Applica ions o geno yping by sequencing in aquacul u e
b eeding and gene ics
Diego Robledo
1
, Ch is os Palaiokos as
1
, Luca Ba gelloni
2
, Paulino Ma 
ınez
3
and Ross Hous on
1
1 The Roslin Ins i u e and Royal (Dick) School o Ve e ina y S udies, Uni e si y o Edinbu gh, Midlo hian, UK
2 Depa men o Compa a i e Biomedicine and Food Science, Uni e si y o Pado a, Legna o, Pado a, I aly
3 Depa men o Zoology, Gene ics and Physical An h opology, Facul y o Ve e ina y, Uni e si y o San iago de Compos ela, Lugo, Spain
Co espondence
Ross Hous on, The Roslin Ins i u e and Royal
(Dick) School o Ve e ina y S udies, The
Uni e si y o Edinbu gh, Midlo hian, EH25 9RG
UK. Email: [email p o ec ed]
Recei ed 12 Oc obe 2016; accep ed 27
Decembe 2016.
Abs ac
Selec i e b eeding is inc easingly ecognized as a key componen o sus ainable
p oduc ion o aquacul u e species. The up ake o genomic echnology in aquacul-
u e b eeding has adi ionally lagged behind e es ial a med animals. Howe e ,
he apid de elopmen and applica ion o sequencing echnologies has allowed
aquacul u e o na ow he gap, leading o subs an ial genomic esou ces o all
majo aquacul u e species. While high-densi y single-nucleo ide polymo phism
(SNP) a ays o some species ha e been de eloped ecen ly, di ec geno yping by
sequencing (GBS) echniques ha e unde pinned many o he ad ances in aqua-
cul u e gene ics and b eeding o da e. In pa icula , es ic ion-si e associa ed
DNA sequencing (RAD-Seq) and subsequen a ia ions ha e been ex ensi ely
applied o gene a e popula ion-le el SNP geno ype da a. These GBS echniques
a e no dependen on p io genomic in o ma ion such as a e e ence genome
assembly o he species o in e es . As such, hey ha e been widely u ilized by
esea che s and companies ocussing on nonmodel aquacul u e species wi h ela-
i ely small esea ch communi ies. Applica ions o RAD-Seq echniques ha e
included gene a ion o gene ic linkage maps, pe o ming genome-wide associa-
ion s udies, imp o emen s o e e ence genome assemblies and, mo e ecen ly,
genomic selec ion o ai s o in e es o aquacul u e like g ow h, sex de e mina-
ion o disease esis ance. In his e iew, we b ie ly discuss he his o y o GBS, he
nuances o he a ious GBS echniques, bioin o ma ics app oaches and applica-
ion o hese echniques o a ious aquacul u e species.
Key wo ds: aquacul u e, geno yping, nex -gene a ion sequencing, es ic ion-si e associa ed
DNA, selec i e b eeding, single nucleo ide polymo phism.
Backg ound
Despi e he c i ical ole o aquacul u e in global ood secu-
i y, he as majo i y o wo ld ish and shell ish p oduc ion
is based on s ocks wi hou ad anced selec i e b eeding p o-
g ammes (Gjed em e al. 2012; Janssen e al. 2016). Aqua-
cul u e b eeding schemes end o lag behind hei e es ial
li es ock coun e pa s in e ms o he up ake o genomic
echnologies, and o many aquacul u e species, molecula
gene ic ools a e only applied o pedig ee econs uc ion
(Cha anne e al. 2016). In compa ison, mos mode n
b eeding p og ammes in li es ock a e now unde pinned by
genomic selec ion (GS, Meuwissen e al. 2001), he bene i s
o which a e well-illus a ed in dai y ca le (Hayes e al.
2009). GS ypically equi es genome-wide gene ic ma ke
da a o a la ge numbe o indi idual animals. Up un il a
ew yea s ago, ob aining gene ic ma ke s was cos ly and
labo ious; hence, la ge numbe s o ma ke s we e only a ail-
able o a hand ul o well-s udied species. Howe e , he
ecen ad ances in nex -gene a ion sequencing (NGS) ha e
g ea ly educed he cos o nucleic acid sequencing, and
he e o e also gene ic ma ke disco e y. This has opened
he doo o apid gene a ion o genome-wide gene ic ma -
ke da ase s, ei he ia gene a ion and applica ion o SNP
a ays, o di ec ly ia geno yping by sequencing (GBS)
echniques (Da ey e al. 2011). GBS echniques ha e e o-
lu ionized he ield o e olu iona y genomics ( e iewed in
And ews e al. 2016) and ha e also led o se e al ad ances
in gene ics and b eeding o aquacul u e species, he subjec
o his e iew.
©2017 The Au ho s. Re iews in Aquacul u e Published by Wiley Publishing Asia P y L d670
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use,
dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Re iews in Aquacul u e (2018) 10, 670–682 doi: 10.1111/ aq.12193
Due o he high ecundi y o aquacul u e species, he
majo i y o b eeding p og ammes a e based on collec ion
o ai da a on close ela i es (e.g. ull siblings) o he selec-
ion candida es, pa icula ly whe e he ai o in e es can-
no be measu ed on he candida es hemsel es (e.g. ille
quali y, disease esis ance). Wi hou gene ic ma ke s, his
se -up enables amily selec ion, whe eby amily-le el es i-
ma ed b eeding alues (EBVs) o selec ion candida es a e
calcula ed using he da a collec ed on he ela i es. How-
e e , o u ilize he wi hin- amily gene ic a ia ion in hese
ai s, gene ic ma ke s a e necessa y o dis inguish be ween
selec ion candida es. Implemen a ion o ma ke s in b eed-
ing can b oadly be spli in o wo ca ego ies; ma ke -assis ed
selec ion (MAS) and GS. MAS is based on he use o a -
ge ed ma ke s linked o majo quan i a i e ai s loci
(QTL) a ec ing he ai , and one o he i s examples in
aquacul u e was hos esis ance o in ec ious panc ea ic
nec osis i us (IPNV) in A lan ic salmon (Salmo sala ,
Hous on e al. 2008; Moen e al. 2009). Fo ai s wi h a
polygenic a chi ec u e, GS is a mo e app op ia e app oach,
whe eby he ela i es o he selec ion candida es become
he ‘ aining’ popula ion wi h geno ypes and pheno ypes,
and hose da a a e used o calcula e genomic b eeding al-
ues (GEBVs) o selec ion candida es wi h geno ype da a
only. This applica ion o genomic selec ion in aquacul u e
b eeding is a a o ma i e s age, and mos examples o da e
ha e ocussed on imp o ed b eeding o esis ance o in ec-
ious diseases (e.g. Ødeg

a d e al. 2014; Tsai e al. 2015,
2016b; Vallejo e al. 2016; Dou e al. 2016; Palaiokos as
e al. 2016). The majo i y o high- esolu ion gene ic s udies
in aquacul u e species, and applica ions o genomic selec-
ion, ha e been unde pinned by GBS echniques, ei he by
di ec ly p o iding geno ype da a o by disco e ing ma ke s
o he design o SNP a ays, which a e cu en ly only a ail-
able o a hand ul o aquacul u e species (e.g. A lan ic sal-
mon, Hous on e al. 2014; Y
a~
nez e al. 2016; Paci ic oys e ,
C assos ea gigas, and Eu opean la oys e , Os ea edulis,
Lap
egue e al. 2014; channel ca ish, Ic alu us punc a us,
Liu e al. 2014; common ca p, Cyp inus ca pio,Xue al.
