Influence of the PCR artifacts on the genotyping efficiency by the microsatellite loci using native polyacrylamide gel electrophoresis

Kulibaba, R. O.; Liashenko, Yuriy

doi:10.3103/s0095452716030087

Cited by 6 publications

(9 citation statements)

References 13 publications

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“…However, PCR amplification of genomic DNA using SSR markers can produce sequence artifacts because of errors in Taq DNA polymerase activity and the formation of chimeric and heteroduplex molecules [ 19 – 21 ]. The production of artifacts, particularly in the case of highly polymorphic SSR markers, can cause difficulties in allele size calling [ 22 ]. Alleles of the same sized products may have different sequences [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular genetic diversity and population structure analyses of rutabaga accessions from Nordic countries as revealed by single nucleotide polymorphism markers

Fredua-Agyeman

Hwang

et al. 2021

BMC Genomics

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Background Rutabaga or swede (Brassica napus ssp. napobrassica (L.) Hanelt) varies in root and leaf shape and colour, flesh colour, foliage growth habits, maturity date, seed quality parameters, disease resistance and other traits. Despite these morphological differences, no in-depth molecular analyses of genetic diversity have been conducted in this crop. Understanding this diversity is important for conservation and broadening the use of this resource. Results This study investigated the genetic diversity within and among 124 rutabaga accessions from five Nordic countries (Norway, Sweden, Finland, Denmark and Iceland) using a 15 K single nucleotide polymorphism (SNP) Brassica array. After excluding markers that did not amplify genomic DNA, monomorphic and low coverage site markers, the accessions were analyzedwith 6861 SNP markers. Allelic frequency statistics, including polymorphism information content (PIC), minor allele frequency (MAF) and mean expected heterozygosity ($$ \overline{H} $$ H ¯ e) and population differentiation statistics such as Wright’s F-statistics (FST) and analysis of molecular variance (AMOVA) indicated that the rutabaga accessions from Norway, Sweden, Finland and Denmark were not genetically different from each other. In contrast, accessions from these countries were significantly different from the accessions from Iceland (P < 0.05). Bayesian analysis with the software STRUCTURE placed 66.9% of the rutabaga accessions into three to four clusters, while the remaining 33.1% constituted admixtures. Three multivariate analyses: principal coordinate analysis (PCoA), the unweighted pair group method with arithmetic mean (UPGMA) and neighbour-joining (NJ) clustering methods grouped the 124 accessions into four to six subgroups. Conclusion Overall, the correlation of the accessions with their geographic origin was very low, except for the accessions from Iceland. Thus, Icelandic rutabaga accessions can offer valuable germplasm for crop improvement.

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Section: Introductionmentioning

confidence: 99%

Molecular genetic diversity and population structure analyses of rutabaga accessions from Nordic countries as revealed by single nucleotide polymorphism markers

Fredua-Agyeman

Hwang

et al. 2021

BMC Genomics

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“…Ideally, a perfect PCR means all the amplified sequences are exactly the same as the original template(s). However, in reality, artificial molecules are frequently produced (Cline et al, 1996; Sharifian, 2010; Kulibaba and Liashenko, 2016). These artifacts include those derived from mutation, recombination (Brakenhoff et al, 1991; Komatsoulis and Waterman, 1997; Haas et al, 2011), and heteroduplex formation when double-stranded DNA contain sites with non-complementary bases such as A–C and G–T pairs (L’Abbé et al, 1992; Thompson et al, 2002; Kulibaba and Liashenko, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…However, in reality, artificial molecules are frequently produced (Cline et al, 1996; Sharifian, 2010; Kulibaba and Liashenko, 2016). These artifacts include those derived from mutation, recombination (Brakenhoff et al, 1991; Komatsoulis and Waterman, 1997; Haas et al, 2011), and heteroduplex formation when double-stranded DNA contain sites with non-complementary bases such as A–C and G–T pairs (L’Abbé et al, 1992; Thompson et al, 2002; Kulibaba and Liashenko, 2016). Previous studies have shown that primer sequence matching, template length, the type of DNA polymerase, annealing temperature, extension time, and the number of PCR cycles could all contribute to the generation of PCR artifacts (Suzuki and Giovannoni, 1996; Polz and Cavanaugh, 1998; Suzuki et al, 1998; Ishii and Fukui, 2001; Kanagawa, 2003; Sharifian, 2010).…”

Section: Discussionmentioning

confidence: 99%

Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom

Zhou

Jiao

et al. 2019

Front. Microbiol.

