Combined Probability Of Identity Research Articles

Progeny Selection and Genetic Diversity in a Pinus taeda Clonal Seed Orchard

The present article describes the development of an improved Pinus taeda clonal seed orchard adapted to the edaphoclimatic conditions of Uruguay. Initially, 2068 hectares distributed in nine companies were prospected, and 124 plus trees were identified based on growth, straightness, and health traits. These trees were clonally propagated via grafting to establish a clonal seed orchard. For the genetic evaluation of the orchard, two progeny tests were carried out in the Rivera and Paysandú municipalities. Quantitative genetic analyses allowed us to identify a simple genotype–environment interaction and an expected genetic gain for volumes of 17%, 13%, and 8% for selection intensities of 12%, 25%, and 50%, respectively. Moreover, the genetic diversity of the 124 clones of the orchard was assessed using 10 microsatellite markers. The fingerprinting profiles allowed us to identify a total of 224 alleles. The polymorphism information content of the different markers was in the range of 0.594 to 0.895. The combined probability of identity and probability of identity among siblings had a discrimination power of 8.26 × 10–14 and 5.91 × 10–5, respectively. Analysis of the genetic structure demonstrated that the seed orchard population was not structured by the supplier.

Development and validation of a SNPs panel used for beef traceability throughout the food chain

Meat labelling is important to avoid unfair competition practices of producers, processors and sellers aiming gain an economic advantage from misrepresenting cheaper meat products as premium quality ones. Genetic traceability is an ideal tool for the detection and prevention of fraud in the sale of beef. In this work, a method for validating protected status based on a combination of single nucleotide polymorphisms (SNPs) and a high-throughput real-time PCR protocol was developed. A total of 1,911 beef samples were used to develop and validate an OpenArray® panel consisting of 26 SNPs selected from an ISAG-ICAR panel. The applicability of the method was determined by comparing 143 blind pairs of samples. The combined probability of identity (PI) with the 26 SNPs selected was 2.09-11 with 22 SNPs showing an individual PI lower than 0.4. In all tests, the blind pair of samples were correctly as-signed. The panel designed and validated in this study is of great utility to confirm genetic content of the specific genetic group tested and detect fraud in a routine way that requires a minimum amount of time and is cost effective.

Development and technical application of SSR-based individual identification system for Chamaecyparis\xa0taiwanensis\xa0against illegal logging convictions

Chamaecyparis taiwanensis is an endemic plant suffering illegal logging in Taiwan for its high economic value. Lack of direct evidence to correlate stump and timber remains a hurdle for law enforcement. In this report, 23 polymorphic Genomic Simple Sequence Repeat (gSSR) and 12 Expressed Sequence Tag (EST)-SSR markers were developed and their transferability was assessed. The individual identification system built from selected non-linkage 30 SSR markers has a combined probability of identity as 5.596 × 10–12 equivalents to identifying an individual in a population of up to 18 million C. taiwanensis with 99.99% confidence level. We also applied the system in an actual criminal case by selecting 19 of these markers to correlate illegally felled timbers and victim trees. Our data demonstrate that molecular signals from three timbers hit with three victim trees with confidence level more than 99.99%. This is the first example of successfully applying SSR in C. taiwanensis as a court evidence for law enforcement. The identification system adapted advanced molecular technology and exhibits its great potential for natural resource management on C. taiwanensis.

WITHDRAWN: Genetic structure and diversity of Santa Inês sheep flocks in Brazilian Mid-North.

Ahead of Print article withdrawn by publisher.

Evaluation of ten SNP Markers for Human Identification and Paternity Analysis in Persian Population

Background: DNA markers are inevitable tools of human identification in forensic science. Single Nucleotide Polymorphisms (SNPs) are one category of these markers which is concerned to use especially in the case of degraded DNA because of their short amplicons.Objectives: Detection of highly informative SNPs by the criteria is the essential step to develop a useful panel of SNP markers. The purpose of this work is to get high informative SNPs for human identification in Persian ethnic of the Iranian population.Material and Methods: Genotype and allele frequencies of 10 SNPs from the SNPforID browser were determined by a PCR-RFLP method on 100 samples that was taken from 100 unrelated Persian people.Results: These ten SNPs were in Hardy-Weinberg equilibrium (P value > 0.1) except rs1355366 (P value = 0.02) and Heterozygosity of seven SNPs is greater than 0.45 but minor allele frequency of only four SNPs is more than 0.45. According to criteria only three SNPs rs1454361, rs2111980 and rs2107612 can pass all standards and are highly informative in population for forensic uses.Conclusions: Our data showed that the CPI (Combined probability of Identity) and CPE (Combined Power of Exclusion) for ten SNPs are 1.13 E-04 and 0.809 respectively. It was also showed based on the criteria only three SNPs (rs2107612, rs1454361 and rs2111980) are highly informative in Persian population. If we can find 39 SNPs with PE and PI close to PE and PI of these three SNPs (rs2107612, rs1454361 and rs2111980), we will be able to use of these 39 SNPs in human identification with sufficient power of discrimination.

