Novel Set Of Microsatellite Markers Research Articles

Development of 32 novel microsatellite loci in Juglans sigillata using genomic data.

PremiseA novel set of microsatellite markers was developed for Juglans sigillata (Juglandaceae), an endemic walnut species in southwestern China, to facilitate cultivar identification and future investigations into the genetic structure and domestication history of this species and its close relatives.Methods and ResultsWe developed 32 microsatellite loci for J. sigillata using genomic data and used them to examine 60 individuals from three natural populations. A high level of polymorphism was detected by these primers, with up to eight alleles observed per locus, and an average of four alleles across populations. The levels of observed and expected heterozygosity ranged from 0.000–1.000 and 0.000–0.785, respectively. All but two of the loci were also successfully amplified in three closely related Eurasian Juglans species (J. regia, J. cathayensis, and J. mandshurica).ConclusionsThe microsatellite loci identified here provide a powerful resource for examining the genetic structure and domestication history of Juglans, as well as identification of its cultivars.

Characterization and cross-species transferability of a novel set of microsatellites derived from root transcriptomes of Camellia oleifera

AbstractCamellia oleifera is an important woody plant producing healthy edible oils. People need a large number of molecular markers, especially microsatellite, in breeding of C. oleifera. In this study, we sequenced the root transcriptomes of C. oleifera, and then designed a novel set of microsatellite markers based on the root-expressed genes. We assembled a total of 57,121 unigenes with a length of 42.63 Mb, which harboured 15,902 microsatellites. Among these microsatellites, di-nucleotide repeat motifs were the most abundant group (56.45%), then followed by tri- (25.20%), mono- (12.12%), hexa- (3.21%), penta- (2.18%) and quad-nucleotide ones (0.84%). In total, 6738 primer pairs were designed successfully to amplify the microsatellite loci. To test these microsatellite markers, 48 primer pairs were randomly selected and synthesized and validated in C. oleifera and its eight relatives. Up to 75% of the primer pairs amplified in C. oleifera and its relatives, and 62.5% displayed polymorphism. The transferability and diverse alleles across its eight relatives were detected for each polymorphic primer pair. The novel set of microsatellites derived from the root transcriptomes here provided a useful resource for future molecular genetics improvement of C. oleifera and its relatives.

Population genetic structure and interspecific differentiation between Acer davidii Franchi. and A. morrisonense Hayata (Aceraceae) based on SSR markers

Acer davidii Franch. and A. morrisonense Hayata are two important forest tree species (Aceraceae) endemic to mainland China and the Taiwan area, respectively. To investigate population structure and interspecific differentiation between them, we characterized a set of novel microsatellite markers using Illumina sequencing technology. The cross-species amplification analysis showed that 11 out of 21 polymorphic SSR primers of A. davidii also exhibited polymorphisms in A. morrisonense. At the species level, A. davidii has a slightly higher genetic diversity (mean observed heterozygosity, HO = 0.180) than A. morrisonense (HO = 0.119). AMOVA showed that most of the variations in A. davidii occurred among individuals within populations and within individuals. Bayesian clustering analyses demonstrated that the two maple species formed two clear genetic lineages. PCoA showed that A. davidii and A. morrisonense were significantly divided into two genetic groups. In addition, asymmetrical weaker gene flow was detected among the two forest tree species. These results suggest that the long term geographic isolation between the mainland and Taiwan may have resulted in a high level of genetic differentiation between these two important maple species.

A set of novel microsatellite markers developed for an economically important tree, Dracontomelon duperreanum, in China.

Dracontomelon duperreanum, the most representative species of the family Anacardiaceae, is an important multipurpose tree in China and Vietnam. However, no genetic diversity studies have been reported on this species. In this study, we identified 11 microsatellite markers for D. duperreanum by using the restriction-site-associated DNA sequencing (RAD-seq) method and examined their polymorphisms in 22 samples obtained from the South China Botanical Garden, South China. We could detect only two or three alleles for each microsatellite marker. The mean observed and expected heterozygosities were 0.41 and 0.39, respectively, which were lower than those reported for the species with similar life history forms. These relatively low genetic diversities in this common plant species are unexpected and might have resulted from its extensive cultivation. To our knowledge, this is the first report of microsatellite markers in the genus Dracontomelon. These microsatellite markers will be valuable for studying the genetic diversities and structures in D. duperreanum and other Dracontomelon species.

