Ci a ion: Shi, Y.; Ackah, M.; Amoako,
F.K.; Zhao, M.; an de Puije, G.C.;
Zhao, W. The Mechanism o he
De elopmen and Main enance o
Sexual Dimo phism in he Dioecious
Mulbe y Plan (Mo us alba). Biology
2024,13, 622. h ps://doi.o g/
10.3390/biology13080622
Academic Edi o : Wenqiang Li
Recei ed: 15 June 2024
Re ised: 8 Augus 2024
Accep ed: 13 Augus 2024
Published: 15 Augus 2024
Copy igh : © 2024 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/).
biology
A icle
The Mechanism o he De elopmen and Main enance o Sexual
Dimo phism in he Dioecious Mulbe y Plan (Mo us alba)
Yisu Shi 1,2, Michael Ackah 1,2 , F ank Kwa eng Amoako 3, Mengdi Zhao 4,*, G ace C. an de Puije 5
and Weiguo Zhao 1,2,*
1Jiangsu Key Labo a o y o Se icul u e Biology and Bio echnology, School o Bio echnology,
Jiangsu Uni e si y o Science and Technology, Zhenjiang 212100, China; [email p o ec ed] (Y.S.);
[email p o ec ed] (M.A.)
2
Key Labo a o y o Silkwo m and Mulbe y Gene ic Imp o emen , Minis y o Ag icul u e and Ru al A ai s,
The Se icul u al Resea ch Ins i u e, Chinese Academy o Ag icul u al Sciences, Zhenjiang 212100, China
3Ins i u e o Plan Nu i ion and Soil Science, Kiel Uni e si y, He mann-Rodewald-S aße 2, 24118 Kiel,
Ge many; [email p o ec ed]
4Depa men o Ma e ials Science and Enginee ing, Suzhou Uni e si y o Science and Technology,
Suzhou 215011, China
5Depa men o C op Science, School o Ag icul u e, College o Ag icul u e and Na u al Sciences,
Uni e si y o Cape Coas , Cape Coas P.O. Box 5007, Ghana; [email p o ec ed]
*Co espondence: [email p o ec ed] (M.Z.); [email p o ec ed] (W.Z.)
Simple Summa y: This s udy highligh s he impo ance o in e sexual di e en ia ion in dioecious
plan specia ion and main enance. Using Mo us alba plan s, we iden i ied 1543 di e en ially exp essed
genes (DEGs) be ween male and emale indi iduals. No ably, 333 genes we e exclusi ely exp essed
in male in lo escences, while 66 we e unique o emale in lo escences. Th ough compa a i e an-
sc ip omics, 841 genes wi h signi ican ly highe exp ession in males we e linked o de ense- ela ed
pa hways, and 702 genes wi h emale-biased exp ession we e ela ed o g ow h and de elopmen
pa hways. Signals o selec ion we e de ec ed in bo h male- and emale-biased genes, indica ing hei
ole in e olu ion. These indings ad ance ou unde s anding o he molecula basis o in e sexual
di e en ia ion and shed ligh on sex e olu ion in M. alba plan s.
Abs ac : In e sexual di e en ia ion is c ucial o he specia ion and main enance o dioecious plan s,
bu he unde lying mechanisms, including he genes in ol ed, a e s ill poo ly unde s ood. He e, we
ocused on a ypical dioicous plan Mo us alba, o explo e he molecula oo p in s ele an o sex
e olu ion by e ealing he di e en ially exp essed genes (DEGs) be ween wo sexes and he es ing
signals o selec ion o hese DEGs. F om he esul s, we ound a o al o 1543 DEGs. In e es ingly,
333 and 66 genes exp ession we e de ec ed only in male and emale in lo escences, espec i ely. Using
compa a i e ansc ip omics, he exp ession o 841 genes we e ound o be signi ican ly highe in
male han in emale in lo escences and we e mainly en iched in de ense- ela ed pa hways including
he biosyn hesis o phenylp opanoids, cu in, sube ine and waxes. Meanwhile, he exp ession o
702 genes was emale-biased and la gely en iched in pa hways ela ed o g ow h and de elopmen ,
such as ca bohyd a e me abolism, auxin signaling and cellula esponses. In addi ion, 16.7% and
17.6% signals o selec ion we e signi ican ly de ec ed in emale- and male-biased genes, espec i ely,
sugges ing hei non-negligible ole in e olu ion. Ou indings expanded he unde s anding o he
molecula basis o in e sexual di e en ia ion and con ibu e o u he esea ch on sex e olu ion in
dioecious plan s.
Keywo ds: sex-biased gene exp ession; sexual dimo phism; sexual selec ion; pollen compe i ion;
haploid selec ion; a i icial selec ion; species di e si y
Biology 2024,13, 622. h ps://doi.o g/10.3390/biology13080622 h ps://www.mdpi.com/jou nal/biology
Biology 2024,13, 622 2 o 17
1. In oduc ion
Ongoing global change poses a se ious h ea o he conse a ion o plan biodi e -
si y and ecosys em unc ioning [
1
]. Fo conse a ion pu poses, i is he e o e essen ial o
elucida e how plan s espond o changing en i onmen s, which equi es a comp ehensi e
unde s anding o hei li e his o y [
2
]. Dioecious plan s ha e many di e en ep oduc i e
ai s ha ha e e ol ed unde di e en selec i e o ces, and he i ness o indi iduals
wi h di e en sexes can be in luenced by di e en ac o s [
3
–
6
]. Howe e , al hough such
in e sexual di e ences call o sex-speci ic conse a ion s a egies wi hin a single dioecious
plan species [
3
,
7
], he de ails o ai di e ences and he unde lying molecula basis and
e olu iona y ajec o ies a e s ill poo ly unde s ood. The e o e, s udies on he gene exp es-
sion pa e ns ele an o he ai s cons i u ing sexual dimo phism a e o g ea impo ance
o dioecious plan s.
