The Mechanism of the Development and Maintenance of Sexual Dimorphism in the Dioecious Mulberry Plant (Morus alba)

Shi, Yisu; Ackah, Michael; Amoako, Frank, Kwarteng; Zhao, Mengdi; van der Puije, Grace, C; Zhao, Weiguo; Amoako, Frank Kwarteng; van der Puije, Grace C

doi:10.3390/biology13080622

The Mechanism of the Development and Maintenance of Sexual Dimorphism in the Dioecious Mulberry Plant (Morus alba)

Shi, Yisu,Ackah, Michael,Amoako, Frank Kwarteng,Zhao, Mengdi,van der Puije, Grace C,Zhao, Weiguo

Published: 2024-01-01
DOI: 10.3390/biology13080622
Repository: macau.uni-kiel.de
Language: en

Abstract

Intersexual differentiation is crucial for the speciation and maintenance of dioecious plants, but the underlying mechanisms, including the genes involved, are still poorly understood. Here, we focused on a typical dioicous plant Morus alba, to explore the molecular footprints relevant to sex evolution by revealing the differentially expressed genes (DEGs) between two sexes and the testing signals of selection for these DEGs. From the results, we found a total of 1543 DEGs. Interestingly, 333 and 66 genes expression were detected only in male and female inflorescences, respectively. Using comparative transcriptomics, the expression of 841 genes were found to be significantly higher in male than in female inflorescences and were mainly enriched in defense-related pathways including the biosynthesis of phenylpropanoids, cutin, suberine and waxes. Meanwhile, the expression of 702 genes was female-biased and largely enriched in pathways related to growth and development, such as carbohydrate metabolism, auxin signaling and cellular responses. In addition, 16.7% and 17.6% signals of selection were significantly detected in female- and male-biased genes, respectively, suggesting their non-negligible role in evolution. Our findings expanded the understanding of the molecular basis of intersexual differentiation and contribute to further research on sex evolution in dioecious plants.

