Trinucleotide Simple Sequence Repeats Research Articles

Genome-Wide Characterization and Development of Simple Sequence Repeat Markers for Molecular Diversity Analyses in Yellowhorn (Xanthoceras sorbifolium Bunge).

Yellowhorn (Xanthoceras sorbifolium Bunge) is a valuable ornamental, medicinal, and woody oilseed species that is indigenous to China. The breeding improvement of yellowhorn has been hindered by a lack of suitable markers and sufficient information regarding the molecular diversity of this species. In this study, we conducted a comprehensive analysis of the yellowhorn genome to characterize the simple sequence repeat (SSR) loci. A total of 4,007,201 SSRs were successfully identified. Among these markers, mono-nucleotide SSRs were most abundant in the genome, while the tri-nucleotide SSRs accounted for the highest proportion in coding sequences. The GO and KEGG function enrichment analysis revealed that most SSR loci in coding sequences were associated with potential biological functions. Additionally, we used 30 pairs of primers to amplify SSR markers to gain a better understanding of the genetic variation in yellowhorn germplasms. The average values of observed heterozygosity and polymorphism information content were 0.625 and 0.517, respectively. Population structure, phylogeny and principal component analyses identified two distinct subclusters. Furthermore, yellowhorn germplasms with the same geographical distribution tended to group together. Moreover, a total of 26 yellowhorn core collections, which accounted for approximately 14.94% of the total yellowhorn germplasms, effectively represented the genetic diversity of all original germplasms. Our findings not only unveiled the genetic diversity and population structure of yellowhorn germplasms but also investigated the yellowhorn core collection, which will serve as a strong basis for yellowhorn management and genetic improvement.

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.

Studying Human Pathogenic Cryptococcus Gattii Lineages by Utilizing Simple Sequence Repeats to Create Diagnostic Markers and Analyzing Diversity.

In this study, we compared the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) among the lineages of human pathogenicCryptococcus gattii using an in-silico approach to gain a deeper understanding of the structure and evolution of their genomes. C. gattii isolate MF34 showed the highest RA and RD of SSRs in both the genomic and transcriptomic sequences, followed by isolate WM276. In both the genomic (50%) and transcriptomic (65%) sequences, trinucleotide SSRs were the most common SSR class. A motif conservation study found that the isolates had stronger conservation (56.1%) of motifs, with isolate IND107 having the most (5.7%) unique motifs. We discovered the presence of SSRs in genes that are directly or indirectly associated with disease using gene enrichment analysis. Isolate-specific unique motifs identified in this study could be utilized as molecular probes for isolate identification. To improve genetic resources among C. gattii isolates, 6499 primers were developed. These genomic resources developed in this study could help with diversity analysis and the development of isolate-specific markers.

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.

Microsatellite diversity and complexity in the viral genomes of the family Caliciviridae

BackgroundMicrosatellites or simple sequence repeats (SSR) consist of 1–6 nucleotide motifs of DNA or RNA which are ubiquitously present in tandem repeated sequences across genome in viruses: prokaryotes and eukaryotes. They may be localized to both the coding and non-coding regions. SSRs play an important role in replication, gene regulation, transcription, and protein function. The Caliciviridae (CLV) family of viruses have ss-RNA, non-enveloped, icosahedral symmetry 27–35 nm in diameter in size. The size of the genome lies between 6.4 and 8.6 kb. ResultsThe incidence, composition, diversity, complexity, and host range of different microsatellites in 62 representatives of the family of Caliciviridae were systematically analyzed. The full-length genome sequences were assessed from NCBI (https://www.ncbi.nlm.nih.gov), and microsatellites were extracted through MISA software. The average genome size is about 7538 bp ranging from 6273 (CLV61) to 8798 (CLV47) bp. The average GC content of the genomes was ~ 51%. There are a total of 1317 SSRs and 53 cSSRs in the studied genomes. CLV 41 and CLV 49 contain the highest and lowest value of SSRs with 32 and 10 respectively, while CLV16 had maximum cSSR incidence of 4. There were 29 species which do not contain any cSSR. The incidence of mono-, di-, and tri-nucleotide SSRs was 219, 884, and 206, respectively. The most prevalent mono-, di-, and tri-nucleotide repeat motifs were “C” (126 SSRs), AC/CA (240 SSRs), and TGA/ACT (23 SSRs), respectively. Most of the SSRs and cSSRs are biased toward the coding region with a minimum of ~ 90% incident SSRs in the genomes’ coding region. Viruses with similar host are found close to each other on the phylogenetic tree suggesting virus host being one of the driving forces for their evolution. ConclusionsThe Caliciviridae genomes does not conform to any pattern of SSR signature in terms of incidence, composition, and localization. This unique property of SSR plays an important role in viral evolution. Clustering of similar host in the phylogenetic tree is the evidence of the uniqueness of SSR signature.

