Simple Sequence Repeats Sequences Research Articles

Developing Novel Microsatellite Markers for Kaempferia parviflora by Microsatellite Capture Sequencing (MiCAPs)

Kaempferia parviflora, a medicinal plant widely used in Southeast Asia, has been validated clinically for its diverse pharmaceutical applications. Despite extensive research in pharmacology, there is a notable lack of cytogenetic and genomic research, primarily due to limited genetic information. Simple Sequence Repeat (SSR) is considered a robust class of molecular markers frequently used in biodiversity studies. In this study, we adopted Microsatellite Capture Sequencing (MiCAPs) to obtain SSR sequences for marker development. We identified 13,644 SSRs and developed and validated ten sets of SSR markers through capillary electrophoresis. The ten primer sets generated 27 alleles, with an average Polymorphism Information Content (PIC) of 0.36. Principle Coordinate Analysis (PCoA) distinguished two types of K. parviflora, consistent with classification by leaf margin color (red and green). A neighbor-joining dendrogram of seven Zingiberaceae species was constructed with the SSR-containing sequences. The 2-c value of K. parviflora is first reported here as 3.16 ± 0.03; the genome size is estimated at 3090.48 Mbp. The newly developed molecular markers are crucial for variety identification and the conservation of wild resources. Additionally, the cytogenetic and phylogenetic information provides valuable insights into the genetic diversity and evolutionary relationships.

Genome-wide simple sequence repeat analysis and specific molecular marker development of rye

BackgroundRye (Secale cereale L.) is the most widely used related species in wheat genetic breeding, and the introduction of its chromosome fragments into the wheat genome through distant hybridization is essential for enriching the genetic diversity of wheat. Rapid and accurate detection of rye chromatin in the wheat genome is important for distant hybridization. Simple sequence repeats (SSRs) are widely distributed in the genome, and SSRs of different species often exhibit species-specific characteristics.ResultsIn this study, genome-wide SSRs in rye were identified, and their characteristics were outlined. A total of 997,027 SSRs were selected, with a density of 115.97 SSRs/Mb on average. There was no significant difference in the number of SSRs on each chromosome. The number of SSRs on 2R was the highest (15.29%), and the number of SSRs on 1R was the lowest (13.02%). The number of SSRs on each chromosome is significantly correlated with chromosome length. The types of SSR motifs were abundant, and each type of SSR was distributed on 7 chromosomes of rye. The numbers of mononucleotide simple sequence repeats (MNRs), dinucleotide simple sequence repeats (DNRs), and trinucleotide simple sequence repeats (TNRs) were the greatest, accounting for 46.90%, 18.37%, and 22.64% of the total number, respectively. Among the MNRs, the number of G/C repeats and the number of 10 bp motifs were the greatest, accounting for 26.24% and 31.32% of the MNRs, respectively. Based on the SSR sequences, a total of 657 pairs of primers were designed. The PCR results showed that 119 pairs of these primers were rye-specific and could effectively detect rye chromatin in the wheat genome. Moreover, 86 pairs of the primers could also detect one or more specific rye chromosomes.ConclusionThese results lay a foundation for both genomic evolution studies of rye and molecular breeding in wheat.

Development, Identification and Validation of a Novel SSR Molecular Marker for Heat Resistance of Grapes Based on miRNA

The adverse effects of high temperatures on fruit quality and yield have been reported in many studies of grapevines. MiRNAs play crucial roles in plant growth and development and also fulfill functions in regulating the high-temperature response. In this research, miRNA-based SSR (simple sequence repeat) polymorphisms were analyzed according to the grape genome sequence and then combined with polymerase chain reaction (PCR) amplification and Sanger sequencing methods to analyze the miRNA-SSR diversity of different heat-resistant grape varieties. A statistical analysis of SSR sequences showed that 391 and 12 SSRs were specific to pri-miRNA and pre-miRNA, respectively. In pri-miRNA containing SSR, hexa-nucleotide repeats were the most abundant (52.69%), followed by tetra-nucleotide (13.04%) and minimum penta-nucleotide (4.09%), which were also observed in pre-miRNA sequences. On the other hand, differences in heat resistance among grape varieties were observed. Based on the results of leaf Fv/Fm images, values and phenotypic changes under high-temperature stress, 20 heat-resistant (e.g., Niagara Rosada and Grand Noir) and 20 heat-sensitive (e.g., Shine Muscat and Jumeigui) grape varieties were identified. Further, PCR-Sanger sequencing was used to screen SSR differences in four thermos-tolerant and four thermos-sensitive grape varieties, and finally, eight SSR differential primers were found to be able to distinguish these varieties. Of these, four pairs of primers were selected for validation in 40 grape germplasm resources (20 thermo-tolerance and 20 thermo-sensitivity). The VMIRSSR167c3 highlights that the ratio of SSR (CT) repeat values greater than 17 in heat-resistant varieties was 90%. In conclusion, the VMIRSSR167c3 marker can accurately distinguish between heat-resistant and heat-sensitive grape varieties. The results provide a novel molecular marker for the genetic improvement of grape germplasm resources and will be beneficial to the breeding of heat-resistant varieties in the future.

