Ci a ion: Ramí ez, D.; Rod íguez, M.E.;
C oss, I.; A ias-Pé ez, A.; Me lo, M.A.;
Anaya, M.; Po ela-Bens, S.; Ma ínez,
P.; Robles, F.; Ruiz-Rejón, C.; e al.
In eg a ion o Maps Enables a
Cy ogenomics Analysis o he
Comple e Ka yo ype in Solea
senegalensis.In . J. Mol. Sci. 2022,23,
5353. h ps://doi.o g/10.3390/
ijms23105353
Academic Edi o s: Robe Has e ok,
Na alia Bo owska-Zuchowska,
Ewa Robaszkiewicz and
Igo Rogozin
Recei ed: 15 Ma ch 2022
Accep ed: 9 May 2022
Published: 11 May 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
In eg a ion o Maps Enables a Cy ogenomics Analysis o he
Comple e Ka yo ype in Solea senegalensis
Daniel Ramí ez 1,† , Ma ía Es he Rod íguez 1,† , Ismael C oss 1, Albe o A ias-Pé ez 1,
Manuel Alejand o Me lo 1, Ma co Anaya 1, Sil ia Po ela-Bens 1, Paulino Ma ínez 2, F ancisca Robles 3,
Ca melo Ruiz-Rejón3and Lau eana Rebo dinos 1,*
1Á ea de Gené ica, Facul ad de Ciencias del Ma y Ambien ales, INMAR, Uni e sidad de Cádiz,
11510 Cádiz, Spain; [email p o ec ed] (D.R.); ma iaes he . [email p o ec ed] (M.E.R.);
[email p o ec ed] (I.C.); [email p o ec ed] (A.A.-P.); [email p o ec ed] (M.A.M.);
[email p o ec ed] (M.A.); [email p o ec ed] (S.P.-B.)
2Depa amen o de Zoología, Gené ica y An opología Física, Uni e sidad de San iago de Compos ela,
27002 Lugo, Spain; [email p o ec ed]
3Depa amen o de Gené ica, Uni e sidad de G anada, 18071 G anada, Spain; obles@ug .es (F.R.);
ca melo@ug .es (C.R.-R.)
*Co espondence: lau [email p o ec ed]; Tel.: +34-956-016181
† These au ho s con ibu ed equally o his wo k.
Abs ac :
The Pleu onec i o mes o de , which includes se e al comme cially-impo an species, has
unde gone ex ensi e ch omosome e olu ion. One o hese species is Solea senegalensis, a la ish wi h
2n= 42 ch omosomes. In his s udy, a cy ogenomics app oach and in eg a ion wi h p e ious maps
was applied o cha ac e ize he ka yo ype o he species. Syn eny analysis o S. senegalensis was
ca ied ou using wo la ish as a e e ence: Cynoglossus semilae is and Scoph halmus maximus. Mos
S. senegalensis ch omosomes (o ch omosome a ms o me acen ics and subme acen ics) showed a
one- o-one mac osyn enic pa e n wi h he o he wo species. In addi ion, we s udied how epe i i e
sequences could ha e played a ole in he e olu ion o S. senegalensis bi-a med (3, and 5–9) and
ac ocen ic (11, 12 and 16) ch omosomes, which showed he highes ea angemen s compa ed
wi h he e e ence species. A highe abundance o TEs (T ansposable Elemen s) and o he epea ed
elemen s was obse ed adjacen o elome ic egions on ch omosomes 3, 7, 9 and 16. Howe e , on
ch omosome 11, a g ea e abundance o DNA ansposons was de ec ed in in e s i ial BACs. This
ch omosome is syn enic wi h se e al ch omosomes o he o he wo la ish species, sugges ing
ea angemen s du ing i s e olu ion. A simila si ua ion was also ound on ch omosome 16 ( o
mic osa elli es and low complexi y sequences), bu no o TEs ( e oelemen s and DNA ansposons).
These di e ences in he dis ibu ion and abundance o epe i i e elemen s in ch omosomes ha
ha e unde gone emodeling p ocesses du ing he cou se o e olu ion also sugges a possible ole o
simple epea sequences in ea anged egions.
Keywo ds:
Solea senegalensis; pleu onec i o mes; gene ic maps; cy ogenomics; ch omosome e olu ion;
ka yo ype; epe i i e sequences; compa a i e genomics
1. In oduc ion
The s udy o e eb a e ka yo ypes has p esen ed esea che s wi h di e se challenges
in sys ema ics and e olu ion. In ish, cy ogene ics has played a less signi ican ole, pa ly
because o he di icul y o ob aining adequa e li e samples (indi iduals, issues o cells)
o , o example, deep-sea ish o ob ain hei ka yo ypes. E en when sui able samples a e
a ailable, he applica ion o cy ogene ic echniques is complex and labo ious, and he e
is no gua an ee o ob aining good ch omosomal obse a ions. Howe e , he applica ion
o ka yo ype da a oge he wi h mo phology, genome size and sequence da a can p o-
duce mo e obus esul s in he e olu ion and classi ica ion o o ganisms. In pa icula ,
In . J. Mol. Sci. 2022,23, 5353. h ps://doi.o g/10.3390/ijms23105353 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 5353 2 o 14
he applica ion o luo escence in si u hyb idiza ion (FISH) and de i ed echniques has
been e y use ul, o example, o s udy he o igin and e olu ion o sex ch omosomes in
eleos species [1–3].
