SCIENTIFICREPRTS心HDifferent autosomes evolved into sexOPENchromosomes in the sister genera of SalixSUBJECTAREAS:MOLECULAR EVOLUTIONand PopulusGENOMEPLANTEVOLUTIONJing Hou*, Ning Ye*, Defang Zhang*, Yingnan Chen, Lecheng Fang, Xiaogang Dai & Tongming YinReceivedThe Southem Modern Forestry Collaborafive Innovafion Center,Nanjing Forestry UniversityNanjing 210037,Chin8October2014AcceptedWillows (Salix)and poplars (Populus) are dioecious plants in Salicaceaefamily.Sex chromosome in poplar16 February 2015genome wasconsistentlyreportedtobeassociated with chromosomeXiX.In contrast topoplar,this studyrevealed that chromosomeXV was sex chromosome in willow.Previous studies revealed that both ZZ/ZWPublishedandXX/XY sex-determining systems could bepresent in some species of Populus.Inthis study,sex of S.13March2015suchowensis was found to be determined bythe ZW system in which thefemalewas the heterogameticgender.Gene syntenicand collinear comparisons revealed macrosyntenybetween sexchromosomes and thecorresponding autosomes between these two lineages.By contrast, no syntenic segments were found to beshared between poplar's and willow's sex chromosomes.Syntenic analysis also revealed substantialCorrespondence andchromosome rearrangementsbetweenwillow's alternate sexchromatids.Sincewillowand poplaroriginaterequests for materialsfrom a common ancestor,we proposed that evolution of autosomes into sex chromosomes in these twoshould be addressedtolineagesoccurredaftertheirdivergence.ResultsofthisstudyindicatethatsexchromosomesinSalicaceaeT.Y. (tmyin@njfu.com.are still at the early stageofevolutionarydivergence.Additionally,this studyprovided valuable informationcn)for better understanding the genetics and evolution of sex chromosome in dioecious plants.* These authorsex determination has theintriguing aspect in evolutionary and developmental biology.Incontrast to themore completed sex chromosome evolution in animals, sex chromosomes in many plants are still atcontributed equally todifferent evolutionary stagest-, and thus afford the opportunity of investigating the early stage of sexthis work.chromosome evolution.It is believed that sex chromosomes originate from a pair of autosomess.6,and the sex-determiningsvstemsindioeciousplantsalmostcertainlyevolvedindependentlyfromancestralhermaphroditesthatlackedsexchromosomes23Onlyabout4%ofhigherplantsshowfulldioecism,withindividualsofseparatesexesSalicaceae is a family of dioecious woody plants,and the male or female flowers are arranged in morphologically different catkins (Figure 1) on the male or female treesio. Salix and Populus are the sister genera inSalicaceae (Figure 2).Genome analysis revealed that these two lineages originated from a common paleotetra-polyploidancestor-3ThechomosomesfSalicaceaearetypicalymacentricandsmall4Basedoncyiogical studies,there is no evidence of morphologically differentiated sex chromosomes in the Salicaceaespeciesi6-1s Various mechanisms have been proposed to explain the expression of gender in Salicaceae.Alstrom-Rapaport et al.proposed a multi-locus sexdetermination in the Salix,and that thepresence of sexchromosomes was unlikelyl9. In contrast, Semerikov et al. reported that a single locus governed the sex deter-mination and thefemale was theheterogametic gender in basket willow2o.More recently,several studies revealedthattherewas a singlelocuslocatedon chromosomeXiXof Populus, with a peritelomericlocalization inmembersoftheAigeiros subgenera2122 anda centromericlocalization in subgenera ofLeuce2-2,appearedtocontainagene(genes)thatcontrolledgenderdetermination.Multiplelinesofevidence suggested thatpoplar'schromosomeXIXwas intheprocessofevolving intoan incipient sex chromosome22.27.The sex-determiningregion inpoplar showssome interestingfeatures, including location ofgender locus,highly divergenthaplotypes,severerecombinationsuppression,distinctivepattern of sRNAoccurrence, and significantlyfewersingle nucleotidepolymorphisms(SNP) than the rest of Populus genomell127. Mapping studies on P. deltoides22 and P. alba2 revealed that sexdetermination occurred through a ZW system in which thefemalewas the heterogametic gender2.However, themalesex wasreportedastheheterogameticgenderinP.nigra'andPtremuloides-hus,itispossiblethatboth ZZ/ZW (femaleheterogamety)and XX/XY (male heterogamety) sex-determining systems could be presentin somemembers of the family Salicaceae27.Ithas been confirmed that the sistergenera of Salix and Populusoriginate from a common ancestorl-13 Since different gender determining systems probably evolve separately-SCIENTIFICREPORTS15:9076/DOI:10.1038/srep09076
Different autosomes evolved into sex chromosomes in the sister genera of Salix and Populus Jing Hou*, Ning Ye*, Defang Zhang*, Yingnan Chen, Lecheng Fang, Xiaogang Dai & Tongming Yin The Southern Modern Forestry Collaborative Innovation Center, Nanjing Forestry University, Nanjing 210037, China. Willows (Salix) and poplars (Populus) are dioecious plants in Salicaceae family. Sex chromosome in poplar genome was consistently reported to be associated with chromosome XIX. In contrast to poplar, this study revealed that chromosome XV was sex chromosome in willow. Previous studies revealed that both ZZ/ZW and XX/XY sex-determining systems could be present in some species of Populus. In this study, sex of S. suchowensis was found to be determined by the ZW system in which the female was the heterogametic gender. Gene syntenic and collinear comparisons revealed macrosynteny between sex chromosomes and the corresponding autosomes between these two lineages. By contrast, no syntenic segments were found to be shared between poplar’s and willow’s sex chromosomes. Syntenic analysis also revealed substantial chromosome rearrangements between willow’s alternate sex chromatids. Since willow and poplar originate from a common ancestor, we proposed that evolution of autosomes into sex chromosomes in these two lineages occurred after their divergence. Results of this study indicate that sex chromosomes in Salicaceae are still at the early stage of evolutionary divergence. Additionally, this study provided valuable information for better understanding the genetics and evolution of sex chromosome in dioecious plants. Sex determination has the intriguing aspect in evolutionary and developmental biology. In contrast to the more completed sex chromosome evolution in animals, sex chromosomes in many plants are still at different evolutionary stages1–4, and thus afford the opportunity of investigating the early stage of sex chromosome evolution. It is believed that sex chromosomes originate from a pair of autosomes5,6, and the sexdetermining systems in dioecious plants almost certainly evolved independently from ancestral hermaphrodites that lacked sex chromosomes2,3. Only about 4% of higher plants show full dioecism, with individuals of separate sexes7–9. Salicaceae is a family of dioecious woody plants, and the male or female flowers are arranged in morphologically different catkins (Figure 1) on the male or female