2014a; ainbow ou , Onco hynchus mykiss, Pal i e al.
2015a).
The mos common GBS echniques in ol e lib a y
p epa a ion s eps ha esul in deep sequence da a a a
epea able subse o si es dispe sed h oughou he genome,
ypically using one o wo es ic ion enzymes (RE),
al hough also new GBS echniques based on a ge ed
sequencing ha e been ecen ly de eloped (i.e. GT-Seq, dis-
cussed below). The eason behind his genome complexi y
educ ion is ha high-co e age sequencing o a ypical
aquacul u e species’ genome wi h enough dep h o con i-
den ly call geno ypes is s ill p ohibi i ely expensi e o he
numbe o animals equi ed o high- esolu ion gene ic
s udies and b eeding p og amme applica ions. Genome
complexi y educ ion ia RE is as and inexpensi e.
Indeed, RE-based echniques ha e been commonplace in
geno yping o many yea s, wi h RFLP and AFLP being
widely applied o gene a e geno yping assays o limi ed
numbe s o gene ic ma ke s. The ma iage o hese ideas
wi h NGS has enabled a majo b eak h ough o gene ic
s udies o complex ai s in nonmodel o ganisms, and hei
applica ion o imp o e aquacul u e p oduc ion.
RAD sequencing
Res ic ion-si e associa ed DNA sequencing (RAD sequencing
o RAD-Seq) co e s a ange o GBS echniques which com-
bine he use o genome complexi y educ ion wi h REs and
he high sequencing ou pu o NGS echnologies. RAD-Seq
was i s desc ibed by Bai d e al. (2008), ollowing on om a
simila idea based on mic oa ays (Mille e al. 2007). Some
o he main easons o i s ins an success a e ha RAD-Seq
does no equi e any p io genomic knowledge, i allows gen-
e a ion o popula ion-speci ic geno ype da a (i.e. no asce -
ainmen bias) and i o e s lexibili y in e ms o desi ed
ma ke densi y ac oss he genome. The use o di e en REs
o inno a i emodi ica ions o hebase echniqueallowsa
high le el o con ol o e he numbe o ma ke s ob ained
o a speci ic s udy. RAD-Seq and simila echniques a e also
amenable ools o aquacul u e b eeding, whe e gene ic
ma ke s ha e ypically been used in amily assignmen and
pedig ee econs uc ion (Vandepu e & Ha ay 2014). Mass
spawning species a e common in aquacul u e, whe e mixed
ea ing and unknown pa en al con ibu ion necessi a e he
use o geno yping o amily-based b eeding. RAD-Seq po en-
ially acili a es a single expe imen whe eby pedig ees a e
econs uc ed, gene ic di e si y is quan i ied, QTL can be
mapped and genomic b eeding alues calcula ed (Palaiokos-
as e al. 2016). Since he o iginal RAD-Seq pape by Bai d
e al. (2008), se e al a ian s o his me hodology ha e been
desc ibed. Th ee o hem ha e been ex ensi ely used in aqua-
cul u e gene ics esea ch: he o iginal RAD-Seq (Bai d e al.
2008), 2b-RAD (Wang e al. 2012) and ddRAD (Pe e son
e al. 2012). O he RAD-based echniques like ezRAD (Too-
nen e al. 2013) o SLAF-seq (Sun e al. 2013) in oduced
mino modi ica ions, which do no con e a majo ad an age
o aquacul u e applica ions. All a ailable RAD-based ech-
niques ha e been ecen ly e iewed in dep h elsewhe e
(And ews e al. 2016); he e o e, he e we ha e ocused on
hose mos ele an in aquacul u e b eeding. The main ea-
u es o o iginal RAD-Seq, 2b-RAD and ddRAD a e shown
in Table 1, and hey a e b ie ly desc ibed below.
O iginal RAD-Seq
In o iginal RAD-Seq (Bai d e al. 2008), genomic DNA
samples om se e al animals a e indi idually diges ed wi h
Re iews in Aquacul u e (2018) 10, 670–682
©2017 The Au ho s. Re iews in Aquacul u e Published by Wiley Publishing Asia P y L d 671
RAD sequencing in aquacul u e
a RE o choice. The diges ed DNA is hen andomly shea ed
and pooled a e liga ion o adap o s wi h nucleo ide ba -
codes o unique iden i ica ion o each sample. The esul ing
es ic ion agmen s a e selec ed o sui able size ange (i.e.
o Illumina sequencing, ypically 300–600 bp), and a e a
subsequen polyme ase chain eac ion (PCR) s ep, he ag-
men s a e sequenced. The esul is high-co e age sequence
da a o lanking egions o he RE cu si es, which a e ypi-
cally dispe sed qui e e enly h oughou he genome. As such,
a genome-wide gene ic ma ke da ase can be p oduced
ac oss a popula ion o indi iduals a a ac ion o he cos o
whole genome esequencing. Illumina sequencing o sho
agmen s ei he in ol es sequencing one (one ead, single
end) o bo h ( wo eads, pai ed end) ends o each agmen
and cu en ly gi es eads o up o 300 bp in leng h. Each
lanking sequence o he RE cu si e is e e ed o as a RAD
locus (o RAD- ag), and he high co e age o RAD ags acil-
i a es simul aneous SNP de ec ion and geno yping. The
numbe o RAD ags, and he e o e SNPs, gene a ed in he
expe imen is uneable ia he choice o a e o mo e e-
quen cu ing RE. The mos commonly used enzyme o da e
is Sb I which has an eigh base ecogni ion si e and he e o e
cu s ela i ely in equen ly h oughou he genome. Online
ools a e a ailable o guide he choice o he mos app op i-
a e RE acco ding o he equi emen s and budge o he
s udy (Lepais & Wei 2014). In addi ion o sequencing and
geno yping indi iduals, he app oach is also amenable o
geno yping pooled popula ions o bulk-seg egan analysis
(Bai d e al. 2008; Hohenlohe e al. 2010). One o he main
d awbacks o he o iginal echnique is ha shea ing by soni-
ca ion is andom and a iable, po en ially hinde ing he e i-
ciency and he ep oducibili y o RAD-Seq (Da ey e al.
