High Polymorphism Information Content Research Articles

Genetic signatures of polymorphic microsatellite loci in the Ambiguous silver pomfret, Pampus argenteus (Teleostei, Stromateidae)

Pampusargenteus is a broadly exploited pelagic fish species, commonly misidentified as Pampusechinogaster. Genetic variation and population structure in Pampusargenteus was studied based on seven microsatellite loci. The observed high average allele number, heterozygosity values, and polymorphism information content of P.argenteus suggested high genetic diversity. No population genetic differentiation was detected based on the results of pairwise Fst, three-dimensional factorial correspondence analysis (3D-FCA) and STRUCTURE analysis, which implied continuous gene flow. Wilcoxon signed rank tests did not indicate significant heterozygosity excess, and recent genetic bottleneck events were not detected. Coupled with previous mitochondrial DNA results, the findings presented here indicate that high gene flow characterizes the current phylogeographic pattern of the species.

Identification and adaptability of hatchery-origin Japanese flounder released into the Bohai Sea after the summer fishing moratorium

Because the artificial cultivation environment differs from the natural habitat of the Bohai Sea, it is essential to study the adaptability of hatchery-reared juveniles released into this area. Hatchery-origin Paralichthys olivaceus stocks were identified via morphological observation and genetic tags (i.e., mitochondrial DNA and microsatellites). Seven genomic microsatellite loci with a high PIC (polymorphic information content, 0.885–0.945) and a high exclusion probability (0.930–0.981) were selected and arranged into two multiplex PCRs. In total, 46.75% of the captures were assigned to our broodstocks. There were no significant differences in genetic diversity, such as Ar (allelic richness, P = .754) and He (expected heterozygosity, P = 0. 658), among the hatchery-origin stock (HS), their parents (P), the wild stock (WS) and the captured broodstocks (CB) in 2016. However, losses of Ar in the HS indicated that more attention should be given to the number of broodstocks in a reared population. The overall genetic divergence among these four groups was low (FST = 0.005), and the results of AMOVA also showed no genetic differences among them (P = .500), indicating that no pronounced genetic differentiation was found following the stock enhancement program in the Bohai Sea. There was no significant difference between HS and WS in terms of growth parameters (P > .05) and feeding ecology (P > .05). Furthermore, the stomach contents of the two groups were basically identical and consisted mainly of fish. Therefore, overall, the hatchery-origin juveniles had already adapted to the natural area after the summer fishing moratorium.

Evaluation of 17 microsatellite markers for parentage testing and individual identification of domestic yak (Bos grunniens).

BackgroundYak (Bos grunniens) is the most important domestic animal for people living at high altitudes. Yak ordinarily feed by grazing, and this behavior impacts the accuracy of the pedigree record because it is difficult to control mating in grazing yak. This study aimed to evaluate the pedigree system and individual identification in polled yak.MethodsA total of 71 microsatellite loci were selected from the literature, mostly from the studies on cattle. A total of 35 microsatellite loci generated excellent PCR results and were evaluated for the parentage testing and individual identification of 236 unrelated polled yaks. A total of 17 of these 35 microsatellite loci had polymorphic information content (PIC) values greater than 0.5, and these loci were in Hardy–Weinberg equilibrium without linkage disequilibrium.ResultsUsing multiplex PCR, capillary electrophoresis, and genotyping, very high exclusion probabilities were obtained for the combined core set of 17 loci. The exclusion probability (PE) for one candidate parent when the genotype of the other parent is not known was 0.99718116. PE for one candidate parent when the genotype of the other parent is known was 0.99997381. PE for a known candidate parent pair was 0.99999998. The combined PEI (PE for identity of two unrelated individuals) and PESI (PE for identity of two siblings) were >0.99999999 and 0.99999899, respectively. These findings indicated that the combination of 17 microsatellite markers could be useful for efficient and reliable parentage testing and individual identification in polled yak.DiscussionMany microsatellite loci have been investigated for cattle paternity testing. Nevertheless, these loci cannot be directly applied to yak identification because the two bovid species have different genomic sequences and organization. A total of 17 loci were selected from 71 microsatellite loci based on efficient amplification, unambiguous genotyping, and high PIC values for polled yaks, and were suitable for parentage analysis in polled yak populations.