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers.

Durian (Durio zibethinus) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, HE = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10−3. Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called “clones”, “varieties”, or “cultivars”. Such matters have a direct impact on the regulation and management of durian genetic resources in the region.

A set of tetra-nucleotide core motif SSR markers for efficient identification of potato (Solanum tuberosum) cultivars.

Simple sequence repeat (SSR) is a popular tool for individual fingerprinting. The long-core motif (e.g. tetra-, penta-, and hexa-nucleotide) simple sequence repeats (SSRs) are preferred because they make it easier to separate and distinguish neighbor alleles. In the present study, a new set of 8 tetra-nucleotide SSRs in potato (Solanum tuberosum) is reported. By using these 8 markers, 72 out of 76 cultivars obtained from Japan and the United States were clearly discriminated, while two pairs, both of which arose from natural variation, showed identical profiles. The combined probability of identity between two random cultivars for the set of 8 SSR markers was estimated to be 1.10 × 10−8, confirming the usefulness of the proposed SSR markers for fingerprinting analyses of potato.

An efficient identification strategy of clonal tea cultivars using long-core motif SSR markers.

Microsatellites, or simple sequence repeats (SSRs), especially those with long-core motifs (tri-, tetra-, penta-, and hexa-nucleotide) represent an excellent tool for DNA fingerprinting. SSRs with long-core motifs are preferred since neighbor alleles are more easily separated and identified from each other, which render the interpretation of electropherograms and the true alleles more reliable. In the present work, with the purpose of characterizing a set of core SSR markers with long-core motifs for well fingerprinting clonal cultivars of tea (Camellia sinensis), we analyzed 66 elite clonal tea cultivars in China with 33 initially-chosen long-core motif SSR markers covering all the 15 linkage groups of tea plant genome. A set of 6 SSR markers were conclusively selected as core SSR markers after further selection. The polymorphic information content (PIC) of the core SSR markers was >0.5, with ≤5 alleles in each marker containing 10 or fewer genotypes. Phylogenetic analysis revealed that the core SSR markers were not strongly correlated with the trait ‘cultivar processing-property’. The combined probability of identity (PID) between two random cultivars for the whole set of 6 SSR markers was estimated to be 2.22 × 10−5, which was quite low, confirmed the usefulness of the proposed SSR markers for fingerprinting analyses in Camellia sinensis. Moreover, for the sake of quickly discriminating the clonal tea cultivars, a cultivar identification diagram (CID) was subsequently established using these core markers, which fully reflected the identification process and provided the immediate information about which SSR markers were needed to identify a cultivar chosen among the tested ones. The results suggested that long-core motif SSR markers used in the investigation contributed to the accurate and efficient identification of the clonal tea cultivars and enabled the protection of intellectual property.

Cross-species amplification of microsatellite loci for non-invasive genetic monitoring of the snowy owl (Bubo scandiacus)

Genetic analysis has shown to be an important and efficient method for monitoring species, but the lack of genetic markers constrains such monitoring for many species. Here, we cross-amplified microsatellite loci in the snowy owl (Bubo scandiacus) using primers previously characterized in other species. Among polymorphic loci, we selected 12 loci that amplified relatively short fragments (<250 base pairs) to facilitate genotyping of moulted feathers and other non-invasively collected samples. These 12 loci, and a sex-typing marker, were amplified in two multiplex PCR sets and used to screen 49 snowy owls from a northern European population. The number of alleles observed at single loci was 2 to 17. Expected and observed heterozygosity ranged from 0.12 to 0.91 and from 0.12 to 0.96, respectively. The combined probability of identity for the 12 loci was 5.7 × 10−11. Our multiplex PCR assays are expected to be useful for genetic monitoring, parentage analysis, and population genetic studies of the snowy owl.

Microsatellites for Carpotroche brasiliensis (Flacourtiaceae), a useful species for agroforestry and ecosystem conservation.