Draft genome sequencing of Tilletia caries inciting common bunt of wheat provides pathogenicity-related genes.

Common bunt of wheat caused by Tilletia caries is an important disease worldwide. The T. caries TC1_MSG genome was sequenced using the Illumina HiSeq 2500 and Nanopore ONT platforms. The Nanopore library was prepared using the ligation sequencing kit SQK-LSK110 to generate approximately 24 GB for sequencing. The assembly size of 38.18 Mb was generated with a GC content of 56.10%. The whole genome shotgun project was deposited at DDBJ/ENA/GenBank under the accession number JALUTQ000000000. Forty-six contigs were obtained with N50 of 1,798,756 bp. In total, 10,698 genes were predicted in the assembled genome. Out of 10,698 genes, 10,255 genes were predicted significantly in the genome. The repeat sequences made up approximately 1.57% of the genome. Molecular function, cellular components, and biological processes for predicted genes were mapped into the genome. In addition, repeat elements in the genome were assessed. In all, 0.89% of retroelements were observed, followed by long terminal repeat elements (0.86%) in the genome. In simple sequence repeat (SSR) analysis, 8,582 SSRs were found in the genome assembly. The trinucleotide SSR type (3,703) was the most abundant. Few putative secretory signal peptides and pathogenicity-related genes were predicted. The genomic information of T. caries will be valuable in understanding the pathogenesis mechanism as well as developing new methods for the management of the common bunt disease of wheat.

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.

Microsatellite Variation in the Most Devastating Beetle Pests (Coleoptera: Curculionidae) of Agricultural and Forest Crops.

Weevils, classified in the family Curculionidae (true weevils), constitute a group of phytophagous insects of which many species are considered significant pests of crops. Within this family, the red palm weevil (RPW), Rhynchophorus ferrugineus, has an integral role in destroying crops and has invaded all countries of the Middle East and many in North Africa, Southern Europe, Southeast Asia, Oceania, and the Caribbean Islands. Simple sequence repeats (SSRs), also termed microsatellites, have become the DNA marker technology most applied to study population structure, evolution, and genetic diversity. Although these markers have been widely examined in many mammalian and plant species, and draft genome assemblies are available for many species of true weevils, very little is yet known about SSRs in weevil genomes. Here we carried out a comparative analysis examining and comparing the relative abundance, relative density, and GC content of SSRs in previously sequenced draft genomes of nine true weevils, with an emphasis on R. ferrugineus. We also used Illumina paired-end sequencing to generate draft sequence for adult female RPW and characterized it in terms of perfect SSRs with 1–6 bp nucleotide motifs. Among weevil genomes, mono- to trinucleotide SSRs were the most frequent, and mono-, di-, and hexanucleotide SSRs exhibited the highest GC content. In these draft genomes, SSR number and genome size were significantly correlated. This work will aid our understanding of the genome architecture and evolution of Curculionidae weevils and facilitate exploring SSR molecular marker development in these species.

