Chromosome-level assembly and analysis of three hydroxy fatty acid-producing Physaria species

Domain: biology
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
Several Physaria species (Brassicaceae) produce abundant hydroxy fatty acids in their seeds, with industrial applications. Here, we report three chromosome-level genomes of Physaria species: P. lindheimeri, P. pallida and P. fendleri, with sizes of 344 Mb, 329 Mb and 452 Mb, respectively. Comparative genome analysis reveals that these three Physaria species diverged from Arabidopsis thaliana approximately 14.10-14.46 million years ago and underwent two consecutive Physaria-specific whole-genome duplication events. Their centromeres harbor an 111-bp satellite repeat and two retrotransposon classes (Gypsy/CRM, Copia/Ale). Transcriptomic analysis identifies seed-highly expressed lipid synthesis genes potentially underlying unique fatty acid profiles. Furthermore, we pinpoint the two residues in FAH12 variants that cause the disparity in hydroxylation activity among the three Physaria species. Taken together, this study provides important genomic resources for investigating the evolution of Physaria species and developing industrial oil crops for sustainable production of hydroxy fatty acids.

Introduction
Plantoilsrepresentanimportantnaturalresourceofhumansociety,providingfood,essentialnutrientsorindustrialfeedstockdependingontheirfattyacidcomposition.Mostoilseedcropsproduceoilscontainingcommonfattyacids,suchaspalmiticacid(C16:0),stearicacid(C18:0), oleic acid (C18:1), linoleic acid (C18:2), and α-linolenic acid (C18:3),andcanbeusedinfoodornon-foodapplications,manyspeciesproduceunusualfattyacidsthatmaybevaluableforindustrialapplicationsduetotheiruniquechemicalstructures,includingvariationsincarbonchainlength,positionsofdoubleandtriplebonds,andthe presence of functional groups such as hydroxyl, epoxy, and cyclopropanegroups1.Forexample,castor(Ricinuscommunis)accumulates approximately 90% ricinoleic acid (12-hydroxyoctadeccis-9-enoicacid;C18:1-OH)initsseedoil2,3.Whileitprovidesavitalsourceofhydroxyfattyacid(HFA)forvariousindustrialapplications4,castorisunsuitableforlarge-scaleagriculturalproductionduetothepresenceoftoxinricinandthehyper-allergic2Salbumins5.AsimilarHFA,lesquerolicacid(14-hydroxyeicos-cis-11-enoicacid;C20:1-OH),isfoundatabundance(50–85%)inPhysariaseeds6.Theoilisacolorlesstopale-yellowliquidthatexhibitsexcellentstabilityandbiodegradability,andit may provide an alternative source of HFA for making lubricants, motor oils, desiccants, nylon, plastics, cosmetics, pharmaceuticals, andpolishes6–11.

Physaria,whichwaspreviouslyclassifiedasLesqueralla,isagenusoftheBrassicaceaefamilyandiscloselyrelatedtoPaysonia12.SomePhysariaspeciesexhibitvaluabletraits,includingnon-dehiscentseeds,neutralphotoperiodresponse,uprightgrowthhabit,andhighseedoilyield6(Fig.1a).ExplorationofthePhysariaspeciesfoundhigher(85%)contentsoflesquerolicacidinP.lindheimeriandP.pallida13,makingthemvaluablegeneticresourcestoenhanceHFAproductioninagriculturalcrops.P.fendleriisbeingdevelopedtoprovideasafesourceofHFA14,15.However,comparedtocastorwithhighoilcontent(38–55%ofseedweight)andnearly90%HFA,P.fendleriseedoilcontentisgenerallyloweratabout27–33%andcontainsupto60%C20:1-OH16.Itisnecessary to improve the productivity of HFA by plant breeding. MetabolicengineeringoffersanotherattractiveapproachtoproduceHFAinestablishedagriculturaloilcrops17.TheHFAsynthesisoccursonthephosphatidylcholine(PC),whereC18:1isconvertedintoC18:1-OHby an oleic acid Δ12-hydroxylase (FAH12)18–20. Ensuing elongationC18:1-OHproduceslesquerolicacid(C20:1-OH).Thisstepiscatalyzedbythefattyacidcondensingenzyme3-ketoacyl-CoAsynthase3(KCS3) in a fatty acid elongase (FAE) complex. Physaria KCS3 is highly homologous to AtKCS18 (or AtFAE1) in Arabidopsis but specifically catalyzestheelongationofC18-HFA21.HFAarepredominantlyincorporatedintotriacylglycerol(TAG)throughtheKennedypathwayandacyleditingreactions22–24.Despitetheprogressinunderstandingoilbiosynthesis pathways, engineering plants with high levels of HFA remainslargelyunsuccessful25.

Inthisstudy,weassemblechromosome-levelgenomesofthreerepresentativePhysariaspecies(P.lindheimeri,P.pallida,andP.fendleri)byPacBiosequencingandHi-Ctechnologies.Basedonthehigh-qualitygenomes,weinvestigatetheevolutionaryhistoryofthePhysariagenus.FollowingthedivergenceofPhysariafromArabidopsis,the species-specific whole-genome duplication event is observed. Transcriptomicanalysisunveilsthehighexpressionofkeygenessuchas FAH12and KCS3 that areinvolved inHFAsynthesisinseeds. We showthattheactivityofPfFAH12islowerthanthatofP.lindheimeriandP.pallida,whichmayexplainthevaryingcontentsoflesquerolicacidinthesethreespecies.Takentogether,thehigh-qualityassemblyandanalysisofthreechromosome-levelgenomes,aswellasthestudyofrepresentativeenzymes,wouldprovidevaluableinsightsintotheevolutionary history of Physaria and genetic tools for engineering plants for HFA production and for establishing Physaria species as economicallysignificantoilcrops.

Results
High-qualityassemblyandannotationofthreePhysariagenomes

MultifacetedsequencingapproacheswereusedtoassemblethegenomesofPhysariagenus.About30.46Gb(~92.58×)PacBiohigh-fidelity(HiFi) reads for P. pallida, 28.81Gb (~65.28×) for P. fendleri, and 167.78Gb(~488×)PacBiocontinuouslongreads(CLR)forP.lindheimeri were used to de novo assembly and generate draft genome assemblies.Illuminashortreadsof115.43Gb(~335×)forP.lindheimeri, 110.10 GB (~335×) for P. pallida and 55.73Gb (~126×) for P. fendleri wereusedtocorrectgenomeassemblies.Inaddition,117.52Gb(~341×) high-throughputchromosomeconformationcapture(Hi-C)readsfor P.lindheimeri,50.36Gb(~153×)forP.pallidaand112.37Gb(~254×)for P. fendleri were used in genome assembly (Supplementary Data 1). Through these approaches, we assembled 344.42Mb chromosome-level P. lindheimeri genome with contig N50 size of 6.08Mb and scaffoldN50sizeof53.59Mb,forwhichthetotallengthofpseudochromosomes was324.41Mb with pseudochromosome lengths ranging from 46.30Mb to 66.59Mb. For P. pallida, we generated 329.03Mb chromosome-level genome with the contig N50 size of 30.42Mb,scaffold N50sizeof51.55Mb,and atotallength ofpseudochromosomes amounting to 314.31Mb with pseudochromosome lengthsrangingfrom41.77Mbto65.95Mb.ForP.fendleri,451.62Mb chromosome-levelgenomewasgeneratedwiththecontigN50sizeof 33.69Mb,scaffoldN50sizeof69.75Mb,andatotallengthofpseudochromosomesamountingto429.99Mbwithpseudochromosome lengthsrangingfrom66.60Mbto81.59Mb(Fig.1b,c,Table1,Supplementary Data 2). The sizes of the three genome assemblies are consistentwiththeestimatedsizesbasedonk-merstatisticswithshort reads (Supplementary Fig. 1, Supplementary Data 3). Moreover, we identified6,10and10seven-basetelomeric(5’-TTTAGGG-3’)repeatsin theP.lindheimeri,P.pallidaandP.fendleri(2n=12)genomeassemblies(Fig.1c).

Toassessaccuracyandcompletenessofthegenomeassemblies ofP.lindheimeri,P.pallidaandP.fendleri,theIlluminapair-endreads were mapped back to three genome assemblies with the alignment rates of 99.28%, 97.50% and 96.26%, respectively (Supplementary Data4).Atotalof98.5%,99.1%and98.6%coreeukaryoticgenesfrom theBenchmarkingUniversalSing-CopyOrthologs(BUSCO)werecapturedbythethreegenomeassemblies,respectively(Supplementary Data5).Inaddition,thelongterminalrepeatassemblyindex(LAI)of the genome assemblies was 18.60, 20.80, and 23.79, respectively (Table1),indicatingthatallthreegenomeassembliesmeetthecriteria forhigh-qualityreferencegenomes.Notably,thegenomeassemblies of P. pallida and P. fendleri meet the gold standard for genome assembly quality26. In summary, all assessment suggested the high-qualityreferencegenomesofthethreePhysariaspecies.