Some ai s wi hou a di ec ela ion o game e de elopmen bu show signi ican
sexual dimo phism a e called seconda y sexual cha ac e is ics [
7
]. These ai s in ol ed in
sexual dimo phism in dioecious species mainly include lowe ing phenology, lo al cha ac-
e s (s uc u e, size, and numbe o lowe s in an in lo escence), lo al chemical composi ion
such as nu ien con en and he bi o e- esis an chemicals, and physical de enses [
7
–
9
].
Fu he mo e, hese ai s a e hough o in luence i ness by a ec ing pollina ion, ma ing,
ecundi y, and seed iabili y [
10
]. P e ious s udies ha e shown ha in mos dioecious plan s,
males end o p oduce mo e lowe s wi h sho e lowe longe i y and less synch onous low-
e ing compa ed o emales, and hei e olu ion is likely d i en by compe i ion o pollina ion
e iciency and ma ing compe i ion [
11
]. In addi ion, he e may be s onge chemical and
physical de enses o male lowe s in wind-pollina ed plan s han insec -pollina ed species,
which may help o ensu e su icien pollen o pollen dispe sal [
11
–
13
]. As o emales, hei
lowe s end o syn hesize mo e auxins o ini ia e he ep oduc i e p og am and s imula e
seed de elopmen , as well as nu ien s ha can bo h a ac pollina o s and acili a e seed
g ow h [
14
,
15
]. Howe e , he molecula basis o hese di e en ia ed ep oduc i e ai s
is s ill unclea . Al hough some pa hways in ol ed in plan me abolism ha e been iden-
i ied [
7
,
16
,
17
]. Fo una ely, he apid de elopmen o ansc ip omics and compa a i e
genomics has shed ligh on un a elling he molecula mechanisms unde lying ai a ia ion
by de ec ing a iable exp ession and signals o posi i e selec ion o candida e genes [5,18].
Species in he genus Mo us a e widely dis ibu ed and p o ide an impo an pool
o species o ag icul u e, bu many wild species a e h ea ened [
19
]. Mulbe y (M. alba)
(2n = 28) is a dioecious wind-pollina ed pe ennial woody plan , cul i a ed in Asia [
20
,
21
].
M. alba exhibi s appa en sexual dimo phism in lowe s uc u e, numbe and size pe
in lo escence and lowe ea lie in males han in emales [
21
–
23
]. This p o ides an excellen
model o sc eening genes associa ed wi h sexual dimo phism. In addi ion, a high-quali y
assembly wi h a genome size o 346.39 Mb has been epo ed o his species [
21
], p o iding
a p o ound basis o ansc ip ome s udies. Fu he mo e, he e a e many dioecious and
monoecious species in he genus Mo us [
24
], so he p esence o signi ican signals o posi i e
selec ion in he sc eened genes can be easily es ed by in e speci ic compa isons wi h closely
ela ed monoecious species.
In his s udy, we used compa a i e ansc ip ome analysis o sc een di e en ially
exp essed genes (DEGs) be ween wo sexes o M. alba in lo escences collec ed om h ee
male and h ee emale ees and pe o med unc ional en ichmen on selec ed DEGs ele-
an o ai s o sexual dimo phism, wi h u he e olu iona y analysis o unco e hei
e olu iona y his o y. We aim o answe wo scien i ic ques ions: (1) whe he DEGs in
male and emale in lo escences a e in ol ed in egula ing he de elopmen o sex-speci ic
ai s, and (2) whe he hese genes a e unde posi i e selec ion. Ou esul s will p o ide
heo e ical e idence and elucida e he molecula basis o he o igin and main enance o
sexual dimo phism in plan s and will aid u u e conse a ion e o s.
