Unique Simple Sequence Repeat Research Articles

A survey in natural olive resources exposed to high inoculum pressure indicates the presence of traits of resistance to Xylella fastidiosa in Leccino offspring.

The epidemic spread of the harmful bacterium Xylella fastidiosa causing the "olive quick decline syndrome", decimating olive trees in southern Italy, in the region of Apulia, prompted investigations to search for olive genotypes harbouring traits of resistance. A prospecting survey was carried out to identify, in the heavily infected area of Apulia, olive genotypes bearing resistance. Given the limited genetic diversity in the commercial olive groves with few cultivars widely cultivated, surveys targeted predominantly spontaneous olive genotypes in natural and uncultivated areas. Trees, selected for the absence of symptoms, were subjected to diagnostic tests and parentage analysis to disclose their genetic background. Transcriptomic analyses were also employed to decipher the molecular pathways in resistant genotypes. Artificial inoculations were carried out to confirm the resistant phenotypes of four open-pollinated seedlings of the cultivar Leccino. Among the 171 olive collected genotypes, 139 had unique simple sequence repeat (SSR) profiles, with the cultivars Leccino, Cellina di Nardò, and Ogliarola salentina being the most frequent candidate parents. Among the Leccino progeny (n. 61), 67% showed a highly resistant (HR), resistant (R), or tolerant (T) phenotype to infection by X. fastidiosa. The occurrence of such phenotypes among those deriving from Cellina di Nardò and Ogliarola salentina was 32% and 49%, respectively. Analyses of the transcriptomic profiles of three Leccino-bearing genotypes, naturally infected and not showing symptoms, unravelled that a total of 17,227, 13,031, and 4,513 genes were found altered in the expression, including genes involved in photosynthesis, cell wall, or primary and secondary metabolism. Indeed, transcriptomic analyses showed that one of these genotypes (S105) was more resilient to changes induced by the natural bacterial infection than the remaining two (S215 and S234). This study consolidates the evidence on the presence and heritage of resistance traits associated with the cv. Leccino. Moreover, valuable insights were gathered when analysing their transcriptomic profiles, i.e., genes involved in mechanisms of response to the bacterium, which can be used in functional genetic approaches to introduce resistance in susceptible cultivars and initiate strategies in olive-breeding programs through marker-assisted selection.

Morphological and Molecular Characterisation of Nineteen (19) Tomato Cultivars from Northern Nigeria

Tomato is an autogamous vegetable crop whose genetic variability has been reduced by selection and breeding processes. Characterisation of tomato germplasm, therefore, aids the documentation of the genetic variability existing in a population. To morphologically characterise tomato varieties from Northern Nigeria, and molecularly characterise the varieties using (Simple Sequence Repeat (SSR) markers, nineteen (19) tomato varieties were collected from major markets in seven states in Northern Nigeria using a purposive sampling method. Tomato seeds were extracted, prepared, sowed in screen houses and morphologically characterised using cross-sectional shapes, core, pericarp, matured fruit colour, overall fruit shape, size and firmness. Leaves of tomato seedlings four weeks after sowing were pooled and used for genomic DNA extraction. The polymerase chain reaction (PCR) was performed and PCR products were visualized through electrophoresis. SSR fragment analysis was scored as the presence or absence of a specific fragment size from the Electropherograms.
 Results showed that 44% of the colour of tomato core was white, 17% were red and pink, 5% were orange and yellow and 11% were variants of other colours, showing wide variations in the qualitative traits. Similarities among the nineteen tomato samples ranged from 14 (Dan India with Derica sweet, Kaduna Tangino, Nassarawa Roma 1 and 2, Niger Roma2, UTC01 and UTC 02) to 86% (between Mai mara and Derica small). SSR gave three unique SSR bands that identified and characterised the cultivars. 
 The result of this study revealed genetic diversity within a group of tomato genotypes of different origins cultivated in Northern Nigeria using molecular (SSR) analysis. This confirmed the efficacy of the SSR markers as highly variable markers that detect the codominant single locus and are suitable to distinguish between the genetically related cultivars.

