Dinucleotide Simple Sequence Repeats Research Articles

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.

First Karyotypic Insights into Potamotrygon schroederi Fernández-Yépez, 1958: Association of Different Classes of Repetitive DNA

Introduction: Currently, there are 38 valid species of freshwater stingrays, and these belong to the subfamily Potamotrygoninae. However, cytogenetic information about this group is limited, with studies mainly using classical techniques, Giemsa, and C-banding. Methods: In this study, we used classical and molecular cytogenetic techniques – mapping of 18S and 5S rDNA and simple sequence repeats (SSRs) – in order to investigate the karyotypic composition of Potamotrygon schroederi and reveal the karyoevolutionary trends of this group. Results: The species presented 2n = 66 chromosomes with 18m + 12sm + 16st + 20a, heterochromatic blocks distributed in the centromeric regions of all the chromosomes, and terminal blocks in the q arm of pairs 2 and 3. Mapping of 18S rDNA regions revealed multiple clusters on pairs 2 and 7 and a homolog of pair 24. The 5S rDNA region was found in the pericentromeric portion of the subtelocentric pair 16. Furthermore, dinucleotide SSRs sequences were found in the centromeric and terminal regions of different chromosomal pairs, with preferential accumulation in pair 17. In addition, we identified conspicuous blocks of (GATA)n and (GACA)n sequences colocalized with the 5S rDNA (pair 16). Conclusion: In general, this study corroborates the general trend of a reduction in 2n in the species of Potamotrygoninae subfamily. Moreover, we found that the location of rDNA regions is very similar among Potamotrygon species, and the SSRs accumulation in the second subtelocentric pair (17) seems to be a common trait in this genus.

Distribution and conservation of simple sequence repeats in plant pathogenic species of Zymoseptoria and development of genomic resources for its orphaned species.

To better understand the structure and evolution of the genomes of four plant pathogenic species of Zymoseptoria, we analyzed the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) in their whole genome and transcriptome sequences. In this study, SSRs are defined as repeats of more than 12 bases in length. The genome and transcriptome sequences of Zymoseptoria ardabiliae show the highest RA (201.1 and 129.9) and RD (3229.4 and 1928.2) of SSRs, while those of Zymoseptoria pseudotritici show the lowest RA (167.2 and 118.5) and RD (2482.2 and 1687.0). The majority of SSRs in the genomic and transcriptome sequences of species were trinucleotide SSRs, while dinucleotide SSRs were the least common. The most common trinucleotide motifs in the transcriptomic sequences across all species were those that encoded the amino acid arginine. As per our motif conservation study, Zymoseptoria tritici (12.4%) possessed the most unique motifs, while Z. pseudotritici (3.9%) had the fewest. Overall, only 38.1% of the motifs were found to be conserved among the species. Gene enrichment studies reveal that three of the species, Z. ardabiliae, Zymoseptoria brevis, and Z. pseudotritici, have SSRs in their genes related to cellular metabolism, while the remaining Z. tritici harbors SSRs in genes related to DNA synthesis and gene expression. In an effort to improve the genetic resources for the orphan species of pathogenic Zymoseptoria, a total of 73,134 primers were created. The genomic resources developed in this study could help with analyses of genetic relatedness within the population and the development of species-specific markers.

Diosgenin biosynthesis investigation in medicinal herb (Tribulus terrestris) by transcriptome analysis