Teleos s a e a g oup o ishes ex emely di e se in hei mo phology, beha iou and
gene ics. This di e si y could be ela ed o a whole-genome duplica ion ha ook place in
his g oup be o e i s di e si ica ion, in addi ion o he wo duplica ions ha occu ed du ing
he o igin o e eb a es. Di e en lines o e idence, such as he numbe o ch omosomal
ea angemen s, he unc ionalisa ion o duplica ed genes, he a e o p o ein e olu ion,
and conse a ion o non-coding elemen s, show a highe a e o e olu ion in eleos s han
in o he e eb a es [
4
]. This g oup has small ch omosomes and an ances al ka yo ype o
48 ac ocen ic ch omosomes [5,6].
Wi hin he eleos s, he genome size o he o de Pleu onec i o mes is among he
smalles o all ishes, anging in size om 400 o 650 Mb [
7
–
9
]. Thei ch omosomes a e e y
small [
10
], wi h diploid ch omosome numbe s anging om 2n= 28 o 2n= 48 [
11
]; and
hey ha e unde gone ex ensi e ch omosome e olu ion, as i is shown by he ka yo ype
o mula o he h ee species compa ed in his s udy: S. senegalensis has n= 21 ch omosomes
composed o 3 MT + 2 SMT + 4 STL + 12 TL [
12
]; C. semilae is has n= 21 TL ch omo-
somes [
13
]; and S. maximus has n= 22 ch omosomes composed o 2 MT + 1 SMT/STL +
5 STL + 14 TL [14].
The phylogeny o he o de Pleu onec i o mes has been dispu ed, wi h some wo ks
suppo ing a monophyle ic o igin [
15
,
16
] and o he s a poly/pa aphyle ic one [
17
–
19
].
Howe e , genome analysis o 11 la ish species sugges s ha he subo de s Pleu onec oidei
and Pse odoidei o igina ed om dis inc Pe coid ances o s, making he Pleu onec i o mes
a polyphyle ic g oup [20].
Cy ogenomics appea s o be pa icula ly use ul in he s udy o ch omosome o gan-
isa ion and e olu ion in non-model o ganisms [
1
,
2
,
21
]. Fu he mo e, in ish, physical
mapping o genes is especially impo an since he size o ch omosomes makes hei
indi idual iden i ica ion di icul and causes a high deg ee o gene ic in e e ence [22].
The combina ion o in o ma ion om di e en ypes o maps, such as linkage and
physical maps, allows he c ea ion o so-called “in eg a ed” maps. These maps acili a e
ine mapping o quan i a i e ai loci (QTL), posi ional cloning, genome sequencing and
assembly, and make genome-wide compa a i e s udies possible [
23
–
25
]. Among he
di e en me hodologies ha enable he c ea ion o in eg a ed maps is he combina ion
o FISH and bac e ial a i icial ch omosome (BAC) genomic lib a ies. BAC clones ca y
long DNA agmen s ha allow he de elopmen o mul iple p obes pe ch omosome.
This, oge he wi h hei sequencing by Nex Gene a ion Sequencing (NGS), acili a es he
ancho ing o linkage and genomic da a o ch omosomes [
22
]. BAC lib a ies ha e p o en
o be essen ial o he iden i ica ion o comple e genomic sequences o he in eg a ion o
gene ic and physical maps and compa a i e genomic s udies [24].
Wi h echnological ad ances, genome sequencing has become almos ou ine, e en
o non-model o ganisms. Wi hin he Pleu onec i o mes, he numbe o genomes a ailable
o s udy has inc eased conside ably in ecen yea s [
20
], including species o comme cial
in e es such as u bo [
26
], Japanese lounde [
27
] and sole [
7
]. Only o a ew species has
a le el o ch omosome assembly been eached. Al hough he in o ma ion p o ided by
hese genomes is ele an a bo h basic (e.g., ch omosomal e olu ion and me amo phosis
mechanisms) and applied le els (e.g., g ow h pe o mance and disease esis ance), u he
e inemen is needed o make hem use ul o associa ion s udies, gene mapping and
compa a i e genomics.