Atotalof33,677,36,997and39,517protein-codinggeneswere annotatedinP.lindheimeri,P.pallidaandP.fendlerigenomeswithan average coding sequences length of 1166, 1208 and 1205bp, which weresimilartootherspeciesofBrassicaceae(SupplementaryFig.2, SupplementaryData6,SupplementaryData7).Mappingthesegenes to multiple protein databases, it was found that 95.95%, 98.72 and 98.80%ofthegeneswerefunctionalineachgenome(Supplementary Data 8). Transposable elements (TEs) occupy 57.39%, 53,93% and 62.62%ofP.lindheimeri,P.pallidaandP.fendlerigenomesandmost abundant category of TEs was the long-terminal retrotransposon (LTR),withLTR/CopiaandLTR/Gypsyaccounting15.17%and18.22%in P.lindheimerigenome,12.05%and12.08%inP.pallidagenome,and 13.59% and 23.58% in P. fendleri genome (Supplementary Data 9). Furthermore,10,394,10,382and13,143non-codingwerepredictedin P.lindheimeri,P.pallidaandP.fendlerigenomes,containing152,154 and155microRNAs,1647,2647and1,864transferRNAs,3927,5457, and 6494 ribosomal RNAs and 4668, 2124 and 4,631 small nuclear RNAsineachgenome,respectively(Table1).

Species-specificwhole-genomeduplicationeventsinthethree Physariaspecies

A phylogenetic tree was constructed to elucidate the evolution of Physaria genus, based on 276 single-copy gene families from three Physaria species and 15 other representative species (Fig. 2a). This revealedthatthegenusPhysariaismostcloselyrelatedtoA.thaliana, divergingfromitabout15.11millionyearsago(Mya).Subsequently,P. fendleridivergedfromtheothertwoPhysariaspeciesapproximately 5.36Mya,followedbyP.lindheimeriandP.pallidaaround3.39Mya (Fig.2a).Analysisofgenefamilyexpansionandcontractionrevealed 769,884and997expandedgenefamilies,aswellas1181,628and732 contracted gene families in P.lindheimeri,P.pallida and P.fendleri, respectively(Fig.2a).Furthergeneontology(GO)enrichmentanalysis indicatedthattheseexpandedgenesweresignificantlyenrichedinthe terms of‘regulationofprotein ubiquitination’,‘photosyntheticelectrontransportchain’,and‘ATPsynthesiscoupledwithprotontransport’(SupplementaryFig.3),respectively.

To identify the whole-genome duplication event in Physaria genus, KsvalueswereanalyzedusingA.thalianaasarepresentative species of Brassicaceae.Ouranalysisindicatedthat,inadditionto thepreviousreportedαwhole-genomeduplication(WGD)eventin Brassicaceae (with a peak Ks value around 0.92–0.95), Physaria genusexhibitedadistinctpeakataKsvalueof0.42–0.43(Fig.2b). Thesesuggestedthespecies-specificWGDinPhysariafollowingits divergence from A. thaliana, estimated to occur approximately 14.10–14.46Mya.TofurtherinvestigateWGDspecificinPhysaria,we identified1298,1387,and1387syntenicblocksinP.lindheimeri,P. pallida and P. fendleri compared to A. thaliana, respectively. SyntenicblocksatKs<0.5revealeda1:4relationshipbetweenA.thaliana and Physaria, suggesting that two whole-genome duplicated events occurred following their divergence (Fig. 2c, SupplementaryFig.4).

Thesynonymoussubstitutionratesexhibitedaunimodalpattern, indicating that two whole-genome duplication events took place withinarelativelynarrowtimeframe,makingitchallengingtodistinguishthembasedondivergencetimesalone.Todifferentiatebetweentwowhole-genomeduplication eventswithinthe Physariagenus,A. thalianawasdividedinto8blocksbasedonsyntenicrelationships,andhomologous gene pairs in each block were used to construct maximumlikelihoodtrees(SupplementaryFig.5).Theaimwastoseparate 1:4syntenicblocksfromeachotherbasedonthetopologyofthetree. Unfortunately,whenweaddedupthemaximumfrequencyofthetree topologyforeachblock,wefoundthatP.lindheimeri,P.pallidaandP. fendleriwereonly40.63%,39.15%,and39.94%,respectively(SupplementaryData10).Thisindicatedthatitwasnotpossibletoseparateall pairwisesyntenicblocksbythismethod.Thiscouldbeduetothatthe Physaria genus underwent two diploidization events with a short interval,whichmakesthemindistinguishable.Furthermore,analysisof genome-wide synteny revealed that P. lindheimeri and P. pallida, as wellasP.lindheimeriandP.fendleri,sharea1:1syntenicrelationship, indicatingthatthreePhysariaspeciesunderwentthesameWGDevent and did not experience further WGD event after their divergence (Fig.2d,e).

By comparing with the ancestral eudicot karyotype (AEK)27, we wereabletotracethekaryotypeevolutionrouteofPhysariagenus. After diverging from A. thaliana (which has 5 chromosomes), the chromosomes of Physaria genus underwent multiple fissions and fusions, resulting in the current six chromosomes (Fig. 3a, SupplementaryFig.6).Thelongterminalrepeatretrotransposon(LTR-RT)is themostabundanttypeofrepetitivesequenceinthePhysariagenus (SupplementaryData9).EstimationaburstofLTR-RTinsertiontimes for P. lindheimeri, P. pallida and P. fendleri indicated that they approximately0.0296Mya,0.0219Myaand0.0096Mya,respectively (SupplementaryFig.7).ThisimpliedthattheLTR-RTsremainhighly active in the Physariagenus,with theiractivitypredominantlylocalizedtochromosomeends(SupplementaryFig.8).Inaddition,when comparing the three Physariaspecies with A.thaliana and Capsella Rubella,weobservedthat15,890genefamiliesweresharedamongfive species,while375,357and518genefamilieswereuniquetoP.lindheimeri,P.pallidaandP.fendleri,respectively(Fig.3b).KEGGenrichmentanalysisoftheseuniquegenefamiliesrevealedtheirsignificant enrichment in pathway such as “DNA replication”, “DNA repair and recombination proteins”, “Mismatch repair” and “Nucleotideexcision repair”.TheseresultssuggestedthataftertherecentWGDandLTR-RTsamplificationeventsinPhysaria,theregenefamiliesmayplayakey role in maintaining normal cellular function and genome stability (Fig.3c–e)28.

SequencearchitectureofthecentromericregionofPhysaria species

Throughouttheintricateprocessofcelldivision,centrosomesassume apivotalroleinupholdingtheintegrityofchromosomesandensuring the precise transmission of genetic information29. Functioning as a pivotalconstituentessentialforfirmlyanchoringchromosomestothe mitoticspindleduringcelldivision,theassemblyofcentromereposes inherent challenges, attributed to the abundance of repetitive DNA elements. These repetitive sequences are mainly composed of high copynumbertandemrepeats(TR)andcentromericretrotransposons (CR).Thedistinctivesatelliterepeatunitsofcentromereshavebeen recognizedacrossavarietyofspecies,includingthepresenceof155bp CentO satellite repeat sequence in rice, the abundance of 178bp CEN180satelliterepeatsequenceinArabidopsis,andtheenrichment of156bpCentCsatelliterepeatsequenceinmaize30–32.Basedonthis characteristic,weidentifiedthecentromericregionsofchromosomes inthegenomesofP.lindheimeri,P.pallida,andP.fendleri(Fig.4aand SupplementaryFig.9).InP.lindheimerigenome,centromeresexhibita lengthrangeof2.18Mbto3.14Mb,withanaveragelengthof2.62Mb, andeachcentromerecontainsanaverageof4308satelliterepeat sequences.InP.pallidagenome,centromeresspanfrom1.52Mbto 4.96Mb, with an average length of 3.55Mb, and each centromere containsanaverageof4664satelliterepeatsequences.Similarly,inP. fendlerigenome,centromerelengthsrangefrom1.48Mbto3.26Mb, withanaveragelengthof2.36Mb,andeachcentromerecontainsan averageof5439satelliterepeatsequences(SupplementaryData11).

Furthermore,weidentifiedsatelliterepeatsequencewithalengthof 111bpinallthreegenomes.Wedesignatedthecentromericsatellite repeatofthethreePhysariaasCentPl,CentPp,andCentPf(Fig.4b).Itis noteworthythatthesatelliterepeatunitsamongthethreePhysaria species are highly similar. The identity between the satellite repeat sequences of P. lindheimeri and P. pallida reaches 94%, while the averagesequenceidentityofcentromericsatellite sequenceswithin chromosomesisas high as96%.Thisresultsuggeststhesignificant conservation of satellite repeat units in the centromeric regions of these plant genomes.