Assessment of simple sequence repeats signature in hepatitis E virus (HEV) genomes

BackgroundHepatitis E virus (HEV) is small (27–34 nm diameter) non-enveloped with positive sense ssRNA genome. Microsatellites or simple sequence repeats (SSR) are short tandem repeat sequences present across coding and non-coding regions of both prokaryotes and eukaryotes. They are involved with genome function and evolution at multiple levels. ResultsThe complete genome sequences of 22 HEV genomes of the family Hepeviridae and genus Orthohepevirus (21 species) and Piscihepevirus (1 species) were extracted from NCBI database (http://www.ncbi.nlm.nih.gov/). The extraction of microsatellites was done using Imperfect Microsatellite Extractor (IMEx) in ‘Advance-Mode’. The average genome size of the studied HEV genomes was 7003nt and it ranged from 6649nt (HEV11) to 7310nt (HEV22). The average GC content of the genomes was ~ 55%. A total of 519 SSRs and 21 cSSRS were extracted from the HEV genomes with an average incidence of 24 per genome ranging from 14 (HEV13) to 34 (HEV19). The cSSR incidence ranged from 0 (eight species) to 4 (HEV19). The genomes with no cSSR incidence had an SSR incidence range from 14 to 28. There were just four hexa-nucleotide repeat motifs and 5 penta-nucleotide repeat motifs observed. The most prevalent mono-, di-, and tri-nucleotide repeat motifs were “C”, “GT/TG”, and “GAC/CTG” respectively. The studied genomes had a minimum of ~ 90% incident SSRs present in the coding regions. Viruses with same or similar hosts are placed together on the phylogenetic tree implicating viral host being one of the driving forces for evolution. ConclusionsHost range in viruses is being decided by multiple factors aided by the unique genome SSR signature and genomes of varied compositions need to be analyzed to forge a widely acceptable rule for predicting the same.

Comparison of the Chloroplast Genome Sequences of 13 Oil-Tea Camellia Samples and Identification of an Undetermined Oil-Tea Camellia Species From Hainan Province.

The comparison of chloroplast genome (cpDNA) sequences among different plant species is an important source of plant molecular phylogenetic data. In this paper, the cpDNA sequences of 13 different oil-tea camellia samples were compared to identify an undetermined oil-tea camellia species from Hainan Province. The cpDNA of the samples was sequenced and resequenced, and divergence hotspots and simple sequence repeat (SSR) variations were analyzed. Bayesian inference (BI) and maximum-likelihood (ML) phylogenetic trees were constructed based on the full cpDNA sequences. The cpDNA sequences were 156512∼157089 bp in length and had the circular tetrad structure typical of angiosperms. The inverted repeats (IRs) of different species included varying contractions and expansions. The cpDNA sequences of the samples of the undetermined species of oil-tea camellia from Hainan Province and Camellia gauchowensis from Xuwen County were identical. In total, 136 genes were annotated, including 91 protein-coding genes (PCGs), 37 tRNA genes and 8 rRNA genes. The GC content of the cpDNA was 37.3%. The small single-copy (SSC)/IR boundary was rich in variation. Divergence hotspots were mainly located in the intergenic space (IGS) and coding sequences (CDSs), and there were obvious differences in divergence hotspots among species. The same divergence hotspots were found in Camellia vietnamensis, Camellia gauchowensis and the undetermined species of oil-tea camellia from Hainan Province. A total of 191∼198 SSR loci were detected. Most of the SSRs included A or T, and the distribution of SSRs in the cpDNA was uneven. Different species shared common SSRs and exhibited unique SSRs. Based on the full cpDNA sequences, the evolutionary relationships of different species of Camellia were well identified. The thirteen samples were classified into 2 clades and 6 subclades, and the different sections of Camellia clustered on the same branch in 2 clades and 2 subclades. Camellia vietnamensis was more closely related to the undetermined species of oil-tea camellia from Hainan Province and the sample of Camellia gauchowensis from Xuwen County than to the sample of Camellia gauchowensis from Luchuan County. Camellia osmantha was closely related to Camellia gauchowensis and Camellia vietnamensis. In conclusion, the cpDNA of different oil-tea camellia species has a conserved tetrad structure with certain length polymorphisms. SSRs are expected to be developed as “barcodes” or “identity cards” for species identification. SSR variations and other factors result in abundant divergence hotspots in the CDSs and IGS (one non-CDS region), indicating that full cpDNA sequences can be used for the species identification and phylogenetic analysis of Camellia. Accordingly, the undetermined species of oil-tea camellia from Hainan Province is likely Camellia vietnamensis, Camellia vietnamensis and Camellia gauchowensis may be the same species, and additional genetic evidence is needed to determine whether Camellia osmantha is a new independent species. The previous division of related sections of Camellia may need readjustment based on full cpDNA sequences.