The Senegalese sole (Solea senegalensis (Kaup, 1858)) is a la ish belonging o he o de
Pleu onec i o mes. The species is widely dis ibu ed in he wa e s o he A lan ic Ocean,
om he Gul o Biscay o he no hwes coas o A ica, and in he Medi e anean Sea,
om he S ai o Gib al a o Tunisia. I is a sole o g ea comme cial alue based on high
demand and a easonable p ice [
28
]. Howe e , some p oblems hinde i s p oduc ion [
28
]:
(1) high la al mo ali y, (2) sub-op imal la al weaning s a egies, and (3) inadequa e
In . J. Mol. Sci. 2022,23, 5353 3 o 14
disease con ol. The e o e, in o de o ensu e sus ainabili y and imp o ed p oduc ion, i
is necessa y o s udy he in luence o gene ics on he p oduc i e ai s, physiology and
immunology o he species.
Conside able e o has been made in ecen yea s o unde s and he cy ogene ic
and genomic aspec s o his species. Molina-Luzón e al. [
29
] de eloped he i s haploid
gene ic map o Senegalese sole using gynogene ic amilies and 129 mic osa elli es, wi h
he consensus map consis ing o 27 linkage g oups (LGs). Se e al s udies ha e used a
BAC-FISH app oach o loca e and map genes on he ch omosomes, as well as o s udy
aspec s such as his one e olu ion [
30
], sex de e mina ion and di e en ia ion [
31
–
33
], epe -
i i e DNA amilies [
34
], and ch omosomal e olu ion [
35
]. This has made i possible o
imp o e he in eg a ed gene ic map [
36
] and o p opose ha he e olu ion o se e al Sene-
galese sole ch omosomes (e.g., he la ges me acen ic ch omosome, i.e., ch omosome 1,
and ch omosomes 2 and 4) is due o Robe sonian usions, pe icen ic in e sions, and
o he ch omosomal ea angemen s by ansposable elemen s. Gue e o-Cóza e al. [
37
]
analysed a d a genome o iden i y mic osa elli es. By combining he mic osa elli es iden-
i ied in ha wo k wi h mic osa elli es om Molina-Luzón e al. [
29
] i was possible o
cons uc an in eg a ed map con aining 21 linkage g oups, which ma ches he ch omosomal
numbe o he species. Subsequen ly, a ch omosome-le el assembly was p esen ed [
7
] o
he genome o a male S. senegalensis, ob ained by de no o assembly and cons uc ion o a
linkage map wi h ddRAD ma ke s. Howe e , he linkage g oups iden i ied in hese la e
wo wo ks ha e no been ela ed o ch omosomes.
Hence, he pu pose o his wo k is o ob ain new in o ma ion, in eg a e i wi h p e ious
maps and he genome sequence o S. senegalensis, and ela e hese esul s o he knowledge
a he ch omosomal le el, hus ad ancing he s udy o he comple e ka yo ype o he
species and i s e olu ion.
2. Resul s
2.1. Cy ogene ic Map
A o al o 126 BAC clones we e mapped o he Senegalese sole ch omosome comple-
men (Table S1 [
38
–
41
], Figu es 1and S1). Six een o hese clones con ain candida e genes
ela ed o he immune sys em, eigh een ela ed o sex de e mina ion o ep oduc ion, eigh
ela ed o me amo phosis, o y-six we e andomly aken om he BAC lib a y (anony-
mous), and hi y-eigh con ain mic osa elli e ma ke s used by Molina-Luzón e al. [
29
]
o gene a e he gene ic map (Table S2). Fi e BAC clones we e loca ed on wo o h ee
ch omosomes, and disca ding hese, an a e age o 4.7 BAC clones we e posi ioned pe ch o-
mosome a m (conside ing me acen ic and subme acen ic ones as bi-a med). Howe e ,
some a ms p esen ed a highe numbe o BAC clones, such as he p a m o
ch omosome 2
,
he q a m o ch omosome 4, and ch omosomes 6, 16 and 19. Howe e , o one o he
la ges ch omosomes in he S. senegalensis ka yo ype, ch omosome 5, only one BAC clone
was ound in each a m. A Megablas sea ch in he ch omosome le el-sca olding o
S. senegalensis male [
7
] allowed a p ecise mapping o he BACs and he co ec ion o
p e ious loca ions o some BACs (Table S3).
2.2. Sequencing and Gene Anno a ion
All BAC clones had been sequenced in p e ious s udies, excep hose con aining
mic osa elli e ma ke s, which we e sequenced o he p esen s udy. To summa ise, a o al
o 6,726,594 bp ha e been sequenced; he N50 alue anged om 327 o 124,351 bp, and he
L50 anged om 1 o 31 (Table S4). Up o 279 genes we e anno a ed om he hi y-eigh
new BAC clones (Table S5).