trees10. Salix and Populus are the sister genera in Salicaceae (Figure 2). Genome analysis revealed that these two lineages originated from a common paleotetrapolyploid ancestor11–13. The chromosomes of Salicaceae are typically metacentric and small14,15. Based on cytological studies, there is no evidence of morphologically differentiated sex chromosomes in the Salicaceae species16–18. Various mechanisms have been proposed to explain the expression of gender in Salicaceae. Alstro¨m-Rapaport et al. proposed a multi-locus sex determination in the Salix, and that the presence of sex chromosomes was unlikely19. In contrast, Semerikov et al. reported that a single locus governed the sex determination and the female was the heterogametic gender in basket willow20. More recently, several studies revealed that there was a single locus located on chromosome XIX of Populus, with a peritelomeric localization in members of the Aigeiros subgenera21,22 and a centromeric localization in subgenera of Leuce23–26, appeared to contain a gene (genes) that controlled gender determination. Multiple lines of evidence suggested that poplar’s chromosome XIX was in the process of evolving into an incipient sex chromosome22,27. The sex-determining region in poplar shows some interesting features, including location of gender locus, highly divergent haplotypes, severe recombination suppression, distinctive pattern of sRNA occurrence, and significantly fewer single nucleotide polymorphisms (SNP) than the rest of Populus genome11,22,27. Mapping studies on P. deltoides22 and P. alba26 revealed that sex determination occurred through a ZW system in which the female was the heterogametic gender22. However, the male sex was reported as the heterogametic gender in P. nigra21 and P. tremuloides23–25. Thus, it is possible that both ZZ/ZW (female heterogamety) and XX/XY (male heterogamety) sex-determining systems could be present in some members of the family Salicaceae27. It has been confirmed that the sister genera of Salix and Populus originate from a common ancestor11–13. Since different gender determining systems probably evolve separately OPEN SUBJECT AREAS: MOLECULAR EVOLUTION GENOME PLANT EVOLUTION Received 8 October 2014 Accepted 16 February 2015 Published 13 March 2015 Correspondence and requests for materials should be addressed to T.Y. (tmyin@njfu.com. cn) * These authors contributed equally to this work. SCIENTIFIC REPORTS | 5 : 9076 | DOI: 10.1038/srep09076 1
www.nature.com/scientificreportsMOMFemaleMalesedoFemaleMaleFigure 1Flowers of the female and male trees in Salicaceae species. On willow and poplar, the male or female flowers are separately arranged inmorphologically different catkins on themale or femaletrees.Photos taken byTongming Yin and Jing Hou.Oryza sativaVitis viniferaSalix suchowensis100Populoustrichocarpa100100RicimuscommunisboArabidopsisthaliana100100Carica papayaPrunus persicaSubstitutions/site00.30.60.91.2Figure 2Phylogenetic tree of selected plant species. The phylogenetic tree was constructed with 1,881 single-copy genes on 4-fold degenerate sites.Thebranch length represents the neutral divergence rate. The posterior probabilities (credibility of the topology) for inner nodes are all 100%.2SCIENTIFICREPORTS15:90761DOI:10.1038/srep09076
Figure 1 | Flowers of the female and male trees in Salicaceae species. On willow and poplar, the male or female flowers are separately arranged in morphologically different catkins on the male or female trees. Photos taken by Tongming Yin and Jing Hou. Figure 2 | Phylogenetic tree of selected plant species. The phylogenetic tree was constructed with 1,881 single-copy genes on 4-fold degenerate sites. The branch length represents the neutral divergence rate. The posterior probabilities (credibility of the topology) for inner nodes are all 100%. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9076 | DOI: 10.1038/srep09076 2
www.nature.com/scientificrepand quiterecentlyin species ofthisfamily,Salicaceae is adesirableof 38.4Mb.Chromosome identities of the willowgenomeweresystem to study the genetics and evolution of sex chromosomes indesignated byblasting willow sequencescontaining the mappeddioecious plants.SNPs against the P.trichocarpa genome sequences. It was found thatS. suchowensis is an early flowering shrub willow that belongs tothe linkage group containing the gender locus was chromosome XVsubgenus Vetrix2sRecently,its genomehasbeen sequenced andin the willow genome (Figure 3a).This chromosome is an autosomepublically available. To gain further insight into the origin andin poplars.Thus,thewillow's sex chromosome correspondsto aevolutionof sexchromosomes inSalicaceaespecies, weidentifiedpoplar's autosome.Previous studies revealed that chromosomeXIXwas the sex chromosome in poplars?1-26,by contrast, chromosomethe sex chromosome in S. suchowensis, as well as compared the sexXIX was identified as an autosome in willow.chromosomes between the sistergenera of Salixand Populus.Onwillow'schromosomeXVandchromosomeXIX,18and40sequence scaffolds were mapped,respectively,along the correspondResultsandDiscussioning chromosome based on thematernal map (Figure 3a).Because theTo map the gender locus of S. suchowensis, we established a largesequenced individuals of both P.trichocarpa and S. suchowensis weremapping pedigree in which the sequenced individual was the mater-femalesiiis, we first conducted syntenic analysis between sex chro-nal parent. Among the progeny, 374 individuals were randomlymosomes and the corresponding autosomes in these two lineagesselected for mapping and locating the gender locus in the willowbased on the willow's female map. Syntenic analysis revealed highgenome.Investigation of sex phenotype confirmed constant gendercollinearity on chromosome XIX(Figure 4a)and on chromosomeforallofthemappingprogenyacrossthreelocations intwocontinu-XV (Figure4b)between willow and poplar.In P.trichocarpa, com-ous years. Among these individuals, 183 werefemale,188 were male,plex haplotype divergence was observed between its alternate sexand 3remained sexually immature.Statistical analysis indicated thatchromatidsii27. To explore whether the Z and W sex chromatidssegregation ofgenderfitfor the expected1:1Mendelian segregationdiverged with each other in willow, we examined the sequence scaf-ratio (x = 0.067, where (x = 3.84 for α ≤ 0.05). Subsequently,folds anchored on willow's chromosome XV based on themale map,geneticmaps were separatelyconstructed for the maternal and pater-which included 45 sequence scaffolds (Figure 3b). Contrary to thenal parents by using amplified fragment length polymorphismconsistency revealed based on thefemale map (Figure 4b), syntenic(AFLP) markers and the pseudo-testcross strategy29. Totally, 1,137analysis based on the male map revealed substantial chromosome1:1 segregating markers were generated by90 primer combinationsrearrangements (Figure 4c), suggesting the occurrence of significantwith the374mappingprogeny.Among thesemarkers, 494 testcrosschromosomedivergencebetween