Biology 2024,13, 622 3 o 17
2. Ma e ials and Me hods
2.1. Plan Ma e ials
The lowe buds we e collec ed om he ees o wo M. alba pu i ied b eeding lines,
h ee BaiTiao (BT), which p oduces only male lowe s, and h ee Y20 which p oduces
only emale lowe s [
25
]. These b eeds a e g own a he Se icul u e Resea ch Ins i u e
o he Chinese Academy o Ag icul u al Sciences in Zhenjiang, Jiangsu P o ince, China
(N 32
◦
11
′
45.80
′′
, E 119
◦
23
′
45.80
′′
). Main enance condi ions o he M. alba ee a e as ollows:
minimum and maximum empe a u es (9–20 °C), ela i e humidi y (40–60%), i iga ion
( ain all), day leng h (12 h), and ligh in ensi y (15,000 lx). Addi ionally, in o ma ion abou
he appea ance condi ions o he ees includes age (5 yea ), diame e (5 cm), and heigh
(2 m). BT and Y20 ha e cha ac e is ics such as la ge in lo escences, a g ea e numbe o
lowe s, and wide adap abili y and a e ypical cul i a s o M. alba. The e o e, hese wo
pu i ied lines we e used as he s udy ma e ials o compa e he di e ences in gene exp ession
be ween male and emale in lo escences o M. alba. Male and emale in lo escence (1.3 m
om he g ound) samples we e collec ed ( ha is, male in lo escence om BT pu i ied
lines and emale in lo escence om he pu i ied Y20 lines). The samples we e a mix u e o
di e en in lo escences om he same plan , and six ees (3 om BT and Y20 each) we e
selec ed o sampling, ep esen ing h ee biological eplica es. In all, a o al o six ca kins
we e sampled. In lo escence sampling was conduc ed om 9:00 p.m. o 11:00 p.m. Only
ully de eloped adul ca kins (0.8–1.5 cm long and 0.3–0.5 cm in diame e ) we e collec ed,
when he ca kins we e ully expanded, bu be o e he lowe s had opened (Figu e 1). Whole
ca kins we e cu and immedia ely ozen in liquid ni ogen o RNA ex ac ion.
Biology2024,13,xFORPEERREVIEW3o 17


2.Ma e ialsandMe hods
2.1.Plan Ma e ials
The lowe budswe ecollec ed om he eeso  woM.albapu i iedb eedinglines,
h eeBaiTiao(BT),whichp oducesonlymale lowe s,and h eeY20whichp oducesonly
emale lowe s[25].Theseb eedsa eg owna  heSe icul u eResea chIns i u eo  he
ChineseAcademyo Ag icul u alSciencesinZhenjiang,JiangsuP o ince,China
(N32°11′45.80″,E119°23′45.80″).Main enancecondi ions o  heM.alba eea eas ollows:
minimumandmaximum empe a u es(9–20℃), ela i ehumidi y(40–60%),i iga ion
( ain all),dayleng h(12h),andligh in ensi y(15,000lx).Addi ionally,in o ma ionabou 
heappea ancecondi ionso  he eesincludesage(5yea ),diame e (5cm),andheigh 
(2m).BTandY20ha echa ac e is icssuchasla ge in lo escences,ag ea e numbe o 
lowe s,andwideadap abili yanda e ypicalcul i a so M.alba.The e o e, hese wo
pu i iedlineswe eusedas hes udyma e ials ocompa e hediffe encesingeneexp es-
sionbe weenmaleand emalein lo escenceso M.alba.Maleand emalein lo escence
(1.3m om heg ound)sampleswe ecollec ed( ha is,malein lo escence omBTpu i-
iedlinesand emalein lo escence om hepu i iedY20lines).Thesampleswe eamix-
u eo diffe en in lo escences om hesameplan ,andsix ees(3 omBTandY20each)
we eselec ed o sampling, ep esen ing h eebiological eplica es.Inall,a o alo six
ca kinswe esampled.In lo escencesamplingwasconduc ed om9:00p.m o11:00p.m.
Only ullyde elopedadul ca kins(0.8–1.5cmlongand0.3–0.5cmindiame e )we ecol-
lec ed,when heca kinswe e ullyexpanded,bu be o e he lowe shadopened(Figu e
1).Wholeca kinswe ecu andimmedia ely ozeninliquidni ogen o RNAex ac ion.

Figu e1.Physicalappea anceo collec edadul Mo usalbaca kins.(a)Femaleca kinswi hp o ud-
ingpis illa e lowe s.(b)Maleca kinswi hp o udings amina e lowe s.
2.2.RNAEx ac ionandIlluminaSequencing
Using heRNAisoPlus eagen (Taka a,Shanghai,China), e e se ansc ip ionand
o alRNAex ac ionwe epe o medoneachsampledca kininacco dancewi h heman-
u ac u e ’sins uc ions.UsinganAgilen 2100Bioanalyze (Agilen Technologies,Palo
Al o,CA,USA)andNanoD op1000spec opho ome e (IMPLEN,CA,USA), hequan i y
andpu i yo  o alRNAwe eassessed.Fu he mo e,1µgo  o alRNApe samplewas
used ocons uc anRNAsequencelib a yusing heNEBNex
®
Ul aTMRNALib a y
P epKi  o Illumina(NEB,USA)acco ding o hemanu ac u e ’sspeci ica ions.Subse-
quen ly, ansc ip omesequencingwasconduc edusinganIlluminaNo aseq2500pla -
o m,whichp oducedpai ed-end eadso 150bp.
Figu e 1. Physical appea ance o collec ed adul Mo us alba ca kins. (a) Female ca kins wi h p o uding
pis illa e lowe s. (b) Male ca kins wi h p o uding s amina e lowe s.