2.3. In eg a ion o Maps
The gene ic and physical maps ob ained by Molina-Luzón e al. [
29
] and Gue e o-
Cóza e al. [
7
], espec i ely, we e in eg a ed wi h he cy ogene ic map (Table 1). The
In . J. Mol. Sci. 2022,23, 5353 4 o 14
21 sca olds ob ained by Gue e o-Cóza e al. [
7
] we e co ela ed wi h he 21 ch omosomes
o he cy ogene ic map.
In .J.Mol.Sci.2022,23,xFORPEERREVIEW4o 15



Figu e1.Cy ogene icmapo Soleasenegalensisob ainedwi hBACp obes:Yellowcolo indica es
BACscon ainingmic osa elli es;pinkcolo indica esBACswi hgenes ela ed osexual ep oduc‐
ion/di e en ia ion;g eencolo indica esBACswi hgenes ela ed ome amo phosis;clea blue
colo indica esBACscon aininggenes ela ed o heimmunesys em;andda kbluecolo indica es
BACswi hgenesbelonging oo he ca ego ies.Sho a msa epandlonga eq(uppe ispandlowe 
isqinme acen ics).*BACs oundonmo e hanonech omosome.
2.2.SequencingandGeneAnno a ion
AllBACcloneshadbeensequencedinp e iouss udies,excep  hosecon ainingmi‐
c osa elli ema ke s,whichwe esequenced o  hep esen s udy.Tosumma ise,a o al
o 6,726,594bpha ebeensequenced; heN50 alue anged om327 o124,351bp,and
heL50 anged om1 o31(TableS4).Up o279geneswe eanno a ed om he hi y‐
eigh newBACclones(TableS5).
2.3.In eg a iono Maps
Thegene icandphysicalmapsob ainedbyMolina‐Luzóne al.[29]andGue e o‐
Cóza e al.[7], espec i ely,we ein eg a edwi h hecy ogene icmap(Table1).The21
sca oldsob ainedbyGue e o‐Cóza e al.[7]we eco ela edwi h he21ch omosomes
o  hecy ogene icmap.
Table1.In eg a iono  hecy ogene icmapo Soleasenegalensiswi hpublishedlinkagemapand
physicalmap.
Ch omosomeo Soleasenega‐
lensisLinkageG oup*Ch omosomeLe el‐Sca old‐
ing**
121,271
27,12,153
31,4,1516
418,252
59,1121
Figu e 1.
Cy ogene ic map o Solea senegalensis ob ained wi h BAC p obes: Yellow colo indica es
BACs con aining mic osa elli es; pink colo indica es BACs wi h genes ela ed o sexual ep oduc-
ion/di e en ia ion; g een colo indica es BACs wi h genes ela ed o me amo phosis; clea blue
colo indica es BACs con aining genes ela ed o he immune sys em; and da k blue colo indica es
BACs wi h genes belonging o o he ca ego ies. Sho a ms a e pand long a e q(uppe is pand lowe
is qin me acen ics). * BACs ound on mo e han one ch omosome.
Table 1.
In eg a ion o he cy ogene ic map o Solea senegalensis wi h published linkage map and
physical map.
Ch omosome o
Solea senegalensis Linkage G oup * Ch omosome
Le el-Sca olding **
1 21, 27 1
2 7, 12, 15 3
3 1, 4, 15 16
4 18, 25 2
5 9, 11 21
6 4, 27 5
7 3, 4, 14 7
8 6 10
9 1, 11, 13 12
10 19 20
11 11 17
12 19 18
13 10 11
14 1 6
15 14 19
16 16, 19, 20 9
17 - 13
18 - 15
19 2 4
20 22, 24 8
21 8 14
* Linkage map desc ibed by Molina-Luzón e al. [29]. ** Physical map desc ibed by Gue e o-Cóza e al. [7].
In . J. Mol. Sci. 2022,23, 5353 5 o 14
FISH analysis o BAC clones allowed co ela ion o 23 ou o he 27 Linkage G oups
(LGs) ob ained by Molina-Luzón e al. [
29
] wi h he excep ion o LG5, LG17, LG23 and LG26.
These LGs we e loca ed on 19 ch omosomes as no BAC clones con aining mic osa elli e
ma ke s hyb idized on ch omosomes 17 and 18. Fou ch omosomes co ela ed one- o-one
wi h ou LGs (ch omosomes 8, 13, 19 and 21); he emaining i een ch omosomes had one,
wo o h ee LGs ha a e epea ed on one o mo e addi ional ch omosomes.