The centromeric region is characterized not only by the abundanceofhigh-copy-numbertandemrepeatsbutalsobyasignificant presence of specific types of retrotransposons. Notably, the centromeres of rice are abundant in Ty3/gypsy type retrotransposons, while those of Arabidopsis are rich in ATHILA long terminal repeat retrotransposons.Similarly,inmaize,thecentromeresareabundance ofCRMandnon-CRMGypsytyperetrotransposons32–34.Withinspecies ofthePhysariagenus,weidentifiedanenrichmentoftwoclassesof retrotransposons, Gypsy/CRM and Copia/Ale, in the centromeric regions(Fig.4aandSupplementaryFig.9).Notably,Gypsy/CRM-type retrotransposonsexhibitthehighestabundance,followedbyCopia/Ale-typeretrotransposons.Interestingly,thereisnoevidentcorrelationbetweenthecopynumberofretrotransposonsoneachchromosome and the length of the centromere (Supplementary Data 12). AmongthethreePhysariaspecies,thecentromeresoftheP.lindheimeri genome exhibit the highest copy numbers for both classes of retrotransposons.Specifically,thecopynumbersofGypsy/CRMtype retrotransposonsrangefrom387to577oneachchromosome,while thecopynumbersofCopia/Aletyperetrotransposonsrangefrom147 to 297. Despite P. fendleri having the largest genome among three species, we observed the lowest copy numbers of Gypsy/CRM and Copia/Aleretrotransposonsinitscentromericregions.Notably,inthe centromericregionsofchromosomes3and4,Copia/Ale-typeretrotransposonsarepresentonlyonce(SupplementaryData12).

Despitetheenrichmentofsatelliterepeatsequencesandspecific retrotransposons in the centromeric regions, protein-coding genes havebeensuccessfullyidentifiedinallthreePhysariaspecies.P.lindheimericontainsthehighestgenecountinitscentromeres,totaling111 genes,followedbyP.pallidawithacountof68genes.Notably,despite P.fendlerihavingthelargestgenome,itscentromericregioncomprises only19protein-codinggenes(SupplementaryData13).Weestablished acriterionforgeneexpression,consideringTPMvaluesgreaterthan1 inatleastonetissue.InP.lindheimeri,P.pallida,andP.fendleri,we discovered48,9,and9expressedgenes,respectively(Supplementary Data 13). However, it’s noteworthy that their expression levels are notably low. This phenomenon could be attributed to potential heterochromatin-mediated transcriptional suppression or the presenceofpseudogenesthatarenotactivelytranscribed35.

SegmentalduplicationsandtandemlyduplicatedgenesofPhysariaspecies

Weidentifiedsegmentalduplications(SD)withinthegenomesofP. lindheimeri,P.pallida,andP.fendleri,comprising86.88Mb,73.41Mb, and 166.66 MB, respectively. These sequences constitute 26.78%, 23.38%,and38.76%oftheirrespectivegenomes(Fig.4c,SupplementaryFig.10,andSupplementaryData14).Notably,inP.fendleri,the contentofSDissignificantlyhighercomparedtotheotherspecies. Differingfromthegenomesofriceandasparagusbean,thefrequency ofSDisrelativelyuniformacrosseachchromosomeinthreePhysaria specie35,36(SupplementaryData14).WithintheSDregionsofthethree Physariaspecies,atotalof4055,3813,and6824geneswereidentified, respectively.GOenrichmentanalysisrevealedthatthegeneswithin theseSDregionsarepredominantlyassociatedwithprocessessuchas “DNA integration” and “DNA metabolic process” (Supplementary Fig. 11). These genes are likely instrumental in preserving genome integrity and stability across the three Physaria species and are essentialfornormalfunctioningandsurvivalofcells.

Tandem duplication is a widespread occurrence in plant genomes, exerting a crucial influence on evolutionary processes and adaptation to dynamic environments. Tandemly duplicated genes, linked to specific functions, contribute to the expansion of gene familiesandleadtoanincreaseingenedosageintheformofgene clusters37.InP.lindheimeri,P.pallida,andP.fendleri,atotalof1446, 1872, and 1718 tandemly duplicated gene clusters were individually identified,collectivelycomprising3700,5139,and4617genes(Fig.4c andSupplementaryData15).Inordertodissectthefunctionalimplications ofthese tandemlyduplicated geneswithinthegenome,GO enrichment analysis was performed. The results unveiled a notable enrichmentofthesegenesincategoriesrelatedto“defenseresponse,” “responsetostress,”and“responsetostimulus”(Fig.4d).Thisfinding suggeststheirpotentialroleinaidingspeciestomaintainbiological homeostasisandadapttoenvironmentalfluctuations.Analyzingthe expression patterns of genes within these three GO categories revealedtissue-specificexpression,indicatingthefunctionalengagementofthesegenesacrossdiversetissues(SupplementaryFig.12). WeconductedananalysisofNLR(nucleotide-bindingleucine-rich repeat) resistance genes in the three species. This gene category is distinguished by the abundance of nucleotide-binding leucine-rich repeatsequences(NLR).NLRgenesplayapivotalroleinhostresistanceresponsesagainstpathogens,astheyrecognizepathogenproteins and activate mechanisms of resistance38. In P. lindheimeri, P. pallida,andP.fendleri,atotalof177,221,and167NLRresistancegenes wereidentified,respectively.Notably,asignificantproportionofthese resistance genes were observed to be tandemly duplicated genes. Specifically, 73 (41.24%), 98 (44.34%), and 66 (39.52%) of the NLR resistancegenesinthethreePhysariaspecieswereidentifiedastandemlyduplicatedgenes.

Highexpressionofkeygenesdrivesaccumulationofhydroxy fattyacidsinseedsofthreePhysariaspecies

TheseedsofthreePhysariaspeciesarecharacterizedbytheiroilswith abundantHFA6,13.Greenhouse-grownplantsproducedseedscontainingoilsfromP.lindheimeri,P.pallidaandP.fendleriat24.23%,18.32% and17.96%,respectively(SupplementaryFig.13andSupplementary Data16).Notably,theprimaryfattyacidislesquerolicacid(C20:1-OH), whichaccountsfor80.78%,84.56%and51.19%oftotalfattyacidsinP. lindheimeri, P. pallidaand P. fendleri, respectively (Fig. 5a and SupplementaryData17).

ToelucidatethegeneticmechanismsofoilbiosynthesisandHFA accumulation in the three Physaria species, we compared transcriptomes between developing seeds at different stages (early developingseed,middledevelopingseed,andlatedevelopingseed) andleafandstem.Atotalof4703and5636up-regulateddifferentially expressedgenes(up-DEGs)wereidentifiedinP.lindheimeri,3194and 3758 up-DEGs in P. pallida, 3923 and 3370 up-DEGs in P. fendleri between developing seed and leaf (seed vs leaf) or stem (seed vs. stem),respectively(SupplementaryFig.14).KEGGfunctionalenrichmentanalysisoftheseDEGsdemonstratedthatmanywereassignedto “lipid metabolism”, “linoleic acid metabolism”, “lipid biosynthesis proteins”, “fatty acid elongation”, “alpha-linolenic acid metabolism” and“biosynthesisofunsaturatedfattyacids”(SupplementaryFig.14). Furthermore,weidentified197,195and182genesinP.lindheimeri,P. pallidaandP.fendleri,respectively,orthologoustoatotalof111fatty acid andlipidbiosynthesisgenes previouslyreportedin A.thaliana (SupplementaryData18,SupplementaryData19).

The unique character of the three Physaria species is the high concentrationsofHFAinseedoils,particularlylesquerolicacid(C20:1-OH)2,39,40.TheHFAsynthesisgenesFAH12andKCS3inthethreePhysaria species all showed substantially higher expression levels in middle and late-developing seed as compared to early-developing seed,leaf,andstem,wheretheirexpressionswerenegligible(Fig.5b, c).Othermajorgenesinvolvedinthelipidsynthesispathwaywerealso predominantly expressed in seeds with low expression in leaf and stem,suchasSAD,akeydesaturasethatcatalyzestheconversionof C18:0-ACP to C18:1-ACP in the plastid, and genes encoding TAG assembly enzymes including lysophosphatidic acid acyltransferase (LPAT), diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) (Fig. 5b). LPAT specifically incorporatesacylgroupsintothesn-2positionoftheglycerolbackbone41,42, andhasbeenshowntobethemainlimitforHFAaccumulationinthe TAG molecules in P. fendleri43. LPAT genes are highly expressed in developingseedsattheearlytomiddlestages(Fig.5b).DGATsarealso highlyexpressedinmiddletolatestagesofdevelopingseeds(Fig.5b), whichisconsistentwiththepreviousreportsthatDGATvariantswith strong preference to HFA contribute to their accumulation in seed oil25,44.Besidesthesebiosyntheticgenes,wealsoidentifiedfivetranscriptionfactors(TFs),ABSCISICACIDINSENSITIVE3(ABI3),WRINKLED1 (WRI1),FUSCA3(FUS3),LEAFYCOTYLEDON1(LEC1),andLEAFYCOTYLEDON2(LEC2),whichplaycentralrolesinregulatinglipidbiosynthesis and the accumulationof seed oil45–50. TheseTFs also showed much higherexpressionindevelopingseedsthaninleafandstem(Fig.5b).

TheelevatedexpressionofkeygenesandTFsinvolvedinlipidbiosynthesisinseedmaycontributetothesynthesisofHFA-containing oilsinthethreePhysariaspecies.