2013). Howe e , his andom shea ing s ep can also be a
bene i , as he a iable size o he genomic agmen s
ancho ed a he RE cu si e acili a es he assembly o a con-
ig based on he pai ed-end eads. This augmen s anno a ion
o he RAD loci when he e is no e e ence genome a ailable,
and also he design o speci ic p ime s o e-geno yping o
a ge ed SNPs. In addi ion, he pai ed-end da a om
RAD-Seq allow iden i ica ion and emo al o pu a i e PCR
duplica es ( eads o igina ed om he same o iginal DNA
agmen , he e o e p esen ing iden ical sequences), which
can hinde analysis and in e p e a ion o Illumina sequenc-
ing da a (Schweyen e al. 2014). While he e a e se e al
sou ces o po en ial bias and e o in RAD-Seq echniques
(see e iew by And ews e al. 2016), se e al heo e ical and
empi ical s udies ha e demons a ed ha RAD-Seq does en-
de ep oducible geno yping da a ac oss di e en labo a o-
ies, popula ions and e en species (e.g. DaCos a & So enson
2014; Gonen e al. 2015).
2b-RAD
The i s majo modi ica ion o he o iginal RAD echnique
was e med 2b-RAD (Wang e al. 2012). The main inno a-
ion in 2b-RAD is he use o ype IIB REs, which sha e he
ea u e o cu ing he genomic DNA a bo h sides o he
ecogni ion si e a a ixed dis ance, esul ing in p o uding
noncohesi e ends. The esul is sho genomic DNA ag-
men s o iden ical size a each IIB RE si e in he genome.
Lib a y cons uc ion in he 2b-RAD p o ocol is simple. Fol-
lowing DNA diges ion, adap o s a e liga ed o he agmen s,
and speci ic ba codes a e added o each sample h ough
PCR ampli ica ion using degene a ed linke s. Samples a e
hen pooled and sequenced ypically using Illumina echnol-
ogy, bu allowing o uns o sho e ead leng h due o he
smalle size o he agmen s in compa ison o o iginal RAD
(2b-RAD agmen s a e 33–36 bp). The use o ype IIB REs
heo e ically acili a es he sampling and sequencing o iden-
ical si es ac oss indi iduals, ci cum en ing he po en ial bias
o RAD-Seq caused by he andom shea ing s ep. I also
a oids he ime-consuming and po en ially e o -p one size-
selec ion s ep, which cha ac e izes he majo i y o o he
RAD me hods. Addi ionally, 2b-RAD is cu en ly he only
membe o he RAD amily ha allows emo al o loci
exhibi ing s and bias (Pu i z e al. 2014a). The possibili y o
p oduce indi idually ba coded lib a ies allows a ge ed
adjus men be o e pooling o ob ain mo e equal ep esen a-
ion o indi idual samples. The main ca ea o his me hod
is ha i p oduces sho sequencing eads (33–36 bp), which
a e less amenable o alignmen o e e ence genome assem-
blies, and hinde s ollow-up applica ions such as he design
o indi idual SNP assays (due o lack o SNP lanking
sequence). Howe e , his is no an issue i a d a genome
sequence is a ailable o he species, as is becoming he case
in many aquacul u e ish species.
Table 1 Summa y o he di e en geno yping by sequencing (GBS)
echniques
Technique Key ea u es Ad an ages Disad an ages
RAD-Seq Diges ion wi h
one RE
•Pai ed-end con igs
•PCR duplica e
emo al
•Complex
lib a y
p epa a ion
2bRAD Diges ion wi h
ype IIB REs
•No size-selec ion
s ep
•High ep oducibili y
•Easy lib a y
p epa a ion
•S and bias
de ec ion
•Sho
agmen s
•Remo al o
PCR
duplica es
no possible
ddRAD Diges ion wi h
wo di e en
REs
•Can mul iplex
many samples
•Easy lib a y
p epa a ion
•Flexibili y o e
SNP densi y
•Repea abili y
dependen
on size-
selec ion s ep
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672
D. Robledo e al.
ddRAD
Pe e son e al. (2012) de eloped a new RAD-Seq pla o m
using a double diges ion o genomic DNA wi h wo REs
(ddRAD), hus elimina ing he shea ing s ep o o iginal
RAD. The ddRAD p o ocol is mo e lexible han RAD-Seq
o 2b-RAD in e ms o a ge ed ma ke densi y; he numbe
o agmen s and SNPs can be eadily ailo ed by combin-
ing di e en RE pai s. Due o he ypical use o a a e and a
common cu ing enzyme, ddRAD esul s in ewe
sequenced si es han RAD-Seq, acili a ing highe sequence
co e age and/o mo e indi iduals mul iplexed wi hin a sin-
gle sequencing lane. Highe mul iplexing is possible due o
combina ional mul iplex indexing, whe eby a i s ba code
is in oduced in he liga ion s ep and a second du ing he
PCR. The e o e, a la ge numbe o samples can po en ially
be sequenced in a single lane han wi h he o he RAD ech-
niques. Compa ed o he RAD-Seq p o ocol, he wo k low
o p epa a ion o ddRAD lib a ies is simple , quicke and
also subs an ially cheape . Howe e , he wo k low is s ill
mo e complex han he 2b-RAD p o ocol and equi es a
size-selec ion s ep. To ensu e epea abili y o sampled
ddRAD loci ac oss samples and lib a ies, consis ency o size
selec ion is pa amoun (And ews e al. 2016). A simpli ied
a ia ion o he ini ial ddRAD p o ocol, whe e bo h P1 and
P2 adap o s wi h indi idual ba codes a e liga ed p io o
size selec ion (Palaiokos as e al. 2015a), u he educes
hands-on ime o lib a y p epa a ion.
RAD bioin o ma ic analyses
The ad en o NGS posed impo an challenges in e ms o
da a s o age, ans e and analysis, which necessi a ed he
de elopmen o specialized ha dwa e and so wa e. Conse-
quen ly, he imp o emen o NGS-based sequencing pla -
o ms occu ed in andem wi h con inuous de elopmen
and imp o emen o sui able bioin o ma ics ools o anal-
yse he la ge da ase s. A weal h o so wa e is a ailable o
analysing da a o igina ing om he RAD amily o ech-
niques. In he cu en e iew, a gene al amewo k o da a
analysis will be desc ibed, a he han a emp ing o p o-
ide a comp ehensi e o e iew o all a ailable ools.