Next-generation sequencing (NGS) has revolutionized the analysis of specific genes, pathways, and their regulation in various species. Tribulus terrestris L., an annual medicinal herb of Zygophyllaceae family, has gained significant attention due to its diverse medicinal properties, including anti-inflammatory, antimicrobial, and anti-cancer effects. Diosgenin, a steroidal saponin, is the major bioactive compound responsible for the medicinal importance of T. terrestris. However, there is a paucity of information regarding the genes involved in the diosgenin biosynthetic pathway in T. terrestris. To address this gap, this study aimed to identify candidate genes associated with diosgenin biosynthesis through whole transcriptome profiling. A total of ∼7.9 GB of data, comprising 482 million reads, was obtained and assembled into 148,871 unigenes. Subsequently, functional annotations were assigned to 50 % of the unigenes using sequence similarity searches against the NCBI non-redundant (NR), Uniprot, KEGG, Pfam, GO, and COG databases, primarily based on Gene Ontology and KEGG-KAAS pathways. The majority of unigenes associated with the biosynthesis of the steroidal diosgenin backbone exhibited up-regulation in the fruit, leaf, and root tissues, except the SQE gene in root. The differential expression of selected genes was further validated through quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the study identified 21,026 unigenes related to transcription factors and 15,551 unigenes containing simple sequence repeats (SSR). Notably, di-nucleotide SSR motifs exhibited a high repeat frequency. These findings greatly enhance our understanding of the diosgenin biosynthesis pathway and provide a basis for future research in molecular investigation and metabolic engineering, specifically for boosting diosgenin content.

Genome-wide discovery of di-nucleotide SSR markers based on whole genome re-sequencing data of Cicer arietinum L. and Cicer reticulatum Ladiz

Simple sequence repeats (SSRs) are valuable genetic markers due to their co-dominant inheritance, multi-allelic and reproducible nature. They have been largely used for exploiting genetic architecture of plant germplasms, phylogenetic analysis, and mapping studies. Among the SSRs, di-nucleotide repeats are the most frequent of the simple repeats distributed throughout the plant genomes. In present study, we aimed to discover and develop di-nucleotide SSR markers by using the whole genome re-sequencing (WGRS) data from Cicer arietinum L. and C. reticulatum Ladiz. A total of 35,329 InDels were obtained in C. arietinum, whereas 44,331 InDels in C. reticulatum. 3387 InDels with 2 bp length were detected in C. arietinum, there were 4704 in C. reticulatum. Among 8091 InDels, 58 di-nucleotide regions that were polymorphic between two species were selected and used for validation. We tested primers for evaluation of genetic diversity in 30 chickpea genotypes including C. arietinum, C. reticulatum, C. echinospermum P.H. Davis, C. anatolicum Alef., C. canariense A. Santos & G.P. Lewis, C. microphyllum Benth., C. multijugum Maesen, C. oxyodon Boiss. & Hohen. and C. songaricum Steph ex DC. A total of 244 alleles were obtained for 58 SSR markers giving an average of 2.36 alleles per locus. The observed heterozygosity was 0.08 while the expected heterozygosity was 0.345. Polymorphism information content was found to be 0.73 across all loci. Phylogenetic tree and principal coordinate analysis clearly divided the accessions into four groups. The SSR markers were also evaluated in 30 genotypes of a RIL population obtained from an interspecific cross between C. arietinum and C. reticulatum. Chi-square (χ2) test revealed an expected 1:1 segregation ratio in the population. These results demonstrated the success of SSR identification and marker development for chickpea with the use of WGRS data. The newly developed 58 SSR markers are expected to be useful for chickpea breeders.

Comparative genomics reveals the presence of simple sequence repeats in genes related to virulence in plant pathogenic Pythium ultimum and Pythium vexans