Pattern and variation in simple sequence repeat (SSR) at different genomic regions and its implications to maize evolution and breeding

BackgroundRepetitive DNA sequences accounts for over 80% of maize genome. Although simple sequence repeats (SSRs) account for only 0.03% of the genome, they have been widely used in maize genetic research and breeding as highly informative codominant DNA markers. The genome-wide distribution and polymorphism of SSRs are not well studied due to the lack of high-quality genome DNA sequence data.ResultsIn this study, using data from high-quality de novo-sequenced maize genomes of five representative maize inbred lines, we revealed that SSRs were more densely present in telomeric region than centromeric region, and were more abundant in genic sequences than intergenic sequences. On genic sequences, tri- and hexanucleotide motifs were more abundant in CDS sequence and some mono- and dinucleotide motifs were more abundant in UTR sequences. Median length and chromosomal density of SSRs were both narrowly range-bound, with median length of 14-18 bp and genome-wide average density of 3355.77 bp/Mbp. LTR-RTs of < 0.4 Mya had higher SSR density (4498-4992 bp/Mbp). The genome-specific and motif-specific SSR polymorphism were studied. Their potential breeding applications were discussed.ConclusionsWe found that the median length of SSR sequences of different SSR motifs was nearly constant. SSR density in genic regions was much higher than intergenic regions. In addition, SSR density at LTR-RTs of different evolutionary ages varied in a narrow range. The SSRs and their LTR-RT carriers evolved at an equal rate. All these observations indicated that SSR length and density were under control of yet unknown evolutionary forces. The chromosome region-specific and motif-specific SSR polymorphisms we observed supported the notion that SSR polymorphism was invaluable genome resource for developing highly informative genome and gene markers in maize genetic research and molecular breeding.

DendroSSR: SSRs and sequence alignment as tools for building phylogeny trees.

This study introduces a new method to construct phylogenetic trees by combining both of the Simple Sequence Repeats (SSRs) and sequence alignments. The purpose of this work is to present the DendroSSR program and show it via a case study involving diverse Aspergillus species. To show how the DendroSSR program works to resolve complicated species relationships in phylogenetic trees, we employed the Aspergillus species as an example of a research case. The DendroSSR employs a technique containing multiple phases beginning with, detecting SSRs, computing SSRs similarities, sequences alignment, building a distance matrix based on SSRs similarity and sequences alignments, and then hierarchical clustering, and presenting the findings in a dendrogram. Sometimes sequence alignments alone may not give adequate information to generate a phylogenetic tree to resolve complicated species relationships. Therefore, establishing a distance matrix that is formed of addition of SSRs similarity across sequences to the traditional sequence alignment helps the process substantially and resolves the connections of complex species on phylogenetic trees. Additionally, it may be hard to distinguish complex relationships across species when studying conserved sequences, which could lead to an incomplete representation of their evolutionary relationships. These limitations are addressed by DendroSSR, which offers a technique to produce phylogenetic trees by incorporating SSRs similarity across species into the approach of generating phylogenetic trees. As it is known, SSRs are extensively scattered across the genomes of species and exhibit a great variation. Therefore, SSRs may support the knowledge gathered from sequence alignments by providing more information on genetic variation and even evolutionary relationships. The use of DendroSSR analysis might be considered for creating phylogenetic trees as a complementary or secondary strategy among the species under examination in circumstances where traditional phylogenetic analysis fails to clarify the species complex phylogenetic relationships.