Acco dingly, he mos popula , s aigh o wa d o use and
egula ly upda ed o he a ailable ools a e highligh ed in
e ms o a sugges ed o de o usage ha migh o m a com-
ple e RAD analysis pipeline.
Expe imen al design and simula ion
Sequencing and lib a y cons uc ion ypically accoun o
he bulk o he cos o any expe imen u ilizing NGS. This
leads o a balancing exe cise, whe eby esea che s s i e o
include as many samples as possible pe sequencing lane
(mul iplexing), wi hou comp omising he ead co e age
equi ed o accu a e SNP geno ype calling. The e o e, wo
key a iables o a RAD expe imen a e he choice o he
RE (a ec ing how many si es a e sequenced), and he
desi ed ead co e age pe locus. In silico simula ion is a
aluable ool o any well-designed RAD expe imen . The
R-based package SimRAD (Lepais & Wei 2014) can be u i-
lized o simula ion-based p edic ion o he expec ed num-
be o loci o each RE (o hei combina ion) and he
genome o s udy. Al hough simula ion es ima es a e likely
o di e om he empi ical da a, aluable in o ma ion can
be gained o op imize expe imen al design be o e commi -
ing o he high cos associa ed wi h lib a y cons uc ion
and sequencing.
Demul iplexing lib a ies
The iles ha a e gene a ed by he sequence ( ypically
Fas Q iles) equi e demul iplexing in o indi idual samples
based on nucleo ide ba codes. The mos popula packages
o his ask include S acks (Ca chen e al. 2011) and
pyRAD (Ea on 2014). S anda d quali y con ol p ocedu e is
o disca d sequence eads below use -de ined accep able
quali y sco es, e oneous ba codes and eads missing he
cha ac e is ic sequence pa e n ob ained om he RE. Fol-
lowing demul iplexing, sequence iles co esponding o
each indi idual a e gene a ed o downs eam analyses,
including SNP calling and geno yping.
SNP iden i ica ion
One o he key ad an ages o RAD-Seq app oaches o non-
model o ganisms (including many aquacul u e species) is
he abili y o iden i y and geno ype SNPs wi hou equi ing
a e e ence genome o he o ganism unde s udy. This
app oach, commonly de ined in he li e a u e as de no o
assembly, can be pe o med using ei he S acks (Ca chen
e al. 2011), pyRAD (Ea on 2014) o dDocen (Pu i z e al.
2014b); howe e , he la e is limi ed o ddRAD o ezRAD
da a. The de no o app oach in ol es iden i ica ion and
assembling o RAD loci in each indi idual, based on use -
de ined pa ame e s ela ed o ead co e age equi ed pe
locus, and sequence di e gence be ween loci (Ca chen e al.
2011). Iden i ica ion o SNPs and in e ence o alleles wi hin
RAD loci is pe o med using a maximum-likelihood-based
algo i hm (Hohenlohe e al. 2010), which unde akes s a-
is ical es s a each nucleo ide posi ion o assess he likeli-
hood o a pa icula diploid geno ype. In doing so, he
model implici ly es ima es and accoun s o sequencing
e o a e (Ca chen e al. 2011). The S acks so wa e does
no cu en ly suppo SNP iden i ica ion and geno yping in
he pai ed-end (P2) ead, unless ancho ed o a second RE
(e.g. in ddRAD). The e o e, in o iginal RAD expe imen s
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RAD sequencing in aquacul u e
using S acks, he P2 ead is ypically used o quali y con-
ol (e.g. emo al o PCR duplica es), and o cons uc ing
pai ed-end ‘mini-con igs’ which acili a e BLAST alignmen
and geno yping assay design (E e e al. 2011). The simul-
aneous use o P1 and P2 eads in he case o dDocen , and
he applica ion o an alignmen -clus e ing algo i hm in he
case o pyRAD, allow he iden i ica ion o inse ion/dele ion
polymo phisms (indels) and iden i ica ion o SNPs in he
P2 eads.
Due o he dec easing cos o NGS, e e ence genome
sequences a e becoming a ailable o many impo an
aquacul u e species. The numbe o species wi h e e ence
genome assemblies is apidly inc easing (A lan ic cod,
Gadus mo hua, S a e al. 2011; Paci ic oys e , Zhang e al.
2012; Eu opean sea bass, Dicen a chus lab ax, Tine e al.
2014; ainbow ou , Be helo e al. 2014; Japanese eel,
Anguilla japonica, Kai e al. 2014; hal -smoo h ongue sole,
Cynoglossus semilae is, Chen e al. 2014; common ca p, Xu
e al. 2014b; No he n pike, Esox lucius, Rondeau e al.
2014; Nile ilapia, O eoch omis nilo icus, B awand e al.
2015; Asian sea bass, La es calca i e , Vij e al. 2016;
Medi e anean mussel, My ilus gallop o incialis, Mu ga ella
e al. 2016; u bo , Scoph halmus maximus, Figue as e al.
2016; A lan ic salmon, Lien e al. 2016; channel ca ish,
Chen e al. 2016), and new sequencing da a will imp o e
genome quali y and anno a ion. The e o e, e e ence-
guided RAD-Seq app oaches a e likely o be inc easingly
u ilized. Bo h S acks and dDocen can u ilize e e ence gen-
ome in o ma ion, using s anda d alignmen ools ollowed
by simila SNP calling algo i hms o he de no o app oach
desc ibed abo e.
Po en ial bias and sou ces o e o
While he bioin o ma ic pipelines o he RAD-like
app oaches a e becoming inc easingly s anda dized, he e
emains po en ial in insic ba ie s ha mus be o e -
come o ensu e he gene a ion o accu a e and epea able
SNP da ase s. One example ha is pa icula ly ele an o
he aquacul u e esea ch communi y is dis inguishing
be ween genuine allelic SNPs and pa alogous a ian s
esul ing om ances al whole genome duplica ion. This
is pa icula ly a challenge o salmonid species, and
s a egies o accoun o his include (i) assessing ead
co e age o pa e ns sugges i e o pa alogous a ia ion,
(ii) checking o excessi e he e ozygosi y a loci and (iii)
sequencing (double) haploid indi iduals as he basis o
il e ing ou pa alogous sequence a ian s (e.g. E e e &
Seeb 2014; Hous on e al. 2014; Pal i e al. 2015a,b).
Ano he po en ial sou ce o e o o all RAD-Seq s udies
is he p oblem o RAD allele d opou (Gau ie e al.