2.4. Syn eny Analysis
The analysis was pe o med o each ch omosome o S. senegalensis in compa ison wi h
he o hologous egions o C. semilae is and S. maximus (Figu es S2–S37). This allowed us o
p oduce a compa a i e map among he h ee species (Figu e 2and Tables S6 and S7). In
his map, he BAC clones o each ch omosome and each ch omosome a m ( o me acen ics
and subme acen ics) o S. senegalensis ha e hei own colou o mo e easily dis inguish
be ween hem and o ace mo e easily he syn enic posi ions in he o he species. One o
he main esul s is ha , o mos o he S. senegalensis ch omosomes (o ch omosome a ms,
o me acen ics and subme acen ics), he e is a co esponding o hologous ch omosome
in he o he wo species.
The i s me acen ic pai o S. senegalensis has wo o hologue ch omosomes in
C. semilae is (3 and 20) and h ee in S. maximus (7, 18 and 21), and mos o he a m-speci ic
BAC clones a e on one ch omosome o he o he wo species, wi h he excep ion o only wo
BAC clones. In he second me acen ic pai , i s BAC clones a e mo e dispe sed h oughou
he ch omosome complemen o bo h C. semilae is and S. maximus. Howe e , hose o he
p a m (yellow blocks in Figu e 2) appea o be mo e conse ed a a majo o hologous
locus (ch omosome 1 in C. semilae is and ch omosome 2 in S. maximus), al hough some
BACs a e pa ially loca ed on o he ch omosomes. The BAC clones o he q a m (g een
blocks in Figu e 2) do no ha e a majo o hologous locus; ins ead, BAC clones a e mo e
widely dis ibu ed. The BAC clones o he hi d me acen ic pai a e on a single o hologous
ch omosome in bo h C. semilae is (ch omosome 1) and S. maximus (ch omosome 17).
The BAC clones ound on each a m o subme acen ic pai 4 a e p esen on wo
di e en ch omosomes in he o he species. Speci ically, he BAC clones o he p a m a e
ound on ch omosome 14 o C. semilae is and on ch omosome 1 o S. maximus, whe eas
hose on he q a m a e ound on ch omosomes 16 and 14 o C. semilae is and S. maximus,
espec i ely. The si ua ion is di e en in he case o subme acen ic pai 5 o S. senegalensis.
Fi s , his is he ch omosome wi h he lowes numbe o BAC clones ound (only wo, one
on each a m), and second, hey a e loca ed on only one ch omosome in he o he species,
i.e., ch omosome 14 in C. semilae is and ch omosome 16 in S. maximus. Howe e , he
ela i e posi ion o hese wo BAC clones di e s among he h ee species; in S. senegalensis
and S. maximus he wo BAC clones a apa , bu in C. semilae is hey a e close .
As o he ou sub elocen ic pai s ( om pai 6 o 9), all o hem show ha he BAC
clones o each ch omosome a e loca ed on an o hologous ch omosome in each o he o he
wo species. Speci ically, sub elocen ic pai 6 co esponds o ch omosomes Z and 9 o
C. semilae is and S. maximus, espec i ely; sub elocen ic pai 7 o ch omosomes 5 and 5;
sub elocen ic pai 8 o 6 and 10; and sub elocen ic pai 9 o 15 and 13. S ikingly, he BAC
clones o sub elocen ic ch omosome 6 localize o he Z ch omosome o C. semilae is, bu
no o he W ch omosome, apa om a ew genes.
The elocen ic ch omosomes o S. senegalensis can be di ided be ween hose in which
i s BAC clones a e sca e ed on di e en ch omosomes o bo h C. semilae is and S. maximus,
and hose in which he BAC clones a e all oge he on one o hologous ch omosome.
Figu es S38 and S39 o he supplemen a y ma e ial show he dis ibu ion o BAC clones on
each elocen ic ch omosome among he ch omosome complemen o he o he wo species.

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In .J.Mol.Sci.2022,23,xFORPEERREVIEW6o 15
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Figu e2.Compa a i emappingo BACcloneslocalizedin(a)Soleasenegalensiswi h(b)Cynoglossus
semilae is,and(c)Scoph halmusmaximus.BACclonesloca edin hesamech omosomea m(consid‐
e ingme acen icandsubme acen icch omosomesasbi‐a med)o S.senegalensisa e ep esen ed
wi h hesamecolo .
The i s me acen icpai o S.senegalensishas woo hologuech omosomesinC.
semilae is(3and20)and h eeinS.maximus(7,18and21),andmos o  hea m‐speci ic
BACclonesa eononech omosomeo  heo he  wospecies,wi h heexcep iono only
woBACclones.In hesecondme acen icpai ,i sBACclonesa emo edispe sed
h oughou  hech omosomecomplemen o bo hC.semilae isandS.maximus.Howe e ,
hoseo  hepa m(yellowblocksinFigu e2)appea  obemo econse eda amajo 
Figu e 2.
Compa a i e mapping o BAC clones localized in (
a
)Solea senegalensis wi h (
b
)Cynoglossus
semilae is, and (
c
)Scoph halmus maximus. BAC clones loca ed in he same ch omosome a m (conside -
ing me acen ic and subme acen ic ch omosomes as bi-a med) o S. senegalensis a e ep esen ed wi h
he same colo .