Simple Sequence Repeat and S-Locus Genotyping to Assist the Genetic Characterization and Breeding of Polyploid Prunus Species, P. spinosa and P. domestica subsp. insititia

Polyploid Prunus spinosa (2n = 4 ×) and P. domestica subsp. insititia (2n = 6 ×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programs. In Hungary, 16 cultivar candidates and a recognized cultivar ‘Zempléni’ were selected from wild-growing populations including ten P. spinosa, four P. domestica subsp. insititia and three P. spinosa × P. domestica hybrids (2n = 5 ×) were also created. Genotyping in eleven simple sequence repeat (SSR) loci and the multiallelic S-locus was used to characterize genetic variability and achieve a reliable identification of tested accessions. Nine SSR loci proved to be polymorphic and eight of those were highly informative (PIC values ˃ 0.7). A total of 129 SSR alleles were identified, which means 14.3 average allele number per locus and all accessions but two clones could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified and the complete and partial S-genotype was determined for 10 and 7 accessions, respectively. The DNA sequence was determined for a total of 17 fragments representing 11 S-RNase alleles. ‘Zempléni’ was confirmed to be self-compatible carrying at least one non-functional S-RNase allele (SJ). Our results indicate that the S-allele pools of wild-growing P. spinosa and P. domestica subsp. insititia are overlapping in Hungary. Phylogenetic and principal component analyses confirmed the high level of diversity and genetic differentiation present within the analysed accessions and indicated putative ancestor–descendant relationships. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species but non-related accessions sharing common S-alleles may distort phylogenetic inferences.

Phylogenic position and marker studies using cpDNA of C. wightii: A critically endangered and medicinally important plant in India

Complete chloroplast genome of Commiphora wightii (family Burseraceae), a medicinally important-critically endangered plant, was assembled using next generation sequencing. Genome annotation was conducted using CpGAVAS web server. The genome is 156,064 bp in length, presenting a typical quadripartite structure of large (LSC; 93,841 bp) and small (SSC; 19,897 bp) single-copy regions separated by a pair of inverted repeats (IRs; 21,163 bp). The genome encodes 125 genes including 86 protein genes, 29 tRNAs, and eight rRNAs. Total 16 simple sequence repeats (SSR), detected in the plastome include the di, tri, tetra, and pentanucleotide repeat. When compared with the plastome of other members of Burseraceae it showed one unique SSR. Total twenty-two hypervariable regions of loci were found in the genome, which could serve as DNA barcodes for species identification. The newly sequenced complete cp genome identified SSRs, nucleotide diversity, phylogenetic analysis, and IR contraction will help in understanding the plastome evolution and genetic conservation of this critically endangered medicinal plant in the future.