Characterization and defining of a core set of novel microsatellite markers for use in genotyping and diversity study of Adriatic fig (Ficus carica L.) germplasm

To support studies of the genetic diversity and population structure of Ficus carica L., 16 additional microsatellite markers were developed from genomic libraries enriched for GA/TC and GT/AC motifs. A total of 87 alleles were amplified in 83 analyzed fig samples. The number of alleles per locus ranged from three to eight, with a mean value of 5.44 alleles per locus. Mean values of observed and expected heterozygosity were 0.575 and 0.626, respectively. Calculated PIC values varied among loci in a range from 0.369 to 0.761. All markers were successfully cross-amplified in five closely related species belonging to the Moraceae family: Ficus (F. barteri Sprague, F. rubiginosa Desf. ex Vent., F. benjamina L.) and Morus (M. alba L. and M. nigra L.). These novel microsatellite markers represent a new valuable tool for further genome investigation, population studies, and management of genetic resources of wild, feral, and domesticated Ficus carica L. samples and other related species of Moraceae family, distributed mostly in tropical and subtropical regions throughout the world.

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis.

BackgroundSafflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing.Principal FindingsLow throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower.ConclusionNext generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection.

A novel set of microsatellite markers for the European Grapevine MothLobesia botranaisolated using next-generation sequencing and their utility for genetic characterization of populations from Europe and the Middle East

Using a high-throughput 454 pyrosequencing approach a novel set of microsatellite markers was developed for one of the key grapevine insect pests, the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae). 20 primer pairs flanking a microsatellite motif were designed based on the sequences obtained and were subsequently evaluated in a sample of 14 L. botrana populations from Europe and the Middle East. 11 markers showed stable and reproducible amplification patterns; however, one of the 11 markers was monomorphic in all L. botrana populations analysed. Estimated frequencies of null alleles of more than 20% were evident for two of the markers tested, but varied substantially depending on the respective L. botrana population. In 12 of the 14 L. botrana populations observed heterozygosities were lower to those expected under Hardy-Weinberg equilibrium, indicating a deficiency of heterozygotes in the respective populations. The overall F ST value of 0.075 suggested a moderate but significant genetic differentiation between the L. botrana populations included in this study. In addition, a clear geographic structure was detected in the set of samples, evident through a significant isolation by distance and through results from structure analysis. In structure analysis, L. botrana populations were grouped in two clearly separated clusters according to their European (Spain, Italy, Germany) or Middle Eastern (Israel, Syria, Turkey) origin. This novel set of microsatellite markers can now be applied to study the evolutionary ecology of this species including host shifts and host adaptation as well as spread of individuals across worldwide viticulture.

Rapid Changes in the Genetic Composition of Stagonosporopsis tanaceti Population in Australian Pyrethrum Fields

A novel set of microsatellite markers were developed and employed for geographical and temporal population analyses of Stagonosporopsis tanaceti, the cause of ray blight of pyrethrum in Australia. Genotyping of 407 isolates, using 13 markers, suggested an asexual mode of reproduction with significant linkage disequilibrium and high levels of clonality. Low geographical differentiation and widespread distribution of a few multilocus genotypes (MLGs), in the absence of airborne ascospores, suggested the role of human-mediated movement of seed as a major means of long-distance pathogen dispersal. The genetic composition of S. tanaceti was stable for a decade then changed rapidly in only 2 years. Bayesian clustering analyses and minimum spanning networks determined only two major clonal lineages in and prior to 2010. However, in 2012, a previously unobserved cluster of MLGs was detected, which significantly increased in frequency and displaced the historically dominant MLGs by 2013. This rapid change in the genetic composition of S. tanaceti could indicate a second introduction then a selective sweep, or strong selection pressures from recently introduced fungicides or pyrethrum varieties. These results may have serious implications for durability of management strategies for this disease.