2.5. S udy o Repea ed Sequence
To gain u he insigh s in o how epe i i e sequences migh ha e played a ole in he
e olu ion o bi-a med ch omosomes (ch omosomes 1–9) in S. senegalensis, he abundance
and co e age o ansposons, sa elli es, simple epea s, small RNA and low complexi y
sequences on ch omosomes we e analysed (Figu e S40.1–13; Tables S8 and S9). The epe i-
In . J. Mol. Sci. 2022,23, 5353 7 o 14
i e elemen s on ch omosomes 1, 2 and 4 we e analysed p e iously [33–35]; he e o e, he
emaining bi-a med ch omosomes (3, 5–9) a e analysed in he p esen wo k. In addi ion,
ch omosomes 11 and 16 we e also analysed o de e mine he epe i i e elemen s in ol ed in
ch omosome ansloca ions and ea angemen s and hei e olu ion. Finally, S. senegalensis
p esen s an XX/XY de e mina ion sys em bu lacks he e omo phic sex ch omosomes. The
p esence o a ecen pu a i e sex-de e mining gene desc ibed on ch omosome 12 [
7
] makes
his ch omosome o in e es o s udy he possible abundance o epe i i e elemen s, as is
obse ed in sex ch omosomes [42,43].
I can be es ima ed ha he co e age o he six epe i i e elemen s analysed in his
s udy is 20% and he numbe o loci pe Mb is 2500 in S. senegalensis ch omosomes
(Figu es 3and S40; Tables S8 and S9).
In .J.Mol.Sci.2022,23,xFORPEERREVIEW8o 15
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Figu e3.Numbe o locipe Mb(NL/Mb)andco e age(%)o  epea elemen sinBACs omch o‐
mosomes3,5–9,11,12and16o Soleasenegalensis:DNA ansposons, e oelemen s,smallRNA,
sa elli es,simple epea s,lowcomplexi y.
Asumma yo  hemos  ele an  esul sob aineda e  heanalysiso  epe i i eele‐
men sin heBACsmappedalong heeigh ch omosomesisshowninTableS10.
Conside ing heBACsasgenomicsampleso  hech omosomes,andmeasu ing he
mean alueso  henumbe o locipe ch omosome,i canbeseen ha ch omosome12
has hehighes meannumbe o DNA ansposonso allch omosomesanalysed,andalso
haslow a iance(Figu eS40.12).Thehighes meannumbe o  e oelemen sis oundon
ch omosome8, ollowedbych omosomes12and16.A  he amilyle el, hehighes mean
numbe so hobo‐Ac i a o andTc1‐IS630‐Pogoa e oundonch omosome12.In ela ion
o epe i i eelemen sco e age, hehighes meannumbe o DNA ansposonsis ound
onch omosomes16and12.Re oelemen sha e,ona e age, hehighes co e ageonch o‐
mosomes8,9and12.Ch omosome12has hehighes meanco e age alues o hobo‐
Ac i a o ,L2/C1/RexandTc1‐IS630‐Pogoelemen s.Themos abundan LINEs,ona e ‐
age,a eon8and12,andLTRsa eon8and9(Figu eS40.13).
3.Discussion
Thedensi yo  hecy ogene icmapo S.senegalensishasbeeninc easedby33BAC
cloneswi h espec  o ha ob ainedbyMe loe al.[36],andconsequen ly,ma ke sa e
a ailable o allch omosomeso  hecomplemen .Inaddi ion,BLASTanalysiso BACs
Figu e 3.
Numbe o loci pe Mb (NL/Mb) and co e age (%) o epea elemen s in BACs om
ch omosomes 3, 5–9, 11, 12 and 16 o Solea senegalensis: DNA ansposons, e oelemen s, small RNA,
sa elli es, simple epea s, low complexi y.
A summa y o he mos ele an esul s ob ained a e he analysis o epe i i e
elemen s in he BACs mapped along he eigh ch omosomes is shown in Table S10.
Conside ing he BACs as genomic samples o he ch omosomes, and measu ing he
mean alues o he numbe o loci pe ch omosome, i can be seen ha ch omosome 12 has
he highes mean numbe o DNA ansposons o all ch omosomes analysed, and also
has low a iance (Figu e S40.12). The highes mean numbe o e oelemen s is ound
on ch omosome 8, ollowed by ch omosomes 12 and 16. A he amily le el, he highes
In . J. Mol. Sci. 2022,23, 5353 8 o 14
mean numbe s o hobo-Ac i a o and Tc1-IS630-Pogo a e ound on ch omosome 12. In
ela ion o epe i i e elemen s co e age, he highes mean numbe o DNA ansposons is
ound on ch omosomes 16 and 12. Re oelemen s ha e, on a e age, he highes co e age
on ch omosomes 8, 9 and 12. Ch omosome 12 has he highes mean co e age alues o
hobo-Ac i a o , L2/C1/Rex and Tc1-IS630-Pogo elemen s. The mos abundan LINEs, on
a e age, a e on 8 and 12, and LTRs a e on 8 and 9 (Figu e S40.13).