Premise of the study:We developed microsatellite markers for Carpotroche brasiliensis (Flacourtiaceae), a dioecious tree that is used as a food resource by midsize animals of the Brazilian fauna.Methods and Results:We designed 30 primer pairs using next-generation sequencing and classified 25 pairs as polymorphic. Observed heterozygosity ranged from 0.5 to 1.0, and expected heterozygosity ranged from 0.418 to 0.907. The combined probability of exclusion was greater than 0.999 and the combined probability of identity was less than 0.001, indicating that these microsatellites are appropriate for investigations of genetic structure, individual identification, and paternity testing.Conclusions:The developed molecular tools may contribute to future studies of population genetics, answering ecological and evolutionary questions regarding efficient conservation strategies for C. brasiliensis.

Informativeness of minisatellite and microsatellite markers for genetic analysis in papaya.

The objective of this study was to evaluate information on minisatellite and microsatellite markers in papaya (Carica papaya L.). Forty minisatellites and 91 microsatellites were used for genotyping 24 papaya accessions. Estimates of genetic diversity, genetic linkage and analyses of population structure were compared. A lower average number of alleles per locus was observed in minisatellites (3.10) compared with microsatellites (3.57), although the minisatellites showed rarer alleles (18.54 %) compared with microsatellite (13.85 %). Greater expected (He = 0.52) and observed (Ho = 0.16) heterozygosity was observed in the microsatellites compared with minisatellites (He = 0.42 and Ho = 0.11), possibly due to the high number of hermaphroditic accessions, resulting in high rates of self-fertilization. The polymorphic information content and Shannon-Wiener diversity were also higher for microsatellites (from 0.47 to 1.10, respectively) compared with minisatellite (0.38 and 0.85, respectively). The probability of paternity exclusion was high for both markers (>0.999), and the combined probability of identity was from 1.65(-13) to 4.33(-38) for mini- and micro-satellites, respectively, which indicates that both types of markers are ideal for genetic analysis. The Bayesian analysis indicated the formation of two groups (K = 2) for both markers, although the minisatellites indicated a substructure (K = 4). A greater number of accessions with a low probability of assignment to specific groups were observed for microsatellites. Collectively, the results indicated higher informativeness of microsatellites. However, the lower informative power of minisatellites may be offset by the use of larger number of loci. Furthermore, minisatellites are subject to less error in genotyping because there is greater power to detect genotyping systems when larger motifs are used.

Mining microsatellites in the peach genome: development of new long-core SSR markers for genetic analyses in five Prunus species.

A wide inventory of molecular markers is nowadays available for individual fingerprinting. Microsatellites, or simple sequence repeats (SSRs), play a relevant role due to their relatively ease of use, their abundance in the plant genomes, and their co-dominant nature, together with the availability of primer sequences in many important agricultural crops. Microsatellites with long-core motifs are more easily scored and were adopted long ago in human genetics but they were developed only in few crops, and Prunus species are not among them. In the present work the peach whole-genome sequence was used to select 216 SSRs containing long-core motifs with tri-, tetra- and penta-nucleotide repeats. Microsatellite primer pairs were designed and tested for polymorphism in the five diploid Prunus species of economic relevance (almond, apricot, Japanese plum, peach and sweet cherry). A set of 26 microsatellite markers covering all the eight chromosomes, was also selected and used in the molecular characterization, population genetics and structure analyses of a representative sample of the five diploid Prunus species, assessing their transportability and effectiveness. The combined probability of identity between two random individuals for the whole set of 26 SSRs was quite low, ranging from 2.30 × 10−7 in peach to 9.48 × 10−10 in almond, confirming the usefulness of the proposed set for fingerprinting analyses in Prunus species.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1098-0) contains supplementary material, which is available to authorized users.

DNA fingerprinting of Japanese plum (Prunus salicina) cultivars based on microsatellite markers

Forty-seven Japanese plum (Prunus salicina) cultivars were genotyped with eight microsatellite markers, aiming at obtaining the DNA fingerprinting profiling, distinguishing and characterizing a representative set of Japanese plum cultivars. The eight SSR loci amplified 104 alleles (8 to 21 alleles per locus, mean 13). Polymorphism Information Content (PIC) ranged from 0.680 to 0.886 (mean 0.803). The observed heterozigozity (Ho) ranged from 0.529 to 0.915 (mean 0.770). Probability of Identity (I) of each locus ranged from 0.019 to 0.113 (mean 0.054). The combined Probability of Identity was 2.66 x 1011, and the Power of Exclusion of the eight loci was 99.99976%. 57 out of 104 alleles showed frequency lower than 0.05. These low allele frequencies contributed to raise the distinguish ability of plum cultivars. These results will contribute, as excellent descriptors, to select parental for crossings, to perform early identification of segregating clones with potential to be cultivars, and to protect the cultivars.

