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A first AFLP-Based Genetic Linkage Map for Brine Shrimp
Artemia franciscana and Its Application in Mapping the
Sex Locus
Stephanie De Vos1'2'3, Peter Bossier1, Gilbert Van Stappen1, Ilse Vercauteren2'3, Patrick Sorgeloos1,
M arnik Vuylsteke2'3*
1 Laboratory of Aquaculture, Artemia Reference Center (ARC), D epartm ent o f Animal Production, Ghent University, Gent, Belgium, 2 D epartm ent o f Plant Systems Biology,
VIB, Gent, Belgium, 3 D epartm ent o f Biotechnology and Bioinformatics, Ghent University, Gent, Belgium
Abstract
We r e p o rt o n t h e c o n stru c tio n o f sex-specific linkage m aps, t h e identification o f sex-linked m arkers a n d t h e g e n o m e size
e stim a tio n for t h e brine sh r im p A rtem ia franciscana. Overall, from t h e analysis o f 433 AFLP m arkers s e g r e g a t in g in a 112 fullsib family w e identified 21 m ale a n d 22 f e m a le linkage g r o u p s (2n = 42), c o verin g 1,041 a n d 1,313 cM respectively. Fifteen
putatively h o m o l o g o u s linkage g ro u p s, including t h e sex linkage g r o u p s, w e re identified b e t w e e n t h e f e m a le a n d m ale
linkage m ap . Eight sex-linked AFLP m ark er alleles w e r e inh erited from t h e fe m a le pa ren t, s u p p o r t i n g t h e h y p o th e s is o f a
WZ-ZZ se x -d e te rm in in g system . The haplo id A rtem ia g e n o m e size w as e s t im a te d t o 0.93 Gb by flow c ytom e try. The
p r o d u c e d A rtem ia linkage m a p s provide t h e basis for f u rth e r fine m a p p i n g a n d explo ring o f t h e se x -d e te rm in in g region a n d
are a p ossib le m ark e r r e so u rce for m a p p i n g g e n o m i c loci underlying p h e n o ty p i c differences a m o n g A rtem ia species.
C itation : DeVos S, Bossier P, Van Stappen G, Vercauteren I, Sorgeloos P, e t al. (2013) A first AFLP-Based Genetic Linkage Map for Brine Shrimp Artemia franciscana
and Its Application in Mapping th e Sex Locus. PLoS ONE 8(3): e57585. doi:10.1371/journal.pone.0057585
Editor: Zhanjiang Liu, Auburn University, United States o f America
Received O ctober 25, 2012; A ccepted January 22, 2013; Published March 4, 2013
C opyrigh t: © 2013 De Vos e t al. This is an open-access article distributed under th e term s of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided th e original author and source are credited.
Funding: The authors acknow ledge th e sup p o rt o f Ghent University for th e Bijzonder Onderzoeksfonds (BOF) of Stephanie De Vos. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
C om peting Interests: The authors have declared th a t no com peting interests exist.
* E-mail: [email protected]
Introduction
[16], A F L P [17,18], m icrosatellite m arkers [19] a n d the 15,822 b p
m itochondrial genom e sequence [20,21].
In crustaceans, three m ajo r genetic sex d eterm in atio n systems
have b e en suggested b y cytogenetics a n d sex-linked m arkers: W Z Z Z (females are the h eterogam etic sex), X X -X Y (males are the
heterogam etic sex) a n d androdioecy (a m ix o f Z Z m ales a n d W Z
he rm aphrodites, as in Eulimnadia texana) [22]. E xam ples o f
crustaceans w ith an X X -X Y sex-determ ining system are decapods
such as the C hinese m itten c rab Eriocheir sinensis [23,24], terrestrial
isopods a n d the am phipods Orchestia cavimana a n d 0. gammarellus
[25]. Flow ever, a W Z -Z Z sex-determ ining system has b e en found
in decapods such as Litopenaeus vannamei [26], tiger shrim p Penaeus
monodon [27], Macrobrachium rosenbergii [28], k u ru m a p ra w n Penaeus
japonicus [29], A ustralian re d claw crayfish Cherax quadricarinatus
[24] a n d in isopods like Armadillidium vulgare a n d all V alvifera,
except Saduria entomon [30]. In bisexual Artemia, fem ale heterogam ety has b e en suggested previously b y observation o f sexual
hétérochrom osom es i n -4. salina [31], A. franciscana and A. persimilis
[8]; b y crossing experim ents w ith A. franciscana show ing a recessive
sex-linked trait called “w hite eye” [32] a n d b y karyotyping a n d
h ete ro ch ro m a tin experim ents show ing one h e tero ch ro m atic block
in fem ale a n d tw o in m ale -4. persimilis [8].
