Microsatellite Primer Pairs Research Articles

ANALYSIS OF STABILITY OF TRINUCLEOTIDE TTC MOTIFS IN COMMON FLAX PLANTED IN THE CHERNOBYL AREA

Flax (Linum usitatissimum L.) is one of the oldest domesticated plants — it was cultivated as early as in ancient Egypt and Samaria 10,000 years ago to serve as a source of fiber and oil, whence it later spread around the world. Compared with other plants, the flax genome consists of a high number of repetitive sequences, middle repetitive sequences and small repetitive sequences of nucleotides. The aim of the study was to analyze the stability of the existing trinucleotides motifs of microsatellite DNA of the flax genome (genotype Kyivskyi), growing in the Chernobyl conditions. The Chernobyl area is the most extensive “natural” laboratory suitable for the study of radiation effects. Over the last 20 years, the researches collected important knowledge about the effects of low and high radiation doses on the DNA isolated from the plant material growing on the remediated fields near Chernobyl and the plant material from fields contaminated by radioactive cesium 137Cs and strontium 90Sr. Using eight pairs of microsatellite primers, we successfully amplified the samples from the remediated fields. For each primer in the control samples and remediated samples, we detected 1 to 3 fragments per locus, each in size up to 120 to 250 base pairs. The applied microsatellite primers confirmed the monomorphic condition of microsatellite loci.

English

Sclerotinia sclerotiorum , infection of bean fields, has increased in Brazil. Fungicides application is the control strategy used due to lack of cultivars with complete disease resistance. To guide the use of isolates in resistance screening 25 S. sclerotiorum isolates from Brazilian dry bean fields were characterized using microsatellite markers, mycelial compatibility groups (MCGs) and aggressiveness. Microsatellite primer pairs were used to identify polymorphisms among the S. sclerotiorum isolates and MCGs were determined from interaction of all isolates grown side-by-side. Aggressiveness was derived from a straw test where fungal mycelium was placed over a cut bean stem and rated for disease progress. Data from microsatellite profiles grouped the 25 isolates into four clusters and seven MCGs were identified. No association among host cultivar and cluster or MCG of isolates was observed. For MCGs, 57% contained isolates sampled frequently over multiple locations and 43% contained isolates unique to locations. There were significant differences among isolates in aggressiveness within and between MCGs. The most aggressive isolates in resistance screening will be helpful in the identification of higher levels of resistance in bean germplasm/lines.

Microsatellites for genetic and taxonomic research on thyme (Thymus L.)

Microsatellites are considered the marker of choice in modern research. However, there is no application of microsatellites in the research on the genus Thymus due to the scarcity of specific primer pairs. Using in silico data of several genera in the family Lamiaceae, 23 microsatellite primer pairs (LT) were developed and evaluated in 48 samples representing 9 species and subspecies of the genus Thymus. Codominant and multiallelic LT microsatellite markers were not only useful in the determination of genetic structure and variation within and between the species but also were powerful taxonomic aids in the molecular phylogeny of Thymus L. Moreover, some LT primer pairs successfully cross-amplified microsatellite markers in Salvia and Origanum, indicating that they could be used in other genera in the family Lamiaceae. To the best of our knowledge, this is the first report of genic microsatellite markers for the genus Thymus.

Fingerprinting Based on Microsatellite Markers for the Identification of Rice Varieties in Chhattisgarh

Molecular markers are useful tools for determining cultivar identity. The purpose of this study to establish fingerprints of four rice varieties in Chhattisgarh. Microsatellite combines several features of an ultimate molecular marker and they are being used increasingly in various plant genetic studies and applications. The varieties were evaluated utilizing 115 microsatellite markers distributed over the whole rice genome. A total of 160 alleles were detected using 24 microsatellite primer-pairs, and the number of alleles/marker ranged from 2 to 4 with an average of 2.95 alleles/locus. The result suggested that a relatively rapid and small number of microsatellite markers could be used for the identification of rice varieties.