Molecular characterization of Spanish Prunus avium plus trees

Aim of the study: The Breeding Program of wild cherry (Prunus avium) developed by Lourizán Forest Research Center (NW Spain), aims for the creation of the Main Breeding Population, that is formed by a large number of plus trees and for obtaining an Elite Population generated from controlled crosses of a number of plus trees selected by, at least, one trait of economic importance. The aim of this study was to genotype 131 accessions of Prunus avium plus trees, included in the breeding program.Area of study: The Prunus avium plus trees are located in the North, Northwest and Central Spain.Material and Methods: The Prunus avium plus trees were genotyped with nine microsatellites. Several genetic parameters were calculated. Genetic data were analyzed with STRUCTURE and the genetic distance between the plus trees were calculated.Main results: A total of 122 multilocus genotypes were detected. Several accessions with the same genotype were identified, which could be due to clonality or to labelling errors. The nine microsatellites are useful for identifying individuals because the combined probability of identity was low (PI = 5.19X10-9). Bayesian methods detected two genetic clusters in the sampled plus trees.Research highlights: The unique genotypes identified in this work are suitable for being included in the elite breeding population for economic traits.Keywords: Prunus avium; breeding program; microsatellite; genetic distance.

Discrimination of common bean cultivars using multiplexed microsatellite markers.

Analysis of DNA polymorphisms allows for the genetic identification and precise discrimination of species with a narrow genetic base such as common bean. The primary objectives of the present study were to molecularly characterize commercial common bean varieties developed at various research institutions using microsatellite markers and to determine the degree of genetic diversity among the bean varieties analyzed. Fifty cultivars representing 12 grain classes and 64 genitors, i.e., accessions used to develop these cultivars, were characterized. Based on an analysis of 24 simple sequence repeats, the estimates for the average number of alleles and genetic diversity were 8.29 and 0.646, respectively. The combined probability of identity was estimated at 7.05 x 10(-17), indicating a high individual discriminatory power. Thirty-two percent of the cultivars exhibited heterogeneity for multiple loci that reflected either homozygosity for different alleles of a given locus in different individuals or heterozygosity for the locus. The average genetic diversity for the groups of cultivars and genitors was 0.605 and 0.660, respectively, with no genetic differentiation (FST) between these groups. Although similar estimates of expected heterozygosity were observed when the cultivars were grouped by release date, a greater number of private alleles was observed in the most recent cultivars. The genetic differentiation among cultivars originating from different institutions was not different from zero (FST = 0.01). The molecular profile database derived from these analyses may increase the statistical power of genetic estimates and may be incorporated into breeding programs for common bean. Furthermore, the profiles obtained for the different cultivars may be used as molecular descriptors to complement traditional descriptors used in distinctiveness, uniformity and stability tests, thereby improving the traceability of samples and their derivatives and helping to protect the intellectual property rights of breeders.

Isolation, characterization and predicted genome locations of Eurasian eagle-owl (Bubo bubo) microsatellite loci

We isolated 134 eagle-owl (Bubo bubo) microsatellite sequences. Eight of these newly isolated loci were characterized in 38 Eurasian eagle-owls in a northern European population. Sequence homology was used to assign a predicted chromosome location for the eight loci. We also redesigned primers for four previously isolated eagle-owl sequences and cross-amplified two published primer sets previously characterized in other owl species. These 14 loci were amplified in three multiplex PCR sets and displayed 2–9 alleles with expected and observed heterozygosities ranging from 0.34 to 0.85 and from 0.42 to 0.97, respectively. Estimated frequencies of null-alleles were low and only one locus deviated from Hardy–Weinberg equilibrium. After correcting for multiple tests, linkage disequilibrium was found for a single pair of loci. The combined probability of identity for the 14 loci was 3.5 × 10−12. These microsatellite loci are expected to be useful for genetic monitoring, parentage analysis and population genetic studies.

Development of twenty-three novel microsatellite markers for the seagrass, Zostera muelleri from Australia