2013), whe e mu a ions wi hin he ecogni ion sequence
o he RE seg ega ing in he popula ion a e a common
sou ce o null alleles. The ex en o he issue is ela ed o
he leng h o he RE ecogni ion sequence, and i is
he e o e po en ially mo e o a p oblem o ddRAD
(which equi es wo REs) e sus o he me hods (Gonen
e al. 2015; And ews e al. 2016). Bo h ead co e age
le els and assessmen o seg ega ion dis o ion in pedi-
g eed c osses can assis in iden i ying and emo ing, o
accoun ing o , hese null alleles. Finally, he concep o
PCR duplica es is aised abo e, and his is due o p e e -
en ial ampli ica ion o ce ain clonal DNA agmen s
de i ed om he o iginal genomic DNA agmen s. PCR
duplica es can gi e ise o he si ua ion whe e one allele
is o e ep esen ed in he esul ing sequence da a and
causes p oblems wi h di e en ia ing homozygous and
he e ozygous indi iduals a ha locus (Schweyen e al.
2014).
Applica ions o RAD sequencing in aquacul u e
Since i s i s desc ip ion by Bai d e al. (2008), RAD-Seq
has quickly sp ead h ough di e en ields o gene ic
esea ch, and i has been used in di e en aquacul u e spe-
cies o cons uc gene ic maps (e.g. Recknagel e al. 2013;
Gonen e al.2014), o compa a i e genomics (e.g. Kakioka
e al. 2013; Manousaki e al. 2015), o mapping genes
associa ed wi h p oduc ion ai s (e.g. Hous on e al. 2012;
Shao e al. 2015; Fu e al. 2016), mapping sex de e mining
loci (e.g. Palaiokos as e al. 2013a,b), s udying popula ion
dynamics (e.g. B adic e al. 2013), o ishe ies manage-
men (e.g. Ogden e al. 2013), assembling e e ence gen-
omes (e.g. Tine e al. 2014) o gene a ing SNP esou ces
o u u e SNP a ay de elopmen (e.g. Hous on e al.
2014; Pal i e al. 2014). A summa y o he s udies pe -
o med di ec ly ele an o aquacul u e is de ailed below
and in Table 2.
Gene ic ma ke disco e y o SNP a ay de elopmen
Ea ly s udies using RAD-Seq ypically ocussed on sim-
ply gene a ing a gene ic ma ke esou ce o nonmodel
o ganisms. When he genome size o he a ge species
is la ge, hen whole genome ( e)sequencing is a guably
no cos -e ec i e o SNP disco e y ac oss many indi-
iduals, and genome complexi y educ ion is ad an a-
geous. As such, RAD-Seq and simila echniques
enabled a s ep change in he numbe o gene ic ma ke s
(SNPs) a ailable o se e al species (e.g. s u geon, Aci-
pense genus, Ogden e al. 2013; o ainbow ou , Pal i
e al. 2014), and hese ha e subsequen ly been used o
se e al high- esolu ion gene ic s udies. SNPs gene a ed
by RAD echniques ha e also been applied o p oduce
SNP a ays o se e al aquacul u e species, including
A lan ic salmon (Hous on e al. 2014), ainbow ou
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674
D. Robledo e al.

(Pal i e al. 2015a) and Paci ic oys e (Lapegue e al.
2014). Wi h he educ ion in sequencing cos s o e
ecen yea s, whole genome ( e)sequencing (i.e. pool-
sequencing, Schl€
o e e e al. 2014) has become inc eas-
ingly iable. Howe e , RAD-like echniques s ill hold a
signi ican ad an age o SNP disco e y when (i) he e
is no e e ence genome a ailable, and (ii) only a med-
ium densi y SNP esou ce is equi ed.
Linkage maps and e e ence genome assembly
Res ic ion-si e associa ed DNA sequencing echniques
ha e been widely used in aquacul u e species o cons uc -
ing gene ic maps based on ecombina ion e en s in de ined
c osses. Such medium densi y SNP linkage maps a e use ul
ools o downs eam applica ions such as QTL mapping,
compa a i e genomic and gene mining, o popula ion
Table 2 Summa y o aquacul u e-o ien ed s udies using es ic ion-si e associa ed DNA sequencing (RAD-Seq)
S udy Species Aim Technique Samples SNPs Families
Salmonids
Hous on e al. (2012) Salmo sala Disease esis ance QTL (IPNV) RAD 32 6712 Two amilies
Gonen e al. (2014) Salmo sala Linkage map RAD 96 8257 Two amilies
Campbell e al. (2014) Onco hynchus mykiss Disease esis ance QTL
(BCWD and IHNV)
RAD 456 4661 40 amilies
Pal i e al. (2014) Onco hynchus mykiss SNP esou ce RAD (92) 19 145 168 19 gene ic lines
Pal i e al. (2015b) Onco hynchus mykiss Disease esis ance QTL (BCWD) RAD 252 5612/4946 Two amilies
Liu e al. (2015b) Onco hynchus mykiss Co isol esponse o
c owding QTL
RAD 234 4874 One amily
Liu e al. (2015b) Onco hynchus mykiss Disease esis ance QTL (BCWD)
and spleen size QTL
RAD 301 7849 Two hal -sib
amilies
Vallejo e al. (2016) Onco hynchus mykiss Genomic selec ion (BCWD) RAD 711 24 465 81 amilies
E e e and Seeb (2014) Onco hynchus shawy scha The mo ole ance and
g ow h QTL
RAD 422 3534 Six amilies
La son e al. (2016) Onco hynchus ne ka The mo ole ance and
g ow h QTL
RAD 491 11 457 Fi e amilies
Nonsalmonid ish
Palaiokos as e al. (2013b) O eoch omis nilo icus Sex de e mina ion QTL RAD 88 3904/4477 Two amilies
Palaiokos as e al. (2015a) O eoch omis nilo icus Sex de e mina ion QTL ddRAD 372 1279 Fi e amilies
Palaiokos as e al. (2013a) Hippoglossus hippoglossus Sex de e mina ion QTL RAD 93 7572/5954 2 hal -sib
amilies
Palaiokos as e al. (2015b) Dicen a chus lab ax Sex de e mina ion QTL RAD 187 6706 4 +4 hal -sib
amilies
Wang e al. (2015a,b) Scoph halmus maximus Sex de e mina ion and
g ow h QTL
RAD 151 6647 One amily
B own e al. (2016) Polyp ion oxygeneios Sex de e mina ion and
g ow h QTL
ddRAD 59 1609 One amily
Manousaki e al. (2015) Pagellus e y h inus Linkage map ddRAD 99 920 One amily
Shao e al. (2015) Pa alich hys oli aceus Disease esis ance QTL
(Vib io anguilla um)
RAD 218 13 362 One amily
Palaiokos as e al. (2016) Spa us au a a Disease esis ance
genomic selec ion
2b-RAD 777 12 085 75 amilies