SSR-based population structure, molecular diversity and identity cards of Ziziphus species from Pakistan and China

Research work on the genetic diversity within Pakistani and Chinese Ziziphus species is limited, and to date, no single report on the application of DNA barcoding attempt for identification of Ziziphus species is available in literature. Therefore, the current study was designed to assess diversity and relationships among Ziziphus species genotypes and to develop a unique identity card and for their conservation. The study has used 99 simple sequence repeats (SSR) markers for gaining insights into the genetic diversity within 200 genotypes of four Ziziphus species. Successful amplification was achieved with 59 SSRs; and was applied for understanding the population structure and relationships among the Ziziphus species/genotypes. Unique SSR fingerprints of three highly polymorphic loci were used for developing molecular identity cards for each genotype. A total of 158, 165, 168, 157 and 168 alleles were amplified from Z. jujuba Mill., Z. nummularia (Burm.f.) Wight & Arn., Z. oxyphylla Edgew. (Pakistani origin), Z. mauritiana Lam. and Z. jujuba Mill., (Chinese origin) respectively. Alleles per locus ranged from 2 to 6, averaging 2.83, 2.82, 3.366, 2.80 and 3.09. Analysis of molecular variance (AMOVA) revealed high genetic variability (83%) whereas; fixation index (0.014) and gene flow estimates (1.32) indicated frequent gene flow within Ziziphus species. STRUCTURE and PCA analysis resolved all genotypes into five populations. Further, molecular identity cards were developed for all genotypes. The overall results indicated prevalence of genetic variability and relationships among Ziziphus species; yet the same Z. jujuba species across geographical boundaries has retained unique alleles and this may facilitate development of agronomically desirable and genetically improved Ziziphus cultivars.

Implications of genome simple sequence repeats signature in 98 Polyomaviridae species.

The analysis of simple sequence repeats (SSRs) in 98 genomes across four genera of the family Polyomaviridae was performed. The genome size ranged from 3962 (BM87) to 7369 bp (BM85) but maximum genomes were in the range of 5–5.5 kb. The GC% had an average of 42% and ranged between 34.69 (BM95) and 52.35 (BM81). A total of 3036 SSRs and 223 cSSRs were extracted using IMEx with incident frequency from 18 to 56 and 0 to 7, respectively. The most prevalent mono-nucleotide repeat motif was “T” (48.95%) followed by “A” (33.48%). “AT/TA” was the most prevalent dinucleotide motif closely followed by “CT/TC”. The distribution was expectedly more in the coding region with 77.6% SSRs of which nearly half were in Large T Antigen (LTA) gene. Notably, most viruses with humans, apes and related species as host exhibited exclusivity of mono-nucleotide repeats in AT region, a proposed predictive marker for determination of humans as host in the virus in course of its evolution. Each genome has a unique SSR signature which is pivotal for viral evolution particularly in terms of host divergence.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13205-020-02583-w.

An efficient strategy for developing genotype identification markers based on simple sequence repeats in grapevine

Microsatellites are valuable resources for breeding and genetic studies of crop species. The development of simple sequence repeat (SSR) markers, which are applied across closely related cultivars, has been slow, labor-intensive, and expensive, with a low success rate. In this study, we demonstrate an efficient strategy for developing cross-species, amplifiable polymorphic SSR markers from grapevine (Vitis vinifera) based on sequence comparison and in silico polymerase chain reaction amplification of the genome sequences of grape cultivars. A total of 21 primer sets targeting unique SSR loci were screened from polymorphism and heterozygosity analyses of 13 grape cultivars and four wild species. The number of alleles varied from 3 to 12, with a mean of 7.52 alleles per locus. The lowest and highest observed heterozygosity values were 0.24 and 0.82, respectively, with an average of 0.54. Polymorphic information content values for the primer sets ranged from 0.32 to 0.86, with an average of 0.70. We successfully identified genetic relationships among 17 grape genotypes using the unweighted pair group method with arithmetic average and principal coordinates analysis of the SSR markers. The results suggest that the strategy developed in this study is applicable to high-throughput cross-amplifiable SSR marker development for the identification of genotype, genetic study, and examination of genetic diversity in grapevines.