Unraveling the genetic background of the Yangambi Research Center cacao germplasm collection, DR Congo

The Democratic Republic of the Congo (DR Congo or DRC) has some of the most fertile soils in Africa to cultivate cacao, an important cash crop and source of income for many smallholder farmers. Although cacao is currently produced there on small scale, DRC has the potential to grow as a cacao-producing country, thereby supplying the increasing cacao demand in the global market. Since the introduction of cacao varieties in the late nineteenth century, selection and breeding experiments have been carried out based on phenotype, without any knowledge on the genetic background of the cultivars. Therefore, this study analyzes 62 Congolese accessions of the Centre de Recherche de Yangambi (CRY) and 51 accessions of international collections representing 10 reference groups and Trinitario cultivars using 14 microsatellite markers. Descriptive statistics revealed a high gene diversity and polymorphic information content (PIC) for most of the markers, of which mTcCIR 12, 37, and 60 were the most discriminative. Both Bayesian clustering and principal coordinate analysis (PCoA) revealed high-admixed ancestry of the CRY cultivars. The collection was divided in two clusters, of which the first formed a hybrid population, linked to Amelonado, Trinitario, Maranόn, and Nanay, and the second was assigned to predominantly Amelonado, followed by Nanay, Contamana, and Nacional. This high admixture level resulted from numerous hybridization and recombination events that took place in the previous century. The obtained knowledge is essential for efficient conservation, utilization, and selection of high-quality cacao cultivars, which are agronomical favored, disease resistant, and of superior flavor quality.

Inter Simple Sequence Repeat markers for analysis of molecular diversity and genetic structure of eighteen Dendrobium cultivars in Sri Lanka

The genus Dendrobium is one of the largest genera in the family Orchidaceae having more than thousand species over the world with diverse morphological characters. Dendrobium is a popular ornamental plant with complex genetic background which emphasize on the species identification at molecular level. The present study was aimed to identify Inter-Simple Sequence Repeat (ISSR) markers capable of detecting genetic polymorphism to characterize 18 hybrid, commercially available Dendrobium cultivars. Genomic DNA of each cultivar was extracted using CTAB method. A total of 17 different ISSR primers were evaluated. Only the reproducible bands were scored and number of different alleles (Na), number of effective alleles (Ne), Shannon’s Information Index (I), Expected heterozygosity (He), Unbiased expected heterozygosity (UHe), polymorphic percentage and polymorphic information content (PIC) of each primer were calculated. The highest Shannon’s Information Index (0.537±0.08) was recorded by the primer UBC 826 while the highest polymorphic information content (PIC) was generated by primer UBC 807. The PIC values of the primers were ranged from 0.0068 to 0.451, indicating that primers are moderately informative. In total, 631 bands representing 120 loci were amplified showing 85.71% - 100% polymorphism. The genetic similarities between individuals were compiled in the Nei’s genetic identity matrix in order to construct the UPGMA dendrogram. Principle component analysis (PCA) and clustering analysis were done to divide different cultivars into groups. The analysis revealed the presence of four major clusters and two minor clusters among the cultivars. The study suggested that the ISSR markers originated from eight primers 12, 155, UBC 807, UBC 812, UBC 826, UBC 835, UBC 841 and UBC 842 can be used in the detection of molecular variation among cultivars in the genus Dendrobium.

Simulated Disperser Analysis: determining the number of loci required to genetically identify dispersers.

