Single Sequence Repeats Loci Research Articles

Genetic diversity of common bean (Phaseolus vulgaris L.) germplasm from Serbia, as revealed by single sequence repeats (SSR)

Genetic diversity and structure of common bean (Phaseolus vulgaris L.) germplasm from Serbia, comprising 118 landraces and 18 cultivars, was assessed with the application of 27 Single Sequence Repeats (SSR) markers. Thirteen accessions from Agricultural Institute of Slovenia were used as references for gene pool determination. Main parameters of genetic diversity were calculated for each SSR loci, i.e. number of different and rare alleles, number of effective alleles, Shannon's information index, observed and expected heterozygosity and polymorphic information content. A total of 445 allelic variants, with 16.5 alleles per locus on average, were detected. Mean gene diversity (He = 0.79) indicated sufficient reservoir of genetic variation preserved in studied bean germplasm. Landraces displayed higher variability compared to cultivars (405 in relation to 233 allelic variants). Genetic structure and relatedness of accessions was assessed by model-based method and hierarchical clustering method in combination with genetic distance calculation. The Bayesian clustering model implemented in STRUCTURE software, on the primary level (K = 2), revealed clear separation of accessions into two groups, corresponding to gene pool affiliation. Mesoamerican gene pool (M) was represented with 23.5% of accessions, while Andean (A) was larger, composed of 68.4% of studied germplasm. Small group (8.1%) showed admixed genetic structure between two gene pools. Additional variation in respect to two recognized gene pools was revealed (K = 3), whose basis was acknowledged to be within Andean gene pool. Further subdivision of accessions (K = 8), mainly according to the seed forms, was observed. Genetic distance analysis associated with Neighbour-joining clustering method revealed grouping pattern of landraces and cultivars corresponding to the gene pool and their seed phenotypes. Classification and structuring of the bean accessions according to and beyond the gene pool of origin should facilitate conservation strategies and breeding of this material. Combining the information of phenotypic variation obtained in previous research and molecular data reveled in this study will assist in selection of parental components for breeding, or in the choice of smaller sample in order to further acknowledge their breeding value. In addition, obtained results of this work should serve as an additional information on common bean germplasm variation in Western Balkans and beyond, in Europe.

Research Article Development and transferability of microsatellite loci for <i>Spondias</i> <i>tuberosa</i> (Anacardiaceae: Sapindales), a species endemic to the Brazilian semi-arid region

The umbu tree (Spondias tuberosa) is one of the best known plants of the Brazilian semi-arid region; it has great potential for the fruit market due to excellent consumer acceptance. This tree is not presently cultivated; fruit commercialization is based on extrativism. Consequently, there has been little research on the genetics of this species. Our objective was to develop, evaluate and transfer single sequence repeat (SSR) loci to S. tuberosa to support work on genetic resources and agronomic improvement of this species. SSR loci for the umbu tree were developed from a new enriched genomic library and evaluated by PCR. Fourteen SSR loci developed for S. radlkoferi were evaluated for use in S. tuberosa, as well as 18 SSR loci previously identified for this species. DNA was extracted from leaf tissue of eight umbu trees available that are part of a germplasm collection located in Petrolina, PE, Brazil. Of the 14 pairs of primers that were tested, six yielded amplicons, and two showed polymorphism in the genotyped samples. All SSR loci of S. radlkoferi transferred to the umbu tree species, yielding amplicons; however, only four were polymorphic in this sample. Among the eighteen available species-specific SSR loci, six were polymorphic for these eight trees. Among the 38 loci that we evaluated, 12 were polymorphic in the sample, including two new loci for S. tuberosa. The number of alleles ranged from 2 to 12, and 10 of them were moderately to highly informative (PIC>0.50), while nine had heterozygosity greater than 0.50. The six new SSR loci and the 14 SSR loci transferred from S. radlkoferi increase the number of available loci for population studies, germplasm collection and resource management for the development of new umbu tree cultivars.

The transferability of microsatellite loci from a homoploid to a polyploid hybrid complex: an example from fine-leaved Festuca species (Poaceae).