ActivitiesofPhysariaFAH12sweresubstantiallyaffectedbytwo keyaminoacids

ItisnotablethatthelesquerolicacidcontentisremarkablyhigherinP. lindheimeriandP.pallidaseedscomparedtoP.fendleri(Fig.5a).FAH12 and KCS3 are two key enzymes directly involved in biosynthesis of lesquerolic acid, and they are upstream of acyltransferases, which incorporatelesquerolicacidintoTAG.Basedonthefattyacidprofileof threePhysariaspecies,ricinoleicacid(C18:1-OH)isalmostcompletely convertedintolesquerolicacid(Fig.5a).WespeculatedthatKCS3is not a limiting metabolic step of lesquerolic acid in three species. Interestingly,inP.fendleri,theexpressionleveloftheFAH12washigher inmiddleandlatedevelopingseedsthaninP.lindheimeriandP.pallida (Fig.6a).Toexplainthisdiscrepancy,wehypothesizedthatdespiteits hightranscriptionallevel,theactivityofthePfFAH12enzymemaybe considerablylowerthanPlFAH12andPpFAH12.Totestthishypothesis, weexpressedthreeFAH12genesinmammaliancellsandcompared theirabilitiestoproduceHFA(Fig.6b,andSupplementaryData20). WepurifiedtheFAH12proteinsandobservedthattheexpressionof PfFAH12 was higher than PlFAH12 and PpFAH12 (Supplementary Fig. 15a). However, cells hosting PfFAH12 produced much lower amount of ricinoleic acid than PlFAH12 and PpFAH12 (Fig. 6b). No detectablericinoleicacidwasfoundinnegativecontrolpurifiedFAH12 or in the extracts offatty acid desaturase 2(FAD2) (Supplementary Fig. 16), which catalyzes the desaturation of oleic acid to produce linoleic acid2. These results suggest that the better performance of PlFAH12andPpFAH12thanPfFAH12mayplayanimportantroleinthe much higher contents of lesquerolic acids in P. lindheimeri and P. pallidaseedsthanP.fendleri.

SequencecomparisonindicatedthatthePlFAH12hasabout90% sequenceidentitywithPfFAH12,andabout95%sequenceidentitywith PpFAH12 (Supplementary Fig. 17).The residues among the catalytic pockets of PlFAH12 and PpFAH12 are highly conserved. To provide structuralcontextforthesubstratebindingpocketofFAH12,weran AlphaFold351forthesethreePhysariaFAH12,thendockedoleicacid into the AlphaFold prediction structures, respectively(Fig. 6c). The dockingmodels52showthatthenegativelychargedcarboxylheadof oleicacidisdirectedtowardsthepositivelychargedhistidinemotifs. Superposingthesemodelsrevealshighlysimilararchitecturesamong them(Fig.6d).Basedonsequencecomparisonandstructureprediction, we suspected that the weaker activity of PfFAH12 might be associated with its residues H111 and Q225 (Fig. 6d, Supplementary Fig. 17) instead of Q111 and H225 in PlFAH12 or PpFAH12 (Fig. 6d, SupplementaryFig.17).

TotestwhetheranyofthesetworesiduesaffectPfFAH12activity, site-directed mutagenesis was performed by replacing either one residue or both residues of PfFAH12 (PfFAH12H111Q, PfFAH12Q225H, PfFAH12H111Q/Q225H). Visual inspection of the SDS-PAGE analyses of all mutantsrevealedsimilarexpressionasthewildtype(Supplementary Fig.15a).However,theactivitiesofallPfFAH12mutantssignificantly increased and the highest was observed in PfFAH12H111Q/Q225H which ricinoleicacidcontentincreasedbynearly2.5-foldcomparedtothatof wild type (Fig. 6b). These results demonstrated that both Q111 and H225playedcrucialrolesinPfFAH12.Theresults,togetherwiththe genome and evolution analyses, indicated that the divergence of FAH12samongthethreePhysariaspeciesmighthavedeterminedthe rateofricinoleicacidsynthesisandledtodifferentcontentsoflesquerolicacid.Additionally,thereplacementoftwokeyresiduescan enhancetheactivityofPfFAH12.

TofurthertestthefunctionofFAH12forthebiosynthesisofricinoleicacid,wegeneratedtransgenicArabidopsislinesoverexpressing threePhysariaFAH12sdrivenbytheseed-specificnapinpromoter.We obtained three independent homozygous overexpression (OE) lines for each FAH12. The results showed that PlFAH12 had the highest expressionlevelcomparedwithPfFAH12andPpFAH12.PfFAH12and PpFAH12generallyhadcomparableexpressionlevels(Supplementary Fig.18andSupplementaryData21).ThericinoleicacidcontentinT3 matureseedswasquantifiedintheseFAH12-OElines.Comparedwith PfFAH12-OEseeds,thoseexpressingPlFAH12andPpFAH12exhibiteda significantlyhigherricinoleicacidcontent(Fig.6eandSupplementary Data22),consistentwiththehigherinvitroenzymaticactivityofthese twoenzymes.Theseresultsdemonstratethatvariationsinricinoleic acidaccumulationaremainlyinfluencedbythespecificFAH12gene expressed, highlighting differences in enzyme activity among these threeFAH12s.

Methods
Plantgrowthconditionandsamplecollection
TheseedsofP.lindheimeri,P.pallidaandP.fendleriweresowninthe pots with nutrient soil: vermiculite = 1:1 (v/v) in a growth chamber (Conviron) at 26/24 °C under 16-h light/ 8-h dark. Before the seed germinationofP.fendleri,theseedsinpotsweretreatedwithvernalization about half month. The plants in pots were transported to NovogenetoextractDNAforgenomesequencingwhentheygrewbig enough but not bolting. P. lindheimeri and P. fendleri are self-incompatibilityplantsafterthestigmaexertion.Hence,artificialcompulsorypollinationwasperformedbeforetheflowerbudopened.The wholesiliqueswith20-23(earlydevelopingseed,EDS),32-35(middle developingseed,MDS),and45-47(latedevelopingseed,LDS)daysafter pollination(DAP)ofP.lindheimeri,15-17(EDS),25-27(MDS)and35-37 (LDS)DAPofP.pallida,10-13(EDS),20-23(MDS)and30-33(LDS)DAP ofP.fendleriwerecollectedfortheRNA-seqexperiments.Thesamples ofleafandstemwerecollectedatthesametimeastheMDSsiliques.

Sequencing
For Illumina paired-end reads sequencing, 0.2μg DNA from young leaves per species was used for DNA library preparations. The NEB Next®Ultra™DNALibrariesPrepKitforIllumina(NEB,USA)wasused to generate DNA libraries according to the manufacturer’s recommendations.IlluminaPEClusterKit(Illumina,USA)wasusedtocluster the index-coded samples performed on a cBot Cluster Generation System,thentheDNAlibrariesweresequencedontheIlluminaplatform,and150bppaired-endreadsweregenerated.

For PacBio sequencing, ~20kb SMRTbell libraries were constructedfromyoungleavesperspeciesusingtheSMRTbellExpress Template Preparation Kit 2.0 for the CLR module and SMRTbell Express Template Prep Kit 2.0 forthe CCS module. Then, Sequel Ⅱ Sequencing Kit 2.0 was used to sequence and generate PacBio longreads.

For RNA sequencing, RNA was extracted from different tissues statedabove.TheTruSeqPEClusterKitv3-cBot-HS(Illumina)wasused toclustertheindex-codedsamplesonacBotClusterGenerationSystem.ThelibrarieswerethensequencedonanIlluminaNovaseqplatform,and150bppaired-endreadsweregenerated.

Estimationofgenomesizeandheterozygosity
The genome size was estimated using k-mer frequencydistribution generatedfromIlluminashortreads.Thejellyfish(v2.3.0)70wasusedto generate 19-mer frequency distribution. Then, GenomeScope71 was usedtoestimategenomesizeandheterozygosity.

Genomeassemblyandassessinggenomeassemblyquality
ForP.lindheimeri,Canu(v2.1)72wasusedfordenovoassemblybased on PacBio continuous long reads (CLR) with the parameters: rawErrorRate = 0.3, minReadLength = 1000, corOutCoverage = 40, correctedErrorRate=0.045,minOverlapLength=500,corMinCoverage=4,maxThreads=200.Therawcontigswerepolishedtworounds using PacBio continuous long reads with Racon73 and polished two rounds using CLR reads with Pilon74. For P. pallida and P. fendleri, HiFiasm (v0.16.1-r375)75 was used for de novo assembly based on PacBiohigh-fidelity(HiFi)reads,-l0parameterwasusedforP.pallida denovoassembly,-s0.5parameterwasusedforP.fendleridenovo assembly,andoneofthephasedcontigsasthefinalassemblyresult. Thenthecontigsweresubjectedtodehybridizationusingpurge_haplotogs (v1.1.0)76. Finally, the polished contigs were anchored into chromosomesbyusingHiC-Pro(v3.1.0)77andALLHiC(v0.9.13)78based onhigh-throughputchromosomeconformationcapture(Hi-C)reads, and then the assembled genome was manually correction by using Juicexbox(v1.11.08)79.