2.2. RNA Ex ac ion and Illumina Sequencing
Using he RNAiso Plus eagen (Taka a, Shanghai, China), e e se ansc ip ion and
o al RNA ex ac ion we e pe o med on each sampled ca kin in acco dance wi h he manu-
ac u e ’s ins uc ions. Using an Agilen 2100 Bioanalyze (Agilen Technologies, Palo Al o,
CA, USA) and NanoD op 1000 spec opho ome e (IMPLEN, Wes lake Village, CA, USA),
he quan i y and pu i y o o al RNA we e assessed. Fu he mo e, 1
µ
g o o al RNA pe
sample was used o cons uc an RNA sequence lib a y using he NEBNex
®
Ul aTM RNA
Lib a y P ep Ki o Illumina (NEB, Ipswich, MA, USA) acco ding o he manu ac u e ’s
speci ica ions. Subsequen ly, ansc ip ome sequencing was conduc ed using an Illumina
No aseq 2500 pla o m (San Diego, CA, USA), which p oduced pai ed-end eads o 150 bp.
Biology 2024,13, 622 4 o 17
2.3. Quali y Con ol o T ansc ip ome Da a and Exp ession Analysis
To ob ain clean RNA-seq da a, he aw RNA-seq eads om each sample we e p o-
cessed using in-house pe l sc ip s o im adap e s and low-quali y bases om he ends o
he eads, wi h u he il e ing o eads con aining poly-N. The clean, high-quali y eads
o each sample we e hen mapped o he M. no abilis e e ence genome [
20
] using Hisa 2
2.0.5, and he mapped eads we e assembled using S ingTie ( 1.3.3b) o no el ansc ip
p edic ion. F agmen s pe kilobase million ( PKM) was used o es ima e gene exp ession
le els om each gene acco ding o he numbe o eads mapped o ha gene egion using
ea u eCoun s 1.5.0-p3. Di e en ial exp ession analysis was pe o med o de ec he sig-
ni ican ly di e en ially exp essed genes (DEGs) be ween he male and emale ca kins using
he DESeq2 R package ( 1.16.1) [
26
]. Fu he mo e, p- alues we e adjus ed based on he
alse disco e y a e (FDR) using he Benjamini and Hochbe g app oach. Genes wi h a old
change di e ence in exp ession (|log2(FoldChange)| > 0) o (|log2(FoldChange)| < 0) and
adjus ed p- alues (p-adj) < 0.05 we e conside ed as DEGs. Among hese DEGs, he alue o
(|log2(FoldChange)| > 0) was de ined as male-biased genes and (|log2(FoldChange)| < 0)
as emale-biased genes. The ex emes we e conside ed as genes exp essed only in emales
and we e de ined as emale-limi ed genes, and he opposi e was de ined as male-limi ed
genes. Gene on ology (GO) en ichmen analysis was pe o med on he DEGs using he
Clus e P o ile R package [
27
]. GO e ms wi h padj < 0.05 we e conside ed signi ican ly
en iched by DEGs. The Clus e P o ile R package was also used o pe o m DEG en ich-
men s a is ics in he KEGG pa hway [
27
]. Pop_ i_ 3 (Populus ichoca pa) was used as
a e e ence (h ps://plan s.ensembl.o g/Populus_ ichoca pa/In o/Index, accessed on
15 Oc obe 2023).
2.4. RT-qPCR Ve i ica ion
Based on he KEGG and GO en ichmen esul s o he DEGs, 10 emale-biased/-limi ed
exp ession genes en iched in g ow h and de elopmen pa hways and 10 male-biased/-
limi ed exp ession genes en iched in de ense- ela ed pa hways (Table S1) we e andomly
selec ed o RT-qPCR analysis. The RNA samples o lowe buds used o RT-qPCR we e
aken om he same indi iduals as he samples used o ansc ip ome sequencing. A
o al o 20 pai s o gene-speci ic p ime s we e cons uc ed based on hei sequences in
he e e ence genome u ilizing P ime 6 so wa e (Table S1), and cDNA syn hesis was
pe o med using M-MLV e e se ansc ip ase (RTase) (Taka a, Beijing, China), wi h 1
µ
g
RNA samples as he empla e. The cDNA solu ion was dilu ed 5- old, and 1
µ
L o cDNA
was used as he empla e o pe o m gene alida ion using he SYBR G een RT-PCR p o ocol
(Roche, Indianapolis, IN, USA). The eac ion sys em con ained SYBR qPCR 2
×
Taq Mix
(10
µ
L), o wa d p ime (1
µ
L), e e se p ime (1
µ
L), ddH2O (7
µ
L) and cDNA (1
µ
L), and
he
β
-ac in gene (as a no maliza ion con ol); he p ime o he
β
-ac in gene is shown in
Table S1. All eac ions we e pe o med in h ee biological eplica es using h ee empla es.
The p ocedu e o he RT-qPCR was as ollows: 95
◦
C o 10 min, ollowed by a cycle
p og am (dena u a ion: 95
◦
C o 10 s, annealing: 50
◦
C o 10 s, and ex ension: 70
◦
C
o 10 s) o 45 imes. The mean ela i e exp ession o each gene was no malized o he
e e ence gene, β-ac in, and calcula ed using he 2−∆∆C me hod [28].
2.5. Analysis o Adap i e E olu ion o Sex-Biased and Unbiased Genes
To es whe he he sex-biased genes exp essed only in males o emales and he sex-
biased DEGs had unde gone signi ican posi i e selec ion, we used he e olu iona y a io
(dN/dS) o all M. alba genes using paml 4.9 [
29
]. Fo each M. alba gene, he nucleo ide
sequences we e ansla ed in o p o ein sequences, and hei coding sequences we e sea ched
in TB ools 11.0.2 [
30
]. As M. alba sha es a common ances o wi h M. no abilis and di e ged
wi h P unus pe sica, which is a he maph odi e [
21
], we compa ed he coding sequences om
ou M. alba ansc ip ome da a wi h he homologous genes in he M. no abilis genome [
20
].