Empirical genetic datasets used for estimating contemporary dispersal in wild populations and to correctly identify dispersers are rarely tested to determine if they are capable of providing accurate results. Here we test whether a genetic dataset provides sufficient information to accurately identify first-generation dispersers. Using microsatellite data from three wild populations of common starlings (Sturnus vulgaris), we artificially simulated dispersal of a subset of individuals; we term this ‘Simulated Disperser Analysis’. We then ran analyses for diminishing numbers of loci, to assess at which point simulated dispersers could no longer be correctly identified. Not surprisingly, the correct identification of dispersers varied significantly depending on the individual chosen to ‘disperse’, the number of loci used, whether loci had high or low Polymorphic Information Content and the location to which the dispersers were moved. A review of the literature revealed that studies that have implemented first-generation migrant detection to date have used on average 10 microsatellite loci. Our results suggest at least 27 loci are required to accurately identify dispersers in the study system evaluated here. We suggest that future studies use the approach we describe to determine the appropriate number of markers needed to accurately identify dispersers in their study system; the unique nature of natural systems means that the number of markers required for each study system will vary. Future studies can use Simulated Disperser Analysis on pilot data to test marker panels for robustness to contemporary dispersal identification, providing a powerful tool in the efficient and accurate design of studies using genetic data to estimate dispersal.

Characterization of olive progenies derived from a Tunisian breeding program by morphological traits and SSR markers

Abstract An olive breeding program was conducted in Tunisia since 1994 using controlled crosses and open pollination on 'Meski' cultivar mainly used for table olives in northern Tunisia. From a total of 200 progenies, thirteen genotypes were subsequently selected and are potentially suitable for both oil processing and table olive uses. The study aimed to identify the aforementioned genotypes as well as the pollen donors used for cross breeding using morphological and molecular approaches. The morphological study showed high variation between genotypes and permitted their repartition into two groups according to the main discriminative parameters, which are the fruit and endocarp weights based on principal component analysis. The parentage of thirteen olive progenies and the molecular identification of 26 olive cultivars, including 8 olive varieties used as the parents in the cross breeding program, were analyzed using a set of six co-dominant polymorphic Simple Sequence Repeats (SSRs) selected among the most suitable markers for olive identification. SSR analysis revealed a high polymorphic information content value and a low probability of identity among genotypes, indicating the efficiency of SSR for detecting genetic diversity among genotypes and their potential use for olive breeding. Progenies were grouped into two groups, according to the crosses. Transmission of alleles from parents to offspring was observed, in which ten of the thirteen progenies sampled were matched consistently to their maternal parent ‘Meski’ and the paternity was confirmed. Three did not match, but their parents were identified and they appear derivatives from other breeding crosses. These genotypes remain to be evaluated for their agronomic performances.

Development and characterization of fifteen polymorphic microsatellite loci in Bryconamericus aff. iheringii (Teleostei: Characidae) and cross-amplification in related Characidae species

ABSTRACT Data on 15 novel microsatellite loci from the Neotropical fish Bryconamericus aff. iheringii are presented here. Analyses of 32 individuals from four different streams revealed 192 different alleles, ranging from four to 32 alleles per locus (mean of 12.8 per locus). Observed and expected heterozygosities ranged from 0.094 to 0.813 and 0.205 to 0.952, respectively. These loci showed high polymorphic information content and will be a resource for genetic studies of B. aff. iheringii. Furthermore, several loci also amplified other small Neotropical Characidae (Piabarchus stramineus and Piabina argentea) and should be useful for these species.

Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in Brassica rapa

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.

Genetic diversity and population structure in tunisian castor genotypes (Ricinus communis L.) Detected using scot markers

Due to the chemical and physical properties of castor oil (Ricinus communis L.) that make it a valuable raw material for numerous industrial applications, including the production of biofuel, interest to develop more and better varieties has been increased. In the present study, the representatives of the genus castor collected from 12 different parts of Tunisia were differentiated by the DNA fingerprinting patterns using 37 SCoT primers. PCR amplification of DNA using 37 primers for SCoT analysis produced 268 DNA fragments that could be scored in all 56 genotypes of Tunisian castor. The number of amplified fragments varied from 4 (SCoT 45, SCoT 31 and ScoT 17) to 10 (SCoT 3, SCoT 11, SCoT 14, SCoT 18 and SCoT 12). Of the 268 amplified bands 230 were polymorphic, with an average of 6.22 polymorphic bands per primer. To determine the level of polymorphism in the analysed group of Tunisian castor genotypes polymorphic information content (PIC) was calculated. The lowest values of polymorphic information content were recorded for SCoT 17 (0.411) and the the highest PIC values were detected for SCoT 14 (0.868) with an average of 0.751. A dendrogram was constructed from a genetic distance matrix based on profiles of the 37 SCoT primers using the unweighted pair-group method with the arithmetic average (UPGMA). According to analysis, the collection of 56 Tunisian castor genotypes were clustered into two main clusters (1 and 2). Of the 56 genotypes of Tunisian castor, 2 unique genotypes were separated (BA-5 and K-4). Genetically the closest were two genotypes from Tunisian region Souassi (S-2 and S-5) in subclaster 2bc. Results showed the utility of SCoT markers for estimation of genetic diversity of castor genotypes leading to genotype identification.

Genetic diversity of Epimedium elatum (Morren & Decne) revealed by RAPD characterization

Epimedium elatum (Morren & Decne) is a rare perennial monotypic medicinal herb of Berberidaceae family, endemic to high altitude shady coniferous forests of Northwestern Himalayas in India. Traditionally, it has been used in local health care system for treatment of many ailments. In the present study, a total of ten RAPD markers were used for assessment of genetic diversity in twenty accessions of E. elatum, collected from diverse ecozones in Jammu and Kashmir. Ten primers amplified a total of 100 polymorphic bands with average of 10 bands per assay unit. Primer OPO-07 generated maximum number of bands (15) & produced highest polymorphic information content (0.39), marker index (5.96) and resolving power (8.9), thereby distinguishing E. elatum genotypes. The study showed moderate level of genetic polymorphism in twenty accessions of E. elatum. RAPD data set revealed association between genetic diversity and eco-geographic distribution of most E. elatum genotypes, and thereby proved useful tool for their genetic/molecular characterization. There is an urgent need for developing quick conservation strategies to save its natural germplasm from extinction in the Northwestern Himalayas.

Characterization of cultivated and wild species of Capsicum using microsatellite markers

Diversity of twenty four genotypes of hot pepper representing cultivated species Capsicum annuum, C. frutescens, C. baccatum and C. chinense as well as a wild species C. chacoense was analyzed in the present study using 99 microsatellite loci distributed uniformly throughout the genome. The 85 polymorphic loci, out of 99 simple sequence repeat (SSR) loci used, amplified a total of 192 alleles among the 24 genotypes with one to five allele per loci. The average number of alleles per loci was found to be 2.25. The highest polymorphism information content (PIC value) was observed to be 0.729 for the marker located on linkage group 6. Principal component analysis provided useful information regarding genetic relationship among genotypes as it distributed all the genotypes studied into three major groups each including different species. All the C. annuum genotypes were grouped together while other cultivated species formed a separate group. The C. chacoenese was the only wild species studied which, although, fell within the first group but was placed separately from C. annuum. Besides, all the Chilli leaf curl resistant genotypes were grouped together.

Genetic diversity analysis using ISSR markers in Black nightshade (Solanum nigrum L. Complex)

The genetic diversity and breeding potential among fifteen black nightshade genotypes (Solanum nigrum L. complex) was assessed using ISSR markers. Out of the 36 ISSR markers used in this study, 28 markers produced 151 amplicons of which 133 were polymorphic. The number of amplicons for each marker varied from 2 to 8. The percentage of polymorphism ranged from 50% to 100%. The polymorphic information content (PIC) ranged between 0.469 and 0.863. The highest PIC value was observed for the primer SN-ISSR26 and the lowest was observed for SN-ISSR21. The other marker efficiency parameters such as average band informativeness, resolving power, effective multiplex ratio and marker index were found be effective for the ISSR primers used in this study. The similarity coefficient value ranged from 0.413 to 1.000. The genetic distance between the genotypes collected from Chinnakalvehali (Sn 47) and Ottampatty (Sn 08) was lowest (0.413) and thus revealed that the genotypes were dissimilar, showing higher variation. Whereas the genotypes Sn 19 (Kallipalayam) and Sn 15 (Salem) showed higher similarity coefficient values (0.863) which indicated close genetic relationship between them. The results obtained in this study, showed that ISSR markers can efficiently differentiate the black nightshade genotypes.

Screening transferable microsatellite markers across genus Phalaenopsis (Orchidaceae)

BackgroundMolecular identification based on microsatellite loci is an important technology to improve the commercial breeding of the moth orchid. There are more than 30,000 cultivars have been enrolled at the Royal Horticultural Society (RHS). In this study, genomic microsatellite primer sets were developed from Phalaenopsis aphrodite subsp. formosana to further examine the transferability of across 21 Phalaenopsis species.Methods and resultsTwenty-eight polymorphic microsatellite markers were obtained using the magnetic bead enrichment method, with high transferability of the 21 species of the genus Phalaenopsis, especially in the subgenus Phalaenopsis. The 28 newly developed polymorphic microsatellite markers with high polymorphism information content values. The best and second fit grouping (K) are inferred as two and four by the ΔK evaluation in the assignment test. This result indicates that these microsatellite markers are discernible to subgenus Phalaenopsis.ConclusionsOur results indicate that these new microsatellite markers are useful for delimiting species within genus Phalaenopsis. As expected, the genetic relationships between species of subgenus Phalaenopsis can be well distinguished based on the assignment test. These molecular markers could apply to assess the paternity of Phalaenopsis as well as investigating hybridization among species of genus Phalaenopsis.

Genetic variability in Italian populations of Drosophila suzukii

BackgroundDrosophila suzukii is a highly destructive pest species, causing substantial economic losses in soft fruit production. To better understand migration patterns, gene flow and adaptation in invaded regions, we studied the genetic structure of D. suzukii collected across Italy, where it was first observed in 2008. In particular, we analysed 15 previously characterised Simple Sequence Repeat (SSR) markers to estimate genetic differentiation across the genome of 278 flies collected from nine populations.ResultsThe nine populations showed high allelic diversity, mainly due to very high heterozygosity. The high Polymorphism Information Content (PIC) index values (ranging from 0.68 to 0.84) indicated good discrimination power for the markers. Negative fixation index (FIS) values in seven of the populations indicated a low level of inbreeding, as suggested by the high number of alleles. STRUCTURE, Principal Coordinate and Neighbour Joining analysis also revealed that the Sicilian population was fairly divergent compared to other Italian populations. Moreover, migration was present across all populations, with the exception of the Sicilian one, confirming its isolation relative to the mainland.ConclusionsThis is the first study characterising the genetic structure of the invasive species D. suzukii in Italy. Our analysis showed extensive genetic homogeneity among D. suzukii collected in Italy. The relatively isolated Sicilian population suggests a largely human-mediated migration pattern, while the warm climate in this region allows the production of soft fruit, and the associated D. suzukii reproductive season occurring much earlier than on the rest of the peninsula.

Venetian Local Corn (Zea mays L.) Germplasm: Disclosing the Genetic Anatomy of Old Landraces Suited for Typical Cornmeal Mush Production

Due to growing concern for the genetic erosion of local varieties, four of the main corn landraces historically grown in Veneto (Italy)—Sponcio, Marano, Biancoperla and Rosso Piave—were characterized in this work. A total of 197 phenotypically representative plants collected from field populations were genotyped at 10 SSR marker loci, which were regularly distributed across the 10 genetic linkage groups and were previously characterized for high polymorphism information content (PIC), on average equal to 0.5. The population structure analysis based on this marker set revealed that 144 individuals could be assigned with strong ancestry association (>90%) to four distinct clusters, corresponding to the landraces used in this study. The remaining 53 individuals, mainly from Sponcio and Marano, showed admixed ancestry. Among all possible pairwise comparisons of individual plants, these two landraces exhibited the highest mean genetic similarity (approximately 67%), as graphically confirmed through ordination analyses based on PCoA centroids and UPGMA trees. Our findings support the hypothesis of direct gene flow between Sponcio and Marano, likely promoted by the geographical proximity of these two landraces and their overlapping cultivation areas. Conversely, consistent with its production mainly confined to the eastern area of the region, Rosso Piave scored the lowest genetic similarity (<59%) to the other three landraces and firmly grouped (with average membership of 89%) in a separate cluster, forming a molecularly distinguishable gene pool. The elite inbred B73 used as tester line scored very low estimates of genetic similarity (on average <45%) with all the landraces. Finally, although Biancoperla was represented at K = 4 by a single subgroup with individual memberships higher than 80% in almost all cases (57 of 62), when analyzed with an additional level of population structure for K = 6, it appeared to be entirely (100%) constituted by individuals with admixed ancestry. This suggests that the current population could be the result of repeated hybridization events between the two accessions currently bred in Veneto. The genetic characterization of these heritage landraces should prove very useful for monitoring and preventing further genetic erosion and genetic introgression, thus preserving their gene pools, phenotypic identities and qualitative traits for the future.

Study of Genetic Diversity among the Seeded Banana Found in the Northeast Region of India by RAPD (Randomly Amplified Polymorphic DNA) Analysis

The RAPD (Random Amplified Polymorphic DNA) technique was used to evaluate the genetic variation among twenty one wild bananas found in the northeastern region. Out of the ten primers used eight give 100% polymorphism. Among the ten primers used UBC-425 gives the highest polymorphic information content, resolving power as well as highest marker index. The highest dissimilarity index was obtained between the sample MZ1 (Changkha from Mizoram) and AR12 (kutikol from Arunachal) scoring the dissimilarity index of 0.85 whilst the lowest/highest dissimilarity/similarity was found between AS13 and AS14 (Athiakol and Bhimkol from Assam) scoring the dissimilarity index of 0.17. This result indicates that AS13 and AS14 were more closely related as compared with all the other accessions studied while MZ1 and AR12 were highly dissimilar. Dendrogram based on Nei’s coefficient reveals two major groups which were again sub-grouped into six minor clusters. The dendrogram was not able to clearly distinguish the different genome groups in the study. The study reveals significant level of diversity among the wild bananas studied and it could be utilized for future conservation and sustainable utilization of the genetic resources.

Investigation of Genetic Variation In Berberis Vulgaris Using ISSR and SSR Molecular Markers

Barberry fruit is a medicinal plant, and it is one of the most important horticultural crops in South Khorasan province, Iran. Genetic diversity has a basic role in the successful breeding of crop varieties with durable resistance to biotic and abiotic stresses. The main objective of this study was to assess the genetic diversity of 20 ecotypes of Berberis, which collected from different regions of South Khorasan province, Iran, using ISSR and SSR markers. In this study, 10 ISSR primers and 5 SSR primer pairs, with the amplification of suitable polymorphic alleles were used. A total of 98 bands for ISSR markers and 43 bands for SSR markers were detected between 300 to 1300 bp and 100 to 1100 bp in size, respectively. Polymorphic ISSR-7 and CA03 primers amplified the highest number of alleles with 17 and 24 bands, respectively, while ISCS50 and CA30 primers amplified only two polymorphic alleles. The ISCS57 and GA31 primers had the highest polymorphic information content (PIC) and ISCS50 and GA04 primers had the lowest PIC. The estimated Nei's and Shannon indices for genetic diversity in ISSR markers were 0.24 and 0.35, while for SSR markers these were 0.23 and 0.34, respectively. Based on cluster analysis, five and six main groups were identified for ISSR and SSR markers, showing high genetic variations among a set of collected barberry ecotypes. Analysis of molecular variances in both ISSR and SSR markers showed that high level of total variation was due to within populations, rather. Therefore, it will be better to select within populations in breeding programs.