O v er the last decade, linkage m aps have b e en developed for a
n u m b e r o f crustaceans such as Daphnia pulex [33], D. magna [34],
Tigriopus californicus [35], P. monodon [36—38], L. vannamei [39—41],
Fenneropenaeus chinensis [42,43] a n d P. japonicus [44], Sex-linked
m arkers have b e en found in m ales o f the isopod Mysis relicta [45]
a n d o f Triops cancrijbrmis [46], In fem ale crustaceans, sex-linked
Artemia, know n as brin e shrim p, is a genus o f small planktonic
crustaceans found w orldw ide in n a tu ra l salt lakes a n d salterns [1].
T h e ir larvae (nauplii) are the m ost com m only used live food in
a q u acu ltu re activities, specifically for larval grow th o f m ore th an
85% o f the m arin e species re are d in aq u acu ltu re [2,3]. A dult
Artemia survive extrem e salinities, while their encysted em bryos
(cysts), p ro d u c ed u n d e r stressful conditions, have a unique
tolerance for high doses o f U V a n d ionizing radiation, anoxia,
th erm al extrem es a n d desiccation-hydration cycles [4-6], Cysts
re m a in viable for years a n d p ro d u c e nauplii w ithin 24 h after
hydration.
A n overview o f Artemia cytogenetics, D N A c o n te n t a n d available
m olecular tools is provided. Six different sexually dim orphic
species can be found in the Artemia genus, am o n g w hich Artemia
franciscana Kellogg, 1906 [4] a n d several obligate parthenogenetic
Artemia populations ra nging in ploidy from 2n to 5n [7]. All
sexually d im orphic Artemia species a re diploids w ith 2n = 42,
except A. persimilis (2n = 44) [8]. T h e Artemia genom e size has been
assessed w ith two different techniques p ro d u c in g discordant
estim ates: 2.93 G b (3 pg) by Feulgen densitom etry [9] a n d 1.47
G b (1.5 pg) by D N A reassociation kinetics [10], D espite the use o f
flow cytom etry in the m ost recent evaluations o f crustacean
genom e size [11-13], so far no flow cytom etry-based estim ates o f
the Artemia genom e have b e en published. T o date, genom ic
resources for Artemia have b e en lim ited to R A P D [14,15], R F L P
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Artem ia Linkage M ap an d Sex-Linked Markers
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Artemia Linkage Map and Sex-Linked Markers
Figure 1. Artemia franciscana autosom al fem ale linkage groups T w en ty-on e linkage groups representing th e Artemia franciscana
autosom al genom e containing m arkers o rigin atin g fro m fem ale p arental strain V in h Chau (ARC1349). Each AFLP m a rk e r is re p re s e n te d
b y (1) a c o d e refe rrin g t o th e c o rre s p o n d in g PC (T able S I ), fo llo w e d b y (2) th e m o le c u la r size o f th e f r a g m e n t in n u c le o tid e s a n d (3) th e ty p e o f
p a re n ta l m a rk e r (fem a le m a rk e r, ta g g e d a s "F"). C u m u la tiv e m a rk e r d is ta n c e s (cM) a re in d ic a te d o n th e left.
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 5 7 5 8 5 .g 0 0 1
m arkers have b e en found in the isopods Paracerceis sculpta [47] a n d
Jaera ischiosetosa [48], in the crab Cancer setosus [49], in p en aeid
shrim ps L. vannamei [41] a n d P. monodon [27] a n d in giant
freshw ater p ra w n M . rosenbergii [50]. M oreover, a h erm ap h ro d itedeterm in in g allele has b e en studied in the androdioecious
b ra n ch io p o d E. texana [51]. So far, n e ith e r linkage m aps, n o r
trait-linked m arkers including sex-linked m arkers have been
identified in Artemia [8].
G enetic linkage m aps are invaluable in forw ard genetic analyses
for the identification o f the genom ic loci responsible for
p h enotypic differences. From this perspective, Artemia offers a
n u m b e r o f m ajo r advantages for tim e-effective gen eratin g o f
experim ental m ap p in g p opulations a n d for m ap p in g n a tu ra l allelic
variation. T h ey have a short g eneration tim e (2—A weeks),
offspring p ro d u c tio n o f several h u n d re d individuals p e r brood,
storability o f cysts for years, easy b reed in g in large num bers a n d
levels o f genetic variability th a t are am o n g the highest w ithin
crustaceans [16,41,52]. In addition, we expect th a t forw ard
genetic ap p ro ach es in Artemia are n o t only restricted to Artemiaspecific traits, b u t are also valuable for m ap p in g traits such as sex,
Vibrio p ath o g e n resistance a n d grow th rate, segregating in
com m ercially im p o rta n t crustaceans. W e believe therefore, th a t
Artemia, could b e a useful m odel species for o th er crustaceans.