Adding “personality” to biocontrol: characterization and suitability of microsatellites for sibship reconstruction in the aphid parasitoid Diaeretiella rapae

Efficiency of natural enemies in pest control in the field frequently shows high degree of variability, making it difficult to propose reliable biocontrol strategies. Due to the small size of many beneficals, underlying mechanisms are difficult to evaluate. In the present study the potential of molecular markers to reconstruct individual foraging decisions of a small parasitoid species unsuitable for direct tracking, i.e. Diaeretiella rapae (M`Intosh) (Hymenoptera: Braconidae), a primary parasitoid of the cabbage aphid Brevicoryne brassicae (Linnaeus) (Hemiptera: Aphididae), was evaluated. Nine newly designed microsatellite primer pairs were tested for their reliability in parentage analysis. Therefore, several families of D.rapae with four to six daughters and high or low degree of inbreeding were established in the lab. Individuals were genotyped and parentage and sibship analysis was calculated with the software COLONY. The results indicated that 91 % of the 85 genotyped offspring from all families have been assigned to the correct family when all nine microsatellite loci have been used and 86 % when only six microsatellite loci have been used. Even in families with high degree of inbreeding up to 87 % of the offspring could be assigned correctly. In conclusion, the sibship analysis of D. rapae provides reliable results, especially with all nine selected microsatellite loci. Therefore it will be a valuable tool to reconstruct individual foraging decisions and characterize parasitoid “personality” in the field to improve biological control strategies.

Construction of DNA Profile Data Base of Strawberry Cultivars Using Microsatellite Markers

국내외에서 재배되고 있는 딸기 100품종의 DNA profile 데이터베이스를 구축하기 위하여 다형성이 높은 microsatellite 마커의 선정과 유전적 유사도 분석을 통한 품종식별력 검정 등에 대한 연구를 수행하였다. 딸기 21품종을 274개의 microsatellite 마커로 검정하여 반복 재현성이 높은 25개의 다형성이 높은 마커를 선정하였다. 이들 마커와 국내외에서 재배되고 있는 딸기 100품종을 검정하였을 때 마커당 평균 대립유전자수는 7.50개로 나타났고, 3-13개까지 다양한 분포를 나타내었다. PIC 값은 마커의 유전자형에 따라 0.333-0.841 범위에 속하였으며 평균값도 0.706으로 높게 나타났다. Microsatellite 마커의 대립유전자를 이용하여 딸기 100 품종에 대한 계통도를 작성하였을 때 품종 육성의 계보 및 육성 지역에 따라 7개의 그룹으로 크게 나누어졌으며 2품종을 제외한 98품종이 microsatellite 마커의 유전자형에 의해 식별이 되는 것으로 나타났다. 본 연구에서 얻어진 딸기 품종별 DNA profile 데이터베이스는 품종보호 출원 품종의 재배심사 및 품종진위성과 관련된 종자분쟁을 해결하는 수단으로 유용하게 활용될 수 있을 것이다. This study was carried out to construct a DNA profile database of 100 strawberry cultivars using microsatellite markers. Two hundred seventy four microsatellite primer pairs were screened with a set of 21 strawberry cultivars with different morphological traits. Twenty five primer pairs were selected because they produced reliable and reproducible fingerprints. These primer pairs were used to develop DNA profiles of 100 strawberry cultivars. Three to thirteen alleles were detected by each marker with an average of 7.50. The average polymorphism information content varied from 0.331 to 841 (average 0.706). Cluster analysis showed that the 100 cultivars were divided into 7 major groups reflecting geographic origin and pedigree information. Moreover, most of the cultivars could be discriminated by marker genotypes. These markers will be useful as a tool for the protection of plant breeders' intellectual property rights in addition to providing the means to intervene seed disputes relating to variety authentication.

Three microsatellite multiplex PCR assays allowing high resolution genotyping of white spruce, Picea glauca

Abstract Fifteen previously published microsatellite primer pairs developed for and tested on Canadian white spruce were screened for amplification and polymorphy in Alaskan populations and tested for their suitability in PCR multiplexing. Eleven loci expressing polymorphisms ranging from 7 to 58 alleles were selected for development and optimization of three multiplex assays. Four natural stands containing a total of 1470 trees were used to characterize the selected loci and demonstrate their applicability for genotyping studies and parentage analysis. These assays can be used for studies focusing on population genetics, parentage analysis, provenance research, or individual genetic fingerprinting. The use of multiplex PCR facilitates large-scale studies by simultaneously enabling high resolution and reducing processing time and per sample cost.

Development and characterization of chloroplast microsatellite markers in a fine-leaved fescue, Festuca rubra (Poaceae).