Mo eo e , he sequences om he P. pe sica genome we e used as an ou g oup. Based on
he bi sco e, e- alue, and local alignmen leng h (Supplemen a y Ma e ials, R sc ip ), we
Biology 2024,13, 622 5 o 17
used blas n 2.12.0 o iden i y he op imal mul idi ec ional ma ching homologs o M. alba
om M. no abilis 2.0 [
20
] and P. pe sica (Peach 1.0) [
31
] genome anno a ion da a. The
coding and p o ein sequences o he h ee species alignmen s we e ex ac ed and compa ed
using Pa aAT 1.0 [
32
]. We es ima ed he dN/dS alues o he M. alba and M. no abilis
lineages wi h codeml (PAML 4.9) [
29
], using a model wi h wo o mo e dN/dS a ios o
b anches, unmode as use ee, and nda a as 13,532, and o he pa ame e s we e se as he
de aul [
33
]. We excluded 4980 alignmen s om he analysis due o un ealis ically high
di e gence (S ×dS + N ×dN > 15% o alignmen ) [7].
In addi ion, we conduc ed a compa ison o he dN/dS alues o he e olu iona y
lineage leading o M. alba and M. no abilis, speci ically ocusing on genes ha exhibi sex-
biased o sex-limi ed exp ession pa e ns, as well as genes ha do no display such biases.
K uskal–Wallis es s we e used o compa e he e olu iona y a io (dN/dS) be ween he
sex-biased and unbiased genes, which is also used in Sande son e al. (2019). Ini ially, a
non-pa ame ic K uskal–Wallis es was employed o assess he di e ences in median alues
o dN/dS be ween sex-biased genes and a andomly selec ed unbiased genes (K uskal. es
and dunn. es in R o pe o m he K uskal–Wallis es and pos hoc es s espec i ely). To
asce ain he a e o neu al e olu ion o hese genes, a compa a i e analysis was conduc ed
on he median dS alues o sex-biased genes and a andomly selec ed se o 3000 unbiased
genes [
7
]. Addi ionally, we conduc ed a compa ison o he 95% quan ile alues o dN/dS
ac oss h ee ca ego ies in o de o assess whe he genes exhibi ing sex-biased o sex-limi ed
cha ac e is ics we e mo e p one o displaying ex eme alues compa ed o he dis ibu ion o
95% quan ile alues de i ed om 5000 boo s ap samples o unbiased genes. This analysis
was pe o med using he boo package in he R p og amming language (h ps://CRAN.
R-p ojec .o g/package=boo , accessed on 12 Augus 2024). To explo e he pa hways and
unc ions o posi i ely selec ed DEGs, KEGG and GO en ichmen analysis o he posi i ely
selec ed DEGs we e pe o med using he clus e P o ile R package (p-adj < 0.05).
3. Resul s
3.1. Sequencing Quali y Con ol and Re e ence Genome Mapping
Six lib a ies we e subjec ed o Illumina sequencing u ilizing samples om male and
emale lowe buds. Each sample was p epa ed in h ee epe i ions. A combined sum o
305,642,716 aw sequencing eads we e acqui ed om he lowe buds o BT (male) and Y20
( emale). A e imming adap e and poo -quali y sequences, 294,445,006 clean eads we e
ob ained. Among hese eads, 139,671,832 (95.6%) we e clean eads om male lowe buds
and 154,773,174 (97.0%) we e clean eads om emale lowe buds. Fo all he lib a ies,
he a e age Q30 was mo e han 92% and he GC con en was mo e han 40% (Table 1).
Fu he mo e, mo e han 64.5% o he clean eads we e mapped o he e e ence genome
(M. no abilis). A o al o 21,155 unigenes we e ob ained om he assembly, wi h he longes
gene being 16,567 bp and he sho es ha ing 72 bp.
Table 1. Summa y o quali y p ep ocessing o RNA sequencing da a.
Sample ID Raw Reads Clean Reads Clean Bases E o Ra e Q30 GC% Mapping Ra io
MBT-1 52,365,500 50,926,984 7.64 G 0.03 93.12 44.31 64.6
MBT-2 44,385,448 41,784,712 6.27 G 0.03 93.55 42.81 58.4
MBT-3 49,325,358 46,960,136 7.04 G 0.03 92.19 40.73 72.8
FY20-1 56,900,778 54,807,454 8.22 G 0.03 92.27 44.23 55.8
FY20-2 48,518,050 47,476,836 7.12 G 0.03 93.46 44.66 66.9
FY20-3 54,147,582 52,488,884 7.87 G 0.03 92.38 43.44 68.5
MBT and FY20: Male and emale mulbe y (Mo us alba) lowe buds used in RNA sequencing. MBT-1, MBT-2,
and MBT-3 and FY20-1, FY20-2, and FY20-3: h ee eplica es o male and emale mulbe y (M. alba) lowe buds,
espec i ely.