theZ and W chromatids in willow.markers fromthematernal parent weremapped into266bins?Wefurthercompared willow's sex chromosome (XV)with poplar'sdistributed on 19 linkage groups, covering a genetic distance ofsex chromosome (XIX),andfound that sex chromosomes between these1924.5 cM (Supplementary Figure 1); Alternatively,549 testcross locitwo lineages shared no syntenic chromosome segments (Figure 4d)from themale were assigned into 263bins on19linkagegroups,indicating that sex chromosomes of poplar and willow originatedspanning a genetic distance of 2223.8 cM (Supplementary Figure 2).fromdifferent ancestral chromosomes.Since sex chromosomes actThecoverageof thefemale andmalemaps was estimated tobe99.99%asautosomesinthealternategeneraofSalicaceae,weproposethatand 99.97% at 20 cM of a marker, respectively. Thus, the establishedturnover of autosomes into sex chromosomes occurred after themaps achieved nearly complete genomecoverage,with linkagegroupdivergence of Salix and Populus.numbers equaled to the 19 haploid chromosome numbers in willow.Because evidence indicated that chromosomeXV and chromosomeBased on the established genetic maps, the gender locus was mappedXIX separately evolved into sex chromosomes after the divergence ofas a 1:1 segregating morphological marker.Mapping results showedwillow and poplar, we proposed that turnover of different autosomesthatgender locus could onlybemapped on the maternal map,but wasinto sex chromosomes might be due to mobile of the sex-determiningunmappable on the paternal map,indicatingthat thefemalewas thegene from chromosomeXIX to chromosomeXV in willow.heterogametic gender in willow, which was in agreement with theAlternatively,thesex-determininggenemayhavetranslocatedfromfindings in P.deltoides2’ and P. alba2that revealed sex determinationchromosome XV to chromosome XIX in poplar.Therefore, there mightoccurred through a ZW system, in which females are heterogametic22.be homologous sex-determining genes within the sex-determiningIt was also noteworthy that marker density in the vicinity of the genderregions between willow and poplar.Mapping of SSR markerslocus (Supplementary Figure 1)was significantly higher than thatrevealed that the gender locus was in between SSR markersexpected by chance alone (P ≤ 0.0001 with a Poisson calculator),SSR151 (151,741bpon scaffold64)andSSR893 (893,817bpon scaf-which indicated severe recombination suppression around the genderfold 64) (Figure 4b). In P. nigra2" and P. deltoides, the close relativeslocus.Recombination suppression has been recognized as a criti-to P. trichocarpa (the sequenced poplar species), gender locus wascal mechanism that triggered the divergence of the alternate sexchromosomes222,3031consistently mapped to the peritelomeric region upper the positionof SSR marker O_206 (Figure 4a).We subsequently searched theTo map the willow sequence scaffolds derived by Dai et al.1 alonghomologous genes shared by these two regions between willow andeach chromosome of the willowgenome, we carried out finemap-poplar,andninewillowgeneswerefoundtohaveatotalof52homo-ping with SNP markers that were generated by resequencing a subsetlogous genes in the sex-determining region of P.trichocarpa. However,of 80progenyfrom the374mapping individuals.AFLPmarkersonall these poplar genes had homologous genes in other regions of thethe established maps were used as landmarks to anchor the SNPmarkers into thecorresponding marker bins. In total, 2,548 testcrosspoplargenome,and noneof thesegenes werefound to be involved inSNP markers fromthematernal parent and3,772testcross SNPsex determination based on gene annotation (Supplementary Table 1).In recent studies, RNA-based sex-determining mechanisms havemarkers from the paternal parent were integrated into the femalebeen found to playa significantrole in higher plants2-34For example(Supplementary Figure 3) and male (Supplementary Figure 4) maps,respectively.Subsequently,SNPmarkers ineachlinkagegroupwerethe tasselseed4 miRNA, ie, miR172, was found to be involved in thesexdeterminationofthemale inflorescence inmaize23Interestinglyused to map the willow sequence scaffolds derived byDai et al.13alongthecorrespondingchromosome.the miR172 family was conserved in Populus's and located on theIn total, 432 sequence scaffolds (covering a physical length ofperitelomeric end of chromosome XIx7.We searched the homolgous194.1 Mb) and 630 sequence scaffolds (covering a physical lengthsequencesof miR172 in willow,and none of them appeared in theof206.2Mb)weremappedbased onthefemale (Figure3a)andmalesex-determiningregion.Morerecently,Akagietal.reported thata(Figure 3b) maps, respectively.There remained 7,009 unmappedsex-determining candidate (OGI) encoded a small RNA targeting asequence scaffolds larger than 2 kb, covering a total physical lengthfeminizing gene (MeGI) in persimmons (Diospyros lotus), and the3SCIENTIFICREPORTS15:9076|DOI:10.1038/srep09076
and quite recently in species of this family, Salicaceae is a desirable system to study the genetics and evolution of sex chromosomes in dioecious plants. S. suchowensis is an early flowering shrub willow that belongs to subgenus Vetrix28. Recently, its genome has been sequenced and publically available13. To gain further insight into the origin and evolution of sex chromosomes in Salicaceae species, we identified the sex chromosome in S. suchowensis, as well as compared the sex chromosomes between the sister genera of Salix and Populus. Results and Discussion To map the gender locus of S. suchowensis, we established a large mapping pedigree in which the sequenced individual was the maternal parent. Among the progeny, 374 individuals were randomly selected for mapping and locating the gender locus in the willow genome. Investigation of sex phenotype confirmed constant gender for all of the mapping progeny across three locations in two continuous years. Among these individuals, 183 were female, 188 were male, and 3 remained sexually immature. Statistical analysis indicated that segregation of gender fit for the expected 151 Mendelian segregation ratio (x2 5 0.067, where (x2 5 3.84 for a # 0.05). Subsequently, genetic maps were separately constructed for the maternal and paternal parents by using amplified fragment length polymorphism (AFLP) markers and the pseudo-testcross strategy29. Totally, 1,137 151 segregating markers were generated by 90 primer combinations with the 374 mapping progeny. Among these markers, 494 testcross markers from the maternal parent were mapped into 266 bins distributed on 19 linkage groups, covering a genetic distance of 1924.5 cM (Supplementary Figure 1); Alternatively, 549 testcross loci from the male were assigned into 263 bins on 19 linkage groups, spanning a genetic distance of 2223.8 cM (Supplementary Figure 2). The coverage of the female and male maps was estimated to be 99.99% and 99.97% at 20 cM of a marker, respectively. Thus, the established maps achieved nearly complete genome coverage, with linkage group numbers equaled to the 19 haploid