Developing Restriction Associated DNA Tags for the Ion Torrent Personal Genome Machine
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Authors
Agyabeng-Dadzie, Fiifi
Issue Date
2014-08
Type
Thesis
Language
en_US
Keywords
Kryptolebias marmoratus , Biology , Kryptolebias ocellatus , Ion Torrent Personal Genome Machine , Restriction Associated DNA Tag , Restriction Fragment Length Polymorphisms , morphological studies , Valdosta State University aquatic lab , genotypic analysis , RAD-Tags , SNP analysis , AFLP , RFLP , cryptic species
Alternative Title
Abstract
The identification and classification of similar cryptic species such as
Kryptolebias marmoratusand Kryptolebias ocellatus,as distinct species has always been
problematic within the biological sciences. Though Kryptolebias marmoratusand
Kryptolebias ocellatusare both known to be self-fertilizing hermaphrodites, out crossing
has been observed resulting in heterozygous offspring. Recently, a viable hybrid (Gitmo)
between K. marmoratusand K. ocellatushas been isolated in the Valdosta State
University aquatic lab. This hybrid questions the proposed species status of these fishes.
Classifications of organisms are usually based on both morphological and
genotypic studies. Based on morphological studies, both fishes were thought to be
synonymous because of very similar anatomical and physiological characteristics.
Mitochondrial studies (genotypic analysis) ofthese fishes have suggested otherwise.
Unfortunately, thorough whole genome studies are very expensive and labour intensive.
Methods such as Restriction Fragment Length Polymorphisms (RFLP), Amplified
Fragment Length Polymorphisms (AFLP) and microsatellite analysis are more commonly used. These methods, though helpful, make analyses based on the length polymorphisms of the DNA fragments and only a small portion of the genome. Furthermore, surveys yield a very limited amount of genetic information.
A more reliable method of analyzing large numbers of these genotypic markers
concurrently would be extremely valuable for taxonomic questions. Developing
Restriction Associated DNA Tag (RADTag) genome sequencing using methods for the
Ion Torrent Personal Genome Machine (PGM) provides a way not only to cut down the
cost of genomic sequencing but also provides a method that performs thousands of Single Nucleotide Polymorphisms (SNP) analyses atonce, without the use of a reference
sequence. Analysis of RADTag sequences on public domains “Stacks” software
displayed thousands of markers present in the Hon9 (Kryptolebias marmoratus) genome
and confirms the Hon9 strain to be highly homozygous (0.999) across all loci. This
achievement paves the way for further genomic studies with high throughputs and
accuracy. This RADtag genome will provide the information needed for resolving the
questionable relationship between K. marmoratusand the presumptive K. ocellatus
cryptic species.