Application of Bioinformatics resources for mining of simple sequence repeats (SSRs) marker in plant genomes: An Overview

In the case of plants, the prediction of simple sequence repeats (SSRs) is important for the purpose of gene mapping, biodiversity study and detection of genes with desired characters. In addition to this, due to their polymorphic nature and distribution throughout the genome, it is considered as an ideal marker in plants. However, the traditional methods for the detection of SSR-based polymorphism cause difficultly. As an alternative method, the bioinformatics approaches have been used extensively in the study of these molecular markers, in a economic way. The next generation sequencing (NGS) techniques generate a huge amount of genomic data which is stored in the public databases in the form of whole genome and EST. This provides the opportunity to implement the bioinformatics tools to predict and annotate the SSR associated with desired gene of the plants. Several computational programs along with the pipelines have been developed to detect the SSR sequences automatically by using the genomic information from the database. In this review, the application and availability of specific bioinformatics resources and the methods of application involved for SSR discovery have been presented by taking suitable examples from the literature.

Simple sequence repeat insertion induced stability and potential 'gain of function' in the proteins of extremophilic bacteria.

Here, we analysed the genomic evolution in extremophilic bacteria using long simple sequence repeats (SSRs). Frequencies of occurrence, relative abundance (RA) and relative density (RD) of long SSRs were analysed in the genomes of extremophilic bacteria. Thermus aquaticus had the most RA and RD of long SSRs in its coding sequences (110.6 and 1408.3), followed by Rhodoferax antarcticus (77.0 and 1187.4). A positive correlation was observed between G + C content and the RA-RD of long SSRs. Geobacillus kaustophilus, Geobacillus thermoleovorans, Halothermothrix orenii, R. antarcticus, and T. aquaticus preferred trinucleotide repeats within their genomes, whereas others preferred a higher number of tetranucleotide repeats. Gene enrichment showed the presence of these long SSRs in metabolic enzyme encoding genes related to stress tolerance. To analyse the functional implications of SSR insertions, three-dimensional protein structure modelling of SSR containing diguanylate cyclase (DGC) gene encoding protein was carried out. Removal of SSR sequence led to an inappropriate folding and instability of the modelled protein structure.

Genome evolution during bread wheat formation unveiled by the distribution dynamics of SSR sequences on chromosomes using FISH

BackgroundDuring the bread wheat speciation by polyploidization, a series of genome rearrangement and sequence recombination occurred. Simple sequence repeat (SSR) sequences, predominately located in heterochromatic regions of chromosomes, are the effective marker for tracing the genomic DNA sequence variations. However, to date the distribution dynamics of SSRs on chromosomes of bread wheat and its donors, including diploid and tetraploid Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum turgidum ssp. dicocoides, reflecting the genome evolution events during bread wheat formation had not been comprehensively investigated.ResultsThe genome evolution was studied by comprehensively comparing the distribution patterns of (AAC)n, (AAG)n, (AGC)n and (AG)n in bread wheat Triticum aestivum var. Chinese Spring and its progenitors T. urartu, A. speltoides, Ae. tauschii, wild tetroploid emmer wheat T. dicocoides, and cultivated emmer wheat T. dicoccum. Results indicated that there are specific distribution patterns in different chromosomes from different species for each SSRs. They provided efficient visible markers for identification of some individual chromosomes and SSR sequence evolution tracing from the diploid progenitors to hexaploid wheat. During wheat speciation, the SSR sequence expansion occurred predominately in the centromeric and pericentromeric regions of B genome chromosomes accompanied by little expansion and elimination on other chromosomes. This result indicated that the B genome might be more sensitive to the “genome shock” and more changeable during wheat polyplodization.ConclusionsDuring the bread wheat evolution, SSRs including (AAC)n, (AAG)n, (AGC)n and (AG)n in B genome displayed the greatest changes (sequence expansion) especially in centromeric and pericentromeric regions during the polyploidization from Ae. speltoides S genome, the most likely donor of B genome. This work would enable a better understanding of the wheat genome formation and evolution and reinforce the viewpoint that B genome was originated from S genome.

Development of SSR markers in Paeonia based on De Novo transcriptomic assemblies.