Microsatellite Signature of Reference Genome Sequence of SARS-CoV-2 and 32 Species of Coronaviridae Family

Background: Simple sequence repeats (SSRs) are 1 - 6 bp repeat motif sequences present across both prokaryotic and eukaryotic genomes with various clinical implications besides being tools for conservation and evolutionary studies. Objectives: Analysis of 33 Coronavirus genomes, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for incidence, distribution, and complexity of SSRs patterns to understand their role in host divergence and evolution. Methods: Full-length genome sequences were extracted from National Center for Biotechnology Information (NCBI). Extraction of microsatellites was done using imperfect microsatellite extractor (IMEx) in “Advanced Mode”. Sequences were aligned with MAFFT v6.861b and the maximum likelihood tree was inferred using RAxML v8.1.20 of the GTR + GAMMA+I model with default specifications. Results: A total of 3,442 SSRs and 136 complex sequence repeats (cSSRs) were extracted from the studied 33 genomes. SSR incidence ranged from 82 (CV09) to 144 (CV60). cSSR incidence ranged from 1 (CV42, CV43, CV53) to 11 (CV32). CV61 (SARS-CoV-2) had 107 SSRs and 6 SSRs. Di-nucleotide motifs were the most prevalent followed by tri- and mono-nucleotide motifs. TG/GT was the most represented di-nucleotide motif, followed by CA/AC. In tri-nucleotide SSRs, ACA/TGT was the most represented motif followed by CAA/GTT, whereas in mono-nucleotide SSRs, T was the most observed nucleotide, followed by A. About 94% of SSRs were localized to the coding region. Twenty species, including CV61 (SARS-CoV-2), exhibit mono-nucleotide repeats exclusively in the A/T region, which were clustered in phylogenetic analysis. The sequence similarity of the genomes was assessed through heat map analysis and revealed similar sequences are expectedly placed in proximity on the phylogenetic tree. Conclusions: Mono-nucleotide exclusivity to A/T region and SSR genome signature can be a possible basis for predicting the evolution of viruses in terms of host range.

Comprehensive identification and characterization of simple sequence repeats based on the whole-genome sequences of 14 forest and fruit trees.

Simple sequence repeats (SSRs) are popular and important molecular markers that exist widely in plants. Here, we conducted a comprehensive identification and comparative analysis of SSRs in 14 tree species. A total of 16, 298 SSRs were identified from 429, 449 genes, and primers were successfully designed for 99.44% of the identified SSRs. Our analysis indicated that tri-nucleotide SSRs were the most abundant, with an average of ~834 per species. Functional enrichment analysis by combining SSR-containing genes in all species, revealed 50 significantly enriched terms, with most belonging to transcription factor families associated with plant development and abiotic stresses such as Myeloblastosis_DNA-bind_4 (Myb_DNA-bind_4), APETALA2 (AP2), and Fantastic Four meristem regulator (FAF). Further functional enrichment analysis showed that 48 terms related to abiotic stress regulation and floral development were significantly enriched in ten species, whereas no significantly enriched terms were found in four species. Interestingly, the largest number of enriched terms was detected in Citrus sinensis (L.) Osbeck, accounting for 54.17% of all significantly enriched functional terms. Finally, we analyzed AP2 and trihelix gene families (Myb_DNA-bind_4) due to their significant enrichment in SSR-containing genes. The results indicated that whole-genome duplication (WGD) and whole genome triplication (WGT) might have played major roles in the expansion of the AP2 gene family but only slightly affected the expansion of the trihelix gene family during evolution. In conclusion, the identification and comprehensive characterization of SSR markers will greatly facilitate future comparative genomics and functional genomics studies.

Full-length transcriptome sequencing analysis and development of EST-SSR markers for the endangered species Populus wulianensis