chromosome numbers in willow. Based on the established genetic maps, the gender locus was mapped as a 151 segregating morphological marker. Mapping results showed that gender locus could only be mapped on the maternal map, but was unmappable on the paternal map, indicating that the female was the heterogametic gender in willow, which was in agreement with the findings in P. deltoides22 and P. alba26 that revealed sex determination occurred through a ZW system, in which females are heterogametic22. It was also noteworthy that marker density in the vicinity of the gender locus (Supplementary Figure 1) was significantly higher than that expected by chance alone (P # 0.0001 with a Poisson calculator), which indicated severe recombination suppression around the gender locus. Recombination suppression has been recognized as a critical mechanism that triggered the divergence of the alternate sex chromosomes2,22,30,31. To map the willow sequence scaffolds derived by Dai et al. 13 along each chromosome of the willow genome, we carried out fine mapping with SNP markers that were generated by resequencing a subset of 80 progeny from the 374 mapping individuals. AFLP markers on the established maps were used as landmarks to anchor the SNP markers into the corresponding marker bins. In total, 2,548 testcross SNP markers from the maternal parent and 3,772 testcross SNP markers from the paternal parent were integrated into the female (Supplementary Figure 3) and male (Supplementary Figure 4) maps, respectively. Subsequently, SNP markers in each linkage group were used to map the willow sequence scaffolds derived by Dai et al. 13 along the corresponding chromosome. In total, 432 sequence scaffolds (covering a physical length of 194.1 Mb) and 630 sequence scaffolds (covering a physical length of 206.2 Mb) were mapped based on the female (Figure 3a) and male (Figure 3b) maps, respectively. There remained 7,009 unmapped sequence scaffolds larger than 2 kb, covering a total physical length of 38.4 Mb. Chromosome identities of the willow genome were designated by blasting willow sequences containing the mapped SNPs against the P. trichocarpa genome sequences. It was found that the linkage group containing the gender locus was chromosome XV in the willow genome (Figure 3a). This chromosome is an autosome in poplars. Thus, the willow’s sex chromosome corresponds to a poplar’s autosome. Previous studies revealed that chromosome XIX was the sex chromosome in poplars21–26, by contrast, chromosome XIX was identified as an autosome in willow. On willow’s chromosome XV and chromosome XIX, 18 and 40 sequence scaffolds were mapped, respectively, along the corresponding chromosome based on the maternal map (Figure 3a). Because the sequenced individuals of both P. trichocarpa and S. suchowensis were females11,13, we first conducted syntenic analysis between sex chromosomes and the corresponding autosomes in these two lineages based on the willow’s female map. Syntenic analysis revealed high collinearity on chromosome XIX (Figure 4a) and on chromosome XV (Figure 4b) between willow and poplar. In P. trichocarpa, complex haplotype divergence was observed between its alternate sex chromatids11,27. To explore whether the Z and W sex chromatids diverged with each other in willow, we examined the sequence scaffolds anchored on willow’s chromosome XV based on the male map, which included 45 sequence scaffolds (Figure 3b). Contrary to the consistency revealed based on the female map (Figure 4b), syntenic analysis based on the male map revealed substantial chromosome rearrangements (Figure 4c), suggesting the occurrence of significant chromosome divergence between the Z and W chromatids in willow. We further compared willow’s sex chromosome (XV) with poplar’s sex chromosome (XIX), and found that sex chromosomes between these two lineages shared no syntenic chromosome segments (Figure 4d), indicating that sex chromosomes of poplar and willow originated from different ancestral chromosomes. Since sex chromosomes act as autosomes in the alternate genera of Salicaceae, we propose that turnover of autosomes into sex chromosomes occurred after the divergence of Salix and Populus. Because evidence indicated that chromosome XV and chromosome XIX separately evolved into sex chromosomes after the divergence of willow and poplar, we proposed that turnover of different autosomes into sex chromosomes might be due to mobile of the sex-determining gene from chromosome XIX to chromosome XV in willow. Alternatively, the sex-determining gene may have translocated from chromosome XV to chromosome XIX in poplar. Therefore, there might be homologous sex-determining genes within the sex-determining regions between willow and poplar. Mapping of SSR markers revealed that the gender locus was in between SSR markers SSR151 (151,741 bp on scaffold 64) and SSR893 (893,817 bp on scaffold 64) (Figure 4b). In P. nigra21 and P. deltoides22, the close relatives to P. trichocarpa (the sequenced poplar species), gender locus was consistently mapped to the peritelomeric region upper the position of SSR marker O_206 (Figure 4a). We subsequently searched the homologous genes shared by these two regions between willow and poplar, and nine willow genes were found to have a total of 52 homologous genes in the sex-determining region of P. trichocarpa. However, all these poplar genes had homologous genes in other regions of the poplar genome, and none of these genes were found to be involved in sex determination based on gene annotation (Supplementary Table 1). In recent studies, RNA-based sex-determining mechanisms have been found to play a significant role in higher plants27,32–34. For example, the tasselseed4 miRNA, i.e., miR172, was found to be involved in the sex determination of the male inflorescence in maize32,33. Interestingly, the miR172 family was conserved in Populus35 and located on the peritelomeric end of chromosome XIX27. We searched the homolgous sequences of miR172 in willow, and none of them appeared in the sex-determining region. More recently, Akagi et al. reported that a sex-determining candidate (OGI) encoded a small RNA targeting a feminizing gene (MeGI) in persimmons (Diospyros lotus), and the www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9076 | DOI: 10.1038/srep09076 3