Evaluation of the diversity of garden potato varieties grown in Canada using simple sequence repeat and morphology

Garden potato varieties grown on more than 1.5 ha are subject to variety registration to be sold as seed potato in Canada. Prerequisites for the registration of varieties include the pedigree and breeding history as well as a morphological description. For most varieties, no pedigree or morphological description is available, and furthermore, some groups are essentially derived from one another. Therefore, a review to determine the genetic and morphological similarities and uniqueness of some of the garden potato varieties was initiated. Seven garden potato varieties grown over the largest areas in Canada were genotyped using a set of nine microsatellite [simple sequence repeat (SSR)] markers, resulting in six distinct SSR profiles. These varieties were also grown out for morphological description, providing supplementary information for the national registration and their monitoring during the seed certification process. To further assess the genetic similarities of garden or heritage varieties, 84 potato varieties were genotyped, and the unique SSR profiles of 62 varieties and six SSR profiles shared among the other 22 varieties were added to the Canadian Food Inspection Agency potato variety reference SSR profile collection. Furthermore, a set of blue varieties were described morphologically to evaluate the nature of morphological characteristic variation of varieties sharing the same SSR profile. Although all 14 varieties grown show some morphological differences, all varieties within each group sharing an SSR profile also clustered together based on morphological characteristics.

Genome-Wide Analysis of Simple Sequence Repeats in Bitter Gourd (Momordica charantia).

Bitter gourd (Momordica charantia) is widely cultivated as a vegetable and medicinal herb in many Asian and African countries. After the sequencing of the cucumber (Cucumis sativus), watermelon (Citrullus lanatus), and melon (Cucumis melo) genomes, bitter gourd became the fourth cucurbit species whose whole genome was sequenced. However, a comprehensive analysis of simple sequence repeats (SSRs) in bitter gourd, including a comparison with the three aforementioned cucurbit species has not yet been published. Here, we identified a total of 188,091 and 167,160 SSR motifs in the genomes of the bitter gourd lines ‘Dali-11’ and ‘OHB3-1,’ respectively. Subsequently, the SSR content, motif lengths, and classified motif types were characterized for the bitter gourd genomes and compared among all the cucurbit genomes. Lastly, a large set of 138,727 unique in silico SSR primer pairs were designed for bitter gourd. Among these, 71 primers were selected, all of which successfully amplified SSRs from the two bitter gourd lines ‘Dali-11’ and ‘K44’. To further examine the utilization of unique SSR primers, 21 SSR markers were used to genotype a collection of 211 bitter gourd lines from all over the world. A model-based clustering method and phylogenetic analysis indicated a clear separation among the geographic groups. The genomic SSR markers developed in this study have considerable potential value in advancing bitter gourd research.

Molecular characterization of pearl millet [Pennisetum glaucum (L.) R. Br] inbreds using microsatellite markers

Studies on genetic diversity in Pennisetum germplasm are the promising opportunities for the use of un-domesticated materials for improving pearl millet varieties. DNA based markers have now emerged as a potential genomic tool for estimation of genetic diversity among various cultivars and varietal identification. In present study, genetic diversity among 49 stay green inbreds of pearl millet was studied using simple sequence repeats (SSRs). Twenty nine polymorphic SSR primers, identified after initial screening of 70, were used to study diversity among these lines. A total of 108 alleles were amplified, collectively yielding unique SSR profiles for all the 49 inbreds. The average number of SSR alleles per locus was 3.72, with a range from 2 to 13. Polymorphic information content (PIC) values of various SSR loci across all the 49 inbreds ranged from 0.14 to 0.87 with an average of 0.51 per lo-cus. This indicated sufficient diversity among the 49 pearl millet inbreds and total 5 out of 29 polymorphic SSR loci, namely Xpsmp2070, Xpsmp2001, Xpsmp2008, Xpsmp2066, Xpsmp2072 revealed PIC values above 0.70, can be considered highly useful for differentiation of pearl millet inbred lines. The lowest PIC value (0.47) for linkage group 7 showed comparatively conserved nature of this linkage group A dendrogram obtained using WARD’s minimum variance method further delineates 49 inbreds into 8 major clusters, and the clustering pattern corroborated with their pedigree and characteristics traits.