Full text

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   (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 4. Elleg en, H.; Pa sch, J. The e olu ion o sex-biased genes and sex-biased gene exp ession. Na . Re . Gene . 2007,8, 689–698. [C ossRe ] 5. Ha ison, P.W.; W igh , A.E.; Zimme , F.; Dean, R.; Mon gome y, S.H.; Poin e , M.A.; Mank, J.E. Sexual selec ion d i es e olu ion and apid u no e o male gene exp ession. P oc. Na l. Acad. Sci. USA 2015,112, 4393–4398. [C ossRe ] 6. Zemp, N.; Ta a es, R.; Muyle, A.; Cha leswo h, D.; Ma ais, G.A.B.; Widme , A. E olu ion o sex-biased gene exp ession in a dioecious plan . Na . Plan s 2016,2, 16168. [C ossRe ] [PubMed] 7. Sande son, B.J.; Wang, L.; Ti in, P.; Wu, Z.; Olson, M.S. Sex-biased gene exp ession in lowe s, bu no lea es, e eals seconda y sexual dimo phism in Populus balsami e a. New Phy ol. 2019,221, 527–539. [C ossRe ] 8. Ba e , S.C.; Hough, J. Sexual dimo phism in lowe ing plan s. J. Exp. Bo . 2013,64, 67–82. [C ossRe ] 9. Zaji schek, F.; Bondu iansky, R.; Zaji schek, S.R.; B ooks, R.C. Sexual dimo phism in li e his o y: Age, su i al, and ep oduc ion in male and emale ield c icke s Teleog yllus commodus unde semina u al condi ions. Am. Na . 2009,173, 792–802. [C ossRe ] [PubMed] 10. Ce an es, C.; Al a ez, A.; Cue as, E. Small bu a ac i e: Female-biased nec a p oduc ion and lo al isi o s in a dimo phic sh ub. Plan Biol. 2018,20, 160–164. [C ossRe ] 11. Tsuji, K.; Kobayashi, K.; Hasegawa, E.; Yoshimu a, J. Dimo phic lowe s modi y he isi a ion o de o pollina o s om male o emale lowe s. Sci. Rep. 2020,10, 9965. [C ossRe ] 12. Ge shenzon, J.; Ullah, C. Plan s p o ec hemsel es om he bi o es by op imizing he dis ibu ion o chemical de enses. P oc. Na l. Acad. Sci. USA 2022,119, e2120277119. [C ossRe ] [PubMed] 13. Wakabayashi, K.; Ogu o, M.; I agaki, T.; Sakai, S. Flo al-induced and cons i u i e de ense agains lo i o y: A compa ison o chemical ai s in 12 he b species. Plan Ecol. 2018,219, 985–997. [C ossRe ] 14. Gao, L.; Yu, G.; Hu, F.; Li, Z.; Li, W.; Peng, C. The pa e ns o male and emale lowe s in lowe ing s age may no be op imal esou ce alloca ion o ui and seed g ow h. Plan s 2021,10, 2819. [C ossRe ] [PubMed] 15. Zhao, Y. Auxin Biosyn hesis and I s Role in Plan De elopmen . Annu. Re . Plan Biol. 2010,61, 49–64. [C ossRe ] [PubMed] 16. Liu, J.; Chen, L.-Y.; Zhou, P.; Liao, Z.; Lin, H.; Yu, Q.; Ming, R. Sex biased exp ession o ho mone ela ed genes a ea ly s age o sex di e en ia ion in papaya lowe s. Ho ic. Res. 2021,8, 147. [C ossRe ] [PubMed] 17. Schenck, C.A.; Maeda, H.A. Ty osine biosyn hesis, me abolism, and ca abolism in plan s. Phy ochemis y 2018,149, 82–102. [C ossRe ] [PubMed] 18. A unkuma , R.; Josephs, E.B.; Williamson, R.J.; W igh , S.I. Pollen-speci ic, bu no spe m-speci ic, genes show s onge pu i ying selec ion and highe a es o posi i e selec ion han spo ophy ic genes in Capsella g andi lo a.Mol. Biol. E ol. 2013,30, 2475–2486. [C ossRe ] 19. Xuan, Y.; Ma, B.; Li, D.; Tian, Y.; Zeng, Q.; He, N. Ch omosome es uc u ing and numbe change du ing he e olu ion o Mo us no abilis and Mo us alba.Ho ic. Res. 2022,9, uhab030. [C ossRe ] [PubMed] 20. He, N.; Zhang, C.; Qi, X.; Zhao, S.; Tao, Y.; Yang, G.; Lee, T.-H.; Wang, X.; Cai, Q.; Li, D.; e al. D a genome sequence o he mulbe y ee Mo us no abilis.Na . Commun. 2013,4, 2445. [C ossRe ] [PubMed] 21. Jiao, F.; Luo, R.; Dai, X.; Liu, H.; Yu, G.; Han, S.; Lu, X.; Su, C.; Chen, Q.; Song, Q.; e al. Ch omosome-le el e e ence genome and popula ion genomic analysis p o ide insigh s in o he e olu ion and imp o emen o domes ica ed mulbe y (Mo us alba). Mol. Plan 2020,13, 1001–1012. [C ossRe ] [PubMed] 22. Jaiswal, V.; Kuma , A. Ac i i y and isozymes o pe oxidase in Mo us nig a L. du ing sex di e en ia ion. Z. P lanzenphysiol. 1981, 102, 299–302. [C ossRe ] 23. Lal, M.; Jaiswal, V.S. Modi ica ion o lowe sex and acid phospha ase ac i i y by ph halimides in emale plan s o Mo us nig a L. Plan G ow h Regul. 