www.nature.com/scientificrFigure 3Anchoring the willow sequence scaffolds along each chromosome in the genomes of Salix suchowensis. (a)anchor with the female map(b)anchor with themalemap.Note: willow sequencescaffolds were obtained fromDai et al's studyls.Sequence scaffolds weremapped according to theintegrated SNP markers on the maternal genetic map of S. suchowensis.The orange bars on the left of each chromosome represented the linkagegroups,and the discretebluebars on theright represented the anchored sequence scaffolds.Themapped scaffolds were separated with evenly sizedspaceswhich did not represent theactual sizes of theuncapturedgaps.Chromosome identities weredesignatedbased onsyntenicrelationshipbetweenthewillow's and poplar's chromosomes.As shown in Figure3a,gender locus was mapped onto scaffold_64 on chromosome XV in the female S.suchowensisand markers cosegregated with gender were indicated with red lines.encoded small RNA acted as a sex determinant in this plant4Searchalso sheds new light on understanding the genetics and evolution ofhomologous genes of OGI and MeGI in poplar and willow showedsexchromosomeindioeciousplantsthatnohomologousgeneswerefoundtobedistributedonthesexchromosomes ineitherlineage(SupplementaryTable2).WithallMethodsof these efforts,wehave not identified the likely sexdeterminantsPlant materials. AnF, mapping pedigree with 2,546 progeny was established in 2010.inSalicaceae.TheinvolvementofsmallRNAsinsexdetermina-The maternal parent of this pedigree was described by Dai et al.s, whereas thepaternal parentwasatwo-year-oldS.suchowensiscollected fromLinshu,Shandongtion might explain why they have been difficult to identify to date**.Province.ofChina.Tomapthegenderlocus,374.offspring.wererandomlyselectedRNA-based sex-determining mechanisms maybe present in Sali-gs of the selected individuals were planted at three different locations incaceae species as well, thus small RNAs will be theprimary focusChina: Sihong and Lishui in Jiangsu Province, as well as Shishou in Hubei Provinceinourfuturestudies.The field trials were established in 2012, and three replicates were planted for eachIn plants, species with heterogametic females areless common36.progenyateachlocationTherefore, Salix species should represent a unique system for studyingMap construction. AFLP genotyping was conducted as described by Yin et al.s7dioecy evolution in flowering plants.Although the sex-determiningGeneMapper Software (Version 3.7, Applied Biosystems) was used to score thegene(genes)hasnotbeenidentifiedwiththe current data,thisworkamplicons in range of50500 bp.Map construction,genomelength estimation,andprovides unique insight into the sex determination in Salicaceae andmapcoveragecalculationwereperformedfollowingthedescriptioninYin etal."7,ASCIENTIFICREPORTS15:9076|DOI:10.1038/srep09076
encoded small RNA acted as a sex determinant in this plant34. Search homologous genes of OGI and MeGI in poplar and willow showed that no homologous genes were found to be distributed on the sex chromosomes in either lineage (Supplementary Table 2). With all of these efforts, we have not identified the likely sex determinants in Salicaceae. The involvement of small RNAs in sex determination might explain why they have been difficult to identify to date34. RNA-based sex-determining mechanisms maybe present in Salicaceae species as well, thus small RNAs will be the primary focus in our future studies. In plants, species with heterogametic females are less common36. Therefore, Salix species should represent a unique system for studying dioecy evolution in flowering plants. Although the sex-determining gene (genes) has not been identified with the current data, this work provides unique insight into the sex determination in Salicaceae and also sheds new light on understanding the genetics and evolution of sex chromosome in dioecious plants. Methods Plant materials.An F1 mapping pedigree with 2,546 progeny was established in 2010. The maternal parent of this pedigree was described by Dai et al. 13, whereas the paternal parent was a two-year-old S. suchowensis collected from Linshu, Shandong Province of China. To map the gender locus, 374 offspring were randomly selected, and cuttings of the selected individuals were planted at three different locations in China: Sihong and Lishui in Jiangsu Province, as well as Shishou in Hubei Province. The field trials were established in 2012, and three replicates were planted for each progeny at each location. Map construction. AFLP genotyping was conducted as described by Yin et al. 37. GeneMapper Software (Version 3.7, Applied Biosystems) was used to score the amplicons in range of 50 , 500 bp. Map construction, genome length estimation, and map coverage calculation were performed following the description in Yin et al. 37. A XI scaffold18181 scaffold27728 scaffold15514 scaffold45339 scaffold128 scaffold80 scaffold248 scaffold163 scaffold89 scaffold110 scaffold1153 scaffold2757 scaffold5721 scaffold28869 scaffold47416 scaffold18422 scaffold4 scaffold22136 scaffold222 scaffold117 scaffold336 scaffold121 scaffold13437 scaffold154 scaffold237 scaffold715 scaffold14511 scaffold137 scaffold1836 scaffold277 scaffold70967 scaffold13998 scaffold38445 scaffold231 scaffold17311 scaffold20511 scaffold94 scaffold430 scaffold1081 scaffold9185 scaffold3316 scaffold63 scaffold306 scaffold3481 scaffold6482 scaffold8129 scaffold24617 scaffold34242 scaffold44674 scaffold80385 scaffold89067 scaffold91023 scaffold12153 scaffold40 scaffold29096 scaffold2534 scaffold97 scaffold53354 scaffold323 scaffold7599 scaffold463 scaffold30 scaffold133 scaffold23871 scaffold164 scaffold143 scaffold30410 scaffold104 scaffold66 scaffold393 scaffold178 scaffold171 scaffold48954 scaffold196 scaffold16239 I scaffold262 scaffold202 scaffold105 scaffold24736 scaffold203 scaffold1616 scaffold22343 scaffold210 scaffold24 scaffold1691 scaffold26 scaffold18 scaffold56 scaffold1145 scaffold146 scaffold36 scaffold176 scaffold3791 scaffold230 scaffold854 scaffold157 scaffold8692 II scaffold51116 scaffold8 scaffold12 scaffold1109 scaffold85 scaffold57 scaffold6314 scaffold304 scaffold60 scaffold303 scaffold80727 scaffold70049 scaffold6867 scaffold2176 scaffold27 scaffold1294 scaffold12174 scaffold250 scaffold15 scaffold921 scaffold3520 III scaffold45 scaffold103 scaffold130 scaffold41 scaffold3 scaffold9 scaffold1656 scaffold252 scaffold10741 scaffold39886 scaffold147 IV scaffold76 scaffold16491 scaffold1658 scaffold25 scaffold4096 scaffold48214 scaffold20 scaffold981 scaffold167 scaffold18408 scaffold267 scaffold55 scaffold233 scaffold15209 scaffold6081 scaffold2852 scaffold68 scaffold52415 scaffold1731 scaffold24583 scaffold34 scaffold36060 scaffold4206 scaffold47 scaffold1445 scaffold169 scaffold4351 scaffold5493 scaffold3529 scaffold78 scaffold113 V scaffold1234 scaffold198 scaffold3416 scaffold9775 scaffold135 scaffold33 scaffold1903 scaffold54 scaffold165 scaffold48626 scaffold96 scaffold77 scaffold24858 scaffold6089 scaffold478 scaffold65 scaffold22 VI scaffold13 scaffold74 scaffold144 scaffold3855 scaffold7214 scaffold1 scaffold173 scaffold469 scaffold6411 scaffold227 scaffold2 scaffold1315 IX scaffold5 scaffold7 scaffold4957 scaffold1141 scaffold4364 XII scaffold160 scaffold5245 scaffold17 scaffold46546 scaffold223 scaffold75 scaffold1487 scaffold82 scaffold39351 scaffold358 scaffold45700 scaffold179 scaffold719 scaffold317 scaffold35840 scaffold28594 scaffold33327 scaffold2067 scaffold1128 scaffold43 VIII scaffold5439 scaffold28307 scaffold48 scaffold31 scaffold168 scaffold243 scaffold3292 scaffold205 scaffold69 scaffold32 X scaffold1341 scaffold289 scaffold1512 scaffold10 scaffold35810 scaffold853 scaffold11 scaffold44 scaffold16 VII scaffold1831 scaffold50 