To assess the quality and completeness of the assembled genomes,BWA-MEM(v0.7.17-r1188,https://github.com/lh3/bwa)wasused to map the Illumina paired-end reads to the assembled genomes, HISAT2 (2.1.0)80 was used to map the RNA sequencing data from multiple tissues to the assembled genomes, and minimap2 (v2.24-r1122)81 was used to map the PacBio long reads to the assembled genomes.Moreover, BUSCO (v4.0.6)82 was used to assess the completeness of the assembled genomes and predicted protein-coding genes based on the Embryophyta Plant database (odb10), and long terminal repeat retrotransposons assembly index (LAI) was used to evaluatetheassemblyqualityusingLTR_retriever(v2.9.0)83.

Genomeannotation
Transposableelementsannotation:First,adenovotransposableelementsdatabasewasbuiltusingRepeatModeler,Piler84,RepeatScout85, Trf86, and LTR_FINDER87,then combined with the Repbasedatabase (http://www.girinst.org/repbase) to generate a consensus library. Finally, RepeatMasker (https://www.repeatmasker.org/) was used to predictrepeatsequencesbasedontheconsensuslibrary.

The protein-coding genes were predicted by combining the homology-based prediction, de novo prediction, and RNA-Seq-assisted prediction. Firstly, the non-redundant proteins from five species (A. thaliana, Arabidopsis lyrate, Brassica rapa, Brassica oleracea,andBrassicanapus)closelyrelatedtothePhysariagenuswere usedasthehomologyannotationlibraryandtheinputforTBLASTNto predicthomologoussequences.Additionally,Augustus88,89,Genscan90, andGilmmerHmm91wereusedfordenovogeneprediction.Thirdly, RNAsequencing data were mapped to the genomeusing HISAT280, andtranscriptomeassemblywasdoneusingtheStringTiepipeline92. Lastly, MAKER93 and HiCESAP were used to combine all prediction resultstoproduceanon-redundantgeneset.Moreover,theprotein databases of SwissProt (https://www.gpmaw.com/html/swiss-prot. html), TrEMBL94, NR (https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/), KEGG95, InterPro96 and GO97 were used to predicted the function ofgenes.

Non-codingRNAannotation:tRNAscan-SE98wasusedtoannotate tRNAbasedonthestructuralfeaturesoftRNA,BLASTNwasusedto predicttherRNAbasedonhomologypredictionmethodbymapping rRNA sequences from closely related species to the genomes. The covariancemodeloftheRfamfamilyandtheINFERNALthatcomes withRfamwereusedtopredictthemiRNAandsnRNA.

Phylogeneticanalysisandgenefamilyexpansion/contraction
To elucidate the evolution of the Physaria genus, 15 other species (Amborellatrichopoda,Oryzasativa,Zeamays,Solanumlycopersicum, Vitisvinifera,Glycinemax,Populustrichocarpa,Jatrophacurcas,Ricinus communis, Theobroma cacao, B. rapa, B. oleracea, A. thaliana, Capsella Rubella, and Camelina neglecta) were selected from the Phytozomedatabase(https://phytozome-next.jgi.doe.gov/)99foranalysis. First, Orthofinder (v2.5.4) was used to identify single-copy orthologousgenes.Next,theproteinsequencesandcodingsequences of 276 sing-copy orthologous genes were aligned using MUSCLE (v5.1.linux64)100. Then the best-fit model (Blosum62 + I+G+F) was predicted using ProtTest (v3.4.2)101. RAxML (v8.2.12)102 was used to constructamaximumlikelihoodtreewith1000replicatesbootstrap. Finally,theiTOLwebsite(https://itol.embl.de/)103wasusedtovisualize thephylogenetictree.

MCMCTreeprograminthePAMLpackage(v4.9.j)104wasusedto calculatethedivergencetimefortheabove18species.Threecalibration points were selected fromthe TimeTree database(http://www.timetree.org/)105toestimatethedivergencetime,andthethreecalibrationpointsareZ.maysandO.sativesplittime(0.45–0.52Mya),A. thalianaandV.viniferasplittime(1.07–1.35Mya),aswellasA.trichopodaandV.viniferasplittime(1.73–1.99Mya).Finally,CAFÉ(v5.0.0)106 wasusedtocalculatetheexpansionandcontractionofgenefamilies basedonthephylogenetictreeandgenefamilystatistics.

Whole-genomeduplicationeventsanalysis
To identify the whole-genome duplicated event in Physaria genus, WGDI (v0.6.2)107 was used for identifying syntenic blocks (-icl parameter)andcalculatingtheKsvaluesofgenepairs(-ksparameter).In addition,itwasusedforvisualizingthedensityofKsdistribution(-kf parameter) and for extracting collinear gene pairs from identified blocks(-aparameter).Finally,WGDIwasusedforconstructingphylogenetictreesoftheidentifiedcollineargenepairs(-atparameter).

Identificationofcentromericregions
Gepard (v2.1)108 was employed for the identification of repetitive sequenceswithinthegenomeandtogenerateadotplot.Thedotplot ofchromosomeswassubsequentlyanalyzedtoidentifythedistinctive “black block” regions, generated as a result of intercomparison of repetitivesequences.Theseregionsareconsideredcharacteristicof thecentromericregions,withthefollowingparameters:Wordlength= 10, Window size = 0, Use matrix = DNA. To discern the position, number,andconsistencyofconservedtandemrepeats(motifs),the TandemRepeatsFindersoftware(TRF)wasemployed86.Thespecific parametersutilizedforthisanalysisweresetasfollows:277801050 500-f-d-m.Subsequently,MEME(v5.5.4)wasutilizedtominemotifs presentintherepeats,specificallyfocusingonsatelliterepeatswithin thecentromericregions.The analysis employed the following parameters:-nostatus-time14400-modzoops-nmotifs3-objfunclassic -revcomp-markov_order0-searchsize3000000.

Identificationofsegmentalduplicationandtandem duplicationgenes
Thegenomicsequenceunderwentsoft-maskingusingRepeatMasker (v4.1.0) with the following parameters: -e rmblast -xsmall -gff -pa 1. Subsequently,theBiSERsoftwarewasappliedtoidentifysegmental duplications within the masked genome. Fragments exhibiting an identityexceeding90%andlengthssurpassing1kbwereretainedas segmental duplications109. Genomic protein sequences were compared using Diamond, and the results werefiltered by applying the followingcriteria:e-value<10e-20,proteincoverage>80%,withthe distancebetweentwogenepairsnotexceeding10genes110,111.Instanceswheregenepairsoverlappedwereconstruedastandemduplicated geneclusters.

Differentiallyexpressedgeneanalysis
Transcriptomicdatafromdifferenttissues,includingseedsatdifferent developmentalstages(earlydevelopingseed,middledevelopingseed, andlatedevelopingseed),aswellasleafandstem,wereusedtofind differentiallyexpressedgenes.HISAT2(v2.1.0)80wasusedtomapRNA- Seqdatatothegenome.StringTie(v2.1.7)92wasthenusedtocalculate theTPM,andfeatureCounts112wasusedtocalculatethereadscounts ofthegenesbasedontheresultofmapping.Finally,RpackageDESeq2 (v1.38.3)113wasusedtoidentifythedifferentiallyexpressedgeneswith anadjustedp-valueof0.05.

ExpressionandpurificationofFAH12inmammaliancell
TheFAH12geneswereclonedintothepMLinkvector114,containingan N-terminal3×Flagtag.Thesite-specificmutationswereintroducedinto thegenesbyoverlappingPCRandwereverifiedbyDNAsequencing. Expi293FTMcells(Invitrogen)wereculturedinUnion-293media (Union-Biotech, Shanghai) in a 37°C ZCZY-CS8 shaker (Zhichu Instrument)with5%CO2.Whenthecelldensityreached2.0×106cells permL,theFAH12geneswastransfectedintothecellswithplasmid, respectively. For 40mLofcell culture,80μg ofplasmids were pre-incubated with 240μg of 4kDa linear polyethylenimine (PEI) (Polysciences) for 20min followed by adding the mixture into the dilutedcells.

After culturing for another 60h, the cells were harvested by centrifugation and resuspended in lysis buffer containing 100mM Tris-HCl(pH7.4),150mMNaCland1%LMNG(Anatrace)at4°Cfor2h. The insoluble component was removed by ultracentrifugation at 14,000gfor30min.Thesupernatantwasincubatedwithanti-FlagG1 affinityresin(Genscript)at4°Cfor1h,andtheresinwaswashedwith lysisbufferwith0.01%LMNG.Thenelutedbylysisbufferwith0.01% LMNGand250μgmL−13×FLAGpeptide(GenScript),andsubsequent analysis by SDS-PAGE and immunoblot. The primer sequences are providedinSupplementaryData23.