3. Discussion
The densi y o he cy ogene ic map o S. senegalensis has been inc eased by 33 BAC
clones wi h espec o ha ob ained by Me lo e al. [
36
], and consequen ly, ma ke s a e
a ailable o all ch omosomes o he complemen . In addi ion, BLAST analysis o BACs on
he ch omosome-le el sca olding allowed p ecise mapping on o ch omosomes. Finding
BAC clones ha hyb idize on wo o mo e ch omosomes (such as BAC clones 44K21, 45L11,
57N7, 72B11 and 73A11) is indica i e o he ound o ances al duplica ion ha occu ed
in he eleos lineage. Due o whole genome duplica ion (WGD) o an o ganism, as could
be he ances o o eleos s, he ch omosomal complemen doubles (polyploidiza ion), and
ediploidiza ion could occu when he duplica ed ch omosomes di e ge om each o he ,
bu he ediploidized genome may lea e aces o he ances al polyploid a angemen ,
as e idenced in ex an eleos s by he p esence o many pa alogs [
44
], making hem a
paleopolyploid g oup. The p esence o many pa alogues makes i mo e di icul o elucida e
he ch omosome a angemen s ha ha e aken place among he species s udied. In addi ion,
pa ially duplica ed sequences we e ound wi hin he same ch omosome o he main BAC
clone locus (such as BAC clones 10K23, 3I18, 64A8, 68G4, 45L11, 38B21 o ch omosomes 1,
5, 6, 10, 11 and 12, espec i ely). Sequence duplica ion has been conside ed an impo an
mechanism o adap i e e olu ion h ough ansc ip ional modula ion [45].
Syn eny s udies showed a la ge o hology be ween he ch omosomes o he h ee
species compa ed, as S. senegalensis sha es up o 15 syn enic ch omosomes wi h C. semilae is
and 14 wi h S. maximus, and is in ag eemen wi h wha has been p e iously epo ed in
closely ela ed species [46–48].
Me acen ic ch omosome 1 has i s BAC clones dis ibu ed among ch omosomes 3 and
20 o C. semilae is and mainly among ch omosomes 7 and 21 o S. maximus, which is
consis en wi h p e ious s udies and ein o ces he iew ha his ch omosome o igina es
om a Robe sonian usion e en [
7
,
30
–
33
]. Simila o igins ha e been pos ula ed o
ch omosomes 2 (me acen ic) and 4 (subme acen ic) [
7
,
35
]. Ou da a sugges a a he
dispe sed dis ibu ion o BAC clones on ch omosome 2, bu when he analysis is pe o med
in ela ion o he numbe o anno a ed genes, hey a e mainly ound in ch omosomes 1 and
8 o C. semilae is and in ch omosomes 2 and 4 o S. maximus. The S. senegalensis lineage
a ose om h ee Robe sonian usions ha led o ch omosomes 1, 2 and 4, hus jus i ying
he educ ion om he p oposed ances al ka yo ype o Pleu onec i o mes (n= 24) [
11
] o
n= 21. Mo eo e , i has been p oposed ha ch omosomal usions acili a e a lineage-speci ic
di e si ica ion, since hey can gene a e and accumula e gene ic incompa ibili ies [48].
The cen ic usions ha occu ed in S. senegalensis accoun o h ee o he nine bi-
a med ch omosomes (conside ing sub elocen ics as bi-a med), so pe icen ic in e sions
ha e played a cen al ole in he e olu ion o he Senegalese sole ka yo ype. In e sions a e
impo an ea angemen s ha lead o polymo phism main enance e en s in popula ions
h ough balancing selec ion and a e also impo an in he specia ion p ocess [
49
,
50
]. In
addi ion, he e is much e idence o suppo he iew ha in e sions a e in ol ed in he
en i onmen al adap a ion o species and ha in e sion polymo phisms wi hin a species
a e ela ed geog aphically [51,52].