scaffold140 scaffold185 scaffold301 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scaffold07280 scaffold39351 scaffold82 scaffold75 scaffold13437 scaffold03196 scaffold65365 scaffold43 scaffold298 XIV scaffold247 scaffold14 scaffold01328 scaffold65881 scaffold19 scaffold30 scaffold11224 scaffold28 scaffold183 scaffold01185 scaffold88 scaffold281 scaffold286 III scaffold01794 scaffold28075 scaffold05260 scaffold9 scaffold10169 scaffold03718 scaffold209 scaffold252 scaffold01726 scaffold13957 scaffold68698 scaffold3 scaffold103 scaffold130 scaffold193 scaffold45 scaffold00799 I scaffold230 scaffold157 scaffold25915 scaffold05877 scaffold02176 scaffold29266 scaffold189 scaffold04380 scaffold176 scaffold14185 scaffold11208 scaffold377 scaffold120 scaffold384 scaffold14584 scaffold32005 scaffold53354 scaffold212 scaffold215 scaffold270 scaffold21241 scaffold46389 scaffold20368 scaffold72 scaffold06991 scaffold52242 scaffold10001 scaffold52 scaffold430 scaffold39918 scaffold41 scaffold02650 scaffold22301 scaffold21 scaffold217 scaffold67 scaffold07430 scaffold26756 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scaffold07537 scaffold306 scaffold22816 scaffold184 scaffold163 scaffold15514 scaffold78847 scaffold01344 scaffold31842 scaffold46590 scaffold01676 scaffold01153 scaffold137 scaffold89 scaffold06263 scaffold110 scaffold117 scaffold29450 scaffold26173 scaffold01081 scaffold03316 scaffold04779 scaffold01404 scaffold26709 scaffold33843 scaffold07215 scaffold54803 scaffold02790 scaffold264 scaffold1135 scaffold63 scaffold336 scaffold121 scaffold48626 scaffold12220 scaffold09189 scaffold149 scaffold94 scaffold01556 scaffold10447 scaffold108 II scaffold13193 scaffold19589 scaffold11193 scaffold8 scaffold512 scaffold369 scaffold146 scaffold251 scaffold147 scaffold776 scaffold116 scaffold84 scaffold295 scaffold47 scaffold00613 scaffold144 scaffold01139 scaffold01487 scaffold01882 scaffold02234 scaffold03413 scaffold03580 scaffold03860 scaffold119 scaffold04425 scaffold07699 scaffold09468 scaffold09638 scaffold10986 scaffold14233 scaffold14184 scaffold22787 scaffold30044 scaffold71 scaffold32826 scaffold39412 scaffold02608 scaffold44819 scaffold26584 scaffold18565 scaffold47847 scaffold172 scaffold09734 scaffold10059 scaffold02260 scaffold19472 scaffold54271 scaffold38759 scaffold03665 scaffold221 scaffold363 scaffold05148 scaffold89928 scaffold115 scaffold77 scaffold260 scaffold159 scaffold21921 scaffold89052 scaffold68530 scaffold236 scaffold24492 scaffold27487 scaffold22722 scaffold12637 scaffold75399 scaffold15805 scaffold13211 scaffold431 scaffold21209 scaffold02990 scaffold01371 scaffold413 scaffold01320 scaffold04954 scaffold22393 scaffold04807 scaffold06855 scaffold302 scaffold78961 scaffold38396 scaffold65 scaffold57963 scaffold01352 scaffold324 scaffold12 scaffold00899 scaffold11676 scaffold30598 scaffold85 scaffold303 scaffold304 scaffold60 scaffold57 scaffold55 scaffold06314 scaffold27 scaffold01294 scaffold250 scaffold64281 scaffold81618 scaffold15 scaffold01624 XIII scaffold83 scaffold05424 scaffold10702 scaffold139 scaffold15667 scaffold53 scaffold06170 scaffold218 scaffold06482 scaffold22176 scaffold100 scaffold06171 scaffold20192 scaffold20123 scaffold20174 scaffold39 scaffold26321 scaffold641 scaffold59 scaffold27778 scaffold43542 scaffold85224 scaffold42230 scaffold308 scaffold37554 scaffold47101 scaffold27757 scaffold127 scaffold31626 scaffold7265 scaffold45745 scaffold21446 scaffold1872 scaffold181 scaffold19030 scaffold608 scaffold21456 scaffold345 scaffold17858 scaffold27071 scaffold04097 scaffold26260 scaffold85791 scaffold46144 scaffold229 scaffold332 scaffold29993 scaffold28304 scaffold165 scaffold141 scaffold134 scaffold131 scaffold40198 scaffold28626 scaffold244 scaffold521 scaffold20294 scaffold21557 scaffold12174 scaffold28109 scaffold28303 scaffold12479 scaffold239 scaffold297 scaffold138 scaffold106 (a) (b) Figure 3 | Anchoring the willow sequence scaffolds along each chromosome in the genomes of Salix suchowensis. (a) anchor with the female map (b) anchor with the male map. Note: willow sequence scaffolds were obtained from Dai et al.’s study13. Sequence scaffolds were mapped according to the integrated SNP markers on the maternal genetic map of S. suchowensis. The orange bars on the left of each chromosome represented the linkage groups, and the discrete blue bars on the right represented the anchored sequence scaffolds. The mapped scaffolds were separated with evenly sized spaces, which did not represent the actual sizes of the uncaptured gaps. Chromosome identities were designated based on syntenic relationship between the willow’s and poplar’s chromosomes. As shown in Figure 3a, gender locus was mapped onto scaffold_64 on chromosome XV in the female S. suchowensis, and markers cosegregated with gender were indicated with red lines. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9076 | DOI: 10.1038/srep09076 4
www.nature.com/scientificreport(a)(b)sex chromosomeautosomePt_XVPt_XIXSu_XVSu_XIx西Femaleautosomesex chromosome(c)(d)autosomesex chromosomePL_XVPt_XIXSu_xV i-Female-Su_XVMalesex chromosomesex chromosomeFigure 4Syntenic analysis between sex chromosomes and the corresponding autosomes across Populus trichocarpa and Salix suchowensis.(a) synteny between poplar's and willows chromosome XIX based on the female map. (b) synteny between poplar's and willow's chromosome XV basedon the female map.(c)synteny between poplar's and willow's chromosome XV based on the male map. (d) synteny between poplar's chromosome XIXand willow's chromosome XV based on the female map.Note: Bar on the top ofeach chromosome pair represents poplar's chromosome. Bar at thebottom corresponds to the anchored sequence scaffolds on willow's chromosome. Only sequence scaffolds containing coding genes were included in thisanalysis. The red portions of the chromosome bars represent the sex-determining regions.Poisson probability test was conducted following the description in Remington(Cannabaceae) revealed by fluorescence in situ hybridization of subtelomericet al.8.The map charts were produced with MapChart 2.130.repeat.Comp.Cytogenet.6,39-247 (2012)Smith, C. A. & Sinclair, A. H. Sex determination: insights from the chicken5.Bioessays 26, 120132 (2004).Generating SNP markers. SNP markers were generated with a subset of 80 progenyCarvalho, A. B. & Clark, A. G.Y chromosome of D. pseudoobscura is notfrom the 374 mapping individuals. The combination of two restriction enzymes,6.EcoRI and Msel (Takara Inc.), were used to reduce the complexity of the willowhomologous to the ancestral Drosophila Y.Science 307, 108-110 (2005).genome. For PCR amplification, the EcoRI primer had no selective nucleotide, andALloyd, D. G & Webb, C. J. Secondary sex characters in plants. Bot. Rev. 43,the Msel primer had a “G" selective nucleotide. The PCR amplification was177216 (1977).conducted as described by Yin et al.s.Guttman, D. S. & Charlesworth, D. An X-linked gene with a degenerate Y-linkedThe sequencing library was constructed