Determinationofoilcontentandfattyacidcontentofseedsand mammaliancells
Forseedoilanalysis,8–9mgmatureseedswereaccuratelyweighted andusedinoilextraction.SeedoilcontentandfattyacidcompositionwereanalyzedwithGC-FID115.Sixreplicateswereperformedfor each plant. To analyze fattyacid compositionof mammaliancells, the biomass was pelleted by centrifugation and fatty acids were extractedin2mLacidmethanolsolution(5%H2SO4inmethanoland 1%BHT),supplementedwith40μLinternalstandard(C17:0,Sigma). Theglasstubesweretightlycappedandheatedat85°Cfor2h.After cooling,2mLofultrapurewaterand2mLofhexanewereadded,and fattyacid methyl esters (FAMES) were recovered by collecting the hexane phase. Reaction products were analyzed by gas chromatography-flameionizationdetector(GC-FID)usingoleicacid (C18:1Δ9) (Sigma) and ricinoleic acid (MedChemExpress) as standards. The GC conditions were as follows: oven temperature was increased from 170°C to 230°C at a rate of 3°C min–1. Fatty acid composition in individual cell samples was identified according to their retention times and calculated based on internal standard contentwithpeakarea.

ModeldockingofFAH12andoleicacid
WepredictedthreePhysariaFAH12modelsbyAlphaFold351,andused the programAutoDock Vina 1.1.2116 to dockthe complex models of FAH12andsubstrateoleicacid.Theoriginalstructureofoleicacidwas generatedormodifiedusingPyMOLandfurtherpreparedwithAutoDocktools1117forthedockingprocess.Duringthedockingcalculation, theproteinstructureswereregardedastherigidbody,whiletheligand wasgivencompletetorsionfreedom.Thedockingboxwassetaround thecatalyticarea.Foreachsubstratedocking,atotaloftenmodels werecreated,andthemodelwiththebestbindingaffinitywaschosen for further analysis. All structural figures were generated using the PyMOLprogram(http://www.pymol.org/).

Agrobacterium-mediatedtransformationofArabidopsis
Aftertheplantsreachedthefloweringstage,agrobacterium-mediated transformationwasperformed.Theconstructswereintroducedinto competent cells of Agrobacterium tumefaciens GV3101 via electroporation.Arabidopsisgenetictransformationwascarriedoutusingthe floral-dipmethod.TransformedAgrobacteriumcellswerecollectedby centrifugation and resuspended in a solution containing 5% (w/v) sucroseand0.05%(v/v)surfactantSilwetL-77,adjustingthebacterial suspensiontoanOD₆₀₀of0.6–0.8.Thesuspensionwasplacedina disposablepetridish,andtheinflorescencesoffloweringArabidopsis plantswereimmersedinthebacterialsuspensionfor30s.Afterinfiltration,theplantswereplacedhorizontallyinthedarkfor16–24h.The infiltration step was repeated one week later. Subsequently, plants were returned to standard growth conditions until seeds were harvestedatmaturity.T₀seedscollectedaftertransformationweresown innutrientsoilandcoveredwithplasticwrapfor4–5days.Theywere thengrownunderconditionsof22°Cwitha16-hourlight/8-hourdark photoperiod.Positivetransformantswereselectedbyscreeningwith glufosinateammonium.The primersequencesareprovided inSupplementaryData23.