In the p re sen t study, we re p o rt o n a first A F L P -based linkage
m ap o f A. franciscana. W e additionally p re sen t eight sex-linked
m arkers th a t disclose the linkage group corresponding to the W
chrom osom e a n d confirm fem ale h e terogam ety in A. franciscana.
Finally, we re p o rt o n the estim ation o f the A. franciscana genom e
size by flow cytom etry.
DNA extraction
D N A was extracted from p a ren ts a n d th eir 112 F t offspring
according to a m odified C T A B -m ethod for shrim p tissue [54],
Briefly: to each sam ple, g ro u n d in liquid N 2 , 150 pi o f C T A B
buffer was added. A fter hom ogenization, 750 pi o f ex tra C T A B
buffer was a d d ed a n d the m ix was left a t 25°C for 30 m in. PC A
solution was a d d ed (600 pi; 25:24:1 p h e n o l/c h lo ro fo rm /iso a m y lalcohol). A fter centrifugation, 800 pi o f the u p p e r aqueous phase
was a d d ed to 600 pi o f C A solution (24:1 c h loroform /isoam ylalcohol) a n d the m ix was hom ogenized. T o 700 pi o f the u p p e r
aqueous phase, 630 pi o f isopropanol was added. T h e m ix was
in cu b a ted for 1 h a t —70°C . A fter centrifugation, the pellet was
w ashed w ith 600 pi o f e thanol 70% , air-d ried in a 60°C oven a n d
resuspended in 20 pi o f sterile distilled w ater. D N A quality a n d
co n cen tratio n w ere assessed on a 1% agarose gel.
S e grega tion analysis a n d linkage m a p p in g
A F L P analysis w ith fluorescent dye detection was p erfo rm ed on
a L I-C O R long re a d -IR 2 4200 (L I-C O R Biosciences) as described
by V uylsteke e t al. [55]. Sixty-five Æ /oR I+ 3/Msel+3 prim er
com binations (PCs) listed in T ab le S I w ere used. A F L P analysis
o f paren ts a n d 112 offspring w as done on two separate 64-lane gels
p e r PC .
T h e degree o f polym orphism b etw een the two p a ren tal strains
was estim ated based o n A F L P fragm ents am plified by four PCs
(E 112M 212, E 112M 213, E 112M 233 a n d E 112M 234).
A F L P m arkers w ere scored using the specific im age analysis
software A FLP- Q u a n ta r Pro (h ttp ://w w w .k ey g e n e -p ro d u c ts.co m )
as described in Vuylsteke et al. [55]. E ach A F L P m ark er was
identified b y (1) a code referring to the corresp onding PC
(T able SI), follow ed by (2) the m olecular size o f the fragm ent in
nucleotides as estim ated b y A F L P -Q uantarPro, a n d (3) a tag
referring to the type o f m arker. P aren tal A F L P m arkers
segregating 1:1 in the F t progeny are heterozygous in either the
fem ale (female m arker, tagged as “ F ” ) o r the m ale p a re n t (male
m arker, tagged as “ M ”) a n d hom ozygous absent in the o th er
p a ren t. A F L P m arkers heterozygous in one o f the p a ren ts a n d
hom ozygous p re sen t in the o th er w ere n o t included in the linkage
analysis, because heterozygotes could n o t b e reliably discrim inated
from individuals hom ozygous for the “ b a n d p re sen t” allele. N o tag
was used for b ip are n ta l m arkers, w hich are heterozygous in b o th
paren ts a n d thus, segregate 1:2:1 in the F t progeny. P aren tal a n d
b ip are n ta l A F L P m arkers w ere scored co-dom inantly based on
relative fragm ent intensities resulting in m ore genetic inform ation
c o m p a red to d o m in a n t (present/absent) scoring a n d hence,
speeding u p the m ap p in g process [55]. H ow ever, b ip aren tal
m arkers w ere scored dom in an tly w hen the heterozygotes could not
reliably be discrim inated from the individuals hom ozygous for the
“ b a n d p re sen t” allele.