according to the manufacturer's standardhomologue in a dioecious plant. Nature 393, 263-266 (1998).protocol (llumina, Inc.). Library quantification was carried out with a Quant-iTTMHeslop-Harrison, J. S. & Schwarzacher, T. Organisation of the plant genome inPicoGreen dsDNA Kit (Invitrogen, Inc.), and library qualify was evaluated with anchromosomes. Plant J.66, 18-33 (2011).Agilent 2100 Bioanalyzer (Agilent, Inc.).Foreach lane ofaflow cell, 12 index-labeled10. Rechinger, K. H. Salix taxonomy in Europe-problems, interpretations,samplesweremultiplexed, whichenabled thesequencingof84samples (80progenyobservations.Proceedings ofthe Royal Societyof Edinburgh.Section B.Biologicaland two replicates of the parents) in a full run, and the eighth lane was used for theSciences 98, 1-12 (1992).control. Sequencing was performed on an Ilumina HiSeq 2000 (llumina, Inc.) at11. Tuskan, G. A.et al. The genome of black cottonwood, Populus trichocarpa (Torr.Nanjing Agricultural University following the manufacturer's protocols (Ilumina,& Gray). Science 313, 1596-1604 (2006).Inc.).12. Berlin, S. et al. High-density linkage mapping and evolution of paralogs andRaw sequencing data generated by the Ilumina platform were analyzed with anorthologs in Salix and Populus. BMC Genomics 11, 129 (2010).integrated bioinformatics pipeline. The SAMTOOLS (http://samtools.sourceforge13.Dai, X.G. et al. The willow genome and divergent evolution from poplar after thenet)and Varscan (http:/ivarscan.source-forge.net) were combined to call the SNPscommon genome duplication. Cell Res. 24, 1274-1277 (2014).with default parameters.14. Blackburn, K. B.& Harrison,J. W.H.A preliminary account of the chromosomesand chromosome behavior in the Salicaceae. Ann. Bot. 38, 361-378 (1924).Anchoring the sequence scaffolds. AFLP markers on the established genetic maps15. Islam-Faridi, M. N., Nelson, C. D., DiFazio, S. P., Gunter, L. E. & Tuskan, G. Awere used to pull out tightly linked SNP markers with an LOD threshold ≥10, andCytogenetic analysis of Populus trichocarpa-ribosomal DNA, telomere repeatthese SNPs were assigned to the corresponding marker bins that showed the strongestsequence,and marker-selectedBACs.Cytognet.Genome Res25,74 (2009)linkage on each map. Subsequently, the mapped SNPs were used to anchor willow16. Peto, F. H Cytology of poplar species and natural hybrids. Can. J. Forest. Res. 16,sequence scaffolds that were produced by Dai et al."s, Finally, willow sequences445-455 (1938).containing these SNPs were blasted against the P. trichocarpa genome sequences17.Van Dillewijn, C.Zytologische Studien in der GattungPopulus L.Genetica 22,(ftp://ftp-jgi-psf.org/pub/compgen/phytozome/v9.0/Ptrichocarpa/)to designate the131182 (1940).chromosome identities for the obtained linkage groups on the male and female maps.18. Van Buijtenen, J. P. & Einspahr, D. W. Note on the presence of sex chromosomesin Populus tremuloides.Bot.Gaz.121,60-61 (1959)Synteny and collinearity analyses. Genome sequences, Gff3 file, and the protein19.Alstrom-Rapaport,C,Lascoux,M& Gullberg,U.Sexdetermination and sexratosequences of P.trichocarpa wereretrieved from JGI website (ftp://ftpjgi-psf.org/pub/in the dioecious shrub Salix viminalis L, Theor.Appl. Genet. 94, 493-497 (1997).compgen/phytozo-me/v9.0/Ptrichocarpa/).The corresponding information for20. Semerikov, V. et al. Genetic mapping of sex-linked markers in Salix viminalis L.willow was downloaded from the S. suchowensis website (115.29.234.170/willow)Heredity 91, 293-299 (2003).Synteny and collinearity analyses were performed with bioinformatics toolkit of21.Gaudet,M.etal.GeneticlinkagemapsofPopulus nigraL.includingAFLPs,SSRs,MCScanx.First, coding genes in willow and poplar genome were blasted by usingSNPs, and sex trait. Tree Genet. Genomes 4, 25-36 (2008)BLASTp with an e-value cutoff of 1e-10, which produced a file in blast8 format.22. Yin, T. M. et al. Genome structure and emerging evidence of an incipient sexThen, an in-house perl script was used to flter the gff3 files and reformat the files tochromosome in Populus. Genome Res. 18, 422-430 (2008).make them compatible with MCScanX Finally, MCScanX produced the collinearity23.Markussen,T,Pakull,B.& Fladung,M.Positioning of sex correlated markers forresults, with the following parameters: k 50, m 25, -e le-5, where k is thePopulus in an AFLP- and SSR-marker based genetic map of Populus tremula Xmatch score, -m is the maximum gaps, and -e is the e value cutoff.tremuloides. Silvae Genet. 56, 180-184 (2007).Dual_synteny_plotter in the MCScanX package was used to draw the collinearity24. Pakull, B, Groppe, K, Meyer, M, Markussen, T. & Fladung, M. Genetic linkagecharts.mapping in aspen (Populus tremula L. and Popuhus tremuloides Michx). TreeGenet. Genomes 5, 505-515 (2009).25. Pakull, B. et al. Genetic mapping of linkage group XIX and identification of sex-1. Jamsari, A., Nitz, L, Reamon-Bittner, S. M. & Jung, C.BAC-derived diagnosticlinked SSR markers in a Populus tremulax Populus tremuloides cross.Can.J.markers for sex determination in asparagus. Theor. Appl. Genet. 108, 1140-1146Forest. Res. 41, 245-253 (2011).(2004).26. Paolucci,Iet al. Genetic linkage maps of Populus alba L. and comparative2. Liu, Z. Y. et al. A primitive Y chromosome in papaya marks incipient sexchromosome evolution. Nature 427,348-352 (2004).mapping analysis of sex determination across Populus species. Tree Genet.3.-Filatov,D.AEvolutionaryhistory of Silene latifolia sex chromosomes revealedbyGenomes 6, 863875 (2010).genetic mapping of four genes. Genetics 170, 975-979 (2005).27.Tuskan, G.A. et al. The obscure events contributing to the evolution of an4. Alexandrov, O. S., Divashuk, M. G., Yakovin, N. A. & Karlov, G. I. Sexincipient sex chromosome in Populus: a retrospective working hypothesis, Treechromosome differentiation in Humulus japonicus Siebold & Zuccarini, 1846Genet. Genomes 8, 559-571 (2012).5SCIENTIFICREPORTS15:90761DOI:10.1038/srep09076
Poisson probability test was conducted following the description in Remington et al. 38. The map charts were produced with MapChart 2.139. Generating SNP markers. SNP markers were generated with a subset of 80 progeny from the 374 mapping individuals. The combination of two restriction enzymes, EcoRI and MseI (Takara Inc.), were used to reduce the complexity of the willow genome. For PCR amplification, the EcoRI primer had no selective nucleotide, and the MseI primer had a ‘‘G’’ selective nucleotide. The PCR amplification was conducted as described by Yin et al. 37. The sequencing library was constructed according to the manufacturer’s standard protocol (Illumina, Inc.). Library quantification was carried out with a Quant-iTTM PicoGreen dsDNA Kit (Invitrogen, Inc.), and library qualify was evaluated with an Agilent 2100 Bioanalyzer (Agilent, Inc.). For each lane of a flow cell, 12 index-labeled samples were multiplexed, which enabled the sequencing of 84 samples (80 progeny and two replicates of the parents) in a full run, and the eighth lane was used for the control. Sequencing