In his sense, Pleu onec i o mes a e ishes ha ha e had o adap o he he e ogeneous
ben hic en i onmen ; he e o e, each species could ha e adap ed o local habi a s h ough
species-speci ic ch omosome ea angemen s. Howe e , he e mus be a mechanism ha
igge s such ch omosome ea angemen s. T ansposable Elemen s (TE) a e conside ed
a key mechanism o ch omosomal ea angemen s [
21
,
35
]. In pa icula , Rex e o ans-
In . J. Mol. Sci. 2022,23, 5353 9 o 14
posons ha e p e iously been desc ibed as a majo playe in he ea angemen o he la ges
me acen ic pai o S. senegalensis [
34
]. TEs a e usually supp essed by a complex epigene ic
silencing pa hway, bu i o ganisms a e aced wi h a new challenging en i onmen al si ua-
ion (such as adap a ion o a new ben hic li es yle), a bu s o TE eac i a ion would occu ,
leading o he gene a ion o genomic s uc u al a ia ions [
53
], as obse ed in he o de
Pleu onec i o mes.
TE ac i i y could also explain he dis ibu ion o ch omosome-speci ic BAC clones
o speci ic ch omosomes (o ch omosome a ms) o S. senegalensis be ween wo o mo e
ch omosomes o he o he wo species. These BAC clones a e hose on he p a m o
me acen ic 1 (only wi h S. maximus), bo h a ms o me acen ic 2, and elocen ic 10 (only
wi h S. maximus), 11, 12 and 16. T ansloca ions and mic o- ea angemen s ha e also been
conside ed o be caused by TEs [35,54–56].
The esul s showed ha on ch omosomes 3, 7, 9 and 16, a highe abundance o
TEs and o he epea ed elemen s (co e age and NL/Mb) has been obse ed nex o he
elome ic egions. This pa e n has also been obse ed on ch omosomes 1, 2 and 4 o
S. senegalensis in a p e ious analysis [
33
,
35
]. Howe e , on ch omosome 11, analysis o
epea ed elemen s in mapped BACs shows a highe abundance o DNA ansposons
in in e s i ial BACs. This ch omosome shows e idence o e olu ion om wo o mo e
ch omosomes o he o he wo la ish species. As desc ibed, TEs may be in ol ed in la ge
s uc u al genomic a ia ions, including ansloca ions and o he s [
57
,
58
]; consequen ly,
TEs such as DNA ansposons (hobo-Ac i a o s wi h he highes numbe in he in e s i ial
BACs) and e oelemen s (LINEs and L2/C1/Rex), wi h he highes numbe in his cen al
egion o he ch omosome, could ha e caused ea angemen s du ing he e olu ion o
his ch omosome in he la ish g oup. This si ua ion also occu s on ch omosome 16 o
simple epea s and low complexi y sequences, bu no o TEs ( e oelemen s and DNA
ansposons). These di e ences in he dis ibu ion and abundance o epea elemen s
in ch omosomes ha ha e unde gone emodeling p ocesses in he cou se o e olu ion
sugges a possible ole o simple epea sequences also in ea anged egions [
59
]. The
mean numbe o epea elemen s pe ch omosome shows ha ch omosome 12 has he
highes mean numbe o DNA ansposons.
Recen ly, a S. senegalensis linkage g oup ( e med SseLG18) has been highligh ed as
a nascen sex ch omosome sys em [
7
]. The ollicle-s imula ing ho mone ecep o ( sh ), a
candida e gene o sex de e mina ion, is loca ed on SseLG18. This LG coincides wi h ou
elocen ic pai 12, one o he ch omosomes implica ed in possible TE-media ed anslo-
ca ions. T ansposable elemen s and o he epe i i e sequences, such as simple epea s,
accumula e on he eme ging sex ch omosome. Supp ession o ecombina ion on sex ch o-
mosomes explains no only he expansion o epe i i e DNA and he accumula ion o TEs,
bu also he accumula ion o dele e ious mu a ions in unc ional genes [
42
]. In he BACs
analysed in his wo k, he accumula ion o epea ed elemen s on ch omosome 12 was
obse ed, o some ex en , in all elemen s, especially in DNA ansposons, bu no in la ge
numbe s. This could indica e ha he p ocess o pseudogeniza ion and accumula ion o
epea ed elemen s on his ch omosome 12 ep esen s a p imi i e s age o he s uc u al
changes associa ed wi h e olu ion as a sex ch omosome.
The homologous egions o all BAC clones p esen on ch omosome 6 a e loca ed on
he Z ch omosome o C. semilae is, bu no on he W ch omosome, wi h he excep ion o
e y ew genes. This inding e eals ha a la ge pseudogeniza ion p ocess has aken place
on he W ch omosome o C. semilae is.
4. Ma e ials and Me hods
4.1. PCR Sc eening, Isola ion o Bac Clones and Map In eg a ion
A o al o 126 clones BACs we e used in his s udy; hey a e shown in Table S1. Ou o
126, 38 we e BACs con aining mic osa elli e ma ke s (Simple Sequence Repea s, SSRs). To
selec hese mic osa elli es, we ini ially selec ed 74 ou o he 129 mic osa elli es belonging
o 27 linkage g oups (LGs) desc ibed by Molina-Luzón e al. [
29
]. The 74 mic osa elli es