Linkage a n d segregation analyses w ere p erfo rm ed using the
software package J o in m a p 4 [56]. T h e m ap p in g po p u latio n type
was set to C P (i.e. a pop u latio n resulting from a cross betw een two
heterogeneously heterozygous a n d hom ozygous diploid parents,
linkage phases originally unknow n). T h e segregation type was
encoded according to Jo in m a p 4 re com m endations [56]. A
lo garithm o f the odds (LOD) threshold range betw een 2.0 a n d
14.0 was initially used to g roup p a ren tal m arkers. O n ly linkage
groups co n tain in g a t least three m arkers w ere considered for m ap
M aterials and Methods
M apping pop ula tio n
C yst m aterial o f the A. franciscana strains from S an Francisco
Bay, U S A (SFB; A R C 1364) a n d V in h C hau, V ietn am (VC;
A R C 1349) was o b tain e d from the L ab o ra to ry o f A quaculture &
A rtem ia R eference C e n te r cyst b a n k (h ttp ://w w w .a q u a c u ltu re .
ugent.be). T h e SFB strain was first in tro d u c ed in V in h C hau,
V ietn am in 1982, eventually resulting in the new V C strain in the
late 1980s [53]. First, cysts from b o th strains w ere h a tc h ed
separately in a e ra te d seaw ater (28°C, salinity 35 g.l- ). T h e instar
I nauplii o f each strain w ere th en harvested a n d re are d for a w eek
in a e ra te d seaw ater w ith a d d ed sea salt (Instant O cean® , 28°C ,
final salinity 70 g.l- ) a n d fed w ith Tetraselmis suecica, a m arine
unicellular green alga. T h e Artemia w ere subsequently transferred
to individual Falcon tubes a n d kept th ere u n d e r the sam e
conditions for seven days until sexual m atu ratio n . A controlled
cross b etw een V C (9) a n d SFB (O ') was th en m ade, resulting in F t
progeny th a t was collected over a sieve every two days a n d grow n
until m atu rity u n d e r the sam e conditions as th e p a ren tal
generation. A dult F[ p rogeny was rinsed w ith sterile distilled
w ater a n d the p h enotypic sex o f each F t offspring individual was
d e term in ed visually. F or gut evacuation before D N A extraction,
the offspring a n d paren ts w ere starved d u rin g 24 h, follow ed by
rem oval o f the b ro o d p o u ch in females. P arents a n d p rogeny w ere
stored individually a t —20°C.
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Artemia Linkage Map and Sex-Linked Markers
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PLOS ONE I w w w .plosone.org
4
March 2013 | V olum e 8 | Issue 3 | e57585
Artemia Linkage Map and Sex-Linked Markers
Figure 2. A rte m ia fran ciscana autosom al m ale linkage groups. T w e n ty lin k a g e g ro u p s re p re s e n tin g th e A rtem ia franciscana a u to s o m a l g e n o m e
c o n ta in in g m a rk e rs o rig in a tin g fro m m a le p a re n ta l stra in San F rancisco Bay (ARCI 364). Each AFLP m a rk e r is r e p re s e n te d b y (1) a c o d e refe rrin g to th e
c o rre s p o n d in g PC (Table S1), fo llo w e d b y (2) th e m o le c u la r size o f th e fr a g m e n t in n u c le o tid e s a n d (3) th e ty p e o f p a re n ta l m a rk e r (m ale m arker,
ta g g e d a s "M "). C u m u la tiv e m a rk e r d is ta n c e s a re in d ic a te d o n th e left (cM).
d o i:1 0.1371 /jo u rn a l, p o n e .0 0 5 7 5 8 5 .g 0 0 2
construction. Segregation distortion o f m arkers was tested by using
a x “‘test as im p lem ented in Jo in m a p 4. G rap h ical presen tatio n o f
linkage groups was done w ith the software M a p C h a rt [57].
1.2.1. T h e h aploid G S o f for each Artemia sam ple was calculated
F S X F (a
according to the follow ing form ula [13] : G S = ----------- , w here F s
Fs
is the m ea n fluorescence o f the sam ple a n d F;s is the m ean
fluorescence o f the in tern al standard.
Artemia g e n o m e size es tim atio n by flow cytom etry
T h e h aploid genom e size (GS) o f Artemia was assessed against the
rainbow tro u t (haploid G S 2.4—3.0 p g or 2.35 —2.93 G b [58]) a n d
the chicken genom e (haploid G S 1.07 p g o r 1.05 G b [59]), b o th
used as in tern al standards.
S e grega tion analysis a n d linkage m a p p in g
T h e consistency o f the used m eth o d was assessed by calibrating
rainbow tro u t nuclei (2 pi o f freshly d raw n hep arin ized Oncoiynchus
mykiss blood) against chicken erythrocyte nuclei (2 pi o f 1Ox diluted
B ioSure® C EN singlet, Gallus gallus domesticus, R h o d e Island R e d
female).
E ac h o f the faut: Artemia individuals (i.e. four full-sib m ales from
the V C (9) X SFB (O ') cross) w ere c h o p p ed to g eth er w ith internal
sta n d ard m aterial using a ra zo r in 1 m l o f G albraiths buffer as
described in D olezel a n d B artos [60], Cell suspensions w ere
filtered th ro u g h a 30 p m m esh, p u t o n ice a n d nuclei w ere co­
stained in the d a rk for 2 m in w ith 50 pi o f fluorescent D N A stain
P ropidium Iodide (Sigm a-A ldrich P I solution in w ater 1 m g/m l).