was performed on an Illumina HiSeq 2000 (Illumina, Inc.) at Nanjing Agricultural University following the manufacturer’s protocols (Illumina, Inc.). Raw sequencing data generated by the Illumina platform were analyzed with an integrated bioinformatics pipeline. The SAMTOOLS (http://samtools.sourceforge. net)40 and Varscan (http://varscan.source-forge.net) were combined to call the SNPs with default parameters41. Anchoring the sequence scaffolds. AFLP markers on the established genetic maps were used to pull out tightly linked SNP markers with an LOD threshold $10, and these SNPs were assigned to the corresponding marker bins that showed the strongest linkage on each map. Subsequently, the mapped SNPs were used to anchor willow sequence scaffolds that were produced by Dai et al. 13. Finally, willow sequences containing these SNPs were blasted against the P. trichocarpa genome sequences (ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/Ptrichocarpa/) to designate the chromosome identities for the obtained linkage groups on the male and female maps. Synteny and collinearity analyses. Genome sequences, Gff3 file, and the protein sequences of P. trichocarpa were retrieved from JGI website (ftp://ftp.jgi-psf.org/pub/ compgen/phytozo- me/v9.0/Ptrichocarpa/). The corresponding information for willow was downloaded from the S. suchowensis website (115.29.234.170/willow). Synteny and collinearity analyses were performed with bioinformatics toolkit of MCScanX42. First, coding genes in willow and poplar genome were blasted by using BLASTP43 with an e-value cutoff of 1e210, which produced a file in blast8 format. Then, an in-house perl script was used to filter the gff3 files and reformat the files to make them compatible with MCScanX. Finally, MCScanX produced the collinearity results, with the following parameters: 2k 50, 2m 25, 2e 1e25, where 2k is the match score, 2m is the maximum gaps, and 2e is the e value cutoff. Dual_synteny_plotter in the MCScanX package was used to draw the collinearity charts. 1. Jamsari, A., Nitz, I., Reamon-Bu¨ttner, S. M. & Jung, C. BAC-derived diagnostic markers for sex determination in asparagus. Theor. Appl. Genet. 108, 1140–1146 (2004). 2. Liu, Z. Y. et al. A primitive Y chromosome in papaya marks incipient sex chromosome evolution. Nature 427, 348–352 (2004). 3. Filatov, D. A. Evolutionary history of Silene latifolia sex chromosomes revealed by genetic mapping of four genes. Genetics 170, 975–979 (2005). 4. Alexandrov, O. S., Divashuk, M. G., Yakovin, N. A. & Karlov, G. I. Sex chromosome differentiation in Humulus japonicus Siebold & Zuccarini, 1846 (Cannabaceae) revealed by fluorescence in situ hybridization of subtelomeric repeat. Comp. Cytogenet. 6, 39–247 (2012). 5. Smith, C. A. & Sinclair, A. H. Sex determination: insights from the chicken. Bioessays 26, 120–132 (2004). 6. Carvalho, A. B. & Clark, A. G. Y chromosome of D. pseudoobscura is not homologous to the ancestral Drosophila Y. Science 307, 108–110 (2005). 7. Lloyd, D. G & Webb, C. J. Secondary sex characters in plants. Bot. Rev. 43, 177–216 (1977). 8. Guttman, D. S. & Charlesworth, D. An X-linked gene with a degenerate Y-linked homologue in a dioecious plant. Nature 393, 263–266 (1998). 9. Heslop-Harrison, J. S. & Schwarzacher, T. Organisation of the plant genome in chromosomes. Plant J. 66, 18–33 (2011). 10. Rechinger, K. H. Salix taxonomy in Europe-problems, interpretations, observations. Proceedings of the Royal Society of Edinburgh. Section B. Biological Sciences 98, 1–12 (1992). 11. Tuskan, G. A. et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596–1604 (2006). 12. Berlin, S. et al. High-density linkage mapping and evolution of paralogs and orthologs in Salix and Populus. BMC Genomics 11, 129 (2010). 13. Dai, X. G. et al. The willow genome and divergent evolution from poplar after the common genome duplication. Cell Res. 24, 1274–1277 (2014). 14. Blackburn, K. B. & Harrison, J. W. H. A preliminary account of the chromosomes and chromosome behavior in the Salicaceae. Ann. Bot. 38, 361–378 (1924). 15. Islam-Faridi, M. N., Nelson, C. D., DiFazio, S. P., Gunter, L. E. & Tuskan, G. A. Cytogenetic analysis of Populus trichocarpa-ribosomal DNA, telomere repeat sequence, and marker-selected BACs. Cytogenet. Genome Res. 125, 74–80 (2009). 16. Peto, F. H. Cytology of poplar species and natural hybrids. Can. J. Forest. Res. 16, 445–455 (1938). 17. Van Dillewijn, C. Zytologische Studien in der Gattung Populus L. Genetica 22, 131–182 (1940). 18. Van Buijtenen, J. P. & Einspahr, D. W. Note on the presence of sex chromosomes in Populus tremuloides. Bot. Gaz. 121, 60–61 (1959). 19. Alstro¨m-Rapaport, C., Lascoux, M. & Gullberg, U. Sex determination and sex ratio in the dioecious shrub Salix viminalis L. Theor. Appl. Genet. 94, 493–497 (1997). 20. Semerikov, V. et al. Genetic mapping of sex-linked markers in Salix viminalis L. Heredity 91, 293–299 (2003). 21. Gaudet, M.et al. Genetic linkage maps of Populus nigra L. including AFLPs, SSRs, SNPs, and sex trait. Tree Genet. Genomes 4, 25–36 (2008). 22. Yin, T. M. et al. Genome structure and emerging evidence of an incipient sex chromosome in Populus. Genome Res. 18, 422–430 (2008). 23. Markussen, T., Pakull, B. & Fladung, M. Positioning of sex correlated markers for Populus in an AFLP- and SSR-marker based genetic map of Populus tremula 3 tremuloides. Silvae Genet. 56, 180–184 (2007). 24. Pakull, B., Groppe, K., Meyer, M., Markussen, T. & Fladung, M. Genetic linkage mapping in aspen (Populus tremula L. and Populus tremuloides Michx.). Tree Genet. Genomes 5, 505–515 (2009). 25. Pakull, B. et al. Genetic mapping of linkage group XIX and identification of sexlinked SSR markers in a Populus tremula3 Populus tremuloides cross. Can. J. Forest. Res. 41, 245–253 (2011). 26. Paolucci, I. et al. Genetic linkage maps of Populus alba L. and comparative mapping analysis of sex determination across Populus species. Tree Genet. Genomes 6, 863–875 (2010). 27. Tuskan, G. A. et al. The obscure events contributing to the evolution of an incipient sex chromosome in Populus: a retrospective working hypothesis. Tree Genet. Genomes 8, 559–571 (2012). Figure 4 | Syntenic analysis between sex chromosomes and the corresponding autosomes across Populus trichocarpa and Salix suchowensis. (a) synteny between poplar’s and willow’s chromosome XIX based on the female map. (b) synteny between poplar’s and willow’s chromosome XV based on the female map. (c) synteny between poplar’s and willow’s chromosome XV based on the male map. (d) synteny between poplar’s chromosome XIX and willow’s chromosome XV based on the female map. Note: Bar on the top of each chromosome pair represents poplar’s chromosome. Bar at the bottom corresponds to the anchored sequence scaffolds on willow’s chromosome. Only sequence scaffolds containing coding genes were included in this analysis. The red portions of the chromosome bars represent the sex-determining regions. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9076 | DOI: 10.1038/srep09076 5