Peony is a famous ornamental and medicinal plant in China, and peony hybrid breeding is an important means of germplasm innovation. However, research on the genome of this species is limited, thereby hindering the genetic and breeding research on peony. In the present study, simple sequence repeat (SSR) locus analysis was performed on expressed sequence tags obtained by the transcriptome sequencing of Paeonia using Microsatellite software. Primers with polymorphism were obtained via polymerase chain reaction amplification and electrophoresis. As a result, a total of 86,195 unigenes were obtained by assembling the transcriptome data of Paeonia. Functional annotations were obtained in seven functional databases including 49,172 (Non-Redundant Protein Sequence Database: 57.05%), 38,352 (Nucleotide Sequence Database: 44.49%), 36,477 (Swiss Prot: 42.32%), 38,905 (Clusters of Orthologous Groups for Eukaryotic Complete Genomes: 45.14%), 37,993 (Kyoto Encyclopedia of Genes and Genomes: 44.08%), 26,832 (Gene Ontology: 31.13%) and 37,758 (Pfam: 43.81%) unigenes. Meanwhile, 21,998 SSR loci were distributed in 17,567 unigenes containing SSR sequences, and the SSR distribution frequency was 25.52%, with an average of one SSR sequence per 4.66 kb. Mononucleotide, dinucleotide, and trinucleotide were the main repeat types, accounting for 55.74%, 25.58%, and 13.21% of the total repeat times, respectively. Forty-five pairs of the 100 pairs of primers selected randomly could amplify clear polymorphic bands. The polymorphic primers of these 45 pairs were used to cluster and analyze 16 species of peony. The new SSR molecular markers can be useful for the study of genetic diversity and marker-assisted breeding of peony.

Characterization and Development of EST-SSR Markers from Transcriptome Sequences of Chrysanthemum (Chrysanthemum ×morifolium Ramat.)

Simple sequence repeat (SSR) markers are valuable for genetic and breeding applications, but SSR resources for the ornamental genus chrysanthemum (Chrysanthemum ×morifolium Ramat.) are still limited. Expressed sequence tags (ESTs) are sources of SSRs that represent an opportunity to develop SSRs to accelerate molecular breeding in chrysanthemum. In total, 4661 SSR loci were identified from 3823 SSR-containing unigenes in the chrysanthemum transcriptome with an average of one SSR per 6.98 kb. Of these SSR sequences, trinucleotide repeats (30.0%) predominated, followed by dinucleotide repeats (17.9%). In total, 1584 primer pairs were subsequently synthesized. By screening the parents and six individuals of the F1 progeny, 831 (52.5%) valid EST-SSR markers were identified, of which 361 (43.4%) were polymorphic. The annotation of unigenes containing polymorphic SSRs indicated that 330 (93.5%) demonstrated significant homology to other plant protein sequences. Twenty-five polymorphic EST-SSR markers were further selected for transferability analysis and exhibited 93% amplification in six Ajania species and six other Chrysanthemum species. Based on genotyping of the 59 samples, neighbor-joining analysis revealed six phylogenetic groupings, which was confirmed by population structure analysis and principal component analysis (PCA). Phylogenetic relationships among the 59 samples revealed by SSRs were highly consistent with the traditional taxonomic classification of Chrysanthemum and Ajania. The polymorphism information content (PIC) values ranged from 0.29 to 0.86, with a mean of 0.67, indicating high levels of informativeness. This research reveals the SSR distribution characteristics of chrysanthemum and provides a large number of new EST-SSR markers for further genetic diversity studies, genetic mapping, and molecular marker-assisted selection breeding for chrysanthemum.

Genetic linkage map and QTL identification for adventitious rooting traits in red gum eucalypts.

The eucalypt species, Eucalyptus tereticornis and Eucalyptus camaldulensis, show tolerance to drought and salinity conditions, respectively, and are widely cultivated in arid and semiarid regions of tropical countries. In this study, genetic linkage map was developed for interspecific cross E. tereticornis × E. camaldulensis using pseudo-testcross strategy with simple sequence repeats (SSRs), intersimple sequence repeats (ISSRs), and sequence-related amplified polymorphism (SRAP) markers. The consensus genetic map comprised totally 283 markers with 84 SSRs, 94 ISSRs, and 105 SRAP markers on 11 linkage groups spanning 1163.4cM genetic distance. Blasting the SSR sequences against E. grandis sequences allowed an alignment of 64% and the average ratio of genetic-to-physical distance was 1.7 Mbp/cM, which strengths the evidence that high amount of synteny and colinearity exists among eucalypts genome. Blast searches also revealed that 37% of SSRs had homologies with genes, which could potentially be used in the variety of downstream applications including candidate gene polymorphism. Quantitative trait loci (QTL) analysis for adventitious rooting traits revealed six QTL for rooting percent and root length on five chromosomes with interval and composite interval mapping. All the QTL explained 12.0-14.7% of the phenotypic variance, showing the involvement of major effect QTL on adventitious rooting traits. Increasing the density of markers would facilitate the detection of more number of small-effect QTL and also underpinning the genes involved in rooting process.

Microsatellite analysis and marker development in garlic: distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae.

Allium vegetables, such as garlic and onion, have understudied genomes and limited molecular resources, hindering advances in genetic research and breeding of these species. In this study, we characterized and compared the simple sequence repeats (SSR) landscape in the transcriptomes of garlic and related Allium (A. cepa, A. fistulosum, and A. tuberosum) and non-Allium monocot species. In addition, 110 SSR markers were developed from garlic ESTs, and they were characterized-along with 112 previously developed SSRs-at various levels, including transferability across Alliaceae species, and their usefulness for genetic diversity analysis. Among the Allium species analyzed, garlic ESTs had the highest overall SSR density, the lowest frequency of trinucleotides, and the highest of di- and tetranucleotides. When compared to more distantly related monocots, outside the Asparagales order, it was evident that ESTs of Allium species shared major commonalities with regards to SSR density, frequency distribution, sequence motifs, and GC content. A significant fraction of the SSR markers were successfully transferred across Allium species, including crops for which no SSR markers have been developed yet, such as leek, shallot, chives, and elephant garlic. Diversity analysis of garlic cultivars with selected SSRs revealed 36 alleles, with 2-5 alleles/locus, and PIC = 0.38. Cluster analysis grouped the accessions according to their flowering behavior, botanical variety, and ecophysiological characteristics. Results from this study contribute to the characterization of Allium transcriptomes. The new SSR markers developed, along with the data from the polymorphism and transferability analyses, will aid in assisting genetic research and breeding in garlic and other Allium.

Transcriptome Analysis and Microsatellite Markers Development of a Traditional Chinese Medicinal Herb Halenia elliptica D. Don (Gentianaceae).

Halenia elliptica is a popular Chinese medicinal herb that is used to treat jaundice disease and virus hepatitis, and its wild populations have been reduced significantly due to overharvesting recently. However, effective conservation could not be implemented because of the lack of genomic information and genetic markers. In this study, a de novo transcriptome of H elliptica was sequenced using the NGS Illumina, and 132 695 unigenes with the length >200 bp (base pairs) were obtained. Among them, a total of 32 109 unigenes were scanned to develop simple sequence repeats (SSRs). Based on NCBI (National Center for Biotechnology Information) nonredundant database (Nr), these SSR sequences were annotated and assigned into gene ontology categories. In addition, we designed 126 pairs of SSR primers for polymerase chain reaction amplification, of which 12 pairs were identified to be polymorphic among 40 individuals from 8 populations. We then used the 12 polymorphic SSRs to construct a UPGMA dendrogram of the 40 individuals. In addition, a significant correlation between the genetic relationship and the geographic distance was found, suggesting a phylogeographic structure in H elliptica. Moreover, 2 of these SSRs were also successfully amplified in a related species Veratrilla baillonii, suggesting their cross-species transferability. Generally, the SSR markers with high polymorphisms identified in this study provide valuable genetic resources and represent an initial step for exploring the genetic diversity and population histories of H elliptica and its related species.

The complete plastome sequence of the endangered orchid Habenaria radiata (Orchidaceae)

In this study, we determined the complete chloroplast sequence of Habenaria radiata (Thunb.) Spreng. (Orchidaceae) (NCBI acc. no. KX871237), an endangered plant species protected by the national law of Korea. The gene order and gene content of the H. radiata plastome are similar to those of typical angiosperm plastomes. The 11 ndh genes, which are usually lost in orchid plastomes, are intact in the H. radiata plastome. The complete plastome is 155,353 bp in length and consists of a large single copy of 84,833 bp and a small single copy of 17,718 bp, separated by two inverted repeats of 26,401 bp. The plastome contains 113 genes, of which 79 are protein-coding genes, 30 are tRNA genes, and four are rRNA genes. Sixteen genes contain one intron and two genes (clpP, ycf3) have two introns. A total of 76 simple sequence repeat (SSR) loci, which consist of 58 mono-SSR, 17 di-SSR, and 1 tri-SSR, are scattered along the H. radiata plastome. Some of these plastome SSR and high sequence divergent regions may be useful for development of genetic markers for the populations of H. radiata and other congeneric species. Phylogenetic analysis identified the sister relationship between H. radiata and H. pantlingiana within the tribe Orchideae.

Molecular Structure and Phylogenetic Analyses of Complete Chloroplast Genomes of Two Aristolochia Medicinal Species.

The family Aristolochiaceae, comprising about 600 species of eight genera, is a unique plant family containing aristolochic acids (AAs). The complete chloroplast genome sequences of Aristolochia debilis and Aristolochia contorta are reported here. The results show that the complete chloroplast genomes of A. debilis and A. contorta comprise circular 159,793 and 160,576 bp-long molecules, respectively and have typical quadripartite structures. The GC contents of both species were 38.3% each. A total of 131 genes were identified in each genome including 85 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene (ycf1). The simple-sequence repeat sequences mainly comprise A/T mononucletide repeats. Phylogenetic analyses using maximum parsimony (MP) revealed that A. debilis and A. contorta had a close phylogenetic relationship with species of the family Piperaceae, as well as Laurales and Magnoliales. The data obtained in this study will be beneficial for further investigations on A. debilis and A. contorta from the aspect of evolution, and chloroplast genetic engineering.

Simple sequence repeats showing ‘length preference’ have regulatory functions in humans

Simple sequence repeats (SSRs), simple tandem repeats (STRs) or microsatellites are short tandem repeats of 1–6 nucleotide motifs. They are twice as abundant as the protein coding DNA in the human genome and yet little is known about their functional relevance. Analysis of genomes across various taxa show that despite the instability associated with longer stretches of repeats, few SSRs with specific longer repeat lengths are enriched in the genomes indicating a positive selection. This conserved feature of length dependent enrichment hints at not only sequence but also length dependent functionality for SSRs. In the present study, we selected 23 SSRs of the human genome that show specific repeat length dependent enrichment and analysed their cis-regulatory potential using promoter modulation, boundary and barrier assays. We find that the 23 SSR sequences, which are mostly intergenic and intronic, possess distinct cis-regulatory potential. They modulate minimal promoter activity in transient luciferase assays and are capable of functioning as enhancer-blockers and barrier elements. The results of our functional assays propose cis-gene regulatory roles for these specific length enriched SSRs and opens avenues for further investigations.

Identification of Expressed Sequence Tag-simple Sequence Repeat Markers from the De Novo Transcriptome Sequence of Red Raspberry (Rubus idaeus L.)

Rubus idaeus has remarkable economic and cultural value. Developing efficient simple sequence repeat (SSR) markers is necessary for the molecular breeding of red raspberry. In this study, SSR mining was performed using the de novo transcriptome sequence of R. idaeus. In total, 14,210 SSR sequences were identified from 11,158 SSR-containing unigenes. In all the SSR sequences, mononucleotide, dinucleotide, and trinucleotide repeats were the most common, and their number and percentage were 1323 (9.31%), 6752 (47.52%), and 4897 (34.46%), respectively. Of the mononucleotide and dinucleotide repeats, A/T, AG/CT, AT/AT, and AC/GT were more abundant and accounted for 9.09%, 37.82%, 6.51%, and 3.14% of the total repeat number, respectively. In the trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats, the nucleotide (NT) patterns AAG/CTT, AAAG/CTTT, AAAAG/CTTTT, and AAGAGG/CCTCTT were the most frequent, and accounted for 14.11%, 0.38%, 0.57%, and 0.23% of the total SSRs, respectively. Of the 480 SSR-containing unigenes with gene ontology (GO) annotation, the classification results showed that they were mainly involved in binding, catalytic, and transporter molecular functions. Most of the 3441 SSR-containing unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation were involved in the following top five pathways: metabolic, RNA transport, spliceosome, protein processing in the endoplasmic reticulum, and mRNA surveillance. Thirty pairs of primers derived from the red raspberry transcriptome were randomly selected to assess their polymorphism by using 15 red raspberry germplasms, in which the polymorphism information content (PIC) values ranged from 0.50 to 0.86, with a mean of 0.73, thereby indicating a high level of polymorphism. The unweighted pair group method with arithmetic mean clustering results indicated that the thirty pairs of primers could precisely distinguish the germplasms. This study reveals the SSR distribution characteristics of red raspberry and provides a scientific basis for further genetic diversity studies and genetic linkage map construction for this species.

Cytogenetic Diversity of Simple Sequences Repeats in Morphotypes of Brassica rapa ssp. chinensis.

A significant fraction of the nuclear DNA of all eukaryotes is comprised of simple sequence repeats (SSRs). Although these sequences are widely used for studying genetic variation, linkage mapping and evolution, little attention had been paid to the chromosomal distribution and cytogenetic diversity of these sequences. In this paper, we report the distribution characterization of mono-, di-, and tri-nucleotide SSRs in Brassica rapa ssp. chinensis. Fluorescence in situ hybridization was used to characterize the cytogenetic diversity of SSRs among morphotypes of B. rapa ssp. chinensis. The proportion of different SSR motifs varied among morphotypes of B. rapa ssp. chinensis, with tri-nucleotide SSRs being more prevalent in the genome of B. rapa ssp. chinensis. We determined the chromosomal locations of mono-, di-, and tri-nucleotide repeat loci. The results showed that the chromosomal distribution of SSRs in the different morphotypes is non-random and motif-dependent, and allowed us to characterize the relative variability in terms of SSR numbers and similar chromosomal distributions in centromeric/peri-centromeric heterochromatin. The differences between SSR repeats with respect to abundance and distribution indicate that SSRs are a driving force in the genomic evolution of B. rapa species. Our results provide a comprehensive view of the SSR sequence distribution and evolution for comparison among morphotypes B. rapa ssp. chinensis.

In silico search, characterization and validation of new EST-SSR markers in the genus Prunus.

BackgroundSimple sequence repeats (SSRs) are defined as sequence repeat units between 1 and 6 bp that occur in both coding and non-coding regions abundant in eukaryotic genomes, which may affect the expression of genes. In this study, expressed sequence tags (ESTs) of eight Prunus species were analyzed for in silico mining of EST-SSRs, protein annotation, and open reading frames (ORFs), and the identification of codon repetitions.ResultsA total of 316 SSRs were identified using MISA software. Dinucleotide SSR motifs (26.31 %) were found to be the most abundant type of repeats, followed by tri- (14.58 %), tetra- (0.53 %), and penta- (0.27 %) nucleotide motifs. An attempt was made to design primer pairs for 316 identified SSRs but these were successful for only 175 SSR sequences. The positions of SSRs with respect to ORFs were detected, and annotation of sequences containing SSRs was performed to assign function to each sequence. SSRs were also characterized (in terms of position in the reference genome and associated gene) using the two available Prunus reference genomes (mei and peach). Finally, 38 SSR markers were validated across peach, almond, plum, and apricot genotypes. This validation showed a higher transferability level of EST-SSR developed in P. mume (mei) in comparison with the rest of species analyzed.ConclusionsFindings will aid analysis of functionally important molecular markers and facilitate the analysis of genetic diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2143-y) contains supplementary material, which is available to authorized users.

Transcriptome Analysis of Two Vicia sativa Subspecies: Mining Molecular Markers to Enhance Genomic Resources for Vetch Improvement

The vetch (Vicia sativa) is one of the most important annual forage legumes globally due to its multiple uses and high nutritional content. Despite these agronomical benefits, many drawbacks, including cyano-alanine toxin, has reduced the agronomic value of vetch varieties. Here, we used 454 technology to sequence the two V. sativa subspecies (ssp. sativa and ssp. nigra) to enrich functional information and genetic marker resources for the vetch research community. A total of 86,532 and 47,103 reads produced 35,202 and 18,808 unigenes with average lengths of 735 and 601 bp for V. sativa sativa and V. sativa nigra, respectively. Gene Ontology annotations and the cluster of orthologous gene classes were used to annotate the function of the Vicia transcriptomes. The Vicia transcriptome sequences were then mined for simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. About 13% and 3% of the Vicia unigenes contained the putative SSR and SNP sequences, respectively. Among those SSRs, 100 were chosen for the validation and the polymorphism test using the Vicia germplasm set. Thus, our approach takes advantage of the utility of transcriptomic data to expedite a vetch breeding program.