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Species-specific DNA markers for improving the genetic management of tilapia

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Penilaian

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he tilapias are a group of African and Middle Eastern cichlid fish that are widely
cultured in both developed and developing countries, with total world aquaculture
production of 4,507,002 t and total value of 7,656,257,000 USD in 2012. With
many different species and sub-species of tilapia, and extensive use of interspecies
hybrids, identification of tilapia species is of importance in aquaculture and in
wild populations where introductions have occurred.
A variety of marker technologies have become important tools for genetics
research: for example, biochemical methods including allozymes and molecular
techniques such as mitochondrial DNA (mtDNA), Randomly Amplified
Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphic (AFLP),
and microsatellites. These markers do, however, have drawbacks. Restriction Site
Associated DNA (RAD) sequencing is a next generation sequencing technique
that provides a reliable tool to determine the presence/absence of Single
Nucleotide Polymorphisms (SNPs) among species and sub-species.
This research set out to discover differences in DNA sequences that distinguish
between tilapia species and sub-species, by retrieving species-specific nuclear
DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions
of the ADA gene (which codes for an isozyme that has been used for species
identification); and (ii) next-generation sequencing (RADseq), both standard RAD
and double-digest (ddRADseq). The mitochondrial DNA (mtDNA) marker
cytochrome c oxidase subunit I (COI) was also used to verify tilapia species
status. The ultimate purpose of this study is to develop markers that can be
applied to important questions, such as identification of tilapia species hybrids,
and introgression between species in aquaculture and in the wild.
The first approach based on ADA gene sequence was partially successful,
generating SNP markers that distinguished some species pairs. Of 10 SNP markers
discovered in Oreochromis spp (O. niloticus, O. mossambicus, O. aureus and O. u.
[Mochamad Syaifudin] [Institute of Aquaculture] [September 2015]
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hornorum) and Tilapia zillii, four work well to distinguish one species from nine
other tilapia species in this study. These were T. zillii (Tzil_3_M170), O. aureus
(Oaur_3_R122), O. aureus (Oaur_7_R626) and O. mossambicus
(Omoss_10_Y879), which indicated homozygous genotypes based on
KBioscience Competitive Allele Specific PCR (KASP) assay. The gene tree
indicated T. zillii was the most genetically distinct with the greatest genetic
distances among samples, while all Oreochromis spp were clustered together but
separated between species.
Most species could be discriminated using the COI sequence, with two exceptions.
O. andersonii and O. macrochir could not be distinguished. West African O.
niloticus exhibited COI haplotypes typical of O. aureus, as previously reported in
the literature, although nuclear markers clearly indicated the differences between
these two species. It also did not resolve the phylogenetic positions of S.
melanotheron and S. galilaeus. As a marker for species discrimination, COI has
limitations as it is a single, maternally inherited marker, and thus of limited use in
analysing cases of hybridization/introgression. However, it is still likely to be
useful in combination with multiple nuclear DNA markers.
Standard RAD sequence data based on reference based analysis (RBA, i.e. using
the subset of RAD loci found in the reference tilapia genome) identified 1,613
SNPs in 1,002 RAD loci shared by 75% of the individual analysed (shared RAD
loci) among seven tilapia species: Oreochromis aureus, O. karongae, O.
mossambicus, O. niloticus, O. urolepis, Sarotherodon galilaeus, T. zillii and an
outgroup cichlid species, Pelvicachromis pulcher. A phylogenetic tree based on
shared SNP markers in the RBA showed a very similar pattern to the consensus
derived from earlier molecular marker-based analyses. Further analysis detected
677 species-specific SNP markers for the seven tilapia species (i.e., allele[s]
unique to a single species). Physical mapping of the species-specific SNP markers
onto the O. niloticus genome assembly showed that they were relatively evenly
distributed across the genome, ranging from 0.47 SNPs/Mb in linkage group
(LG) 3 to 1.53 SNPs/Mb in LG 9.
[Abstract]
[Mochamad Syaifudin] [Institute of Aquaculture] [September 2015]
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The RADseq analysis produced many markers but was limited in the number of
individuals that could be included in a sequencing run, and the cost per run (hence
only a small number of individuals was analysed from a single population per
species). Double Digest Restriction Site Associated DNA (ddRAD) sequencing is
cheaper and the analysis based on this technique included a much broader range of
tilapia species, sub-species and populations. The ddRADseq in de novo based
analysis (DBA) identified 1,358 SNPs in 825 shared RAD loci, while the
reference based analysis (RBA) detected 938 SNPs in 571 shared RAD loci
among 10 tilapia species. A phylogenetic tree based on shared SNP markers
of both analyses (DBA and RBA) indicated a similar pattern to most prior
phylogenies based on other characteristics. Further analysis ascertained only
38 species-specific SNP markers (i.e. with an allele unique to that single
species) with minimum 75% loci present across the 10 tilapia species
(including two subspecies in O. niloticus, and at least two populations of the most
important aquaculture species). These SNP markers were distributed as follows:
O. karongae (2), O. aureus (1), O. macrochir (1), S. galilaeus (3), S.
melanotheron (5) and Tilapia zillii (26) but none for O. mossambicus, O.
niloticus, O. andersonii and O. urolepis hornorum. However, a set of
diagnostic SNP markers were identified from a subset of four economically
important species, which are often involved in hybridization in aquaculture: both
sub-species in O. niloticus (7 SNPs), O. aureus (13), O. mossambicus (10) and
O. u. hornorum (7). These SNP markers distinguished each species from the
other three, and a larger number of SNP markers distinguished between species
pairs within this group
Overall, SNP discovery based on the coding sequence of the ADA gene retrieved
only a few markers that discriminated a few species pairs. The DNA barcode COI
had limitations but is still likely to be useful in combination with multiple nuclear
DNA markers. RADseq, using both standard RAD and ddRADseq in particular,
retrieved many species-specific SNP markers across the 10 tilapia species. These
SNPs would be of value in further investigating hybridization and introgression in
a range of captive and wild stocks of tilapias.

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Series Title

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Call Number

Moc s 2015

Publisher

Inderalaya : Fak. Kedokteran., 2015

Collation

xvi, 196 hlm.:ilus.

Language

Indonesia

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Classification

NONE

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Edition

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Subject(s)

DNA

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