Wang e al. (2015a,b) La es calca i e G ow h QTL ddRAD 144 3349 One amily
Fu e al. (2016) Hypoph halmich hys nobilis G ow h QTL 2b-RAD 119 3323 One amily
In e eb a es
Jiao e al. (2014) Chlamys a e i Sex de e mina ion
and g ow h QTL
2b-RAD 98 7458 One amily
Li and He (2014) Pinc ada uca a G ow h QTL RAD 100 1381 One amily
Shi e al. (2014) Pinc ada uca a G ow h QTL 2b-RAD 98 10 577 One amily
Tian e al. (2015) Apos ichopus japonicas G ow h QTL 2b-RAD 102 11 306 One amily
Lu e al. (2016) Ma supenaeus japonicus The mo ole ance and
g ow h QTL
RAD 152 9829 One amily
Dou e al. (2016) Pa inopec en yessoensis Genomic selec ion
(g ow h)
2b-RAD 349 2364 Fi e amilies
Ren e al. (2016) Halio is di e sicolo G ow h QTL RAD 142 3317 One amily
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RAD sequencing in aquacul u e
genomic s udies. Fo example, RAD-based linkage maps
ha e been c ea ed o A lan ic salmon (Gonen e al. 2014),
channel ca ish (Li e al. 2014), Japanese lounde (Shao
e al. 2015), u bo (Wang e al. 2015b) and Asian seabass
(Wang e al. 2015a). Gene ic maps based on RAD-Seq ha e
also con ibu ed o mapping and o ien a ion o sca olds
o e e ence genome assemblies o key aquacul u e species
such as Eu opean sea bass (Tine e al. 2014), ainbow ou
(Be helo e al. 2014), Japanese eel (Kai e al. 2014), hal -
smoo h ongue sole (Chen e al. 2014) and u bo (Figue as
e al. 2016). While NGS echnology has enabled apid and
cheap e e ence genome assemblies, hey a e ypically ag-
men ed and incomple e. Fu he , assembly e o s a e qui e
common, and linkage maps can also assis wi h esol ing
mis-assemblies (Fie s 2015; Tsai e al. 2016a). Aquacul u e
species ypically ha e an amenable amily s uc u e o
high- esolu ion linkage maps, due o he high ecundi y
esul ing in la ge ull and hal sibling amilies. Linkage
maps can also be used in conjunc ion wi h physical e e -
ence genome sequences o de ec a ia ion in ecombina-
ion a es ac oss he genome, wi h implica ions o
downs eam applica ions (e.g. LD be ween ma ke s and
QTL in associa ion mapping s udies).
Mapping QTL associa ed wi h ai s o economic
impo ance
The a e o applica ion o genomic echnology o aquacul-
u e species ends o e lec he deg ee o scien i ic and
comme cial in e es o hose species. This is ypically mo i-
a ed by he in e es o unde s anding he gene ic basis o
economically-impo an p oduc ion ai s, o example
g ow h, disease esis ance o sex de e mina ion. Resea ch-
e s wo king in he high- alue salmonid species we e
amongs he i s o exploi RAD-Seq echniques, e alua -
ing esis ance o di e en pa hogens causing high economic
losses, including in ec ious panc ea ic nec osis in A lan ic
salmon (Hous on e al. 2012), and in ec ious hema opoi-
e ic nec osis (Campbell e al. 2014) and bac e ial cold wa e
disease (Campbell e al. 2014; Liu e al. 2015a; Pal i e al.
2015b) in ainbow ou . Based on ea ly successes, and
gi en he impo ance o disease esis ance o mode n
aquacul u e b eeding p og ammes (Y
a~
nez e al. 2014),
la ge-scale p ojec s ha e been es ablished o apply RAD-like
echniques o de ec ma ke s, and e en ually he genes and
causal mu a ions in ol ed, o imp o ing esis ance. Fo
example, he Eu opean Union unded FISHBOOST p ojec
(www. ishboos .eu) is using RAD sequencing echniques o
geno ype se e al housand animals om la ge-scale disease
challenge expe imen s in ainbow ou , common ca p,
Eu opean sea bass, gil head sea b eam (Spa us au a a) and
u bo . These geno ype and pheno ype da a will be used o
es ima e gene ic pa ame e s, map disease esis ance QTL
and e alua e genomic p edic ion app oaches o disease
esis ance b eeding.
In addi ion o disease esis ance, RAD-Seq associa ion
s udies ha e been widely applied o mapping QTL a ec -
ing a ange o o he p oduc ion- ele an ai s, pa icula ly
in salmonid species. These include spleen size (Liu e al.
2015a) and co isol esponse (Liu e al. 2015b) in ainbow
ou , and he mal ole ance and g ow h in Onco hynchus
ne ka, he sockeye salmon (La son e al. 2016). Ou wi h
he salmonid gene a, RAD-Seq has been pe o med o map
loci a ec ing disease esis ance in oli e lounde (Pa aly-
ch hys oli aceous, Shao e al. 2015), and g ow h in bighead
ca p (Hypoph halmich hys nobilis,Fue al. 2016) and u -
bo (Wang e al. 2015b). In addi ion, RAD-like echniques
ha e been e y popula o ma ke disco e y and QTL
mapping in bi al e shell ish including Chinese scallop
(A gopec en i adians; Jiao e al. 2014), Akoya pea l oys e
(Pinc a a uca a; Li & He 2014; Shi e al. 2014), a iously
colou ed abalone (Halio is di e sicolo ; Ren e al. 2016;
Yesso scallop (Pa inopec en yessoensis; Dou e al. 2016) and
ha e also been applied in he sh imp ku uma p awn (Ma -
supenaeus japonicas;Lue al. 2016) and one echinode m,
he sea cucumbe (Apos ichopus japonicus; Tian e al.
2015). In e es ingly, 2b-RAD has been he mos common
echnique in bi al es, while in in ish, adi ional RAD has
been mo e widely u ilized.
Using RAD o s udy sex de e mina ion
Sex de e mina ion (SD) is one o he mos c i ical ai s o
many aquacul u e species, as pheno ypic sex is o en no e i-
den in ju eniles and sexual dimo phism in g ow h a e is
commonly obse ed. SD is complex in many ish species,
o en wi h polygenic con ol and an en i onmen al compo-
nen ( e iewed in Ma 
ınez e al. 2014), and he applica ion
o la ge geno yping p ojec s has been s ongly ecommended
o sc een o SD loci in ish (e.g. Pan e al. 2016). RAD-like
echniques ha e clea ly boos ed ou knowledge o SD in
aquacul u e, wi h s udies in Nile ilapia (Palaiokos as e al.