Antipathy of Trichoderma against Sclerotium rolfsii Sacc.: Evaluation of Cell Wall-Degrading Enzymatic Activities and Molecular Diversity Analysis of Antagonists

The fungus Trichoderma is a teleomorph of the Hypocrea genus and associated with biological control of plant diseases. The microscopic, biochemical, and molecular characterization of Trichoderma was carried out and evaluated for in vitro antagonistic activity against the fungal pathogen Sclerotium rolfsii causing stem rot disease in groundnut. In total, 11 isolates of Trichoderma were examined for antagonism at 6 and 12 days after inoculation (DAI). Out of 11, T. virens NBAII Tvs12 evidenced the highest (87.91%) growth inhibition of the test pathogen followed by T. koningii MTCC 796 (67.03%), T. viride NBAII Tv23 (63.74%), and T. harzianum NBAII Th1 (60.44%). Strong mycoparasitism was observed in the best antagonist Tvs12 strain during 6-12 DAI. The specific activity of cell wall-degrading enzymes - chitinase and β-1,3-glucanase - was positively correlated with growth inhibition of the test pathogen. In total, 18 simple sequence repeat (SSR) polymorphisms were reported to amplify 202 alleles across 11 Trichoderma isolates. The average polymorphism information content for SSR markers was found to be 0.80. The best antagonist Tvs 12 was identified with 7 unique SSR alleles amplified by 5 SSR markers. Clustering patterns of 11 Trichoderma strains showed the best antagonist T. virens NBAII Tvs 12 outgrouped with a minimum 3% similarity from the rest of Trichoderma.

A comprehensive characterization of simple sequence repeats in pepper genomes provides valuable resources for marker development in Capsicum.

The sequences of the full set of pepper genomes including nuclear, mitochondrial and chloroplast are now available for use. However, the overall of simple sequence repeats (SSR) distribution in these genomes and their practical implications for molecular marker development in Capsicum have not yet been described. Here, an average of 868,047.50, 45.50 and 30.00 SSR loci were identified in the nuclear, mitochondrial and chloroplast genomes of pepper, respectively. Subsequently, systematic comparisons of various species, genome types, motif lengths, repeat numbers and classified types were executed and discussed. In addition, a local database composed of 113,500 in silico unique SSR primer pairs was built using a homemade bioinformatics workflow. As a pilot study, 65 polymorphic markers were validated among a wide collection of 21 Capsicum genotypes with allele number and polymorphic information content value per marker raging from 2 to 6 and 0.05 to 0.64, respectively. Finally, a comparison of the clustering results with those of a previous study indicated the usability of the newly developed SSR markers. In summary, this first report on the comprehensive characterization of SSR motifs in pepper genomes and the very large set of SSR primer pairs will benefit various genetic studies in Capsicum.

Development of genomewide simple sequence repeat fingerprints and highly polymorphic markers in cucumbers based on next‐generation sequence data

AbstractCucumbers are one of the most important vegetable crops worldwide. The molecular markers have substantial potential to improve the efficiency of conventional cucumber breeding. In this study, a total of 101 157 simple sequence repeats (SSRs) were identified across the cucumber 9930 genome. Most (69 526 of 76 103 or 91.36%) SSRs with unique flanking sequences (unique SSRs) had polymorphisms among 103 cucumber genomes; the number of alleles ranged from 2 to 34 with an average of 4.70. Next, we selected 7486 unique SSR loci with polymorphism information content (PIC) ≥0.5 and major allele differences (the size difference between the alleles with the highest and the second highest frequency) ≥3 bp to design primers. The SSR marker polymorphisms were experimentally validated; 50 highly polymorphic SSR markers had polymorphisms with an average PIC of 0.44. PCR analysis also indicated that these SSR markers could be effectively used as molecular markers. The SSR fingerprints and polymorphic SSR markers with large major allele differences could enhance the efficiency of genetic research and marker‐assisted breeding in cucumbers.