T h e use o f P I staining on A. franciscana (G C % 32) [61], 0. mykiss
(G C% 42) [58] a n d G. domesticus (G C % 47) [62] was chosen to
avoid a G C content-linked bias, as occurs w ith D A P I staining [60].
A t least 5,000 nuclei w ere analyzed for each co-stained sam ple,
using a M o d u lar Flow cytom eter a n d cell sorter (M oFlo Legacy,
C ytom ation) w ith a 488 n m A rgon laser a n d P I em ission bandpass
filter o f 5 8 0 /3 0 nm . In stru m en t calibration was p erfo rm ed using
Flow -check Fluorospheres (Beckm an Coulter) a n d internal stan­
dards. Fluorescence o f the nuclei was re co rd e d linearly w ith the
software Sum m it v4.3. F or each co-stained sam ple, fluorescence
histogram s w ere gen erated a n d m ea n fluorescence values w ere
calculated w ith the flow cytom etry d a ta analysis software Cyflogic
A total o f 65 A F L P PC s resulted in a total o f 531 m arkers, o f
w hich 433 w ere p a ren tal (239 fem ale, 194 m ale) a n d 98 m arkers
w ere bip aren tal. B ased o n only four p rim e r com binations (PCs)
yielding 180 A F L P fragm ents, 36% o f the fragm ents segregated
betw een b o th parents.
First, a p a ren tal m ap including only p a ren tal m arkers was
constructed. S um m ary statistics for the p a ren tal m aps are listed in
T ab le 1. T h e grouping o f p a ren tal m arkers a t a L O D score
ra nging from 5.0 to 6.0 resulted in a n u m b e r o f linkage groups
corresponding w ith the haploid chrom osom e n u m b e r (n = 21).
T h e fem ale m ap, c o ntaining 225 m arkers (Figure 1), resulted in 22
“ fem ale” linkage groups (LG) spanning 1,312.9 cM ; the m ale
m ap, c o ntaining 181 m arkers (Figure 2), resulted in 21 “ m ale” L G
spanning 1,041.3 cM . T w enty-eight p e rce n t o f the analyzed
p a ren tal m arkers show ed significant (p < 0 .05; x~ test) segregation
distortion. M ale m arkers w ere m ore often d istorted th a n fem ale
m arkers (31% resp. 25% ). Som e larger genom ic regions did not
contain any m arkers (e.g. 32.5 cM in L G Fem ale_6, Figure 1;
38.0 cM in L G M ale_2, Figure 2), despite the low m edian interm ark er distances o f 3.9 a n d 3.1 cM for the fem ale a n d the m ale
linkage m ap (T able 1).
N ext, a n integ rated m ap was c rea te d including the 98
b ip are n ta l m arkers a n d 406 previously m ap p e d p a ren tal m arkers
(Figure 3). By including b ip are n ta l m arkers, groups consistent w ith
Results
Table 1. Statistics for t h e f e m a le a n d m ale linkage m ap s.
No. of linkage groups
No. o f markers m apped per linkage group
Size of linkage groups (cM)
Intermarker distance (cM)
Fem ale (Vinh Chau)
M a le (San Francisco Bay)
22
21
Min
3
3
Max
19
17
Median
12
8
Mean
10
9
Total
225
181
Min
15.1
9.5
Max
104.4
123.8
Median
63.7
44.4
52.1
Mean
59.7
Total
1312.9
1041.3
Min
0.0
0.0
Max
32.5
38.0
Median
3.9
3.1
Mean
6.5
6.6
doi:10.1371 /journal.pone.0057585.t001
PLOS ONE I w w w .p lo s o n e .o rg
5
March 2013 | V olum e 8 | Issue 3 | e57585
Artem ia Linkage M ap an d Sex-Linked Markers
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March 2013 | V olum e 8 | Issue 3 | e57585
Artemia Linkage Map and Sex-Linked Markers
Figure 3. A rte m ia fran cis ca n a hom ologous autosom al m ale and fem ale linkage groups. F o u rte e n h o m o lo g o u s a u to s o m a l lin k a g e g r o u p
p airs. Each AFLP m a rk e r w a s id e n tifie d b y (1) a c o d e refe rrin g to th e c o rre s p o n d in g PC (T able S1), fo llo w e d b y (2) th e m o le c u la r size o f th e f r a g m e n t
in n u c le o tid e s a n d (3) th e ty p e o f m a rk e r (fem ale m a rk e r, ta g g e d a s "F", m a le m a rk e r, ta g g e d a s "M ", b ip a re n ta l m a rk e r, n o ta g ). C o m m o n b ip a re n ta l
m a rk e rs a re in d ic a te d in b lu e . C u m u la tiv e m a rk e r d is ta n c e s a re in d ic a te d o n th e left (cM).