BackgroundMicrosatellite loci, or single sequence repeats (SSR), are widely used as powerful markers in population genetics. They represent an attractive tool for studying plants such as grasses, whose evolution is driven by hybridisation and polyploidization. However, the development of microsatellite markers has been challenging and time-consuming, especially for non-model organisms lacking available genome-wide sequence data. One straightforward and low-cost approach is to transfer the SSR loci developed for one species, or complex, to another closely-related one. This work evaluates the transferability of microsatellite loci from homoploid to allopolyploid complexes of fine-leaved Festuca species and to assess their use in two new species. The studied complex (F. amethystina—F. tatrae) is a useful model for research on the local adaptability of grasses with different ploidy levels. Since both species can be considered as rare or threatened (F. tatrae—as a mountain and narrow endemic species and F. amethystina—a mountain species with relict lowland populations), any tool enabling studies on genetic diversity and population genetics, such as SSR markers, could also be very useful in a conservation context.MethodsThe ploidy level within populations was estimated using flow cytometry. One diploid and one tetraploid population of F. amethystina and a diploid population of F. tatrae were chosen to test the transferability of SSR loci. Because our work describes the transfer of SSR nuclear markers designed originally for F. gautieri, a phylogenetic tree was prepared based on the ITS marker to assess the genetic distance between the studied complexes. The PCR products were separated on a high-resolution agarose gel, intended for SSR marker analysis. Appropriate solutions for the allotetraploid population and whole mixed-ploidy complex were implemented.ResultsFlow cytometry confirmed earlier data regarding DNA content in the investigated species and cytotypes. The phylogenetic ITS tree indicated a small genetic distance between F. gautieri complexes and the studied species. Ten microsatellite markers were successfully transferred. All markers were polymorphic. In total, 163 different alleles were scored from the 10 SSR loci. PCoA of accessions revealed well-separated groups corresponding to studied populations. Over 60% of the total variance is explained by differentiation within populations and one third among them.ConclusionsThe transferred markers are valid tools for the study of population genetics and inheritance relationships within cytotypes and species and between them. The presented markers can be used to study inbreeding depression in the Festuca species, and variations in the degrees of genetic diversity between different cytotypes in mountain and lowland areas. Our findings can also be applied to study conservation strategies for ensuring biodiversity at the genetic level in polyploid complexes.

Genetic diversity and linkage disequilibrium in the Argentine public maize inbred line collection

AbstractKnowledge of linkage disequilibrium (LD) patterns is considered a prerequisite for effective association mapping studies. However, no LD analysis in the Argentine public temperate maize collection has been reported to date. In this study, a panel of 111 temperate maize inbreds genotyped at 74 single sequence repeats (SSRs) loci was used to assess LD, genetic diversity and population structure to evaluate the suitability of the panel for association mapping. Mini-core sets were also designed for in-depth phenotyping and allele mining purposes. The panel consisted of: (1) locally developed orange flint germplasm; (2) temperate inbred lines with Iowa Stiff Stalk Synthetic background; and (3) eight historic flint lines, some of them from the Cuarentín race. As a result, four subpopulations were defined. Joint analysis of population structure and combining ability allowed identifying two main heterotic patterns. High molecular diversity, a low extent of LD and a high ratio of linked to unlinked SSR loci pairs in significant LD were detected indicating the suitability of the entire collection for association mapping. The fact that the LD extent in the mini-core sets was similar to that observed in the entire collection and that only a small percentage of allelic richness was reduced suggests that these mini-core sets are suitable to capture diversity, exploit phenotypic variance and discover useful variants representative of the entire collection.

Microsatellite markers: what they mean and why they are so useful.

Microsatellites or Single Sequence Repeats (SSRs) are extensively employed in plant genetics studies, using both low and high throughput genotyping approaches. Motivated by the importance of these sequences over the last decades this review aims to address some theoretical aspects of SSRs, including definition, characterization and biological function. The methodologies for the development of SSR loci, genotyping and their applications as molecular markers are also reviewed. Finally, two data surveys are presented. The first was conducted using the main database of Web of Science, prospecting for articles published over the period from 2010 to 2015, resulting in approximately 930 records. The second survey was focused on papers that aimed at SSR marker development, published in the American Journal of Botany's Primer Notes and Protocols in Plant Sciences (over 2013 up to 2015), resulting in a total of 87 publications. This scenario confirms the current relevance of SSRs and indicates their continuous utilization in plant science.

Development and transferability of two multiplexes nSSR in Scots pine (Pinus sylvestris L.)