Biology 2024,13, 622 6 o 17
3.2. Di e en ially Exp essed Genes be ween Male and Female Flowe Buds
Following he ansc ip ome assembly and anno a ion, di e en ial exp ession analysis
was pe o med. Su p isingly, a o al o 1,543 genes we e di e en ially exp essed in male and
emale lowe buds based on he c i e ia s a ed in Sec ion 2.3. Ou o hese DEGs, 702 genes
we e signi ican in emale lowe buds and 841 genes we e signi ican in male lowe buds
(Figu e 2). A o al o 19,612 genes exhibi ed a ansc ip abundance di e ence ha did no
mee he c i e ia (Figu e 2c). These indings indica e he p esence o biologically signi ican
le els o gene exp ession dimo phism. Genes ha we e exp essed exclusi ely in one sex
ype we e de ined as sex-limi ing genes (DEGs showing some exp ession in one sex ype
bu absolu ely ze o coun s in he o he ). A o al o 399 genes exhibi ed exp ession ha was
sex-limi ed. Ou o he o al, 333 genes (83.5%) we e exclusi ely exp essed in male lowe s,
while 66 genes (16.5%) we e exclusi ely exp essed in emale lowe s, sugges ing ha he
p opo ion o male-biased genes was highe han ha o emale-biased genes. The numbe o
genes exhibi ing male bias was ma ginally highe compa ed o hose exhibi ing emale bias.
Biology2024,13,xFORPEERREVIEW6o 17
3.2.Diffe en iallyExp essedGenesbe weenMaleandFemaleFlowe Buds
Following he ansc ip omeassemblyandanno a ion,diffe en ialexp essionanaly-
siswaspe o med.Su p isingly,a o alo 1,543geneswe ediffe en iallyexp essedin
maleand emale lowe budsbasedon hec i e ias a edinSec ion2.3.Ou o  heseDEGs,
702geneswe esigni ican in emale lowe budsand841geneswe esigni ican inmale
lowe buds(Figu e2).A o alo 19,612genesexhibi eda ansc ip abundancediffe ence
ha didno mee  hec i e ia(Figu e2c).These indingsindica e hep esenceo biologi-
callysigni ican le elso geneexp essiondimo phism.Genes ha we eexp essedexclu-
si elyinonesex ypewe ede inedassex-limi inggenes(DEGsshowingsomeexp ession
inonesex ypebu absolu elyze ocoun sin heo he ).A o alo 399genesexhibi ed
exp ession ha wassex-limi ed.Ou o  he o al,333genes(83.5%)we eexclusi elyex-
p essedinmale lowe s,while66genes(16.5%)we eexclusi elyexp essedin emale
lowe s,sugges ing ha  hep opo iono male-biasedgeneswashighe  han ha o  e-
male-biasedgenes.Thenumbe o genesexhibi ingmalebiaswasma ginallyhighe com-
pa ed o hoseexhibi ing emalebias.
Figu e2.Compa isonso geneexp essionbe weenmaleand emale lowe budso M.alba.(a)
Sha eddiffe en iallyexp essedgenesbe weenmaleand emale lowe buds.(b)Geneexp ession
compa isonbe weenmaleand emale lowe buds.(c)Volcanoplo onsex-biasedgenes ha we e
exp essedsigni ican lydiffe en inmaleand emale lowe buds.
Addi ionally, heex en o diffe en ialexp ession,asmeasu edby(|log2FC|),was
signi ican lyhighe  o geneswi hmalebiascompa ed o hosewi h emalebias(Figu e
3a,Wilcoxon anksum es p<0.01,R4.1.0).Themeanexp essionle elso male-biased
geneswe e ound obeconside ablylowe compa ed o hea e ageexp essionle elso 
emale-biasedgenes(Figu e3b;Wilcoxon anksum es p<0.01,R4.1.0),al hough emale
exp ession o male-biasedgeneswassigni ican lyhighe  hanmaleexp ession o  e-
male-biasedgenes(Figu e3b,Wilcoxon anksum es p<0.01,R4.1.0).The indingso 
hisanalysisindica e ha geneswi habias owa ds emalesmayo igina e omheigh -
enedexp essionin emalein lo escencesand educedexp essioninmalein lo escences.
Figu e 2. Compa isons o gene exp ession be ween male and emale lowe buds o M. alba. (a) Sha ed
di e en ially exp essed genes be ween male and emale lowe buds. (b) Gene exp ession compa ison
be ween male and emale lowe buds. (c) Volcano plo on sex-biased genes ha we e exp essed
signi ican ly di e en in male and emale lowe buds.
Addi ionally, he ex en o di e en ial exp ession, as measu ed by (|log2FC|), was
signi ican ly highe o genes wi h male bias compa ed o hose wi h emale bias (Figu e 3a,
Wilcoxon ank sum es p< 0.01, R 4.1.0). The mean exp ession le els o male-biased
genes we e ound o be conside ably lowe compa ed o he a e age exp ession le els
o emale-biased genes (Figu e 3b; Wilcoxon ank sum es p< 0.01, R 4.1.0), al hough
emale exp ession o male-biased genes was signi ican ly highe han male exp ession o
emale-biased genes (Figu e 3b, Wilcoxon ank sum es p< 0.01, R 4.1.0). The indings o
his analysis indica e ha genes wi h a bias owa ds emales may o igina e om heigh ened
exp ession in emale in lo escences and educed exp ession in male in lo escences. Addi-
ionally, i is seen ha he bias owa ds males p edominan ly s ems om he up egula ion
o emale exp ession.