In this study, we evaluated the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) in the complete genome and transcriptomic sequences of the plant pathogenic species of Pythium to acquire a better knowledge of their genome structure and evolution. Among the species, P. ultimum had the highest RA and RD of SSRs in the genomic sequences, whereas P. vexans had the highest RA and RD in the transcriptomic sequences. The genomic and transcriptomic sequences of P. aphanidermatum showed the lowest RA and RD of SSRs. Trinucleotide SSRs were the most prevalent class in both genomic and transcriptomic sequences, while dinucleotide SSRs were the least prevalent. The G + C content of the transcriptomic sequences was found to be positively correlated with the number (r = 0.601) and RA (r = 0.710) of SSRs. A motif conservation study revealed the highest number of unique motifs in P. vexans (9.9%). Overall, a low conservation of motifs was observed among the species (25.9%). A gene enrichment study revealed P. vexans and P. ultimum carry SSRs in their genes that are directly connected to virulence, whereas the remaining two species, P. aphanidermatum and P. arrhenomanes, harbour SSRs in genes involved in transcription, translation, and ATP binding. In an effort to enhance the genomic resources, a total of 11,002 primers from the transcribed regions were designed for the pathogenic Pythium species. Furthermore, the unique motifs identified in this work could be employed as molecular probes for species identification.

Comprehensive Analyses of Simple Sequence Repeat (SSR) in Bamboo Genomes and Development of SSR Markers with Peroxidase Genes.

Simple sequence repeats (SSRs) are one of the most important molecular markers, which are widespread in plants. Bamboos are important forest resources worldwide. Here, the comprehensive identification and comparative analysis of SSRs were performed in three woody and two herbaceous bamboo species. Altogether 567,175 perfect SSRs and 71,141 compound SSRs were identified from 5737.8 Mb genome sequences of five bamboo species. Di-nucleotide SSRs were the most predominant type, with an average of ~50,152.2 per species. Most SSRs were located in intergenic regions, while those located in genic regions were relatively less. Moreover, the results of annotation distribution indicated that terms with P450, peroxidase and ATP-binding cassette transporter related to lignin biosynthesis might play important roles in woody and herbaceous bamboos under the mediation of SSRs. Furthermore, the peroxidase gene family consisted of a large number of genes containing SSRs was selected for the evolutionary relationship analysis and SSR markers development. Fifteen SSR markers derived from peroxidase family genes of Phyllostachys edulis were identified as polymorphic in 34 accessions belonging to seven genera in Bambusoideae. These results provided a comprehensive insight of SSR markers into bamboo genomes, which would facilitate bamboo research related to comparative genomics, evolution and marker-assisted selection.

Diallelic self-incompatibility is the main determinant of fertilization patterns in olive orchards.

Self‐incompatibility (SI) in flowering plants potentially represents a major obstacle for sexual reproduction, especially when the number of S‐alleles is low. The situation is extreme in the commercially important olive tree, where in vitro pollination assays suggested the existence of a diallelic SI (DSI) system involving only two groups (G1 and G2). Varieties belonging to the same SI group cannot fertilize each other, such that successful fruit production is predicted to require pollination between varieties of different groups. To test this prediction, we explored the extent to which the DSI system determines fertilization patterns under field conditions. One hundred and seventeen olive cultivars were first genotyped using 10 highly polymorphic dinucleotide Simple Sequence Repeat (SSR) markers to ascertain varietal identity. Cultivars were then phenotyped through controlled pollination tests to assign each of them to one of the two SI groups. We then collected and genotyped 1440 open pollinated embryos from five different orchards constituted of seven local cultivars with known group of incompatibility groups. Embryos genotype information were used: (i) to assign embryos to the most likely pollen donor genotype in the neighbourhood using paternity analysis, and (ii) to compare the composition of the pollen cloud genetic among recipient trees in the five sites. The paternity analysis showed that the DSI system is the main determinant of fertilization success under field open pollination conditions: G1 cultivars sired seeds exclusively on G2 cultivars, and reciprocally. No self‐fertilization events were observed. Our results demonstrate that DSI is a potent force determining pollination success among varieties within olive orchards used for production. They have the potential to improve management practices by guiding the selection of compatible varieties to avoid planting orchards containing sets of varieties with strongly unbalanced SI groups, as these would lead to suboptimal olive production.