1988,7, 29–37. [C ossRe ] 24. Jolly, M.S.; Dandin, S.B.; Ra ind an, S.; Kuma , R. Sexual polymo phism in he genus Mo us L. P oc. Plan Sci. 1986,96, 315–320. [C ossRe ] 25. A sumi, R.; Nishiha a, R.; Ta o a, K.; U asaki, N.; Ma sumu a, H. Iden i ica ion o dominan gene ic ma ke s ele an o male sex de e mina ion in mulbe y (Mo us alba L.). Euphy ica 2019,215, 187. [C ossRe ] 26. Ande s, S.; Hube , W. Di e en ial exp ession analysis o sequence coun da a. Na . P eced. 2010, 1. [C ossRe ] 27. Yu, G.; Wang, L.-G.; Han, Y.; He, Q.-Y. clus e P o ile : An R package o compa ing biological hemes among gene clus e s. OMICS J. In eg . Biol. 2012,16, 284–287. [C ossRe ] [PubMed] 28. Li ak, K.J.; Schmi gen, T.D. Analysis o ela i e gene exp ession da a using eal- ime quan i a i e PCR and he 2 −∆∆CT me hod. Me hods 2001,25, 402–408. [C ossRe ] [PubMed] 29. Yang, Z. PAML 4: Phylogene ic analysis by maximum likelihood. Mol. Biol. E ol. 2007,24, 1586–1591. [C ossRe ] [PubMed] 30. Chen, C.J.; Chen, H.; Zhang, Y.; Thomas, H.R.; F ank, M.H.; He, Y.H.; Xia, R. TB ools: An in eg a i e oolki de eloped o in e ac i e analyses o big biological da a. Mol. Plan 2020,13, 1194–1202. [C ossRe ] [PubMed] 31. In e na ional Peach Genome Ini ia i e; Ve de, I.; Abbo , A.G.; Scalab in, S.; Jung, S.; Shu, S.; Ma oni, F.; Zheben yaye a, T.; De o i, M.T.; G imwood, J.; e al. The high-quali y d a genome o peach (P unus pe sica) iden i ies unique pa e ns o gene ic di e si y, domes ica ion and genome e olu ion. Na u e Gene . 2013,45, 487–494. [C ossRe ] [PubMed] 32. Zhang, Z.; Xiao, J.; Wu, J.; Zhang, H.; Liu, G.; Wang, X.; Dai, L. Pa aAT: A pa allel ool o cons uc ing mul iple p o ein-coding DNA alignmen s. Biochem. Biophys. Res. Commun. 2012,419, 779–781. [C ossRe ] 33. Yang, Z.; Rannala, B. Molecula phylogene ics: P inciples and p ac ice. Na . Re . Gene . 2012,13, 303–314. [C ossRe ] [PubMed] Biology 2024,13, 622 17 o 17 34. De ani, R.S.; Sinha, S.; Bane jee, J.; Sinha, R.K.; Bendahmane, A.; Bane jee, A.K. De no o ansc ip ome assembly om lowe buds o dioecious, gynomonoecious and chemically masculinized emale Coccinia g andis e eals genes associa ed wi h sex exp ession and modi ica ion. BMC Plan Biol. 2017,17, 241. [C ossRe ] [PubMed] 35. Yuan, Z.; Zhang, D. Roles o jasmona e signalling in plan in lo escence and lowe de elopmen . Cu . Opin. Plan Biol. 2015,27, 44–51. [C ossRe ] [PubMed] 36. McKown, A.D.; Klápš ˇe, J.; Guy, R.D.; Soolanayakanahally, R.Y.; La Man ia, J.; Po h, I.; Skyba, O.; Unda, F.; Douglas, C.J.; El-Kassaby, Y.A.; e al. Sexual homomo phism in dioecious ees: Ex ensi e es s ail o de ec sexual dimo phism in Populus.Sci. Rep. 2017,7, 1831. [C ossRe ] [PubMed] 37. Robinson, K.M.; Delhomme, N.; Mähle , N.; Schi hale , B.; Önskog, J.; Alb ec sen, B.R.; Ing a sson, P.K.; H ids en, T.R.; Jansson, S.; S ee , N.R. Populus emula (Eu opean aspen) shows no e idence o sexual dimo phism. BMC Plan Biol. 2014,14, 276. [C ossRe ] [PubMed] 38. Ha kess, A.; Me ca i, F.; Shan, H.; Sunse i, F.; Fala igna, A.; Leebens-Mack, J. Sex-biased gene exp ession in dioecious ga den aspa agus (Aspa agus o icinalis). New Phy ol. 2015,207, 883–892. [C ossRe ] [PubMed] 39. Bou denx, B.; Be na d, A.; Dome gue, F.; Pascal, S.; Lége , A.; Roby, D.; Pe en , M.; Vile, D.; Haslam, R.P.; Napie , J.A.; e al. O e exp ession o A abidopsis ECERIFERUM1 p omo es wax e y-long-chain alkane biosyn hesis and in luences plan esponse o bio ic and abio ic s esses. Plan Physiol. 2011,156, 29–45. [C ossRe ] [PubMed] 40. S omme , J. The plan ADH gene amily. Plan J. 2011,66, 128–142. [C ossRe ] [PubMed] 41. Wang, H.; Dong, Q.; Duan, D.; Zhao, S.; Li, M.; an Nocke , S.; Ma, F.; Mao, K. Comp ehensi e genomic analysis o he TYROSINE AMINOTRANSFERASE (TAT) genes in apple (Malus domes ica) allows he iden i ica ion o MdTAT2 con e ing ole ance o d ough and osmo ic s esses in plan s. Plan Physiol. Biochem. 2018,133, 81–91. [C ossRe ] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .