Markers For DNA Fingerprinting Research Articles

SSR marker analysis and DNA fingerprinting of mutant rice variety VTL 10 (Lavanya)

VTL-3 is a tall high yielding saline tolerant rice variety developed by Rice Research Station, Vyttila in 1987, which is having a tendency to lodge at maturity. Researchers hence, developed semi-tall lines of VTL-3 using induced mutagenesis. The selected semi-tall mutants were screened with markers linked to Saltol QTL and it was found that this region was not affected in the mutant. A promising line selected after comparative yield trials was later released as VTL 10, Lavanya in the year 2018. In order to differentiate the variety Lavanya from its parent VTL 3, SSR marker analysis and DNA fingerprinting was carried out. These two varieties were screened with 48 SSR markers for DNA fingerprinting and eight markers were found to be polymorphic between VTL-10 andVTL-3. The polymorphic markers include RM600, RM3362,RM279, RM7, RM85, RM149, RM144 and RM1880. The number of alleles ranged from 1 to 3 and the size of PCR products ranged from 110 to 300bp with Polymorphic Information Content (PIC) from 0 to 0.38. From the present study, it was observed that these eight markers can be used to distinguish the mutant line VTL-10 from its original parent VTL-3.

Transcriptome-derived SSR markers for DNA fingerprinting and inter-populations genetic diversity assessment of Atractylodes chinensis

Atractylodes chinensis (fam. Asteraceae) is an important medicinal plant due to its unique pharmacological activity. The species is widely distributed in most areas of northern China. It is difficult to identify different populations of A. chinensis due to their similarity in characteristics. This study was the first investigation to date that assessed the genetic diversity of A. chinensis from different geographical counties of northern China using simple sequence repeat (SSR) markers. Of the 106 SSR primers in the clusters classified in the sesquiterpenoid biosynthesis pathway in the transcriptomic database of A. chinensis, ten with high polymorphism were used to analyze the inter-populations genetic diversity and construct DNA fingerprinting of 19 A. chinensis populations. A total of 78 alleles were detected, with an average number of 6.5 alleles per primer. The PIC value ranged from 0.4748 to 0.8918 with a mean of 0.6265. The neighbor-joining tree was used to classify 19 populations of A. chinensis into three clusters. DNA fingerprinting was performed according to these ten SSR markers. The results revealed that geographic origin is not exactly related to genetic diversity, as populations belonging to different provinces are grouped in the same cluster. The results of this study confirm that SSR markers are effective for genetic diversity analysis. The inter-populations genetic diversity and fingerprinting of A. chinensis in this study could provide a scientific basis for species identification and selective breeding.

Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough

Molecular markers are valuable tools for assessing the genetic variation in yeast. Here, we investigated the utility of SCoT markers for the genetic characterization of yeast strains at inter and intraspecies levels. A total of 345 endogenous yeast strains were isolated from 65 Type I sourdough samples collected from six different regions of Turkey. The seven SCoT primers produced 221 bands, of which 95.47% were polymorphic. Each primer could successfully differentiate species, supported by PIC and RP values. The ITS sequencing of isolates selected from the UPGMA dendrogram revealed that Saccharomyces cerevisiae predominated the microflora, followed by Kazachstania servazzii, K. humilis, Wickerhamomyces anomalus, Torulaspora delbrueckii, and Pichia kudriavzevii, respectively. The AMOVA revealed a high genetic variation between (49%) and within populations (51%) for S. cerevisiae. The high gene flow observed among S. cerevisiae populations suggests that it may have contributed to the geographical evolution of S. cerevisiae via the transportation of the sourdough samples. The different geographical origins were most likely to group separately on the UPGMA and PCoA. Saccharomyces cerevisiae strains from more distant populations generally displayed more significant genetic variation. SCoT markers can successfully be used alone or with the other existing DNA markers for DNA fingerprinting and analyzing the genetic variation between and within species.

Molecular genotypic diversity of populations of brinjal shoot and fruit borer, Leucinodes orbonalis and development of SCAR marker for pesticide resistance.

The brinjal shoot and fruit borer, Leucinodes orbonalis is a destructive pest of Solanum melongena. The control of L. orbonalis with extensive application of synthetic chemical insecticides resulted in the development of resistance with known genetic heterogeneity among populations. Understanding the genetic diversity of their populations is important in developing strategies for their management. The present investigation was performed to characterize populations of L. orbonalis for their genetic diversity in the entire region of Tamil Nadu, South India using random amplified polymorphic DNA (RAPD) primers as a tool of the molecular marker. Among 60 random 10-mer primers, only ten primers generated reproducible and scorable banding profile. Among the ten different random primers, the primers namely OPG 7, OPG 8, OPS 2 and OPS 7 generated the highest genetic variation with over 80% genetic polymorphism. Phylogram analysis produced 18 clusters with eight major and ten minor clusters. Cluster analysis, statistical fitness, population structure and analysis of molecular variance confirmed the significant genetic variation among different populations. A trait specific marker obtained through RAPD was cloned, sequenced and used to develop a stable diagnostic SCAR marker for DNA fingerprinting to distinguish the populations. Amplification of this locus in the samples of 20 different populations indicated recognition of the trait for pesticide resistance in 12 populations. The results suggest that the biochemical nature of host plant varieties of this insect pest and variation in the application of different insecticides are essential contributing factors for the genotypic variations observed among populations of L. orbonalis.

Assessment of Genetic Purity in Rice Using Polymorphic SSR Markers and Its Economic Analysis with Grow-Out-Test

Genetic purity is conventionally performed through grow-out-test (GOT) with morphological characters. Simple sequence repeat (SSR) markers are independent of G X E interaction. Herewith, 16 high yielding varieties of rice were analysed using 55 SSR markers for DNA fingerprinting and identification of genetic impurities: 14 were found to be polymorphic and amplified 48 alleles with an average of 3.43 alleles per each primer pair. The number of alleles amplified ranged from 2 to 6 and the size of the PCR products amplified from these 14 primer pairs ranged from 80 to 450 bp with polymorphic information content (PIC) from 0.14 (RM 346) to 0.99 (RM 5900). PICs at 0.5 or higher are highly informative SSR markers for genetic studies and the study reported (7/14) markers to be highly informative based on PIC values. Besides, the values of effective multiplex ratio, marker index and resolving power for the selected polymorphic primer pairs disclosed that the SSR markers used in the study are highly informative and could be potentially used for distinctness, uniformity and stability (DUS) testing, genetic purity analysis and DNA fingerprinting of rice varieties. Economic analysis of grow-out-test and SSR marker based genetic purity showed that the GOT process incurs Rs. 5.8 (INR) per seed (USD 0.07), while DNA-based marker analysis incurs Rs. 53.75 (INR) or $ 0.71 (USD).

Utilizing Start Codon Targeted (SCoT) markers for DNA fingerprinting and diversity analysis of Artemisia herba-alba plant species

Utilizing Start Codon Targeted (SCoT) markers for DNA fingerprinting and diversity analysis of Artemisia herba-alba plant species

Development of a core SNP panel for cacao (Theobroma cacao L.) identity analysis.

Single nucleotide polymorphisms (SNPs) are preferred markers for DNA fingerprinting and diversity studies in cacao (Theobroma cacao L.). Yet, a consensus SNP panel with a minimum number of SNPs for optimal identity analysis is unavailable for cacao. An initial set of 146 SNP panels of varying sizes were assembled based on heterozygosity, linkage disequilibrium (LD), linkage group (LG) distribution, major allele frequency, minor allele frequency (MiAF), polymorphism information content (PIC), and random distribution. These panels were assessed to determine their ability to distinguish among a training set of 155 accessions. The panels with the best separation ability were supplemented with additional SNPs to create 16 designer panels, which separated all 155 accessions. The 16 designer SNP panels were then assessed on a dataset of 1220 accessions coming from 10 ancestral groups. Increasing the number of SNPs generally yielded improved resolution of genetic identities with concomitant reduction of synonymous groups. The number and choice of SNPs were critical factors with LD, MiAF, and PIC being important selection attributes but an even LG distribution was unnecessary. A robust set of 96 SNPs is recommended as a minimal core SNP panel for cacao DNA fingerprinting to the international cacao community.

Development and Application of EST-SSR Markers for DNA Fingerprinting and Genetic Diversity Analysis of the Main Cultivars of Black Locust (Robinia pseudoacacia L.) in China

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement. More EST-SSR (Expressed sequence tag simple sequence repeat) markers of black locust can be used as a complement and improvement of Genomic-SSR markers for the identification of the function of gene and the construction of genetic map. Additionally, currently there is no simple method for identifying black locust cultivars. In this study, we obtained 2702 unigenes from 3095 expressed sequence tags (ESTs) from the National Center of Biotechnology Information (NCBI) database to identify simple sequence repeats (SSRs) in R. pseudoacacia samples. A total of 170 SSR loci were found to be distributed in 162 non-redundant sequences with a frequency of 6.29%. Dinucleotide repeats were the most predominant types among microsatellites (62.35%), followed by tri-nucleotide repeats (25.88%); the remaining SSRs accounted for less than 12%. The repeat motifs AG/TC (29.25%) and CT/GA (29.25%) were the most abundant among dinucleotides, and AAT/TTA (15.91%) was the most common among tri-nucleotides. A total of 62 primer pairs were designed to screen polymorphic and stable SSR loci. The resulting 25 EST-SSR markers capable of amplifying polymorphic, stable, and repeatable products. Eight newly developed EST-SSR markers and four published SSR markers were selected for DNA fingerprinting and genetic diversity analysis of the 123 main R. pseudoacacia cultivars in China. The 12 SSR loci amplified 102 alleles, with an average number of alleles per locus of 8.5 (range 4–15). The average polymorphism information content at the 12 SSR loci for the 123 cultivars was 0.670 (range 0.427–0.881). The 123 cultivars clustered into six main groups based on similarity coefficients, with most cultivars in one subgroup. Fingerprinting was performed using eight SSR markers; 110 black locust cultivars were distinguished. The results of this study increase the availability of EST-SSR markers in black locust and make it a simple method for checking the collection, the certification, and the correct attribution of clones and cultivars.

Genetic diversity of Phytophthora palmivora isolates from Indonesia and Japan using rep-PCR and microsatellite markers

Phytophthora palmivora infects hundreds of plant species around the world. Determining the genetic diversity among isolates is necessary to establish more effective control strategies, manage resistance and understand epidemiology, long-term evolution of the pathogen, host–pathogen coevolution, and evolutionary processes. Here genetic diversity among 81 isolates of P. palmivora from various host plants and geographical regions in Indonesia and Japan was evaluated using rep-PCR (BOX, ERIC, REP and M13) and microsatellite markers. With both methods, the isolates clustered into six groups, which corresponded more to geographic regions rather than host plants or mating types, although the respective members of each group were not always consistent between the two methods. Isolates from Indonesia, Japan and other countries clearly clustered into separate groups in the rep-PCR analysis. The rep-PCR consistently grouped the isolates from Sumatera Island with some isolates from Sulawesi, but microsatellite markers grouped them with those from the Java islands. A typical A1-mating type from one region grouped in one cluster using both methods. Both DNA fingerprinting markers also grouped the isolates from Nusa Tenggara and Papua islands with those from Japan. Genetic variation was recognized within and among populations of P. palmivora based on geographical areas, host plants and mating types. Possible explanations for the results and suggested strategies for disease management are discussed.

IMEC: Online Marker Efficiency Calculator.

Premise of the StudyTo accurately design plant genetic studies, the information content of utilized markers and primers must be calculated. Plant genotyping studies should take into account the efficiency of each marker system by calculating different parameters to find the optimal combination of primers. This can be problematic because there are currently no easily accessible applications that can be used to calculate multiple indices together.Methods and ResultsThe program Online Marker Efficiency Calculator (iMEC) was developed using R for the simple computation of seven polymorphism indices (heterozygosity index, polymorphism information content, discriminating power, effective multiplex ratio, marker index, arithmetic mean heterozygosity, and resolving power). These indices are based on dominant and codominant DNA fingerprinting markers, thus allowing comparison and selection of optimal genetic markers for a given data set.ConclusionsiMEC simplifies the calculation of diverse indices for the marker of choice to better enable researchers to measure polymorphism information for individual markers. The program is available at https://irscope.shinyapps.io/iMEC/.

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.

Relationship Among Establishment Durations, Kin Relatedness, Aggressiveness, and Distance Between Populations of Eight Invasive Argentine Ant (Hymenoptera: Formicidae) Supercolonies in Japan.

We investigated kin relatedness and kin-recognition abilities of the Argentine ant, Linepithema humile (Mayr), an invader from North America that has pervaded Japan for 20 yr, using genetic analyses and behavioral bioassays. From these data and interactions among factors, we formulated an eradication and management time-scale pattern diagram. Relatedness within a colony using microsatellite markers was effectively zero, whereas relatedness estimated by multilocus DNA fingerprinting markers was relatively high. Specifically, relatedness of recently invaded populations was estimated at nearly 0.3. From the results of behavioral bioassays on the invading populations of the Argentine ant, all colonies except the Kobe supercolonies did not show clearly aggressive behaviors toward workers belonging to other colonies, even when distantly located. Because they are critical factors for eradicating and managing invasive organisms, we assessed the relationships among kin relatedness using multilocus DNA fingerprinting and microsatellite markers, with aggressiveness, in 2011 and 2012, including the establishment durations, and distances among supercolonies. A generalized linear model (GLM) analysis, with establishment durations as an explanatory variable, strongly contributed to explaining estimated relatedness from the two methods. Specifically, models using kin relatedness for both multilocus DNA fingerprinting and microsatellite markers provided the strongest contribution to explaining the establishment durations. Within 3 yr after establishment in a native area, eradication is possible because of their low genetic diversity and small colony size. After 15 yr, eradication will be more difficult, but it is preferable to just monitor the impact for a nonnative ecosystem.

Evidences towards a Genetic Differentiation and Karyotype Evolution of the Sedges Cyperus odoratus L and C Ligularis L (Cyperaceae)

Cyperaceae is composed of pioneer species and presents a complex taxonomy, with genera like Cyperus presenting specific complexes. The aim of this study was to analyze the genetic diversity between the broadly distributed sedges Cyperus ligularis L. and C. odoratus L using DNA fingerprinting markers and cytogenetic features, focusing on collection sites from an Atlantic Forest reserve and its surroundings in the city of Recife (Pernambuco, Brazil). Both the significative genetic diversity (GST = 0.363) and the low gene flow (Nm = 0.877) were found and indicated a clear genetic differentiation between the two species. Moreover, the clustering analysis showed the presence of two distinct genetic groups, also suggesting no evidence of hybridization between C. ligularis and C. odoratus. Regarding the phenogram topology, the species are also two clear different lineages, and although all individuals of C. odoratus were collected in nearby plots, they showed a greater intraspecific genetic variability than that observed among individuals of C. ligularis, which were sampled in a wider geographic range. However, the variation in chromosome numbers within the two species showed an opposite pattern, indicating higher karyotype stability in C. odoratus. Furthermore, the lowest genetic differentiation in C. ligularis may reflect a founder effect associated with the ability to disperse seeds and colonize multiple areas. Further field observations, as well as an analysis of reproductive biology, should enrich the understanding of the genetic structure of the populations investigated and the role of these plants in successional processes.

Evaluation of the growth traits of Toxicodendron vernicifluum progeny based on their genetic groups assigned using new microsatellite markers

AbstractToxicodendron vernicifluum (Stokes) F. A. Barkley is a tree species cultivated in Japan for production of Japanese lacquer. To facilitate the low-cost improvement of T. vernicifluum, we developed microsatellite markers for DNA fingerprinting and family and lineage reconstruction. Nine useful microsatellites were developed, with 3 to 23 alleles per locus and an expected heterozygosity of 0.162-0.838 based on a commercially planted population that included 783 offspring. Six clusters were detected in this population based on the Bayesian clustering method, and 93 half-sib families were identified using parentage and sibship assignment analysis based on a maximum likelihood method. Many members (40-96%) of large (≥10) half-sib families included specific clusters, and members from different families included the same clusters. The cluster effect for tree height was more significant than the half-sib family effect using a linear mixed model, although these effects were not significant for other traits (diameter at breast height and number of forked trunks). Based on the findings from pedigree reconstruction, backward selection for tree height seems possible. We discuss the direction of research for improving T. vernicifluum using our proposed approach.

Clonal diversity and clone formation in the parthenogenetic Caucasian rock Lizard Darevskia dahli [corrected

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.

Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritordeums and their respective parents.

Hexaploid tritordeum (H(ch)H(ch)AABB; 2n = 42) results from the cross between Hordeum chilense (H(ch)H(ch); 2n = 14) and cultivated durum wheat (Triticum turgidum ssp. durum (AABB; 2n = 28). Morphologically, tritordeum resembles the wheat parent, showing promise for agriculture and wheat breeding. Start Codon Targeted (SCoT) polymorphism is a recently developed technique that generates gene-targeted markers. Thus, we considered it interesting to evaluate its potential for the DNA fingerprinting of newly synthesized hexaploid tritordeums and their respective parents. In this study, 60 SCoT primers were tested, and 18 and 19 of them revealed SCoT polymorphisms in the newly synthesized tritordeum lines HT27 and HT22, respectively, and their parents. An analysis of the presence/absence of bands among tritordeums and their parents revealed three types of polymorphic markers: (i) shared by tritordeums and one of their parents, (ii) exclusively amplified in tritordeums, and (iii) exclusively amplified in the parents. No polymorphism was detected among individuals of each parental species. Three SCoT markers were exclusively amplified in tritordeums of lines HT22 and HT27, being considered as polyploidization-induced rearrangements. About 70% of the SCoT markers of H. chilense origin were not transmitted to the allopolyploids of both lines, and most of the SCoTs scored in the newly synthesized allopolyploids originated from wheat, reinforcing the potential use of tritordeum as an alternative crop.

Clonal Diversity and Clone Formation in the Parthenogenetic Caucasian Rock Lizard Darevskia dahli

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.

Molecular markers and their application in genetic diversity of penaeid shrimp

Penaeidae is a family of shrimp, and it contains many species of economic importance, such as the tiger prawn (Penaeus monodon), white leg shrimp, Atlantic white shrimp and Indian prawn. Identification and population genotype structure of penaeid shrimp have been enhanced by molecular markers that can be classified into three types, namely allozyme, mitochondrial and nuclear markers. The widely used mitochondrial DNA markers are 12S rDNA, 16S rDNA, cytochrome b and control region. Random amplification of polymorphic DNA, amplified fragment length polymorphism, restriction fragment length polymorphism, single-stranded conformational polymorphism and microsatellites are the most commonly used nuclear markers for DNA fingerprinting. Molecular markers play a crucial role in penaeid shrimp to evaluate phenotypic and genetic variation, assess demographic bottleneck, study natural population structure, compare wild and hatchery populations, preserve genetic biodiversity, construct chromosome maps and detect whether genetic tag propagation–assisted rehabilitation programs are effective. Increase in the number of molecular markers, construction of high-density genetic maps and implementation of genomic resources (including genome sequencing) are expected to provide tools for the genetic improvement in these aquaculture species through marker-assisted selection. Molecular markers are versatile tools for the identification of populations with genetic crisis by comparing genetic diversities, which helps to establish management units within these threatened species.

Use of simple sequence repeat markers for DNA fingerprinting and diversity analysis of sugarcane (Saccharum spp) cultivars resistant and susceptible to red rot

Red rod is an economically important disease of sugarcane caused by the fungus Colletotrichum falcatum. We used a simple sequence repeat (SSR)-based marker system to identify and analyze genetic relationships of red rot resistant and susceptible sugarcane cultivars grown in Pakistan. Twenty-one highly polymorphic SSR markers were used for DNA fingerprinting and genetic diversity analysis of 20 sugarcane cultivars. These SSR markers were found to be highly robust; we identified 144 alleles, with 3-11 alleles per marker and a mean of 6.8. Three SSR markers were able to identify all 20 cultivars. DNAMAN(®)-generated homology tree was used to analyze genetic diversity among these cultivars; all cultivars shared 58% or more similarity. We correlated polymorphism information content and resolving power values with marker effectiveness in the process of sugarcane cultivar identification. We concluded that a small number of SSR-derived DNA markers will allow breeders to identify red rot resistant and susceptible cultivars.

Amplified fragment length polymorphism markers for DNA fingerprinting in the genus Salvia

The interspecific relationships among 51 worldwide collected accessions of Salvia have been investigated using the amplified fragment length polymorphism (AFLP) technique. The assessed genetic similarities allowed us to group the genotypes into two main clusters according to their geographical origin. Our results are encouraging for further characterization of the genus with the aim to clarify Salvia taxonomy.