of life. We developed loci from these transcriptomes and tested their utility. Methods and Results: Using software packages and custom scripts, we identified microsatellite loci in 1KP transcriptomes. We assessed the potential for cross-amplification and whether loci were biased toward exons, as compared to markers derived from genomic DNA. We characterized over 5.7 million simple sequence repeat (SSR) loci from 1334 plant transcriptomes. Eighteen percent of loci substantially overlapped with open reading frames (ORFs), and electronic PCR revealed that over half the loci would amplify successfully in conspecific taxa. Transcriptomic SSRs were approximately three times more likely to map to translated regions than genomic SSRs. Conclusions: We believe microsatellites still have a place in the genomic age—they r...