Fine mapping of the locus controlling self-incompatibility in European hazelnut

Incompatibility in European hazelnut (Corylus avellana L.) is sporophytic and under the control of a single locus on linkage group 5 between markers G05-510 and AU02-1350. In this study, two rounds of marker development and a population of 192 seedlings with known S-alleles that showed recombination between the flanking markers were used for fine mapping. Using the sequences of random amplified polymorphic DNA and simple sequence repeat (SSR) markers and bacterial artificial chromosome end sequences, 36 contigs from the genome sequence of “Jefferson” hazelnut were identified for pursuit. Di-nucleotide SSR markers in those contigs were developed, characterized, and mapped. This reduced the size to a region of 500 kb that contained the S-locus and 50 predicted genes, in which single-nucleotide polymorphism and additional SSR markers were developed. When the new markers were used in fine mapping, they fully exploited all recombination in the fine mapping population and reduced the region to 193.5 kb containing 18 genes. This 193.5-kb region most likely contains the S1 haplotype. A second region, 2 Mbp away from the first in the “Jefferson” genome (V3), is predicted to represent the S3 haplotype based on SSR marker allele sizes and the ratio of parental reads that align to them. Although they appear side-by-side in the “Jefferson” genome (V3), the mapped markers appear in both sequences in the same order. Gene annotations in the S1 and S3 haplotypes are highly similar and include five probable leucine-rich repeat receptor-like protein kinases with homology to Arabidopsis thaliana genes At1g35710 and three probable receptor-like serine/threonine protein kinases with homology to At5g15080 (PIX7).

Genome-wide characterization of simple sequence repeats in Palmae genomes

BackgroundMicrosatellites or simple sequence repeats (SSRs) have become the most significant DNA marker technology used in genetic research. The availability of complete draft genomes for a number of Palmae species has made it possible to perform genome-wide analysis of SSRs in these species. Palm trees are tropical and subtropical plants with agricultural and economic importance due to the nutritional value of their fruit cultivars.ObjectiveThis is the first comprehensive study examining and comparing microsatellites in completely-sequenced draft genomes of Palmae species.MethodsWe identified and compared perfect SSRs with 1–6 bp nucleotide motifs to characterize microsatellites in Palmae species using PERF v0.2.5. We analyzed their relative abundance, relative density, and GC content in five palm species: Phoenix dactylifera, Cocos nucifera, Calamus simplicifolius, Elaeis oleifera, and Elaeis guineensis.ResultsA total of 118241, 328189, 450753, 176608, and 70694 SSRs were identified, respectively. The six repeat types were not evenly distributed across the five genomes. Mono- and dinucleotide SSRs were the most abundant, and GC content was highest in tri- and hexanucleotide SSRs.ConclusionWe envisage that this analysis would further substantiate more in-depth computational, biochemical, and molecular studies on the roles SSRs may play in the genome organization of the palm species. The current study contributes a detailed characterization of simple sequence repeats in palm genomes.

Development of SSR Markers Based on Transcriptome Sequences of the Wolf Spider Pardosa pseudoannulata (Araneae: Lycosidae)1

Pardosa pseudoannulata (Boesenberg et Strand, l906) is one of the most common wandering spiders in agricultural fields, and a potential bioindicator for heavy metal contamination. In this study, a high-throughput method was used for discovering the simple sequence repeat (SSR) genetic markers from this spider. In total, 2805 sequences containing a total of 3450 SSRs were identified from the previously constructed transcriptome database, with 516 of the sequences containing more than one SSR. The most abundant SSRs in P. pseudoannulata are di-nucleotide (42.32%) SSRs, followed by mono-nucleotide (41.83%). A/T (41.36%) repeats were the most abundant, followed by AT/AT (28.32%). A total of 6561 pairs of SSR primers were designed using Primer 5.0 software, and a subset of 26 SSR primer pairs was randomly selected and validated by RT-PCR. The SSR markers identified here will be an important resource for measuring genetic diversity and biological monitoring research in P. pseudoannulata.