Microsatellite markers from the 'South American fruit fly' Anastrepha fraterculus: a valuable tool for population genetic analysis and SIT applications.

BackgroundAnastrepha fraterculus Wiedemann is a horticultural pest which causes significant economic losses in the fruit-producing areas of the American continent and limits the access of products to international markets. The use of environmentally friendly control strategies against this pest is constrained due to the limited knowledge of its population structure.ResultsWe developed microsatellite markers for A. fraterculus from four genomic libraries, which were enriched in CA, CAA, GA and CAT microsatellite motifs. Fifty microsatellite regions were evaluated and 14 loci were selected for population genetics studies. Genotypes of 122 individuals sampled from four A. fraterculus populations were analyzed. The level of polymorphism ranged from three to 13 alleles per locus and the mean expected heterozygosity ranged from 0.60 to 0.64. Comparison between allelic and genotypic frequencies showed significant differences among all pairs of populations.ConclusionsThis novel set of microsatellite markers provides valuable information for the description of genetic variability and population structure of wild populations and laboratory strains of A. fraterculus. This information will be used to identify and characterize candidate strains suitable to implement effective pest control strategies and might represent a first step towards having a more comprehensive knowledge about the genetics of this pest.

A set of novel microsatellite markers developed for a traditional Chinese medicinal plant, Sarcandra glabra (Chloranthaceae)

A set of novel microsatellite markers developed for a traditional Chinese medicinal plant, Sarcandra glabra (Chloranthaceae)

Isolation and characterization of polymorphic microsatellite loci in the endangered San Diego fairy shrimp (Branchinecta sandiegonensis)

A set of ten polymorphic microsatellite loci were isolated and characterized for the endangered San Diego fairy shrimp (Branchinecta sandiegonensis). These loci were amplified on a set of 24 fairy shrimp collected from vernal pools located throughout San Diego County. The loci selected are highly variable across a wide sampling range (3–29 alleles per locus, 3–20 heterozygotes observed). After directly testing for Mendelian inheritance through family screens, eight markers did not show evidence of null alleles. This novel set of microsatellite markers will be useful in future genetic studies to assess intraspecific diversity, population connectivity and mating patterns.

Validating the use of colouration patterns for individual recognition in the worm pipefish using a novel set of microsatellite markers

In studies of behaviour, ecology and evolution, identification of individual organisms can be an invaluable tool, capable of unravelling otherwise cryptic information regarding group structure, movement patterns, population size and mating strategies. The use of natural markings is arguably the least invasive method for identification. However, to be truly useful natural markings must be sufficiently variable to allow for unique identification, while being stable enough to permit long-term studies. Non-invasive marking techniques are especially important in fishes of the Family Syngnathidae (pipefishes, seahorses and seadragons), as many of these taxa are of conservation concern or used extensively in studies of sexual selection. Here, we assessed the reliability of natural markings as a character for individual identification in a wild population of Nerophis lumbriciformis by comparing results from natural markings to individual genetic assignments based on eight novel microsatellite loci. We also established a minimally invasive method based on epithelial cell swabbing to sample DNA. All pipefish used in the validation of natural markings, independently of sex or time between recaptures, were individually recognized through facial colouration patterns. Their identities were verified by the observation of the same multilocus genotype at every sampling event for each individual that was identified on the basis of natural markings. Successful recaptures of previously swabbed pipefish indicated that this process probably did not induce an elevated rate of mortality. Also, the recapture of newly pregnant males showed that swabbing did not affect reproductive behaviour.

Development and characterization of a novel set of microsatellite markers for Arisaema serratum (Araceae)

We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum.

A set of novel microsatellite markers developed for the traditional Tibetan medicinal plant Halenia elliptica (Gentianaceae)

Microsatellite primers were developed in the traditional Tibetan medicinal plant Halenia elliptica D. Don to investigate its genetic diversity and population genetic structure. Using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol, 24 primer sets were identified in two wild populations. Of these primers, 12 displayed polymorphisms and 12 were monomorphic. The number of alleles per locus ranged from 2 to 6, with a mean of 3.9. The expected (H(E)) and observed (H(O)) heterozygosities ranged from 0.191 to 0.784 and from 0.417 to 0.917, respectively. All these primers successfully amplified in two close relatives of H. elliptica, Swertia bimaculata (Siebold & Zucc.) Hook. f. & Thomson ex C. B. Clarke and S. tetraptera Maxim. These markers will facilitate further studies on the population genetics of Halenia elliptica and its allied species.