2013a, 2015a), A lan ic halibu (Hippoglossus hippoglossus,
Palaiokos as e al. 2013b), Eu opean sea bass (Palaiokos as
e al. 2015b) and u bo (Wang e al. 2015b) inding pu a i e
sex de e mining loci. Con olling sex a io is no only in e -
es ing o ob ain highe g ow h a es, bu also o a oid size
dispe sion o o delay sexual ma u i y. Fu he , he e a e
some clea examples, like he s u geon, whe e he comme cial
ad an age o ea ing ish o one sex o e he o he is ob ious.
Genomic selec ion app oaches
While QTL mapping and MAS app oaches can be success-
ul when he gene ic a chi ec u e o a ai sugges s a gene
o majo e ec (e.g. IPNV esis ance, Hous on e al. 2008;
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676
D. Robledo e al.
Moen e al. 2009), imp o emen o polygenic ai s using
genomic da a is mo e e ec i ely achie ed using genomic
p edic ion o b eeding alues (Meuwissen e al. 2001).
S udies o genomic selec ion in aquacul u e we e i s ca -
ied ou in salmonid ish, wi h simula ed (Sonesson &
Meuwissen 2009; Lillehamme e al. 2013) and empi ical
(Ødeg

a d e al. 2014; Tsai e al. 2015, 2016b; Vallejo e al.
2016) da a, demons a ing he clea ad an ages o e pedi-
g ee-based me hods. S udies using a ying ma ke densi ies
o p edic ion in salmonids ha e highligh ed ha as ew as
a housand SNPs may be adequa e o achie ing he gain in
selec ion accu acy e sus pedig ee app oaches (Ødeg

a d
e al. 2014; Tsai e al. 2015, 2016b). The e o e, i is eason-
able o assume ha RAD-like echniques may be use ul o
genomic selec ion in aquacul u e b eeding, as ypical RAD
SNP da ase s comp ise a ew housand SNPs. Indeed, he
po en ial o his app oach has al eady been highligh ed o
esis ance o bac e ial cold wa e disease in ainbow ou
(Vallejo e al. 2016), o g ow h in Yesso scallop (Dou e al.
2016), and o esis ance o pas eu ellosis in gil head sea
b eam (Palaiokos as e al. 2016).
Gene ic aceabili y and aquacul u e sus ainabili y
One o he main conce ns o aquacul u e p oduce s and
consume s is o minimize he en i onmen al impac o ish
a ming. In his sense, aceabili y ools a e essen ial o assess
he impac o aquacul u e escapees in na u al popula ions o
dis inguish be ween a med and wild specimens. RAD-Seq
has been u ilized o ob ain SNPs o s u geon aceabili y
and conse a ion (Ogden e al. 2013), which will con ibu e
o en o ce cu en legisla ion on aquacul u e and ishing
p ac ices bu also aid on he handling o wild s ocks, c i ical
o sus ainable aquacul u e. RAD-Seq is also he main ool
o he Eu opean p ojec AquaT ace (aqua ace.eu), he
esul s o which ha e been ecen ly p esen ed in he Eu o-
pean Aquacul u e Socie y mee ing in Edinbu gh (Aquacul-
u e Eu ope 2016). One o he AquaT ace objec i es was o
assess he impac o escapees on na u al popula ions o
Eu opean sea bass, gil head sea b eam and u bo , while also
de eloping o ensically alida ed ools o aceabili y pu -
poses. The esul s highligh ed he u ili y o RAD-Seq
app oaches o cap u e popula ion o amily speci ic a ia ion
making i a sui able ool o gene ic aceabili y and conse -
a ion o na u al popula ions. This is o he ou mos impo -
ance o sus ainable aquacul u e g ow h, leading o las ing
economic bene i s, ood sa e y and social accep ance.
RAD-Seq and SNP a ays, owa ds a peace ul co-
exis ence
The de elopmen o NGS has g ea ly inc eased he amoun
o genomic esou ces a ailable in he mos impo an
aquacul u e species, including genome assemblies o many
o hem. Alongside RNA-Seq and whole genome sequenc-
ing, RAD-Seq has con ibu ed signi ican ly o he a ailabil-
i y o abundan gene ic ma ke s compa ed o a ew yea s
ago. While RAD-Seq and simila echniques a e likely o
emain he geno yping me hod o choice o species wi h
ew genomic esou ces, se e al medium and high-densi y
SNP a ays a e al eady a ailable o aquacul u e species
(A lan ic salmon, Hous on e al. 2014; Y
a~
nez e al. 2016;
channel ca ish, Liu e al. 2014; common ca p, Xu e al.
2014a; ainbow ou , Pal i e al. 2015a; Paci ic oys e
and Eu opean la oys e , Lap
egue e al. 2014), and many
mo e a e unpublished o cu en ly being p oduced and
alida ed.
Single nucleo ide polymo phism a ays a e a ype o
DNA mic oa ay, whe e hyb idiza ion o allele-speci ic
p obes esul s in a luo escen signal which can be mea-
su ed o call a geno ype in a gi en loci. They ha e bo h
ad an ages and disad an ages o e RAD-Seq app oaches
(Table 3). Fo ins ance, he expe imen al p ocedu es and
bioin o ma ic analyses a e much simple o he use o
SNP a ays, equi ing less echnical knowledge and usually
esul ing in a as e u na ound. The geno ype sco ing
me hod is mo e obus and amenable o au oma ion, and
he e o e less p one o e o s (Hong e al. 2012; Wall e al.
2014). The epea abili y and ep oducibili y a e highe o
SNP a ays han RAD-Seq, and geno yped loci a e known
in ad ance. Howe e , ha ing a ixed se o loci on he chip
is also a disad an age, especially in species wi h s ong pop-
ula ion s uc u e, because o asce ainmen bias whe eby
he SNP se is biased o polymo phic ma ke s in he disco -
e y popula ion(s). This p esen s a majo issue whe e aqua-
cul u e s ains o a speci ic species a e highly a iable, and
he u ili y o a SNP a ay will a y hugely depending on he
ela ionship o he disco e y popula ion. RAD-like
app oaches o e come his issue and also o e much g ea e
lexibili y o he esea che in e ms o he a ge ed numbe
o loci. Fu he , RAD-Seq cap u es a ia ion ha is speci ic
o popula ions, amilies and indi iduals ha is likely o be
missed om SNP a ay, which a e ypically biased owa ds
common a ian s. Ano he pu a i e ad an age o RAD-like
Table 3 Gene al compa ison o es ic ion-si e associa ed DNA
sequencing (RAD-Seq) and single nucleo ide polymo phism (SNP) chips
RAD-Seq SNP a ays
Sample p ocessing Labo ious S aigh o wa d
Bioin o ma ic analysis Complex Negligible
Tu na ound ime Long Medium
Accu acy Medium-high High
Repea abili y Medium High
Design Adjus able Fixed
Cos Low Medium
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RAD sequencing in aquacul u e
echniques is ha he di ec cos o he expe imen is
cheape , al hough he addi ional ime equi ed o lib a y
p epa a ion and bioin o ma ics analyses should be consid-
e ed in o any compa ison.