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The polymerase chain reaction (PCR) is widely used in modern biology and medicine. However, PCR artifacts can complicate the interpretation of PCR-based results. The internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster is the consensus fungal barcode marker and suspected PCR artifacts have been reported in many studies, especially for the analyses of environmental fungal samples. At present, the patterns of PCR artifacts in the whole fungal ITS region (ITS1+5.8S+ITS2) are not known. In this study, we analyzed the error rates of PCR at three template complexity levels using the divergent copies of ITS from the mushroom Agaricus subrufescens. Our results showed that PCR using the Phusion® High-Fidelity DNA Polymerase has a per nucleotide error rate of about 4 × 10–6 per replication. Among the detected mutations, transitions were much more frequent than transversions, insertions, and deletions. When divergent alleles were mixed as templates in the same reaction, a significant proportion (∼30%) of recombinant molecules were detected. The in vitro mixed-template results were comparable to those obtained from using the genomic DNA of the original mushroom specimen as template. Our results indicate that caution should be in place when interpreting ITS sequences from individual fungal specimens, especially those containing divergent ITS copies. Similar results could also happen to PCR-based analyses of other multicopy DNA fragments as well as single-copy DNA sequences with divergent alleles in diploid organisms.

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“…The fragments longer then 1000–1200 bp for TBP analysis and 2000 bp for h‐TBP analysis are reproducible and are involved in differentiation of hetero‐ and/or homoduplex DNA populations, what may be a possible explanation for these high‐molecular fragments formation during the amplification. These DNA duplexes are visualized by electrophoresis in a polyacrylamide gel (Kulibaba and Liashenko, ).…”

Section: Discussionmentioning

confidence: 99%

Intron length polymorphism of β‐tubulin genes of Aegilops biuncialis Vis

Rabokon

Demkovych

Sozinov

et al. 2017

Cell Biology International

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Intron-specific DNA polymorphism is present among plant β-tubulin gene family members and is considered to be one of the molecular markers based on the difference of tubulin introns length assayed both separately (TBP: 1st intron) or in combination (h-TBP: 1st and 2nd introns). These two approaches are possibly useful for wheat breeding programs, since TBP and h-TBP help to differentiate between the accessions of Aegilops biuncialis Vis., a wild relative of wheat. PCR-derived polymorphic fragments were resolved by PAGE electrophoresis. The length of amplicons varied significantly (395-3900 bp for TBP and 466-3440 bp for h-TBP), while the numbers of polymorphic bands were 21 for TBP and 23 for h-TBP, respectively. PIC mean value was circa 0.3. Dendrograms constructed on the basis of the Nei and Li coefficient with the high bootstrap support reveal a similar order of hierarchy for the samples analyzed using both methods. Thus, both techniques uncover DNA polymorphism level sufficiently high to distinguish different accessions of Ae. biuncialis Vis.

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Influence of the PCR artifacts on the genotyping efficiency by the microsatellite loci using native polyacrylamide gel electrophoresis

Cited by 6 publications

References 13 publications

Molecular genetic diversity and population structure analyses of rutabaga accessions from Nordic countries as revealed by single nucleotide polymorphism markers

Molecular genetic diversity and population structure analyses of rutabaga accessions from Nordic countries as revealed by single nucleotide polymorphism markers

Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom

Intron length polymorphism of β‐tubulin genes of Aegilops biuncialis Vis

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