Populus wulianensis is an endangered species endemic to Shandong Province, China. Despite the economic and ornamental value of this species, few genomics and genetic studies have been performed. In this study, we performed a relevant analysis of the full-length transcriptome sequencing data of P. wulianensis and obtained expressed sequence tag (EST)-simple sequence repeat (SSR) markers with polymorphisms that can be used for further genetic research. In total, 8.18 Gb (3,521,665) clean reads with an average GC content of 42.12% were obtained. From the corrected 64,737 high-quality isoforms, 42,323 transcript sequences were obtained after redundancy analysis with CD-HIT. Among these transcript sequences, 41,876 sequences were annotated successfully. A total of 23,539 potential EST-SSRs were identified from 16,057 sequences. Excluding mononucleotides, the most abundant motifs were trinucleotide SSRs (47.80%), followed by di- (46.80%), tetra- (2.98%), hexa- (1.58%) and pentanucleotide SSRs (0.84%). Among the 100 designed EST-SSRs, 18 were polymorphic with high PIC values (0.721 and 0.683) and could be used for analyses of the genetic diversity and population structure of P. wulianensis. These full-length transcriptome sequencing data will facilitate gene discovery and functional genomics research in P. wulianensis, and the novel EST-SSRs developed in our study will promote molecular-assisted breeding, genetic diversity and conservation biology research in this species.

Mining and Validation of Polymorphic EST-SSR Markers for Analysing Genetic Diversity among Interspecific Hybrids of Tea

Molecular markers are useful tools for studying gene flow and genetic diversity within a population. With the establishment of online databases, sequence can be now be downloaded for identification of Expressed Sequence Tags- Simple Sequence Repeat (SSR) markers designed to amplify the microsatellite loci present in the genes. Of the 170 microsatellites detected from the downloaded 789 ESTs belonging to the nine Camellia spp, only fourteen showed functional EST-SSR markers. Among them, five polymorphic markers specific to tri-nucleotide SSRs were randomly selected and screened alongside five established primers on four tea genotypes (TRFK 570/2, TRFK 688/1, TRFK 83/1, and TRFK 6/8,) The polymorphism information content (PIC) of the SSR markers had mean of 0.33 that ranged between 0.16 and 0.53, whereas discriminating power (D) had mean value of 0.23 ranging between 0.00 and 0.75 with. A total of 64 alleles were detected with an average of 6.4 alleles per SSR locus. Based on a PIC greater than or equal to 0.40, D value greater than or equal to 0.20 and number of polymorphic bands greater than or equal to 1, five functional SSR markers comprising two novel EST-SSRs (Camjap A1, Camjap A4) and three adapted microsatellite markers (TM 134, A37, and A47) were recommended for use in discriminating interspecific hybrids of tea.

Development of novel transcriptome-based SSR markers in Puccinia triticina and their potential application in genetic diversity studies

In wheat, leaf rust caused by Puccinia triticina Eriks is a major disease that causes major losses in production. In the present study, microsatellite or SSR (simple sequence repeat) markers were developed for P. triticina using the transcriptome data obtained during wheat-P. triticina interactions. A total of 37,268 P. triticina differentially expressed transcripts were obtained in the comparative transcriptomics and of these, 6809 (1.8%) transcripts carried SSRs. Tri-nucleotide SSRs were the most abundant (52%) among the identified SSRs. Primer pairs were synthesized for 50 PtESSRs, of which 28 were found to be polymorphic in P. triticina pathotypes of the Indian subcontinent. The number of alleles per locus ranged from 2 to 12 with an average of 5.72. Polymorphic information content (PIC) ranged from 0.29 to 0.96 with an average of 0.69. The observed heterozygosity ranged from 0.10 to 0.48 with an average of 0.21. Interestingly, one SSR marker, namely PtESSR7, could distinguish between different virulence groups of pathotypes. BLAST analysis and in silico prediction of fungal effectors suggested that some of the PtESSR markers may have the potential to be functional markers in the leaf rust pathology. Dendrogram analyses detected two clusters in the 48 P. triticina pathotypes. Results of this study suggest that the newly developed PtESSR markers are useful for genetic studies in P. triticina pathogen. No relationship was found between the genetic diversity and geographic distributions of the P. triticina populations.

Comprehensive functional analysis and mapping of SSR markers in the chickpea genome (Cicer arietinum L.)