Single sequence repeat (SSR) multiplexing is a semi high-throughput PCR methodology for the analysis of multiple SSRs. We developed two SSR multiplexes selected from SSR loci previously reported in the pine literature and tested the transferability of both SSR multiplexes in nine other pine species. We tested 234 nuclear SSR loci (nSSRs) previously described in the pine literature and selected ten nSSRs following the simple criteria of interpretability and reproducibility. Selected nuclear loci were divided into two nSSRs multiplex sets and their amplification was optimized for three different multiplex PCR methods based on: (a) a custom PCR protocol, (b) a custom protocol with hotstart taq polymerase, and (c) a commercially available kit for SSR multiplexing. To validate their performance, the level of genetic diversity was assessed in three Scots pine natural populations (Hungary, northern Sweden and southern Sweden). In addition, we also tested the transferability of these multiplexes in nine other pine species. We have developed two nSSRs multiplexes of five loci each that will contribute to reduce the costs of nSSR scoring, while increasing the capacity of nSSR loci analysis. Amplification was successful in all three populations (94 % success) and the level of polymorphism (7.1 alleles/marker) was similar to that previously reported for other Scots pine natural populations. Transferability of both multiplexes was successful for those pine species closely related to Scots pine.

Molecular characterization of twenty polymorphic microsatellite markers in the polyploid fruit tree species Syzygium samarangense (Myrtaceae)

Syzygium samarangense (Blume) Merr. & Perry (wax apple) is an important commercial fruit tree in Southeast Asia. Here, microsatellite markers were developed to evaluate genetic diversity and distinguish cultivars in this species. In total, 161 microsatellite loci with sufficient flanking sequences to design primer sets were isolated from wax apple using a magnetic bead-enrichment method. Fifty-eight primer sets were designed based on the flanking sequences of each single sequence repeat (SSR) locus and were tested using 14 wax apple cultivars/lines. Twenty SSR loci were found to be polymorphic and transferable across the 14 wax apple cultivars/lines. The number of alleles and effective number of alleles detected per locus ranged from 4 to 12 and from 1.697 to 9.800, respectively. The expected heterozygosity ranged from 0.150 to 0.595 (mean = 0.414). Polymorphism information content values ranged from 0.502 to 0.866 (mean = 0.763). These new microsatellite loci will be of value for characterization of genetic diversity in wax apples and for the identification of cultivars.

Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris L.) based on genome sequences of a Chinese landrace and diversity evaluation

Microsatellite or single sequence repeat (SSR) markers have been commonly used in genetic research in many crop species, including common bean (Phaseolus vulgaris L.). A limited number of existing SSR markers have been designed from high-throughput sequencing of the genome, warranting the exploitation of new SSR markers from genomic regions. In this paper, we sequenced total DNA from the genotype Hong Yundou with a 454-FLX pyrosequencer and found numerous SSR loci. Based on these, a large number of SSR markers were developed and 90 genomic-SSR markers with clear bands were tested for mapping and diversity detection. The new SSR markers proved to be highly polymorphic for molecular polymorphism, with an average polymorphism information content value of 0.44 in 131 Chinese genotypes and breeding lines, effective for distinguishing Andean and Mesoamerican genotypes. In addition, we integrated 85 primers of the 90 polymorphism markers into the bean map using an F2 segregating population derived from Hong Yundou crossed with Jingdou. The distribution of SSR markers among 11 chromosomes was not random and tended to cluster on the linkage map, with 14 new markers mapped on chromosome Pv01, whereas only four loci were located on chromosome Pv04. Overall, these new markers have potential for genetic mapping, genetic diversity studies and map-based cloning in common bean.

Population genetics structure of glyphosate‐resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.

Foundation characteristics of edible Musa triploids revealed from allelic distribution of SSR markers.