Single-Tube Dodecaplex PCR Panel of Polymorphic Microsatellite Markers Closely Linked to the DMPK CTG Repeat for Preimplantation Genetic Diagnosis of Myotonic Dystrophy Type 1.

Preimplantation genetic diagnosis (PGD) of myotonic dystrophy type 1 (DM1) currently uses conventional PCR to detect nonexpanded dystrophia myotonica protein kinase (DMPK) alleles or triplet-primed PCR to detect the CTG-expanded alleles, coupled with analysis of linked microsatellite markers to increase diagnostic accuracy. We aimed to simplify the process of identification and selection of informative linked markers for application to DM1 PGD. An in silico search was performed to identify all markers within 1-1.5 Mb flanking the DMPK gene. Five previously known (D19S559, APOC2, D19S543, D19S112, and BV209569) and 7 novel (DM45050, DM45178, DM45209, DM45958, DM46513, DM46892, and DM47004.1) markers with potentially high heterozygosity values and polymorphism information content were selected and optimized in a single-tube multiplex PCR panel. Analysis of 184 DNA samples of Chinese and Caucasian individuals (91 from unrelated, anonymized cord blood of Chinese babies born at the National University Hospital, Singapore, and 93 Caucasian DNA samples from the Human Variation Panel HD100CAU) confirmed the high polymorphism indices of all markers (polymorphism information content >0.5), with observed heterozygosity values ranging from 0.62-0.93. All individuals were heterozygous for at least 6 markers, with 99.5% of individuals heterozygous for at least 2 markers on either side of the DMPK CTG repeat. The dodecaplex marker assay was successfully validated on 42 single cells and 12 whole genome amplified single cells. The DM1 multiplex PCR panel is suitable for use in DM1 PGD either as a standalone linkage-based assay or as a complement to DMPK CTG repeat expansion-mutation detection.

THE POTENTIAL USE OF SSR MARKERS TO SUPPORT THE MORPHOLOGICAL IDENTIFICATION OF INDONESIAN MUNGBEAN VARIETIES

<p>Mungbean varieties were mainly characterized based on morphological traits. Molecular genetic approach is expected to help the breeder in identification of mungbean varieties in more detail and to protect intellectual property right. This study aimed to identify Indonesian mungbean varieties based on DNA fingerprint profile using a marker set to support morphological characters. A total of 22 Indonesian mungbean accessions were characterized based on 21 morphological traits and 55 simple sequence repeats (SSRs) primers. Of the total 22 mungbean varieties used in the present study, 16 varieties were improved varieties and remaining six varieties were local varieties originated from Java, Nusa Tenggara and Sulawesi collected in GeneBank of ICABIOGRAD. The results showed that the 21 morphological characters were not sufficient to differentiate 22 mungbean varieties, while SSR analysis revealed that eight multi-alleles markers and high polymorphic information content (PIC) values have been successfully selected for varietal identification. The selected markers enabled to differentiate each mungbean variety according to their genetic marker with the lowest distance of 0.125, demonstrating the robustness of the selected marker set as a tool to identify a specific DNA fingerprint profile as a varietal identity (ID). The genetic identity of a variety was shown by digital barcoding which represented a series of alleles produced by corresponding markers. The DNA fingerprint profile of each variety would be beneficial as reference identities of a mungbean variety. <strong></strong></p>