Geneexpressionanalysis
Total RNA was extracted from developing siliques at 10 days using TRIZOL reagent (Vazyme, R401-01). For reverse transcription, the EasyScript®One-Step gDNARemoval andcDNASynthesis SuperMix (TransGen Biotech, AE311-02) was used to synthesize first-strand cDNA,whichsubsequentlyservedasthetemplateforPCRamplification.ForRT-PCRanalysis,thePCRproductswereseparatedona1% agarosegelstainedwithethidiumbromideandvisualizedunderUV light. The quantitative real-time PCR (qRT-PCR) reactions were performed using the 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711). Fluorescence signals were detected with a LightCycler® 480 II instrument, and the data were analyzed using the LightCycler®480Software(Version1.5.1).Thetranscriptlevelsofthe targetgeneswereexaminedusinggene-specificprimers.Ubiquitin5 (UBQ5, At3g62250) was used as an internal control. The primer sequencesareprovidedinSupplementaryData23.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports whole genome sequencing of three related Physaria species which accumulate industrial useful hydroxy fatty acids. Authors made comparative studies and annotated a list of lipid genes among Arabidopsis and the three physaria species. The methods are up-to-date and the work meets the expected standards in genome sequencing field. Authors obtained novel genomic data, and the informative is critical for future understanding the mechanisms underlying HFA biosynthesis.\nSpecific comments.\n1. Regarding the mechanisms of accumulating different levels of HFA among these three Physaria species, Author mainly focused on the FAH12 genes and enzyme activity. It is known that PfFAH12 is a bi-functional hydroxylase/desaturase, and PlFAH12 is a strict FAH12. Authors didn’t mine the data deeply, nor characterize the other important genes such as LPAT2s. 20:1OH may enter the TAGs through Kennedy pathway, it will be valuable if additional in vitro experiments are performed to characterize the substrate specificity on the three LPAT2s (PfLPAT2, PpLPAT2 and PlLPAT2).\n2. Nomenclature ‘KCS3’. Arabidopsis lipid genes are routinely used as standards to name orthologous genes. The original ‘KCS3’ for the HFA-specific condensing enzyme from P. fendleri is orthologous to AtKCS18 (or AtFAE1). Since Arabidopsis has AtKCS3 which synthesizes very long fatty acids, it is better to clarify the nature of ‘KCS3’ used in the manuscript.", "reviewer_2": "The current submitted manuscript by Wang et al titled \"Chromosome-level assembly and analysis of three hydroxy fatty acids producing Physaria species: P. lindheimeri, P. pallida and P. fendleri\" report the full genome and transcriptome analyses for three Physaria species which showed varying capacity in total seed oil content and hydroxy fatty acid content as well. After comparative transcriptomic analysis, the transcript of the major gene resonponsable for HFA synthesis showed high expression in species that has lower FAH content. Based on this finding, the authors looked into details in FAH12 protein sequence and predicted 3-D structure by alpha-fold. The authors then identfied two amino acid substitutions in this protein, and through some in vitro activity assay, and conclude that the two amino acids changes explain the difference in FAH12 activity then likely the hydroxy fatty acid content. The first genome and transcriptomic part is some-how dis-associated from the second part. The paper is straight forward and well written. The data presented is solide but with a major descriptive part. But I consider that the finding in this paper does not reach the level of Nature Communications. I recommend the authors submit the paper to a more plant-specific journal.", "reviewer_3": "Authors finished chromosome-level assemblies of three plants, including P. lindheimeri, P. pallida and P. fendleri. and then performed comparative genome analyses among them and with other plant genomes. There are several major conclusions that they got. First, three genome reference genomes have been released. Second, they revealed that the three Physaria species diverged from Arabidopsis thaliana approximately 31 14.10-14.46 million years ago and underwent two consecutive Physaria-specific whole-genome duplication events. Third, they presented the evolutionary features of centromere sequences. Forth, they find that duplicated genomic regions are associated with maintenance of species biological homeostasis and adaptation to environmental fluctuations. Fifth, transcriptomic analysis shows gene expression profiles that related to lipid synthesis in the seeds. Last, through wetlab works, they find the two residues in FAH12 that caused the disparity in hydroxylation activity among three Physaria species. This work is fine, however, the genomic analysis part is quite common, and there is no striking finding. The wetlab part only provided indirect evidences to support the conclusion.\nMajor concerns:\n1.I am not sure if it is feasible to assay the activity of FAH12 in the animal cells. Animal cells are not plant cell after all. Instead, author should do the transgenic validation in Physaria. In Fig6a, the content of (C20:1-OH) content is highest in P. pallida, however, in Fig6b, PpFAH12WT show lowest C18:1-OH. Both is not consistent. Additionally, the differences of the content in fig6b among these plants are not obvious.\n2. Author did some analysis at the centromere regions. Actually, assembling quality of centromere regions has been validated by any other ways although long reads to be used. Therefore, it is a risk to do any analysis about the centromere region.\nMinor concerns:\n1. Iine 50 author say” Some Physaria species exhibit valuable traits that are conducive to domestication”. It is not clear if is there any physaria species have been cultivated as crops.\n2. Author should introduce the usage of HFA from Physaria species to industry.\n3. Author should introduce the physical and chemical properties of HFA from Physaria,\n4. The author highlighted the unique, twice-occurring whole genome duplication events in the Physaria genus using Ks plots and genome synteny between P. lindheimeri and A. thaliana. However, the analysis still has several issues that need to be addressed to provide more robust evidence for the conclusions: A 4DTv plot may provide more detailed resolution regarding the WGD events.\n5. Figures 2c, 2d, and 2e should use the same Ks color bar and have P. lindheimeri on the Y-axis. Alternatively, creating a Sankey plot including all related species could make the relationships more evident.\n6. The author did not clearly explain how new centromeres and telomeres formed during species differentiation, given the extensive chromosome breaks and fusions in the ancient Physaria genus over such a short interval. Generally, new centromere loci have structural or sequence preferences."}, "author_response": "Point-by-point response to REVIEWER’S COMMENTS\nReviewer #1:\nThis manuscript reports whole genome sequencing of three related Physaria species\nwhich accumulate industrial useful hydroxy fatty acids. Authors made comparative\nstudies and annotated a list of lipid genes among Arabidopsis and the three physaria\nspecies. The methods are up-to-date and the work meets the expected standards in\ngenome sequencing field. Authors obtained novel genomic data, and the informative is\ncritical for future understanding the mechanisms underlying HFA biosynthesis.\nSpecific comments.\n1. Regarding the mechanisms of accumulating different levels of HFA among these\nthree Physaria species, Author mainly focused on the FAH12 genes and enzyme activity.\nIt is known that PfFAH12 is a bi-functional hydroxylase/desaturase, and PlFAH12 is a\nstrict FAH12. Authors didn’t mine the data deeply, nor characterize the other important\ngenes such as LPAT2s. 20:1OH may enter the TAGs through Kennedy pathway, it will\nbe valuable if additional in vitro experiments are performed to characterize the substrate\nspecificity on the three LPAT2s (PfLPAT2, PpLPAT2 and PlLPAT2).\n[Response]: Thanks for your suggestion. We transfected the FAH12s in mammalian\ncells to assay the enzyme activity, and the results show that the FAH12 genes is mainly\na hydroxylase, the content of product 18:2 is very low (Response Fig 1).\nWe agree that the substrate specificity of three LPAT2s may impact the level of\naccumulation of hydroxy fatty acid. Unfortunately, we are unable to purchase\ncommercial 20:1-OH CoA and 20:1-OH-LPA to carry out the in vitro enzyme activity\nassay. We also checked the literatures and confirmed that this kind of enzyme activity\nassay using commercial 20:1-OH CoA and 20:1-OH-LPA has not been reported.\nAdditionally, to nevertheless assess whether the observed amino-acid differences could\ninfluence catalytic efficiency, we analyzed the LPAT2s (PfLPAT2, PpLPAT2 and\nPlLPAT2) sequence identities and performed the substrate docking models (Response\nFig 2,3), show that amino acids with differences are not located near the active pocket\nand may not affect enzyme activity. We included this part of analysis as supplemental\nfigure, described the results and discussed the possibility of LPAT2’s role.\nResponse Fig 1. GC-FID analysis of three Physaria FAH12 and the mutants of\nPfFAH12 expressed from mammalian cells.\nResponse Fig 2. The sequence alignment of LPAT2 from P. lindheimeri, P. pallida and\nP. fendleri.\nResponse Fig 3. Docked models for PfLPAT2 and substrate oleoyl-CoA. The different\nresidues shown in stick representation.\n2. Nomenclature ‘KCS3’. Arabidopsis lipid genes are routinely used as standards to\nname orthologous genes. The original ‘KCS3’ for the HFA-specific condensing enzyme\nfrom P. fendleri is orthologous to AtKCS18 (or AtFAE1). Since Arabidopsis has\nAtKCS3 which synthesizes very long fatty acids, it is better to clarify the nature of\n‘KCS3’ used in the manuscript.\n[Response]: Thanks for your valuable comment. You're right that the nature of 'KCS3'\nwas not clearly explained in the manuscript. So in lines 82-85, we clarify the nature of\nKCS3 by describing it as \"This step is catalyzed by the fatty acid condensing enzyme\n3-ketoacyl-CoA synthase 3 (KCS3) in a fatty acid elongase (FAE) complex. Physaria\nKCS3 is highly homologous to AtKCS18 (or AtFAE1) in Arabidopsis but specifically\ncatalyzes the elongation of C18-HFA (Moon et al., 2001).\"\nReviewer #2:\nThe current submitted manuscript by Wang et al titled \"Chromosome-level assembly\nand analysis of three hydroxy fatty acids producing Physaria species: P. lindheimeri, P.\npallida and P. fendleri\" report the full genome and transcriptome analyses for three\nPhysaria species which showed varying capacity in total seed oil content and hydroxy\nfatty acid content as well. After comparative transcriptomic analysis, the transcript of\nthe major gene resonponsable for HFA synthesis showed high expression in species that\nhas lower FAH content. Based on this finding, the authors looked into details in FAH12\nprotein sequence and predicted 3-D structure by alpha-fold. The authors then identified\ntwo amino acid substitutions in this protein, and through some in vitro activity assay,\nand conclude that the two amino acids changes explain the difference in FAH12 activity\nthen likely the hydroxy fatty acid content. The first genome and transcriptomic part is\nsomehow dis-associated from the second part. The paper is straight forward and well\nwritten. The data presented is solide but with a major descriptive part. But I consider\nthat the finding in this paper does not reach the level of Nature Communications. I\nrecommend the authors submit the paper to a more plant-specific journal.\n[Response]: We appreciate your careful reading and feedback. Lesquerolic acid (C20:1-\nOH) has very important industrial value. We selected these three Physaria species for\nstudy, because they actively synthesize and accumulate C20:1-OH in the seeds.\nHowever, P. lindheimeri and P. pallida accumulate higher C20:1-OH than that of P.\nfendleri. This project is designed to elucidate the pathway of fatty acid biosynthesis in\nthree Physaria species and figure out why P. fendleri accumulates less C20:1-OH. Thus,\nthe first part is genome assembly and characterization of three Physaria species with\ndifferent fatty acid composition, and the second part is to analyze the fatty acid\nbiosynthesis pathway by identification of the key genes and analysis of their expression\nbased on the genome and transcriptome data. Then, we tried to identify the key\ngenes/enzymes involved in the determination of C20:1-OH level in three Physaria\nspecies. We pinpointed the two residues in FAH12 that caused the disparity in\nhydroxylation activity among three Physaria species. We tried to make each part\nconnected and the major conclusion is well supported.