d o i:1 0.1371 /jo u rn a l, p o n e .0 0 5 7 5 8 5 .g 0 0 3
linkage groups o f the p a ren tal m ap w ere o b tain e d a t a L O D
threshold ran g in g b etw een 6 a n d 10. Sixty-nine p ercen t o f the
b ip are n ta l m ark er loci show ed significant (p < 0 .05; x~ test)
segregation distortion. T hese loci w ere still included in m ap
construction a n d evaluated for quality afterw ards, since significant
segregation distortion is in h ere n t to relatively small experim ental
m ap p in g pop u latio n sizes o f ~ 1 0 0 individuals. Forty-nine
b ip are n ta l m arkers (50%) could be m ap p e d in the fem ale as well
as in the m ale m ap, identifying 15 hom ologous linkage groups
including the sex linkage groups (Figure 3, Figure 4).
assessm ent o f the Artemia G S because th eir fluorescence values did
not overlap w ith those o f Artemia, as was the case w ith fluorescence
values o b tain e d from chicken nuclei. LTsing rainbow tro u t nuclei as
the internal standard, the -4. franciscana h aploid genom e size was
estim ated to 0 .9 3 ± 0 .0 9 G b (0 .9 7 ± 0 .0 9 pg; n = 4). Fluorescence
histogram s for each sam ple a n d for chicken are show n in Figure 5.
Fluorescence peaks w ere relatively b ro a d due to cell debris from
the previously frozen Artemia individuals, b u t average D N A content
estim ates w ere consistent th ro u g h o u t the different sam ples, show n
by the small sta n d ard error.
M apping of th e sex locus
Discussion
Staelens et al. [27] described segregation p a tte rn s o f sex-linked
A F L P m arkers th a t unequivocally differentiate the W Z -Z Z a n d
X X -X Y sex-determ ination system. W e observed eight A FLP
m arkers, spanning a region o f 0.2 cM o n L G F e m ale _ l (m arkers
in green, Figure 4) segregating according to p a tte rn 1 a n d a single
m ark er (E l 12M 122M 167.3F) according to p a tte rn 2. B oth
segregation p a tte rn s are expected u n d e r the assum ption o f fem ale
heterogam ety. N o n e o f the m arkers segregated a ccording to
p attern s 6, 7 a n d 8, expected u n d e r the assum ption o f m ale
heterogam ety. T h e m ale linkage group M ale_10 was identified as
hom ologous to F e m ale _ l (Figure 4). In conclusion, the observed
segregation p a tte rn s o f sex-linked A F L P m arkers strongly favour
fem ale over m ale h e terogam ety in Artemia.
W e presen t the first sex-specific A F L P linkage m aps a n d sexlinked m arkers as well as a consistent genom e size (GS) estim ation
for the brin e shrim p A. franciscana.
T h e linkage analysis o f 433 p a ren tal A F L P m arkers segregating
in a 112 full-sib fam ily identified 21 m ale a n d 22 fem ale linkage
groups, corresponding very well w ith the haploid chrom osom e
n u m b e r in A. franciscana (2n = 42) [8]. M ost likely, the m arkers in
small linkage groups (LG) such as F em ale_20 (Figure 1) w ould jo in
one o f the o th er 21 L G b y ad d in g m ore m arkers to the fem ale
m ap. M o re fem ale th a n m ale m arkers w ere generated, suggesting
th a t m ate rn a l A. franciscana strain V in h C h a u (VC) has m ore
unique alleles c o m p a red to the p a te rn al strain S an Francisco Bay
(SFB). T his seems a logical consequence o f the SFB origins o f V C .
T h e level o f polym orphism betw een the tw o A. franciscana p a ren tal
strains was estim ated at 36% , w hich is in the range o f 9 -5 0 %
estim ated previously by K a p p as et al. [53]. G iven their high
m ark er density, the p ro d u c ed genetic m aps are a d eq u a te for the
a n ch o rin g o f Artemia genom e sequences to facilitate the future
construction o f physical m aps for each o f the 21 chrom osom es.