Plant molecular breeding largely depends on the relationship between molecular markers and major traits. Herein, a total of 32,962 genomic simple sequence repeats (SSRs) were detected in the whole genome of chickpea with an average density of 94.93 SSRs/Mb. Chickpea chromosomes uniformity test indicated that the genomic SSRs (gSSRs) were steadily distributed across the genome. Moreover, 48,667 transcriptome sequences were analyzed and 1949 SSR-containing transcript assembly contigs (TACs) were identified. The analysis showed that di- and trinucleotide SSRs were the most frequent SSR motifs within the transcriptome sequences. Among them, AT and TTA and AG and TTC motifs within the transcriptome showed the highest frequencies among di- and trinucleotide repeat motifs, respectively. The SSRs-containing TACs were compared to the GenBank non-redundant database using BLASTX, and subsequently, gene ontology (GO) analysis was performed using QuickGO browser to reduce complexity and highlight biological processes associated with the SSRs-containing TACs. The identified SSRs-containing TACs were categorized into 35 enriched functional-related gene group. The mapping of characterized SSRs-containing TACs onto chickpea chromosomes was performed using BLASTN. The mapping result showed that, a total of 1798 SSRs-containing TACs were mapped onto the chickpea genome. Based on the functional analysis result, 249 and 242 of the mapped SSRs-containing TACs were found in the genes encoding for putative stress-related proteins and transcription factors, respectively. The results presented here can be applied to improve and speed up the chickpea breeding programs.

Genome-wide identification of simple sequence repeat (SSR) markers in Capsicum chinense Jacq. with high potential for use in pepper introgression breeding

Capsicum chinense, is closely related to Capsicum annuum L., and the two species share a common gene pool. There are a limited number of cultivated C. chinense varieties; yet, it is acknowledged that C. chinense germplasm harbors useful alleles for important traits such as fruit yield, disease resistance, accumulation of secondary metabolites and technological fruit characters. Besides, it is feasible to obtain fertile progeny from interspecific crosses of C. chinense and C. annuum. Consequently, C. chinense has high breeding potential for improvement of several different traits in pepper breeding. In the present work, C. chinense genome assembly was mined for trinucleotide simple sequence repeats (SSRs) and repeat loci were converted to 53,749 PCR markers. Marker transferability to C. annuum genome was analyzed with high-stringency cross-species mapping parameters, identifying 17,992 transferable markers. Selecting for interspecific polymorphisms and eliminating redundant loci resulted in 4994 SSR markers distributed over the 12 pepper chromosomes with high potential for interspecific polymorphisms between the two pepper species. Laboratory experiments with a subset of candidate polymorphic markers (36 markers) resulted in an amplification rate of 100% from both pepper species and 31 markers produced polymorphic alleles. Thus, in silico marker development and cross-species mapping analyses proved highly efficient in accuracy of primer design and detection of interspecific sequence divergence. The large set of genome-anchored SSR markers introduced in the present work has high potential for polymorphisms. Therefore, the marker set constitutes a useful genomic resource for broadening the genetic base of cultivated pepper with favorable C. chinense alleles.

Comprehensive Characterization of Simple Sequence Repeats in Eggplant (Solanum melongena L.) Genome and Construction of a Web Resource.

We have characterized the simple sequence repeat (SSR) markers of the eggplant (Solanum melongena) using a recent high quality sequence of its whole genome. We found nearly 133,000 perfect SSRs, a density of 125.5 SSRs/Mbp, and also about 178,400 imperfect SSRs. Of the perfect SSRs, 15.6% were complex, with two stretches of repeats separated by an intervening block of <100 nt. Di- and trinucleotide SSRs accounted, respectively, for 43 and 37% of the total. The SSRs were classified according to their number of repeats and overall length, and were assigned to their linkage group. We found 2,449 of the perfect SSRs in 2,086 genes, with an overall density of 18.5 SSRs/Mbp across the gene space; 3,524 imperfect SSRs were present in 2,924 genes at a density of 26.7 SSRs/Mbp. Putative functions were assigned via ontology to genes containing at least one SSR. Using this data we developed an “Eggplant Microsatellite DataBase” (EgMiDB) which permits identification of SSR markers in terms of their location on the genome, type of repeat (perfect vs. imperfect), motif type, sequence, repeat number and genomic/gene context. It also suggests forward and reverse primers. We employed an in silico PCR analysis to validate these SSR markers, using as templates two CDS sets and three assembled transcriptomes obtained from diverse eggplant accessions.