Cultivar Identity, Intravarietal Variation, and Health Status of Native Grapevine Varieties in Croatia and Montenegro

Croatia and Montenegro (located in southeast Europe) host numerous native (autochthonous) minor grapevine (Vitis vinifera L.) varieties that might irreversibly disappear in the near future because of limited conservation efforts and lack of virus-free propagation material. To prevent their disappearance and help sustain these native populations, a collaborative project was launched to gather and evaluate extremely rare and neglected native grapevine genotypes by establishing true-to-type and virus-tested stock material, ready to use for nurseries. During several field expeditions, 284 grapevine accessions collected in 22 different locations in Croatia and Montenegro were subjected to ampelographic in situ description, genetic identification by nine simple sequence repeat (SSR) markers, serological testing for the presence of common grape viruses, and intravarietal variability assessment by sequence-specific amplification polymorphism (S-SAP) markers. We observed 53 unique SSR profiles, of which 25 genotypes have not been previously reported and are thus worthy of further research. Comparison of these genetic profiles with those from some very rare and poorly described accessions within the European Vitis Database and other published studies revealed several previously unknown synonyms and homonyms, which helped properly name some of the profiles and provided novel information on the history of these cultivars and their distribution in the region. Virus testing revealed high frequencies of infection with Grapevine leafroll-associated viruses 1 and 3 and indicated the urgent need for sanitary selection. Assessment of intravarietal genetic variation indicated the presence of accumulated mutations, which strongly suggests a need for clonal selection.

Development and Validation of EST-SSR Markers from the Transcriptome of Adzuki Bean (Vigna angularis).

The adzuki bean (Vigna angularis (Ohwi) Ohwi and Ohashi) is an important grain legume of Asia. It is cultivated mainly in China, Japan and Korea. Despite its importance, few genomic resources are available for molecular genetic research of adzuki bean. In this study, we developed EST-SSR markers for the adzuki bean through next-generation sequencing. More than 112 million high-quality cDNA sequence reads were obtained from adzuki bean using Illumina paired-end sequencing technology, and the sequences were de novo assembled into 65,950 unigenes. The average length of the unigenes was 1,213 bp. Among the unigenes, 14,547 sequences contained a unique simple sequence repeat (SSR) and 3,350 sequences contained more than one SSR. A total of 7,947 EST-SSRs were identified as potential molecular markers, with mono-nucleotide A/T repeats (99.0%) as the most abundant motif class, followed by AG/CT (68.4%), AAG/CTT (30.0%), AAAG/CTTT (26.2%), AAAAG/CTTTT (16.1%), and AACGGG/CCCGTT (6.0%). A total of 500 SSR markers were randomly selected for validation, of which 296 markers produced reproducible amplicons with 38 polymorphic markers among the 32 adzuki bean genotypes selected from diverse geographical locations across China. The large number of SSR-containing sequences and EST-SSR markers will be valuable for genetic analysis of the adzuki bean and related Vigna species.

Transcriptome sequencing of mung bean (Vigna radiate L.) genes and the identification of EST-SSR markers.