Genome-wide characterization and analysis of microsatellite sequences in camelid species

Microsatellites or simple sequence repeats (SSRs) are among the genetic markers most widely utilized in research. This includes applications in numerous fields such as genetic conservation, paternity testing, and molecular breeding. Though ordered draft genome assemblies of camels have been announced, including for the Arabian camel, systemic analysis of camel SSRs is still limited. The identification and development of informative and robust molecular SSR markers are essential for marker assisted breeding programs and paternity testing. Here we searched and compared perfect SSRs with 1–6 bp nucleotide motifs to characterize microsatellites for draft genome sequences of the Camelidae. We analyzed and compared the occurrence, relative abundance, relative density, and guanine-cytosine (GC) content in four taxonomically different camelid species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. A total of 546762, 544494, 547974, and 437815 SSRs were mined, respectively. Mononucleotide SSRs were the most frequent in the four genomes, followed in descending order by di-, tetra-, tri-, penta-, and hexanucleotide SSRs. GC content was highest in dinucleotide SSRs and lowest in mononucleotide SSRs. Our results provide further evidence that SSRs are more abundant in noncoding regions than in coding regions. Similar distributions of microsatellites were found in all four species, which indicates that the pattern of microsatellites is conserved in family Camelidae.

De Novo Sequencing and Hybrid Assembly of the Biofuel Crop Jatropha curcas L.: Identification of Quantitative Trait Loci for Geminivirus Resistance

Jatropha curcas is an important perennial, drought tolerant plant that has been identified as a potential biodiesel crop. We report here the hybrid de novo genome assembly of J. curcas generated using Illumina and PacBio sequencing technologies, and identification of quantitative loci for Jatropha Mosaic Virus (JMV) resistance. In this study, we generated scaffolds of 265.7 Mbp in length, which correspond to 84.8% of the gene space, using Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Additionally, 96.4% of predicted protein-coding genes were captured in RNA sequencing data, which reconfirms the accuracy of the assembled genome. The genome was utilized to identify 12,103 dinucleotide simple sequence repeat (SSR) markers, which were exploited in genetic diversity analysis to identify genetically distinct lines. A total of 207 polymorphic SSR markers were employed to construct a genetic linkage map for JMV resistance, using an interspecific F2 mapping population involving susceptible J. curcas and resistant Jatropha integerrima as parents. Quantitative trait locus (QTL) analysis led to the identification of three minor QTLs for JMV resistance, and the same has been validated in an alternate F2 mapping population. These validated QTLs were utilized in marker-assisted breeding for JMV resistance. Comparative genomics of oil-producing genes across selected oil producing species revealed 27 conserved genes and 2986 orthologous protein clusters in Jatropha. This reference genome assembly gives an insight into the understanding of the complex genetic structure of Jatropha, and serves as source for the development of agronomically improved virus-resistant and oil-producing lines.

Diversification of the P genome among Agropyron Gaertn. (Poaceae) species detected by FISH.

The genomes of five Agropyron Gaertner, 1770 species were characterized using all potential di- or trinucleotide simple sequence repeat (SSR) motifs and four satellite DNA repeats as fluorescence in situ hybridization (FISH) probes. The sites of 5S and 45S rDNA were relatively conserved among the diploid and tetraploid species. A number of sites for the dinucleotide SSRs AC, AG, and pSc119.2 was detected in all investigated species except A. mongolicum Keng, 1938. Several different trinucleotide SSRs were identified in different tetraploid species. All Agropyron species were suggested to include the basic P genome, although genome differentiation was still observed. The P genome of A. mongolicum was distinct from that of the diploid A. cristatum (Linnaeus, 1753) Gaertner, 1770. and other tetraploid species, with no hybridizations for AC, AG, or pSc119.2 observed. This finding supports designation of the P genomes of A. cristatum and A. mongolicum as Pc and Pm, respectively. An exceptional 5S rDNA site revealed in one set of homoeologous chromosomes strongly supports the allopolyploid origin of A. desertorum (Fischer ex Link, 1821) Schultes, 1824. However, the diploid donors to A. desertorum need further investigation. Similarly, the unique FISH pattern of a pair of 5S rDNA-carrying chromosomes was indicative of a potential allopolyploid origin for A. fragile (Roth, 1800) Candargy, 1984. The conserved distribution of 5S and 45S rDNA suggests A. cristatum (4x) and A. michnoi Roshevitz, 1929 are closely related. Two forms of B chromosomes were identified among individuals A. mongolicum and A. desertorum plants.