Biology 2024,13, 622 7 o 17
Biology2024,13,xFORPEERREVIEW7o 17


Addi ionally,i isseen ha  hebias owa dsmalesp edominan lys ems om heup eg-
ula iono  emaleexp ession.

Figu e3.Sex-biasedgeneexp essioninM.alba.(a)Thep opo ionand angeo DEGsandunbiased
genesinM.albaca kins.(b)Theuppe -hal shows hecompa isono maleand emaleca kindiffe -
en iallyexp essiongenes(DEGs)a diffe en sexbias oldchange h esholds o alle alua edca kin
male-biasedand emale-biasedgenes.Thenumbe sin heb acke s ep esen  henumbe o DEGs
ineachca kinca ego y.Thelowe -hal shows hea e agemaleand emaleca kinexp essiono all
genesa diffe en sexbias oldchange h esholds o all hee alua edca kinmale-biasedand e-
male-biasedgenes.Signi ican diffe encesbe weenmaleand emaleexp essionbasedonWilcoxon
anksum es sa edeno edbyas e iska alphale el(p<0.0001).Thesex-biasedgenesmen ioned
he eincludesex-limi edgenes.BTandY20a e hemaleand emaleca kins.
3.3.Func ionalAnno a ionAssocia edwi hDEGs
Tode e minewhe he  heseDEGsa eassocia edwi h hede elopmen o sex-spe-
ci ic ai s,GOandKEGGanalysiswasused oiden i ypa e nso en ichmen be ween
heDEGsinmaleand emale lowe buds.Acco ding o heGOen ichmen  esul s, he
mos  emale-biasedgeneswe eanno a edinnucleicacidbinding ansc ip ion ac o ac-
i i y(GO:0003700), ansc ip ion ac o ac i i y,andsequence-speci icDNAbinding
(GO:0001071)(Figu e4a,b).Themos male-biasedgeneswe eanno a ed o ca bohyd a e
Figu e 3. Sex-biased gene exp ession in M. alba. (a) The p opo ion and ange o DEGs and unbiased
genes in M. alba ca kins. (b) The uppe -hal shows he compa ison o male and emale ca kin
di e en ially exp ession genes (DEGs) a di e en sex bias old change h esholds o all e alua ed
ca kin male-biased and emale-biased genes. The numbe s in he b acke s ep esen he numbe o
DEGs in each ca kin ca ego y. The lowe -hal shows he a e age male and emale ca kin exp ession
o all genes a di e en sex bias old change h esholds o all he e alua ed ca kin male-biased and
emale-biased genes. Signi ican di e ences be ween male and emale exp ession based on Wilcoxon
ank sum es s a e deno ed by as e isk a alpha le el (p< 0.0001). The sex-biased genes men ioned
he e include sex-limi ed genes. BT and Y20 a e he male and emale ca kins.
3.3. Func ional Anno a ion Associa ed wi h DEGs
To de e mine whe he hese DEGs a e associa ed wi h he de elopmen o sex-speci ic
ai s, GO and KEGG analysis was used o iden i y pa e ns o en ichmen be ween he
DEGs in male and emale lowe buds. Acco ding o he GO en ichmen esul s, he
mos emale-biased genes we e anno a ed in nucleic acid binding ansc ip ion ac o
ac i i y (GO:0003700), ansc ip ion ac o ac i i y, and sequence-speci ic DNA binding
(GO:0001071) (Figu e 4a,b). The mos male-biased genes we e anno a ed o ca bohyd a e
me abolism p ocesses (GO:0005975). To u he unde s and he en ichmen pa hways
o male-/ emale-biased genes, hese DEGs we e analyzed by KEGG pa hway analysis.
Male-biased genes we e en iched in 111 KEGG pa hways and signi ican ly en iched in
he phenylp opanoid biosyn hesis pa hway (pop00940). Female-biased genes we e as-
Biology 2024,13, 622 8 o 17
signed o 101 KEGG pa hways and we e mainly en iched in he plan ho mone signal
pa hway (pop04075) (Figu e 4c,d). The ole o plan ho mones in plan sex de e mina ion
has been demons a ed in many s udies [
34
,
35
]. Acco ding o he esul o KEGG en ich-
men analysis, 22 DEGs (10 emale-biased and 12 male-biased) we e en iched in plan
ho mone signaling, including auxin, cy okinin, salicylic acid, jasmonic acid, and abscisic
acid signaling pa hways (Figu e 5a).
In e es ingly, he male-biased genes we e mainly anno a ed in he abscisic acid and
jasmonic acid signaling pa hways, whe eas he emale-biased genes we e mainly anno a ed
in he cy okinin and auxin signaling pa hways. The sc eened DEGs con ained 88 ansc ip-
ion ac o s (TFs) ha could be success ully anno a ed o he KEGG pa hway, including
33 emale-biased TFs and 55 male-biased TFs.