In he nea u u e, genomic selec ion (GS) is likely o
be a key echnique o b eeding p og ammes o many
aquacul u e species, due o he demons able inc ease in
selec ion accu acy e sus cu en pedig ee-based me hods.
SNP a ays a e now ou inely used in li es ock b eeding
p og ammes o GS and a e inc easingly u ilized in ech-
nologically ad anced aquacul u e b eeding. Se e al s udies
ha e shown ha only mode a e SNP ma ke densi y is
equi ed o e ec i e GS in salmon (Ødeg

a d e al. 2014;
Tsai e al. 2015, 2016b). Vallejo e al. (2016) compa ed
bo h RAD-Seq and SNP a ays o GS o BCWD esis-
ance in ainbow ou , inding simila selec ion accu a-
cies o bo h echniques despi e highe ma ke densi y
om he SNP chip (~40k SNP a ay e sus ~10k RAD-
Seq). This may e lec high le els o linkage disequilib-
ium in ypical aquacul u e amily selec ion p og ammes,
whe eby ai eco ding is o en pe o med on close ela-
i es o he selec ion candida es. The e o e, he highe
ma ke densi y associa ed wi h SNP chips may be ad an-
ageous when p edic ing b eeding alues in animals mo e
dis an ly ela ed o he aining popula ion (Tsai e al.
2016b), o in species wi h g ea e e ec i e popula ion
sizes and/o lowe le els o linkage disequilib ium.
Howe e , gi en he ela i ely sho genomes o many
nonsalmonid aquacul u e species (i.e. Eu opean sea bass
–~763 Mb, o u bo –~658 Mb; A lan ic salmon
–~2970 Mb), he ypical ma ke densi y gene a ed by
RAD-like echniques may be pe ec ly adequa e o e ec-
i e GS. Howe e , his needs o be es ed, as he ecombi-
na ion equency and pa e ns o linkage disequilib ium
ac oss he genome a e pe inen o he ques ion o ade-
qua e ma ke densi y. Fu he educ ions in ma ke den-
si y equi emen s a e likely o be obse ed when geno ype
impu a ion app oaches a e used, o example geno yping
pa en s a high densi y, and o sp ing o a small subse
o he ma ke s. As al eady men ioned, RAD me hods
allow o subs an ial lexibili y in e ms o numbe o
geno yped ma ke s. In addi ion, lowe ing a e age
sequence co e age in he o sp ing wi h pa en s
sequenced a high co e age could be used o gene a e
geno ype da a a a much lowe cos .
Ta ge ed GBS echniques
Bo h RAD-Seq and SNP a ays will also ha e o com-
pe e wi h ecen ly de eloped geno yping me hods based
on a ge ed geno yping by sequencing. Fo example,
geno yping-in- housands by sequencing (GT-Seq, Camp-
bell e al. 2015) is a me hod o a ge ed sequencing
which ollows a mul iplex PCR app oach, whe e hun-
d eds o housands o loci (amplicons) a e selec ed o
geno yping. In his me hod, a mul iplex PCR using
loci-speci ic p ime s ha also con ain Illumina sequenc-
ing p ime s is used o ampli y he a ge ed egions.
Unique ba codes o each sample a e added wi h a sec-
ond PCR eac ion, ollowed by pooling and sequencing
o samples. Unlike RAD echniques, his me hod equi es
p e ious knowledge o design he assays, and he numbe
o SNPs geno yped in a single un is limi ed o a ew
housand. Simila echnologies a e now p o ided by
majo geno yping echnology p o ide s, and i appea s
likely o become one o he mos cos -e ec i e sys ems o
geno yping a ge ed SNPs. O he GBS a ge ed-sequen-
cing echniques ha e also been ecen ly de eloped, o
example RAD cap u e (Rap u e), whe e p eselec ed RAD
ags a e isola ed using cap u e p obes and hen sequenced
(Ali e al. 2016). These a ge ed GBS echniques ha e he
po en ial o become majo playe s in aquacul u e b eed-
ing and gene ics due o hei simplici y and lexibili y.
Howe e , in pa , hey su e om he same limi a ion as
SNP a ays ha hey equi e p io knowledge and selec-
ion o he SNPs ha a e use ul in he popula ion o
in e es .
Fu u e ou look
Res ic ion-si e associa ed DNA sequencing echniques
ha e d i en a majo inc ease in he applica ion o genomics
o aquacul u e species. While he ca alogue o SNP a ays
o aquacul u e species will inc ease in he coming yea s, i
is likely ha RAD echniques will con inue o be widely
applied. We an icipa e ha bo h echniques will co-exis
o se e al yea s, and he choice o RAD-Seq o SNP chip
will depend on he species and p ojec -speci ic ac o s. Fo
example, i may be ha high- alue aquacul u e species wi h
la ge genomes (e.g. salmonids) a e mo e sui able o SNP
a ays, while lowe - alue species wi h smalle genomes
(and/o highe le els o LD) a e mo e sui able o RAD
echniques, al hough i will also depend on he esou ces
a ailable o each pa icula p ojec . Ta ge ed GBS ech-
niques like GT-Seq a e likely o ind a niche in geno yping
hund eds o se e al housands o p e iously iden i ied
SNPs ac oss many samples. Fu he , RAD echniques a e
likely o emain he gold s anda d o new aquacul u e spe-
cies and/o hose p oduced on a smalle scale, whe e SNP
a ays a e no a ailable, and genomic esou ces a e sca ce.
E en ually he cos o gene a ing and analysing sequence
da a may d op o a le el whe e genome complexi y educ-
ion is no longe equi ed, bu i seems unlikely in he sho
e m. The e o e, RAD sequencing will con inue o lou ish
in aquacul u e esea ch in he ollowing yea s and is likely
o be ou inely applied o deli e he bene i s o genomic
Re iews in Aquacul u e (2018) 10, 670–682
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