Identification and validation of novel EST-SSR markers in olives

The olive (Olea europaea L.) is a leading oil crop in the Mediterranean area. Limited information on the inheritance of agronomic significant traits hinders progress in olive breeding programs, which encourages the development of markers linked to the traits. In this study, we report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project. Sequences were de novo assembled into 98,924 unigenes, which were then used as a source for microsatellites searching. We identified 923 unigenes that contained 984 SSRs among which dinucleotide SSRs (36 %) were the most abundant, followed by tri- (33 %) and hexa- (21 %) nucleotides. Microsatellite repeat motif GA (37 %) was the most common among dinucleotides, while microsatellite repeat motif GAA was the most abundant trinucleotide SSR motif (16 %). Gene ontology annotations could be assigned to 27 % of the unigenes. A hundred and ten expressed sequence tag-derived-simple sequence repeats (EST-SSRs) with annotated genes were selected for primer designing and finally, 46 (42 %) polymorphic EST-SSRs were successfully amplified and used to validate genetic diversity among 24 olive varieties. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Twenty-seven EST-SSRs showed good diversity properties and were recommended for further olive genome investigation.

De novo transcriptome of the muga silkworm, Antheraea assamensis (Helfer)

Antheraea assamensis (Lepidoptera: Saturniidae), is a semi-domesticated silkworm known to be endemic to Assam and the adjoining hilly areas of Northeast India. It is the only producer of a unique, commercially important variety of golden silk called “muga silk”. Herein, we report the de novo transcriptome of A. assamensis reared on Machilus bombycina leaves for the first time. Short reads generated by high throughput sequencing of cDNA libraries from multiple tissues, viz. alimentary canal, silk gland and residual body of the 5th instar of muga silkworm were assembled into transcripts via a de novo assembly pipeline followed by functional annotation and classification. A total of 1,21,433 transcripts were generated from ~231 million raw reads of which ~74% (89,583) were either allocated a functional annotation or categorized under Pfam/COG/KEGG categories. Identification of differentially expressed transcripts and their comparative sequence analysis revealed candidate genes related to silk synthesis, viz. silk gland factor-1 and 3, sericin-like transcript, etc. with conserved forkhead, homeo- and POU domains. Several candidate anti-microbial peptides which may have potential anti-bacterial, anti-fungal or anti-parasitic activity in A. assamensis were also identified. T/A and AT/TA were predicted to be the most abundant mono- and di-nucleotide simple sequence repeat markers in the transcriptome. Transcriptome validation was carried out by quantitative real-time PCR (qPCR) amplification of eight transcripts. The resources generated by this study will expand the periphery of existing genomic data on A. assamensis facilitating future in-depth studies on its unknown aspects.

Development, evaluation, and validation of new EST-SSR markers in olive (Olea europaea L.)