A. franciscana g e n o m e size es tim ation by flow cytom etry
LTsing tro u t b loo d as the internal standard, the hap lo id fem ale
chicken genom e size (GS) d e term in ed by flow cytom etry was 1.05
G b (1.07 pg) as previously re p o rte d for fem ale chicken [59]. W e
p referred rainbow tro u t nuclei as the internal sta n d ard in the
Female_l
0 .0 —f t - E113M132M417.5
12 . 8
-
■ E112M224M11Û.3
12M213M371.1F
12M311M288.7
12M333M286.6F
12M223M143-8F
12M432M180.2F
12M341M405.3F
IE1 12M223M273.0F E112M232M351.5F
32.0'I
12M134M244.0F E112M332M3E0.8F
12M133M245.4F E112M213M88.2F
J E1
32.1 '
- 1 e i 12M142M210.7F
32.2' - - E1 13M214M309.5F
32.6'
- - E112M243M315.0F
33.0'
-E1 12M323M272.1F
36.2'
- E1 12M322M415.9F
38.5'
-E1 12M223M277.7
40 .3 '
r ei 12M231M88.5F
41 .7 '
L ei 12M122M167.3F
26.6'
27.Í
28.228.331.4
31.9'
M a le 10
E1 13M132M417.5
E1 12M244M140.5M
5 7 -U Lr e i 12M224M110.3
^ E1 12M231 M254.9M
6.4
- H E 1 12M334M202.2M E112M123M253.4M
8.6
12M423M88.1M
8 7 "A
12M122M169.8M
9.0 A
10.1 A »- E1 12M342M240.0M
12.1
Ji
E1
12M311
M288.7
:.1 Ji
E1 12M214M109.5M
15.5 J
k E1 13M214M337.1M
20.0
0.0
1.4
rt E
E11
29.232.1 ■
E112M243M238-3M
E112M211 M429.7M
E113M214M156.0M
Figure 4. A rte m ia fran ciscana sex linkage groups. F em ale lin k a g e g r o u p Fem ale_1 c o rre s p o n d s w ith th e W c h ro m o s o m e . T h e h o m o lo g o u s m ale
lin k a g e g r o u p M ale_ 1 0 c o rre s p o n d s w ith th e Z c h ro m o s o m e . Each AFLP m a rk e r is r e p re s e n te d b y (1) a c o d e referrin g to th e c o rre s p o n d in g PC
(T able SI ), fo llo w e d b y (2) th e m o le c u la r size o f th e fr a g m e n t in n u c le o tid e s a n d (3) th e ty p e o f m a rk e r (fem ale m a rk e r, ta g g e d a s "F", m a le m arker,
ta g g e d a s "M ", b ip a re n ta l m a rk e r, n o ta g ). C o m m o n b ip a re n ta l m a rk e rs a re in d ic a te d in b lu e . M arkers fully linked to s e x a re m a rk e d in g re e n .
C u m u la tiv e m a rk e r d is ta n c e s (cM) a re in d ic a te d o n th e left.
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 5 7 5 8 5 .g 0 0 4
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March 2013 | V olum e 8 | Issue 3 | e57585
Artemia Linkage Map and Sex-Linked Markers
FI. < L in
Figure 5. Fluorescence histogram s fo r A rte m ia fran ciscana and chicken D NA con ten t estim ation. F lu o re s c e n c e h is to g ra m o f f o u r d iffe re n t
A. franciscana m a le in d iv id u a ls w ith tr o u t a s th e in te rn a l s ta n d a rd (A, B, C, D) a n d o f c h ic k en CEN w ith tr o u t a s th e in te rn a l s ta n d a rd (E).
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 5 7 5 8 5 .g 0 0 5
T his will b e especially useful, considering the n um erous reports o f
repetitive sequences in Artemia [63]. Artemia linkage m aps will also
allow future linkage studies in Artemia for im p o rta n t crustacean
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traits such as resistance to Vibrio, the m ost co m m o n bacterial
p ath o g e n in w orldw ide m arin e fish a n d shellfish aquaculture,
Fifteen hom ologous linkage groups, including the L G re p re ­
senting the sex chrom osom es, w ere identified b etw een the fem ale
March 2013 | V olum e 8 | Issue 3 | e57585
Artemia Linkage Map and Sex-Linked Markers
a n d m ale linkage m aps b y including b ip are n ta l m arkers in the
linkage analysis. T his study identified eight sex-linked A FLP
m ark er alleles m ap p in g to one locus a n d in h erited from the fem ale
p a ren t, suggesting A. franciscana adopts a genetic W Z - Z Z sexdeterm in in g system. Artemia sex-linked m arkers will enable the
study o f n auplii sex ratios a n d th eir dynam ic in n a tu ra l Artemia
populations. T h ey will also enable the furth er fine-m apping o f the
sex-determ ining locus a n d the subsequent identification o f the
p rim a ry sex-determ ining gene(s). F urth erm o re, based o n sequence
hom ology w ith Artemia, sex-determ ining genes m ight be identified
in com m ercially valuable crustaceans, enabling P C R -b a se d allelespecific assay developm ent in the fram ew ork o f the developm ent o f
m ono-sex cultures in shrim p [64],
T h e clustering o f eight sex-linked m arkers in a 0.2 cM region
suggests red u ced recom bination, w hich is often found in sex-linked
regions [65]. G enes from a region th a t stopped recom b ining in the
early evolution o f sex chrom osom es have a high sequence
divergence, allow ing a n estim ate o f w h en the W a n d Z
chrom osom es first stopped recom bining a n d thus, the age o f the
sex chrom osom e system [65].