• Premise of the study: Chloroplast microsatellite markers were developed for Festuca rubra to examine its population genetic characteristics, taxonomy, and coevolution with its endophyte Epichloë festucae.• Methods and Results: Thirteen polymorphic markers were identified from the chloroplast genome of a F. ovina accession and intergenic chloroplast sequences of F. rubra accessions. They amplified a total of 65 alleles in a sample of 93 individuals of F. rubra originating from six different populations located in the Faroe Islands, Finland, Greenland, Norway, and Spain.• Conclusions: The developed microsatellite primer pairs can be used by researchers in population genetic and taxonomic studies, and by plant breeders in breeding programs on grasses.

Population Structure of Geosmithia morbida, the Causal Agent of Thousand Cankers Disease of Walnut Trees in the United States

The ascomycete Geosmithia morbida and the walnut twig beetle Pityophthorus juglandis are associated with thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut). The disease was first reported in the western United States (USA) on several Juglans species, but has been found more recently in the eastern USA in the native range of the highly susceptible Juglans nigra. We performed a comprehensive population genetic study of 209 G. morbida isolates collected from Juglans and Pterocarya from 17 geographic regions distributed across 12 U.S. states. The study was based on sequence typing of 27 single nucleotide polymorphisms from three genomic regions and genotyping with ten microsatellite primer pairs. Using multilocus sequence-typing data, 197 G. morbida isolates were placed into one of 57 haplotypes. In some instances, multiple haplotypes were recovered from isolates collected on the same tree. Twenty-four of the haplotypes (42%) were recovered from more than one isolate; the two most frequently occurring haplotypes (H02 and H03) represented 36% of all isolates. These two haplotypes were abundant in California, but were not recovered from Arizona or New Mexico. G. morbida population structure was best explained by four genetically distinct groups that clustered into three geographic regions. Most of the haplotypes isolated from the native range of J. major (Arizona and New Mexico) were found in those states only or present in distinct genetic clusters. There was no evidence of sexual reproduction or genetic recombination in any population. The scattered distribution of the genetic clusters indicated that G. morbida was likely disseminated to different regions at several times and from several sources. The large number of haplotypes observed and the genetic complexity of G. morbida indicate that it evolved in association with at least one Juglans spp. and the walnut twig beetle long before the first reports of the disease.

Isolation and characterization of 23 microsatellite loci in the stingless bee Melipona subnitida using next-generation sequencing

We described the isolation and characterization of 23 microsatellite loci from the stingless bee (Melipona subnitida). Out of 52 microsatellite primer pairs screened, 17 loci displayed polymorphism and 6 were monomorphic. The analysis of variability was performed in 56 individuals. The number of alleles per locus ranged from 2 to 22 among populations; values for expected and observed heterozygosities ranged from 0.125 to 1.000 and from 0.121 to 0.923, respectively. These are the first microsatellite markers characterized for M. subnitida and they will be useful in obtaining estimates of population-level genetic diversity studies in a near future.

Isolation and characterization of 11 microsatellite markers for Glochidion acuminatum (Phyllanthaceae).

• Premise of the study: Microsatellite markers were developed for Glochidion acuminatum (Phyllanthaceae) to investigate pollen dispersal distances and thereby to assess the effectiveness of specialized Epicephala moths as pollinators.• Methods and Results: Using next-generation sequencing, 11 polymorphic microsatellite primer pairs were developed for G. acuminatum. The primer pairs were tested on 49 individuals from two populations in Japan. The number of alleles per locus ranged from two to 13, and the expected heterozygosity ranged from 0.12 to 0.85. Probability of identity for all loci combined was lower than 1.27 × 107.• Conclusions: The microsatellite markers developed in this study will be useful for evaluating the benefit of specialized Epicephala moth pollination to Glochidion plants.

Polymorphic microsatellite markers for a wind-dispersed tropical tree species, Triplaris cumingiana (Polygonaceae).

• Premise of the study: Novel microsatellite markers were characterized in the wind-dispersed and dioecious neotropical tree Triplaris cumingiana (Polygonaceae) for use in understanding the ecological processes and genetic impacts of pollen- and seed-mediated gene flow in tropical forests.• Methods and Results: Sixty-two microsatellite primer pairs were screened, from which 12 markers showing five or more alleles per locus (range 5–17) were tested on 47 individuals. Observed and expected heterozygosities averaged 0.692 and 0.731, respectively. Polymorphism information content was between 0.417 and 0.874. Linkage disequilibrium was observed in one of the 66 pairwise comparisons between loci. Two loci showed deviation from Hardy–Weinberg equilibrium. An additional 14 markers exhibiting lower polymorphism were characterized on a smaller number of individuals.• Conclusions: These microsatellite markers have high levels of polymorphism and reproducibility and will be useful in studying gene flow and population structure in T. cumingiana.