Rapid and effective genotyping is an important goal to discriminate among the numerous olive cultivars and their wild related forms. The largely used di-nucleotide simple sequence repeat (SSR) markers show a high level of polymorphism and have strongly contributed to solve many inconsistencies in varietal identity, but many problems related to difficult discrimination of neighboring alleles and low comparability of data among different labs severely reduce their applicability for large-scale screening. The availability of numerous transcriptome libraries, which were developed from different tissues of several olive varieties, has allowed their intensive screening to search for polynucleotide microsatellite regions with long core repeats, potentially polymorphic among varieties. An accurate screening of all these polymorphisms has allowed to select a set of 25 trinucleotide and one tetranucleotide SSRs, showing a good level of discrimination power with a high allele pattern resolution and repeatability. They were preliminarily tested on a group of cultivated varieties then validated on a wider group of cultivated and wild plants, and related species and subspecies, demonstrating a good transferability within the entire Olea taxon. Furthermore, an in silico functional prediction has allowed to assign each transcribed sequence to their gene functions and biological process categories, highlighting their potential application of these new EST-SSRs as functional markers.

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 ‘Sijihua’ longan (Dimocarpus longan L.) based on next-generation sequencing technology

ABSTRACT‘Sijihua’ longan (Dimocarpus longan Lour.) blossoms throughout the year, and hence it is a useful material for studies on the mechanism of flowering. However, few genomic resources such as DNA sequences or markers are currently available for ‘Sijihua’ longan. To increase our understanding of the genetic control of important flowering characteristics in ‘Sijihua’ longan, we constructed a mixed cDNA library of ‘Sijihua’ longan using Illumina paired-end sequencing technology and obtained a total of 90,069,246 high-quality reads. Among the unigenes, 43.16% (59,588) had at least one BLASTX hit against the Arabidopsis thaliana sequence database and 42.83% (59,130) had significant similarity with proteins in the NCBI non-redundant protein database. Of these annotated unigenes, a total of 15,956 (11.56%) were mapped onto 271 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. Furthermore, 11,402 unigenes were converted into 13,236 potential simple sequence repeats (SSRs). Dinucleotide SSRs were the dominant repeat motif, with a frequency of 54.3%, followed by trinucleotide (34.6%) and tetranucleotide (4.7%) repeat motifs. AG/CT repeats (46.29%) were the most frequent motifs in the data set. We focused on gene discovery in the area of regulation of flowering, and transcripts were identified for almost every gene involved in the control of flowering. This study demonstrates that Illumina sequencing technology is a rapid and informative approach to gene discovery and molecular marker development in non-model organisms without a whole genome database. Our results provide a comprehensive sequence resource for investigating the mechanisms of flowering in ‘Sijihua’ longan, and will establish a platform for further studies on longan.

Evolution Analysis of Simple Sequence Repeats in Plant Genome.

Simple sequence repeats (SSRs) are widespread units on genome sequences, and play many important roles in plants. In order to reveal the evolution of plant genomes, we investigated the evolutionary regularities of SSRs during the evolution of plant species and the plant kingdom by analysis of twelve sequenced plant genome sequences. First, in the twelve studied plant genomes, the main SSRs were those which contain repeats of 1–3 nucleotides combination. Second, in mononucleotide SSRs, the A/T percentage gradually increased along with the evolution of plants (except for P. patens). With the increase of SSRs repeat number the percentage of A/T in C. reinhardtii had no significant change, while the percentage of A/T in terrestrial plants species gradually declined. Third, in dinucleotide SSRs, the percentage of AT/TA increased along with the evolution of plant kingdom and the repeat number increased in terrestrial plants species. This trend was more obvious in dicotyledon than monocotyledon. The percentage of CG/GC showed the opposite pattern to the AT/TA. Forth, in trinucleotide SSRs, the percentages of combinations including two or three A/T were in a rising trend along with the evolution of plant kingdom; meanwhile with the increase of SSRs repeat number in plants species, different species chose different combinations as dominant SSRs. SSRs in C. reinhardtii, P. patens, Z. mays and A. thaliana showed their specific patterns related to evolutionary position or specific changes of genome sequences. The results showed that, SSRs not only had the general pattern in the evolution of plant kingdom, but also were associated with the evolution of the specific genome sequence. The study of the evolutionary regularities of SSRs provided new insights for the analysis of the plant genome evolution.