Background and AimsThe production of triploid banana and plantain (Musa spp.) cultivars with improved characteristics (e.g. greater disease resistance or higher yield), while still preserving the main features of current popular cultivars (e.g. taste and cooking quality), remains a major challenge for Musa breeders. In this regard, breeders require a sound knowledge of the lineage of the current sterile triploid cultivars, to select diploid parents that are able to transmit desirable traits, together with a breeding strategy ensuring final triploidization and sterility. Highly polymorphic single sequence repeats (SSRs) are valuable markers for investigating phylogenetic relationships.MethodsHere, the allelic distribution of each of 22 SSR loci across 561 Musa accessions is analysed.Key Results and ConclusionsWe determine the closest diploid progenitors of the triploid ‘Cavendish’ and ‘Gros Michel’ subgroups, valuable information for breeding programmes. Nevertheless, in establishing the likely monoclonal origin of the main edible triploid banana subgroups (i.e. ‘Cavendish’, ‘Plantain’ and ‘Mutika-Lujugira’), we postulated that the huge phenotypic diversity observed within these subgroups did not result from gamete recombination, but rather from epigenetic regulations. This emphasizes the need to investigate the regulatory mechanisms of genome expression on a unique model in the plant kingdom. We also propose experimental standards to compare additional and independent genotyping data for reference.

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon x Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0-34.4 and 28.9-31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14-70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.

Fourteen polymorphic microsatellite markers for the fungal banana pathogen Mycosphaerella fijiensis

Fourteen polymorphic microsatellite markers were developed for Mycosphaerella fijiensis, a fungus causing the black sigatoka disease in banana. The sequenced genome of M. fijiensis was screened for sequences with single sequence repeats (SSRs) using a Perl script. Fourteen SSR loci, evaluated on 48 M. fijiensis isolates from Hawaii, were identified to be highly polymorphic. These markers revealed two to 19 alleles, with an average of 6.43 alleles per locus. The estimated gene diversity ranged from 0.091 to 0.930 across the 14 microsatellite loci. The SSR markers developed would be useful for population genetics studies of M. fijiensis.

Use of genome sequence data in the design and testing of SSR markers for Phytophthora species

BackgroundMicrosatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of Phytophthora population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of P. infestans, P. sojae and P. ramorum were mined for candidate SSR markers that could be applied to a wide range of Phytophthora species.ResultsA first approach, aimed at the identification of polymorphic SSR loci common to many Phytophthora species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to P. sojae (P. alni, P. cambivora, P. europaea and P. fragariae). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times.ConclusionKey studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of Phytophthora species was challenging. That said, many novel SSR loci for species other than the three 'source species' (P. infestans, P. sojae and P. ramorum) are reported, offering great potential for the investigation of Phytophthora populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different Phytophthora species.

Development, characterization, and comparative analysis of polymorphism at common bean SSR loci isolated from genic and genomic sources

Microsatellites or SSRs (single sequence repeats) have been used to construct and integrate genetic maps in crop species, including Phaseolus vulgaris. In the present study, 3 cDNA libraries generated by the Bean EST project (http://lgm.esalq.usp.br/BEST/), comprising a unigene collection of 3126 sequences and a genomic microsatellite-enriched library, were analyzed for the presence of SSRs. A total of 219 expressed sequence tags (ESTs) were found to carry 240 SSRs (named EST-SSR), whereas 714 genomic sequences contained 471 SSRs (named genomic-SSR). A subset of 80 SSRs, 40 EST-SSRs, and 40 genomic-SSRs were evaluated for molecular polymorphism in 23 genotypes of cultivated beans from the Mesoamerican and Andean genetic pools, including Brazilian cultivars and 2 related species. Of the common bean genotypes, 31 EST-SSR loci were polymorphic, yielding 2-12 alleles as compared with 26 polymorphic genomic-SSRs, accounting for 2-7 alleles. Cluster analysis from data using both genic and genomic-SSR revealed a clear separation between Andean and Mesoamerican beans. The usefulness of these loci for distinguishing bean genotypes and genetic mapping is discussed.

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

The genetic variability of 38 grapefruit (Citrus paradisi Macf.) and three pummelos (C. maxima (Burm.) Merr..) accessions was evaluated using RAPD, and single sequence repeat (SSR) analyses. Approximately49% of the 198 RAPD were polymorphic, and 4.6 alleles per SSR loci were identified. PIC values changed from 0.093 to 0.450. A UPGMA phenetic tree was constructed and two main grapefruit groups were identified. The grapefruit accessions `do Cabo' and `Siamesa-Filipinas'clustered very close to the pummelos in Group A. The Group B consisted of three sub-groups, which comprised all of the other grapefruit accessions. The majority of grapefruit accessions showed a narrow genetic base suggesting that the observed morphological polymorphism within the group must be associated with somatic mutations, which were not detected by these molecular markers.