Mung bean (Vigna radiate (L.) Wilczek) is an important traditional food legume crop, with high economic and nutritional value. It is widely grown in China and other Asian countries. Despite its importance, genomic information is currently unavailable for this crop plant species or some of its close relatives in the Vigna genus. In this study, more than 103 million high quality cDNA sequence reads were obtained from mung bean using Illumina paired-end sequencing technology. The processed reads were assembled into 48,693 unigenes with an average length of 874 bp. Of these unigenes, 25,820 (53.0%) and 23,235 (47.7%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases, respectively. Furthermore, 19,242 (39.5%) could be classified into gene ontology categories, 18,316 (37.6%) into Swiss-Prot categories and 10,918 (22.4%) into KOG database categories (E-value < 1.0E-5). A total of 6,585 (8.3%) were mapped onto 244 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. Among the unigenes, 10,053 sequences contained a unique simple sequence repeat (SSR), and 2,303 sequences contained more than one SSR together in the same expressed sequence tag (EST). A total of 13,134 EST-SSRs were identified as potential molecular markers, with mono-nucleotide A/T repeats being the most abundant motif class and G/C repeats being rare. In this SSR analysis, we found five main repeat motifs: AG/CT (30.8%), GAA/TTC (12.6%), AAAT/ATTT (6.8%), AAAAT/ATTTT (6.2%) and AAAAAT/ATTTTT (1.9%). A total of 200 SSR loci were randomly selected for validation by PCR amplification as EST-SSR markers. Of these, 66 marker primer pairs produced reproducible amplicons that were polymorphic among 31 mung bean accessions selected from diverse geographical locations. The large number of SSR-containing sequences found in this study will be valuable for the construction of a high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.

Isolation and characterization of microsatellite markers and analysis of genetic diversity in Chinese jujube (Ziziphus jujuba Mill.).

Chinese jujube (Ziziphus jujuba Mill, 2n = 2× = 24, Rhamnaceae) is an economically important Chinese native species. It has high nutritional value, and its medicinal properties have led to extensive use in traditional oriental medicine. The characterization of genotypes using molecular markers is important for genetic studies and plant breeding. However, few simple sequence repeat (SSR) markers are available for this species. In this study, 1,488 unique SSR clones were isolated from Z. jujuba ‘Dongzao’ using enriched genomic libraries coupled with a three-primer colony PCR screening strategy, yielding a high enrichment rate of 73.3%. Finally, 1,188 (80.87%) primer pairs were amplified successfully in the size expected for ‘Dongzao’. A total of 350 primer pairs were further selected and evaluated for their ability to detect polymorphisms across a panel of six diverse cultivars; among these, 301 primer pairs detected polymorphisms, and the polymorphism information content (PIC) value across all loci ranged from 0.15 to 0.82, with an average of 0.52. An analysis of 76 major cultivars employed in Chinese jujube production using 31 primer pairs revealed comparatively high genetic diversity among these cultivars. Within-population differences among individuals accounted for 98.2% of the observed genetic variation. Neighbor-joining clustering divided the cultivars into three main groups, none of which correspond to major geographic regions, suggesting that the genetics and geographical origin of modern Chinese jujube cultivars might not be linked. The current work firstly reports the large-scale development of Chinese jujube SSR markers. The development of these markers and their polymorphic information represent a significant improvement in the available Chinese jujube genomic resources and will facilitate both genetic and breeding applications, further accelerating the development of new cultivars.

Survey of simple sequence repeats in woodland strawberry (Fragaria vesca)

The use of simple sequence repeats (SSRs), or microsatellites, as genetic markers has become popular due to their abundance and variation in length among individuals. In this study, we investigated linkage groups (LGs) in the woodland strawberry (Fragaria vesca) and demonstrated variation in the abundances, densities, and relative densities of mononucleotide, dinucleotide, and trinucleotide repeats. Mononucleotide, dinucleotide, and trinucleotide repeats were more common than longer repeats in all LGs examined. Perfect SSRs were the predominant SSR type found and their abundance was extremely stable among LGs and chloroplasts. Abundances of mononucleotide, dinucleotide, and trinucleotide repeats were positively correlated with LG size, whereas those of tetranucleotide and hexanucleotide SSRs were not. Generally, in each LG, the abundance, relative abundance, relative density, and the proportion of each unique SSR all declined rapidly as the repeated unit increased. Furthermore, the lengths and frequencies of SSRs varied among different LGs.