Microsatellite primers for the gynodioecious grassland perennial Saxifraga granulata (Saxifragaceae).

• Premise of the study: Nine polymorphic and 12 monomorphic microsatellite loci (simple sequence repeats [SSRs]) were isolated and characterized for the gynodioecious grassland perennial Saxifraga granulata.• Methods and Results: Based on genomic screening of leaf material of four individuals from four populations, a total of 21 microsatellite primer pairs were designed for S. granulata. Nine loci were polymorphic and were optimized into two PCR multiplex reactions and tested on 100 individuals from five riparian populations from central Belgium. The number of alleles of the polymorphic loci ranged from three to 18, and gametic heterozygosity ranged from 0.26 to 0.94.• Conclusions: The markers that are presented here are the first microsatellite markers reported for S. granulata and will be used to assess how river systems shape the spatial genetic structure and diversity of riparian populations of this species.

Use of Microsatellite Markers to Identify Commercial Melon Cultivars and for Hybrid Seed Purity Testing

Abstract: Microsatellite markers were used to identify 58 major commercial melon cultivars, and to assess hybrid seed purit y of a melon breeding line known as ‘10H08’. A set of 412 microsatellite primer pairs were utilized for fingerprinting of the melon cultivars. Twenty-nine markers showed hyper-variability and could discriminate all cultivars on the basis of marker genotypes, representing the genetic variation within varietal groups. Cluster analysis based on Jaccard’s distance coefficients using the UPGMA algorithm categorized 2 major groups, which were in accordance to morphological traits. The DNA bulks of female and male parents of breeding line ‘10H08’ were tested with 29 primer pairs based on microsatellites to investigate purity testing of F 1 hybrid seeds, and 5 primer pairs exhibited polymorphism. One microsatellite primer pair (CMGAN12) produced unambi guous polymorphic bands among the parents. Among 192 seeds tested with CMGAN12, progeny possibly generated by self-pollination of the female parent were clearly distinguished from the hybrid progeny. These markers will be useful for fingerprinting melon cultivars and can help private seed companies to improve melon seed purity.Additional key words:

Assessment of genetic purity of four rice cultivars using microsatellite and ISSR markers

Summary The estimation of rice seed purity is conventionally done by a grow-out-test (GOT). As well as other issues, the time required for GOT is a major limitation. In this study, DNA-based microsatellite and ISSR markers were used to establish fingerprints of four rice varieties and used for assessment of genetic purity. A total of 160 alleles were obtained using 23 microsatellite primer pairs for four rice varieties. Six microsatellite markers (RM 247, RM 303, RM 164, RM 108, RM 72 and RM 19) and eight ISSR markers (UBC 842, UBC 810, UBC 812, UBC 834, UBC 808, UBC 873, UBC 841 and ISSR 2) were found to be the most informative primer sets. These primers sets were used for seed purity analysis of all four varieties. Genetic purity based on a set of three markers revealed that all the tagged plants identified as off-types on the basis of morphological traits were confirmed by the molecular marker in seed samples of the four rice varieties.

Variabilidad genética del robalo común Centropomus undecimalis (Perciformes: Centropomidae) en ambiente marino y ribereño interconectados

The Common Snook, Centropomus undecimalis, inhabits riverine and marine areas of Southern Gulf of Mexico, where it is subject to intense use and exploitation. It has been reported that the genetic identification of fish stocks constitutes a valuable tool for wild population management; nevertheless, there is no available information on the genetic identification on fish stocks of this species in the region. The aim of this study was to determine the genetic relationship between C. undecimalis captured in marine and freshwater environments of the Gulf of Mexico and the San Pedro River. For this, muscle tissue samples of 79 specimens were obtained from areas located more than 300km apart. The genotype of each individual was determined using seven microsatellite primer pairs. Five primers amplified efficiently presenting between six and 28 alleles per locus. High levels of heterozygosis were observed in samples from both environments. Deviation from HWE due to an excess of heterozygotes was observed. The values of genetic difference indicate an absence of population structure (F(ST) = 0.0075 and R(ST) = (0.016, p = 0.051) and similarity in the allele frequencies, defined by Nei's index (0.805). Data showed the existence of a high gene flow due to the number of migrants (Nm = 18.7). Our results suggest that individuals living in these environments belong to the same genetic population. We suggest the development of management and protection plans for this fish species population in the wild.