T h e estim ated Artemia G S in this study (0.93 Gb) is sm aller th an
earlier estim ates: 2.93 G b by Feulgen densitom etry [9] a n d 1.47
G b by D N A reassociation kinetics [10]. “A. salina” used to b e a
general n am e for all Artemia species, p resendy confounding the
identity o f the investigated Artemia in m an y studies [1]. Because the
Artemia D N A c o n te n t m easu red by Feulgen densitom etry o n “A.
salina” is alm ost a twofold o f th a t m easu red by D N A reassociation
kinetics, Feulgen densitom etry m ight have b e e n p erfo rm ed o n a
tetraploid A. parthenogenetica, as suggested b y V a u g h n [10]. Also, the
absolute A. franciscana karyotype size varies betw een 60.68 p m a n d
139.26 p m [8], show ing th a t significant intra-specific v a riation in
D N A c o n ten t could explain the high Feulgen densitom etry values
as well.
V a u g h n [10] calculated the Artemia h aploid G S by D N A
reassociation kinetics, based on an A. franciscana G C c o n te n t o f
42 %. M o re recen t m easurem ents how ever, show a n A. franciscana
(SFB) G C c o n te n t o f 32% d e term in ed by C sC l centrifugation a n d
confirm ed by direct chem ical analysis a n d renew ed therm al
d é n atu ratio n [61]. A n estim ated G C c o n te n t low ered b y 1%
results in a 0.018% low er h aploid D N A c o n te n t estim ated by
D N A reassociation kinetics [66]. Flence, based o n a G C c o n te n t o f
32% , the corrected A. franciscana D N A c o n ten t estim ated by
V au g h n [10] is 1.23 G b, a p p ro x im atin g m ore closely the 0.93 G b
Artemia G S estim ated in this study.
C u rren d y , o u t o f the 50,000 know n C ru stacea species, the G S o f
278 crustaceans has b e en determ ined, covering a 400-fold-w ide
genom e size range b etw een Cyclops kolensis, a C yclopoid copepod
(0.14 pg) a n d Ampelisca macrocephala, a n A rctic A m phipod
(64.62 pg) [67,68]. In com parison, A. franciscana has a relatively
small genom e o f 0.97 pg. T his m akes it a poten tial new m odel
c rustacean for w hich genom e sequencing is c u rre n d y feasible,
unlike for crustaceans w ith a m u ch larger genom e size. T o date,
the only publicly accessible sequenced crustacean genom e is the
b ra n ch io p o d D. pulex, w ith a n average genom e size o f 0.23 p g
[ 6 9 ] .
_
U ltim ately, the fu rth er developm ent o f genom ic resources for
Artemia such as the w hole genom e sequence, will a d d a com pletely
new dim ension to Artemia research a n d its use as live food in
aquaculture. M oreover, know ledge o f the A. franciscana sexdeterm in in g system will facilitate future evolutionary studies o f
sex chrom osom es in sexually dim orphic (WZ fe m a le /Z Z male)
a n d p arth en o g en etic Artemia. C onsidering the presence o f sexual
a n d asexual re p ro d u c tio n strategies, the Artemia genus shows
prom ise as a m odel system for the study o f asexuality, its evolution
a n d its evolutionary purpose. Finally, since Artemia is considered a
poten tial cru stacean m odel species, increasing know ledge ab o u t
Artemia genetics a n d genom ics in general a n d sex-related genetics
in particular, are expected to be valuable to crustacean aq u acu l­
ture, presently lacking in m olecular b re ed in g strategies despite
their con trib u tio n o f 23% to the total aq u acu ltu re pro d u ctio n
value [70].
Supporting Inform ation
Table SI List o f the 65 p rim er com b in ation s used for
AFLP a n a ly sis1 E: EcoRI p rim er w ith three selective
b ases; M: M seI p rim er w ith three selective b a ses (1, 2, 3,
4 correspond to A, C, G, T).
(D O C)
Acknowledgments
T he authors would like to thank C hrist M ahieu, for rearing the Artemia
m apping population and Professor Annem ie Decostere and Annelies
Declercq, for donating rainbow trout blood.
Author Contributions
Conceived and designed the experiments: SDV PB M V. Perform ed the
experiments: SD V IV GV. Analyzed the data: SDV M V. C ontributed
reagents/m aterials/analysis tools: PB IV PS. W rote the paper: SD V PB
GV M V PS.
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March 2013 | V olum e 8 | Issue 3 | e57585
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