Seagrasses are one of the most productive and economically important habitats in the coastal zone, but they are disappearing at an alarming rate, with more than half the world’s seagrass area lost since the 1990s. They now face serious threat from climate change, and there is much current speculation over whether they will survive the coming decades. The future of seagrasses depends on their ability to recover and adapt to environmental change—i.e. their ‘resilience’. Key to this, is understanding the role that genetic diversity plays in the resilience of this highly clonal group of species. To investigate population structure, genetic diversity, mating system (sexual versus asexual reproduction) and patterns of connectivity, we isolated and characterised 23 microsatellite loci using next generation sequencing for the Australian seagrass species, Zostera muelleri (syn. Z. capricorni), which is regarded as a globally significant congeneric species. Loci were tested for levels of variation based on eight individuals sampled from Lake Macquarie, New South Wales, Australia. We detected high to moderate levels of genetic variation across most loci with a mean allelic richness of 3.64 and unbiased expected hetrozygosity of 0.562. We found no evidence for linkage disequilibrium between any loci and only three loci (ZosNSW25, ZosNSW2, and ZosNSW47) showed significant deviations from Hardy–Weinberg expectations. All individuals displayed a unique multi-locus genotype and the combined probability of identity across all loci was low (PID = 1.87 × 10−12) indicating a high level of power in detecting unique genotypes. These 23 markers will provide an important tool for future population genetic assessments in this important keystone species.

Markers for Screening Lynch Syndrome Are Reliable and Useful for Identifying the Specimen Mislabeling

BackgroundDuring specimen processing in surgical pathology laboratories, specimen-related adverse events (SRAEs), such as mislabeling and specimen mixed-up might occur. In these situations, molecular techniques using short tandem repeat (STR) loci are required to identify the personal identity. Microsatellite instability (MSI) test is widely used for screening the hereditary non-polyposis colon cancer (Lynch syndrome) in surgical pathologies using polymorphic STR markers. We tried to evaluate the applicability of the MSI test for SRAEs.MethodsWe obtained 253 MSI test results to analyze the allele frequencies. After calibrating the estimated nucleotide lengths, we calculated the allele frequencies, a random match probability, and a likelihood ratio (LR) of three dinucleotide STR markers (D5S349, D17S250, and D2S123).ResultsThe distribution of LR was 136.38 to 5,606,213.10. There was no case of LR<100. In addition, there were 153 cases (60.5%) of LR ranging from 100 to 10,000 and 100 cases (39.5%) of LR>10,000. Furthermore, the combined probability of identity was 9.23×10-4 and the combined power of exclusion was 0.99908.ConclusionsUsing the three STR markers that are recommended for MSI test, all the cases were positively identified in 1% range and about one-third cases showed high LR (>10,000). These results showed that MSI tests are useful to screen the personal identity in case of SRAE in pathology laboratories.

Efficient human paternity testing with a panel of 40 short insertion-deletion polymorphisms

We developed a panel of 40 multiplexed short insertion-deletion (indel) polymorphic loci with widespread chromosomal locations and allele frequencies close to 0.50 in the European population. We genotyped these markers in 360 unrelated self-classified White Brazilians and 50 mother-child-probable father trios with proven paternity. The average heterozygosity (gene diversity) per locus was 0.48, and the combined probability of identity (matching probability) for the 40-locus set was 3.48 x 10(-17). The combined power of exclusion of the indel panel was 0.9997. The efficiency of the 40 indel set in the exclusion of falsely accused individuals in paternity casework was equivalent to the CODIS set of 13 microsatellites. The geometric mean of the paternity indices of the 50 mother-child-probable father trios was 17,607. This panel of 40 short indels was found to have excellent performance. Thus, especially because of its simplicity and low cost, and the fact that it is composed of genomic markers that have very low mutation rates, it represents a useful new tool for human paternity testing.

Application of for the identification and management of hybrid poplar accessions

Molecular markers are a powerful tool with many potential applications in agriculture and forestry. In particular, can provide information on the relatedness of various clones or varieties that are difficult to distinguish morphologically, thus helping in the management of plant accessions and in breeding programs. The goal of this study is to genotype 15 clones used in the Prairie Farm Rehabilitation Administration (PFRA) breeding programs. Simple Sequence Repeat (SSR, or microsatellite markers) were selected for genotyping using the polymerase chain reaction (PCR). This type of marker is considered the method of choice due to their abundance, polymorphism and reliability compared to other types of . Sixteen previously isolated and characterized in aspen (Populus tremuloides) and other poplar species (Populus spp.) were initially tested. Nine markers were selected based on the “informativeness” and the quality of the amplification products. The nine markers were combined in groups of three to improve the efficiency of the genotyping technique. Using the nine markers, the average number of alleles per locus was 5.1. The expected and observed heterozygosity ranges were 0.32 to 0.80 and 0.13 to 0.92 respectively. The results also show that it is possible to produce a unique “DNA fingerprint” specific to each of the 15 hybrid poplar clones with the nine . In this study it was possible to show that two clones, P. ‘Melville’ and P. x ‘Walker’ used in Saskatchewan have similar DNA profiles with nine markers and a combined probability of identity of 2.23×10−6 suggesting that these clones are identical. This observation will prevent unnecessary duplication of the two accessions in breeding programs.