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.

In silico development and characterization of tri-nucleotide simple sequence repeat markers in hazelnut (Corylus avellana L.).

Plant genomes are now sequenced rapidly and inexpensively. In silico approaches allow efficient development of simple sequence repeat markers, also known as microsatellite markers, from these sequences. A search of the genome sequence of 'Jefferson' hazelnut (Corylus avellana L.) identified 8,708 tri-nucleotide simple sequence repeats with at least five repeat units, and stepwise removal of the less promising sequences led to the development of 150 polymorphic markers. Fragments in the 'Jefferson' sequence containing tri-nucleotide repeats were used as references and aligned with genomic sequences from seven other cultivars. Following in silico alignment, sequences that showed variation in number of repeat units were selected and primer pairs were designed for 243 of them. Screening on agarose gels identified 173 as polymorphic. Removal of duplicate and previously published sequences reduced the number to 150, for which fluorescent primers and capillary electrophoresis were used for amplicon sizing. These were characterized using 50 diverse hazelnut accessions. Of the 150, 132 generated the expected one or two alleles per accession while 18 amplified more than two amplicons in at least one accession. Diversity parameters of the 132 marker loci averaged 4.73 for number of alleles, 0.51 for expected heterozygosity (He), 0.49 for observed heterozygosity (Ho), 0.46 for polymorphism information content (PIC), and 0.04 for frequency of null alleles. The clustering of the 50 accessions in a dendrogram constructed from the 150 markers confirmed the wide genetic diversity and presence of three of the four major geographic groups: Central European, Black Sea, and Spanish-Italian. In the mapping population, 105 loci segregated, of which 101 were assigned to a linkage group (LG), with positions well-dispersed across all 11 LGs. These new markers will be useful for cultivar fingerprinting, diversity studies, genome comparisons, mapping, and alignment of the linkage map with the genome sequence and physical map.

Characterization and Development of EST-SSR Markers from a Cold-Stressed Transcriptome of Centipedegrass by Illumina Paired-End Sequencing

Centipedegrass (Eremochloa ophiuroides) is an important warm-season grass native to the temperate and tropical regions of China. However, only a few centipedegrass molecular markers were available, which hindered genetic studies. Simple sequence repeats (SSRs) can provide abundant genetic information but the traditional method of their development was time-consuming and labor-intensive. Now, RNA-seq platform provides a relatively fast and financially affordable approach for large-scale development of SSRs. Therefore, the transcriptome of centipedegrass under cold stress was determined using Illumina paired-end sequencing in this study. Based on the 56 million paired-end reads, 45,575 unigenes with an average of 748 nt in length were assembled. A total of 8539 SSR loci were then identified. Among them, the tri-nucleotide SSRs were the most dominant repeat type (60.80%, 5192), followed by mono- (16.56%, 1414), di- (14.28%, 1219), hexa- (3.36%, 287), penta- (2.87%, 245), and tetra-nucleotide (2.13%, 182) repeats. The most frequent repeat units from mono- to hexa-nucleotide repeats were A/T, AG/CT, CCG/CGG, AAAG/CTTT, AGGGG/CCCCT, and AGGGCG/CCCTCG, respectively. We successfully designed 3511 primer pairs, and then, randomly selected 99 pairs to evaluate their interspecific transferability and preliminarily analyze the genetic diversity of centipedegrass accessions. There were 90 primer pairs that could successfully amplify targets in at least one centipedegrass samples, and 81 of them could also function in at least three of six other Poaceae relatives and 79 showed polymorphism. Based on the 79 polymorphic markers, all centipedegrass accessions could be divided into two groups, i.e., group 1 from Guangxi province and group 2 from other regions. The EST-SSRs from this study provided a useful tool and foundation for genetic diversity, linkage mapping, comparative mapping, and molecular marker-aided breeding in centipedegrass.