Cross-species Utility of 22 Microsatellite Markers in the Melodious Warbler (Hippolais Polyglotta)

Microsatellites are a valuable tool in the analysis of population genetic structure. Utilising microsatellite markers that were originally isolated from other species (cross-species amplification) can prove an efficient way, in terms of time and cost, to obtain markers for genetic studies. Here, 55 avian microsatellite primer pairs were tested for the cross-amplification in the Melodious Warbler ( Hippolais polyglotta). Thirty-five markers amplified, of which 22 were polymorphic, displaying two to nine alleles in the 15 individuals genotyped. The 35 markers which amplified in the Melodious Warbler were tested in its sister species the Icterine Warbler ( H. icterina). Twenty-four markers were amplified, 14 of which were polymorphic in the five H. icterina individuals genotyped. Thirteen loci were polymorphic in both species. The polymorphic loci identified are suitable for analysing the genetic population structure and assigning parentage.

Genetic diversity of wild population of Pyropia haitanensis based on SSR analysis

Genetic diversity analysis was conducted on 80 individuals of 4 populations of non-bred Pyropia haitanensis by simple sequence repeat (SSR) method. Using 15 pairs of microsatellite primers, 37 polymorphic loci were amplified, representing 94.9% of all loci. At the population level, the percentage of polymorphic bands (P) and polymorphism information content (PIC) were 66.67–84.62% and 0.481–0.488, with average value at 73.72% and 0.483, respectively. Expected heterozygosity (He) and Shannon's information index (I) were 0.279 and 0.434, respectively, at the species level, and 0.233 and 0.356 at population level. According to the coefficient of gene differentiation (GST), a large proportion of genetic variance (83.6%) of P. haitanensis was among individuals within populations, only 16.4% genetic variance was among populations, which was identified with the moderate gene flow value (Nm = 2.542). UPGMA clustered the 4 populations into 3 groups, and no significant correlation was found between the genetic distance and the corresponding geographic distance among the populations.

Isolation and Characterization of Microsatellite Markers for Cotinus coggygria Scop. (Anacardiaceae) by 454 Pyrosequencing

Cotinus coggygria Scop. (Anacardiaceae) is a deciduous shrub or small tree that is native to a large area covering from southern Europe, east across central Asia, and the Himalayas in northern China. Shotgun 454 pyrosequencing was used to develop microsatellite markers from the genome of C. coggygra. In this study, 349 microsatellite loci were identified from 40,074 individual sequence reads produced by one-sixteenth run, and primer pairs were designed for these loci. To test the primer amplification efficiency, 50 microsatellite primer pairs were tested across 12 individuals from two C. coggygria populations (Wuzhi Mountain: 36°30'N, 113°39'E; Tianlong Mountain: 37°42'N, 112°26'E). Among the 50 tested primer pairs, eight were found to be polymorphic. The average allele number of the microsatellites was 3.5 per locus, with a range from two to five. The inbreeding coefficient ranged from −0.478 to 0.222. The observed and expected heterozygosities varied from 0.167 to 0.750 and from 0.163 to 0.743, respectively. This set of markers is potentially useful for assessing the genetic diversity, as well as for understanding the population structure and phylogeographical and landscape genetic patterns, of C. coggygria.

New microsatellite and CAPS-microsatellite markers for clarifying taxonomic and phylogenetic relationships within Origanum L.

Several species in the genus Origanum L. are important among culinary herbs in the world and local markets as raw materials in herb and spices, pharmaceutical and cosmetic industries. Microsatellites also known as simple sequence repeats are routinely being utilized in many plant species. However, the use of microsatellites has still been limited in the genus Origanum due to the scarcity of specific primer pairs. Until recently, there were only 13 microsatellite primer pairs used in two species of Origanum. This study reported 30 primer pairs for development of microsatellite and CAPS-microsatellite markers in 8 different Origanum species. Microsatellite and CAPS-microsatellite markers were utilized to test whether they were useful in species identification and phylogenetic studies in 65 individual samples representing 8 Origanum species. Results indicated that these markers were very useful to clarify taxonomic uncertainties within the genus since some of the markers produced species-specific amplification fashions. Reported DNA markers could be useful in Origanum breeding studies to select desirable chemotypes and tracing adulteration in commercial herbal materials.