Biology2024,13,xFORPEERREVIEW8o 17
me abolismp ocesses(GO:0005975).To u he unde s and heen ichmen pa hwayso 
male-/ emale-biasedgenes, heseDEGswe eanalyzedbyKEGGpa hwayanalysis.Male-
biasedgeneswe een ichedin111KEGGpa hwaysandsigni ican lyen ichedin hephe-
nylp opanoidbiosyn hesispa hway(pop00940).Female-biasedgeneswe eassigned o
101KEGGpa hwaysandwe emainlyen ichedin heplan ho monesignalpa hway
(pop04075)(Figu e4c,d).The oleo plan ho monesinplan sexde e mina ionhasbeen
demons a edinmanys udies[34,35].Acco ding o he esul o KEGGen ichmen anal-
ysis,22DEGs(10 emale-biasedand12male-biased)we een ichedinplan ho monesig-
naling,includingauxin,cy okinin,salicylicacid,jasmonicacid,andabscisicacidsignaling
pa hways(Figu e5a).
In e es ingly, hemale-biasedgeneswe emainlyanno a edin heabscisicacidand
jasmonicacidsignalingpa hways,whe eas he emale-biasedgeneswe emainlyanno-
a edin hecy okininandauxinsignalingpa hways.Thesc eenedDEGscon ained88
ansc ip ion ac o s(TFs) ha couldbesuccess ullyanno a ed o  heKEGGpa hway,
including33 emale-biasedTFsand55male-biasedTFs.
Figu e4.Func ionalanalysiso  heDEGsinM.albamaleand emaleca kins.(a)GOen ichmen 
e mso male-biasedexp essiongenes.(b)GOen ichmen  e mso  emale-biasedexp essiongenes.
Figu e 4. Func ional analysis o he DEGs in M. alba male and emale ca kins. (a) GO en ichmen
e ms o male-biased exp ession genes. (b) GO en ichmen e ms o emale-biased exp ession genes.
(c) KEGG-en iched di e en ial exp ession genes o male-biased exp ession genes. (d) KEGG-en iched
di e en ial exp ession genes o emale-biased exp ession genes. BP; biological p ocess. CC; cellula
componen . MF; molecula unc ion.
Biology 2024,13, 622 9 o 17
Biology2024,13,xFORPEERREVIEW9o 17
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(c)KEGG-en icheddiffe en ialexp essiongeneso male-biasedexp essiongenes.(d)KEGG-en-
icheddiffe en ialexp essiongeneso  emale-biasedexp essiongenes.BP;biologicalp ocess.CC;
cellula componen .MF;molecula  unc ion.

Figu e5.Ahea mapanalysiso  heDEGsinM.albamaleand emaleca kins.(a)Ahea mapdia-
g amo  heDEGsbe weenmaleand emale lowe budso M.albain hephy oho monesignaling
pa hways:abscisicacid(i),jasmonicacid(ii),salicylicacid(iii),cy okinin(i ),andauxin( ).(b)The
ansc ip ion ac o (TF) amiliesdiffe en iallyexp essedinmale-biasedgenes.(c)TheTF amilies
diffe en iallyexp essedinmale-biasedand emale-biasedgenes.Thecolo scale ep esen s helog10-
ans o medFPKM alue.Thesex-biasedgenesmen ionedhe eincludesex-limi edgenes.The
male-biasedgenesa eup egula edand emale-biasedgenesa edown egula edbasedon he igu e
legend.
Among hem, he emale-biased/-limi edTFswe eanno a ed o 29KEGGpa hways,
whichwe e ela ed og ow handde elopmen .Theyincludeme abolicpa hways
(pop01100),biosyn hesiso seconda yme aboli es(pop01110),s a chandsuc oseme ab-
olism(pop00500),andsoon.Howe e , hemale-biased/-limi edTFswe eanno a ed o 
Figu e 5. A hea map analysis o he DEGs in M. alba male and emale ca kins. (a) A hea map diag am
o he DEGs be ween male and emale lowe buds o M. alba in he phy oho mone signaling pa hways:
abscisic acid (i), jasmonic acid (ii), salicylic acid (iii), cy okinin (i ), and auxin ( ). (b) The ansc ip ion
ac o (TF) amilies di e en ially exp essed in male-biased genes. (c) The TF amilies di e en ially
exp essed in male-biased and emale-biased genes. The colo scale ep esen s he log
10
- ans o med
FPKM alue. The sex-biased genes men ioned he e include sex-limi ed genes. The male-biased genes
a e up egula ed and emale-biased genes a e down egula ed based on he igu e legend.
Among hem, he emale-biased/-limi ed TFs we e anno a ed o 29 KEGG pa h-
ways, which we e ela ed o g ow h and de elopmen . They include me abolic pa h-
ways (pop01100), biosyn hesis o seconda y me aboli es (pop01110), s a ch and suc ose
me abolism (pop00500), and so on. Howe e , he male-biased/-limi ed TFs we e anno-
a ed o 66 KEGG pa hways, including me abolic pa hways (pop01100), biosyn hesis o
seconda y me aboli es (pop01110), glyce ophospholipid me abolism (pop00564), plan
ho mone signal ansduc ion (pop04075), glycolysis/Gluconeogenesis (pop00010), glyc-
e olipid me abolism (pop00561), and a y acid me abolism (pop01212). TFs’ egula ion o
gene exp ession and he exp ession p o iles o TF genes be ween male and emale lowe
buds a e shown as a hea map (Figu e 5b,c).
Biology 2024,13, 622 16 o 17
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