High degree of sex chromosome differentiation in stickleback fishes

BackgroundStudies of closely related species with different sex chromosome systems can provide insights into the processes of sex chromosome differentiation and evolution. To investigate the potential utility of molecular markers in studying sex chromosome differentiation at early stages of their divergence, we examined the levels and patterns of genetic differentiation between sex chromosomes in nine-spined (Pungitius pungitius) and three-spined sticklebacks (Gasterosteus aculeatus) using microsatellite markers.ResultsA set of novel microsatellite markers spanning the entire length of the sex chromosomes were developed for nine-spined sticklebacks using the sequenced genomes of other fish species. Sex-specific patterns of genetic variability and male-specific alleles were identified at most of these loci, indicating a high degree of differentiation between the X and Y chromosomes in nine-spined sticklebacks. In three-spined sticklebacks, male-specific alleles were detected at some loci confined to two chromosomal regions. In addition, male-specific null alleles were identified at several other loci, implying the absence of Y chromosomal alleles at these loci. Overall, male-specific alleles and null alleles were found over a region spanning 81% of the sex chromosomes in three-spined sticklebacks.ConclusionsHigh levels but distinct patterns of sex chromosome differentiation were uncovered in the stickleback species that diverged 13 million years ago. Our results suggest that the Y chromosome is highly degenerate in three-spined sticklebacks, but not in nine-spined sticklebacks. In general, the results demonstrate that microsatellites can be useful in identifying the degree and patterns of sex chromosome differentiation in species at initial stages of sex chromosome evolution.

Extrapair paternity in a German population of the Northern Wheatear (Oenanthe oenanthe)

We demonstrate the potential of a set of novel microsatellite markers to investigate kin structure and population genetics in the Northern Wheatear (Oenanthe oenanthe). In this study, based on 242 individuals from a population in Rhineland-Palatinate (Germany), we found that 26% of the offspring in 46% of the broods were sired by other males than the social fathers. We tested different hypotheses why males engage in extrapair copulations and found that almost all identified genetic fathers originated from directly neighbouring territories. Additionally, we detected 2 out of 134 offspring in two broods that could not be assigned to their putative mother and thus were probably the result of intraspecific brood parasitism.

A new set of microsatellite markers for the genetic characterization of Ceratocystis fimbriata, an economically important plant pathogen

Ceratocystis fimbriata is a fungal pathogen which attacks several economically important plants, but occurs in host-associated, morphologically indistinguishable forms. In Brazil, this fungus seriously attacks mango trees (Mangifera indica), causing severe loss of yield. This work aimed to develop and characterize a novel set of microsatellite markers for this important pathogen, providing researchers with new molecular tools for the characterization of isolates. Twenty polymorphic primer pairs were designed from a microsatellite-enriched library. We tested the usefulness of these markers through genotyping thirteen isolates of the fungus. On average, 6.65 alleles per locus were detected, revealing the ability of this set of markers to characterize C. fimbriata isolates associated to mango and to other plant species.

Microsatellite markers for mungbean developed from sequence database

A novel set of microsatellite markers for mungbean [Vigna radiata (L.) Wilczek] was developed from the public sequence database. Seventy-eight primers were designed and evaluated for polymorphism among 22 cultivated accessions. Eight polymorphic loci detected two to three alleles per locus with an average of 2.25. The observed heterozygosity varied from 0.00 to 0.18, while the expected heterozygosity ranged from 0.09 to 0.46. Among them, all eight loci showed significant departuring from Hardy-Weinberg equilibrium, while four pairs of loci displayed significant pairwise linkage disequilibrium values. All eight loci except DMB-SSR1 showed heterozygote deficiency.