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Validation of phylogenetic signals in amplified fragment length data: testing the utility and reliability in closely related taxa.

Wooten JA, Tolley-Jordan LR - BMC Res Notes (2009)

Bottom Line: The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher.We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results.We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Alabama Department of Biological Sciences, Box 870344, Tuscaloosa, AL 35487, USA. wootenj@franklin.edu

ABSTRACT

Background: Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher. Modification of this protocol for new labs is intimidating and time-consuming, particularly for taxa in which AFLP have not been previously developed. Furthermore, determining what constitutes quality output during different stages of fragment generation is not well defined and this may further hinder the use AFLP by new researchers.

Findings: We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results. We sufficiently normalized peak intensity and standardized allele calling across all samples for each primer combination. Repeatability was assessed with a phylogenetic tree in which replicate samples clustered together using the minimum evolution procedure. We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster. To minimize offset allele positions, we suggest that researchers analyze different primer combinations at the same time using multiple dyes with the automated sequencer to minimize mismatched alleles across replicates.

Conclusion: For researchers wanting to use AFLP, this molecular technique is difficult and time-consuming to develop. Clarifying what constitutes quality output for each step in AFLP generation will help to reduce redundant trials in protocol development and, in turn, advance the discipline of population genetics.

No MeSH data available.


Step-by-step protocol for AFLP generation. Schematic representation of each step in the AFLP protocol represented on 1% sodium borate agarose gels. All gel images were generated from undiluted DNA solutions. Each gel image contains lanes that represent acceptable products as indicated by a unique symbol. The 100 bp ladder is denoted by # in all gel images.
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Figure 1: Step-by-step protocol for AFLP generation. Schematic representation of each step in the AFLP protocol represented on 1% sodium borate agarose gels. All gel images were generated from undiluted DNA solutions. Each gel image contains lanes that represent acceptable products as indicated by a unique symbol. The 100 bp ladder is denoted by # in all gel images.

Mentions: Snails (Elimia) and salamanders (Desmognathus) were used to evaluate the standardized AFLP protocol. Two different DNA-extraction techniques were employed to achieve high-quality, whole genomic DNA. The DNA was considered high quality if there was an optical density (OD) of 260/230 between 1.8 – 2.1 and 260/280 between 1.8 – 1.9 using a spectrophotometer (NanoDrop ND – 1000). Each sample was visually inspected on 1% sodium borate agarose gels (Figure 1a).


Validation of phylogenetic signals in amplified fragment length data: testing the utility and reliability in closely related taxa.

Wooten JA, Tolley-Jordan LR - BMC Res Notes (2009)

Step-by-step protocol for AFLP generation. Schematic representation of each step in the AFLP protocol represented on 1% sodium borate agarose gels. All gel images were generated from undiluted DNA solutions. Each gel image contains lanes that represent acceptable products as indicated by a unique symbol. The 100 bp ladder is denoted by # in all gel images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654459&req=5

Figure 1: Step-by-step protocol for AFLP generation. Schematic representation of each step in the AFLP protocol represented on 1% sodium borate agarose gels. All gel images were generated from undiluted DNA solutions. Each gel image contains lanes that represent acceptable products as indicated by a unique symbol. The 100 bp ladder is denoted by # in all gel images.
Mentions: Snails (Elimia) and salamanders (Desmognathus) were used to evaluate the standardized AFLP protocol. Two different DNA-extraction techniques were employed to achieve high-quality, whole genomic DNA. The DNA was considered high quality if there was an optical density (OD) of 260/230 between 1.8 – 2.1 and 260/280 between 1.8 – 1.9 using a spectrophotometer (NanoDrop ND – 1000). Each sample was visually inspected on 1% sodium borate agarose gels (Figure 1a).

Bottom Line: The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher.We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results.We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Alabama Department of Biological Sciences, Box 870344, Tuscaloosa, AL 35487, USA. wootenj@franklin.edu

ABSTRACT

Background: Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher. Modification of this protocol for new labs is intimidating and time-consuming, particularly for taxa in which AFLP have not been previously developed. Furthermore, determining what constitutes quality output during different stages of fragment generation is not well defined and this may further hinder the use AFLP by new researchers.

Findings: We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results. We sufficiently normalized peak intensity and standardized allele calling across all samples for each primer combination. Repeatability was assessed with a phylogenetic tree in which replicate samples clustered together using the minimum evolution procedure. We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster. To minimize offset allele positions, we suggest that researchers analyze different primer combinations at the same time using multiple dyes with the automated sequencer to minimize mismatched alleles across replicates.

Conclusion: For researchers wanting to use AFLP, this molecular technique is difficult and time-consuming to develop. Clarifying what constitutes quality output for each step in AFLP generation will help to reduce redundant trials in protocol development and, in turn, advance the discipline of population genetics.

No MeSH data available.