\nReviewer #3 (Remarks to the Author):\nAuthors finished chromosome-level assemblies of three plants, including P. lindheimeri,\nP. pallida and P. fendleri. and then performed comparative genome analyses among\nthem and with other plant genomes. There are several major conclusions that they got.\nFirst, three genome reference genomes have been released. Second, they revealed that\nthe three Physaria species diverged from Arabidopsis thaliana approximately 31 14.10-\n14.46 million years ago and underwent two consecutive Physaria-specific whole-\ngenome duplication events. Third, they presented the evolutionary features of\ncentromere sequences. Forth, they find that duplicated genomic regions are associated\nwith maintenance of species biological homeostasis and adaptation to environmental\nfluctuations. Fifth, transcriptomic analysis shows gene expression profiles that related\nto lipid synthesis in the seeds. Last, through wetlab works, they find the two residues\nin FAH12 that caused the disparity in hydroxylation activity among three Physaria\nspecies. This work is fine, however, the genomic analysis part is quite common, and\nthere is no striking finding. The wetlab part only provided indirect evidences to support\nthe conclusion.\nMajor concerns:\n1.I am not sure if it is feasible to assay the activity of FAH12 in the animal cells. Animal\ncells are not plant cell after all. Instead, author should do the transgenic validation in\nPhysaria. In Fig6a, the content of (C20:1-OH) content is highest in P. pallida, however,\nin Fig6b, PpFAH12WT show lowest C18:1-OH. Both is not consistent. Additionally,\nthe differences of the content in fig6b among these plants are not obvious.\n[Response]: Thanks for your comments. In Fig 6b, PfFAH12WT show lowest C18:1-\nOH that is consistent with the lowest C20:1-OH in P. fendleri in Fig 5a. The activity of\nPpFAH12WT and PlFAH12WT are both significantly higher than PfFAH12WT, even the\nprotein expression level is not as high as PfFAH12WT.\nWe thank for raising the concern about whether FAH12 activity can be reliably\nmeasured in animal cells and for suggesting that transgenic validation be performed in\nPhysaria. First, it is very common to characterize the activity of different enzymes\ninvolved in fatty acid biosynthesis in animal and yeast cells. Animal cells have similar\nfatty acid biosynthesis pathway as plants and this kind of experiment can provide the\nevidence much faster that performing plant transformation. According to your\nsuggestion, we carried out an independent in-planta validation by generating stable\ntransgenic Arabidopsis lines over-expressing each FAH12. The results are described\nbetween line 418 and 430.\n2. Author did some analysis at the centromere regions. Actually, assembling quality of\ncentromere regions has been validated by any other ways although long reads to be\nused. Therefore, it is a risk to do any analysis about the centromere region.\n[Response]: Thank you for highlighting this important concern. We agree that\ncentromeric repeats are notoriously difficult to assemble and that relying on long-read\ndata alone may not be sufficient to guarantee accuracy in these regions. However, we\ndid indeed find numerous characteristics of centromeres within the centromere\nassembly region. For instance, the distinctive satellite repeat units of centromeres have\nbeen recognized across a variety of species, including the presence of 155-bp CentO\nsatellite repeat sequence in rice, the abundance of 178-bp CEN180 satellite repeat\nsequence in Arabidopsis, and the enrichment of 156-bp CentC satellite repeat sequence\nin maize (Cheng et al., 2002; Naish et al., 2021; Wolfgruber et al., 2009), we also\nidentified a highly similar 111 bp satellite repeat sequence in all three Physaria\ngenomes (Fig. 4b). Based on this characteristic, we identified the centromeric regions\nof chromosomes in the genomes of P. lindheimeri, P. pallida, and P. fendleri (Fig. 4a\nand Fig. S9). In P. lindheimeri genome, centromeres exhibit a length range of 2.18 Mb\nto 3.14 Mb, with an average length of 2.62 Mb, and each centromere contains an\naverage of 4,308 satellite repeat sequences. In P. pallida genome, centromeres span\nfrom 1.52 Mb to 4.96 Mb, with an average length of 3.55 Mb, and each centromere\ncontains an average of 4,664 satellite repeat sequences. Similarly, in P. fendleri genome,\ncentromere lengths range from 1.48 Mb to 3.26 Mb, with an average length of 2.36 Mb,\nand each centromere contains an average of 5,439 satellite repeat sequences. (Table\nS11). Furthermore, we identified satellite repeat sequence with a length of 111 bp in all\nthree genomes. It is noteworthy that the satellite repeat units among the three Physaria\nspecies are highly similar. The identity between the satellite repeat sequences of P.\nlindheimeri and P. pallida reaches 94%, while the average sequence identity of\ncentromeric satellite sequences within chromosomes is as high as 96%. This result\nsuggests the significant conservation of satellite repeat units in the centromeric regions\nof these plant genomes.\nThe centromeric region is characterized not only by the abundance of high copy\nnumber tandem repeats but also by a significant presence of specific types of\nretrotransposons. Notably, the centromeres of rice are abundant in Ty3/gypsy type\nretrotransposons, while those of Arabidopsis are rich in ATHILA long terminal repeat\nretrotransposons. Similarly, in maize, the centromeres are abundance of CRM and non-\nCRM Gypsy type retrotransposons (Chen et al., 2023; Miller et al., 1998; Naish et al.,\n2021). Within species of the Physaria genus, we also identified an enrichment of two\nclasses of retrotransposons, Gypsy/CRM and Copia/Ale, in the centromeric regions\n(Fig. 4a and Fig. S9). Specifically, the copy numbers of Gypsy/CRM type\nretrotransposons range from 387 to 577 on each chromosome, while the copy numbers\nof Copia/Ale type retrotransposons range from 147 to 297. Despite P. fendleri having\nthe largest genome among three species, we observed the lowest copy numbers of\nGypsy/CRM and Copia/Ale retrotransposons in its centromeric regions. Furthermore,\nwe also found the proportion of LTR/Gypsy and LTR/Copia elements in telomeric\nversus non-telomeric regions showed significant differences (Response Figure 4).\nResponse Fig 4. The proportion of several major transposons in the genome within\ncentromeric and non-centromeric regions.\nMinor concerns:\n1. Iine 50 author say” Some Physaria species exhibit valuable traits that are conducive\nto domestication”. It is not clear if is there any physaria species have been cultivated\nas crops.\n[Response]: Thank you for pointing out the problem, and we have removed the claim\nthat “Physaria species have been cultivated as crops.” The revised sentence is “Some\nPhysaria species exhibit valuable traits, including non-dehiscent seeds, neutral\nphotoperiod response, upright growth habit, and high seed oil yield (Fig. 1a) (Dierig et\nal., 2011)”.\n2. Author should introduce the usage of HFA from Physaria species to industry.\n[Response]: Thank you for your valuable suggestion. We have introduced the usage of\nHFA from Physaria species to industry in the manuscript. The details are as follows:\nLesquerolic acid (14-hydroxyeicos-cis-11-enoic acid; C20:1-OH), is found at\nabundance (50-85%) in Physaria seeds (Dierig et al., 2011). The oil is a colorless to\npale-yellow liquid that exhibits excellent stability and biodegradability, and it may\nprovide an alternative source of HFA for making lubricants, motor oils, desiccants,\nnylon, plastics, cosmetics, pharmaceuticals and polishes (Badami and Patil, 1980;\nBarclay et al., 1962; Carlson et al., 1990; Ogunniyi, 2006; Wu and Hojilla-Evangelista,\n2005; Dierig et al., 2011). We also added in depth discussion about biotech engineering\nof HFA between line 508 and 528.\n3. Author should introduce the physical and chemical properties of HFA from Physaria.\n[Response]: Thank you for your helpful suggestion. We have introduced the physical\nand chemical properties of HFA from Physaria. The details are as follows: Lesquerolic\nacid (14-hydroxyeicos-cis-11-enoic acid; C20:1-OH), is found at abundance (50-85%)\nin Physaria seeds (Dierig et al., 2011). The oil is a colorless to pale-yellow liquid that\nexhibits excellent stability and biodegradability, and it may provide an alternative\nsource of HFA for making lubricants, motor oils, desiccants, nylon, plastics, cosmetics,\npharmaceuticals and polishes (Badami and Patil, 1980; Barclay et al., 1962; Carlson et\nal., 1990; Ogunniyi, 2006; Wu and Hojilla-Evangelista, 2005; Dierig et al., 2011).\n4. The author highlighted the unique, twice-occurring whole genome duplication events\nin the Physaria genus using Ks plots and genome synteny between P. lindheimeri and\nA. thaliana. However, the analysis still has several issues that need to be addressed to\nprovide more robust evidence for the conclusions: A 4DTv plot may provide more\ndetailed resolution regarding the WGD events.\n[Response]: Thanks for your valuable advice. The 4DTv plots do provide a more\ndetailed resolution about the WGD events. Based on your suggestion, we performed\n4DTv analysis for Physaria genus and A. thaliana, and the results were consistent with\nthose of the Ks plots. Our findings indicate that after diverging from A. thaliana,\nPhysaria underwent a species-specific WGD. However, due to the relatively recent\ntiming of this event, the 4DTv plot displays only a single peak near the x-axis 0 point.\nConsequently, it is not possible to distinguish the two WGD events in Physaria from\nthe 4DTv plot (Response Figure 5).\nResponse Fig 5. The density distribution of 4DTv values for homologous gene pairs\nwithin Physaria species, within A. thaliana, and between P. lindheimeri and A. thaliana.\n5. Figures 2c, 2d, and 2e should use the same Ks color bar and have P. lindheimeri on\nthe Y-axis. Alternatively, creating a Sankey plot including all related species could make\nthe relationships more evident.\n[Response]: Thanks for your kind suggestion. We have used the same Ks color bar for\nFigures 2c, 2d and 2e and placed P. lindheimeri on the Y-axis. The modified graph is\nas follows:\nFigure 2. Phylogenetic and whole-genome duplication events analysis of Physaria\nspecies. (a) The phylogenetic tree of Physaria species and 15 other species, alongside\nthe number of gene family expansion (green numbers) and contraction (orange\nnumbers). The number at the root of the tree (13,676) represents the total number of\ngene families predicted in the most recent common ancestor (MRCA). (b) The density\ndistribution of Ks values for homologous gene pairs within Physaria species, within A.\nthaliana, and between P. lindheimeri and A. thaliana. (c) Syntenic comparative analysis\nbetween A. thaliana and P. lindheimeri. The red dotted frame marks some syntenic\nblocks at ks<0.5, and the syntenic relationship of 1:4 can be clearly seen. Points of\ndifferent colors represent different Ks values of collinear gene pairs. (d) Syntenic blocks\nbetween P. lindheimeri and P. pallida, as well as P. lindheimeri and P. fendleri (e),\nshare a 1:1 syntenic relationship.\n6. The author did not clearly explain how new centromeres and telomeres formed during\nspecies differentiation, given the extensive chromosome breaks and fusions in the\nancient Physaria genus over such a short interval. Generally, new centromere loci have\nstructural or sequence preferences.\n[Response]: Thanks for your kind suggestion. In the original manuscript we did indeed\nprovide insufficient detail on the mechanism underlying the rapid formation of new\ncentromeres and telomeres. However, we identified seven-base telomeric repeats (5'-\nTTTAGGG-3') in all three genomes, which are telomere-specific sequences (Fig 1c).\nMoreover, we also identified satellite repeat sequence with a length of 111 bp in all\nthree genomes, which was confirmed in rice, Arabidopsis, and maize (Cheng et al.,\n2002; Naish et al., 2021; Wolfgruber et al., 2009). Based on this characteristic, we\nidentified the centromeric regions of chromosomes in the genomes of P. lindheimeri,\nP. pallida, and P. fendleri (Fig. 4a and Fig. S9). In P. lindheimeri genome, centromeres\nexhibit a length range of 2.18 Mb to 3.14 Mb, with an average length of 2.62 Mb, and\neach centromere contains an average of 4,308 satellite repeat sequences. In P. pallida\ngenome, centromeres span from 1.52 Mb to 4.96 Mb, with an average length of 3.55\nMb, and each centromere contains an average of 4,664 satellite repeat sequences.\nSimilarly, in P. fendleri genome, centromere lengths range from 1.48 Mb to 3.26 Mb,\nwith an average length of 2.36 Mb, and each centromere contains an average of 5,439\nsatellite repeat sequences. (Table S11). Furthermore, we identified satellite repeat\nsequence with a length of 111 bp in all three genomes. It is noteworthy that the satellite\nrepeat units among the three Physaria species are highly similar. The identity between\nthe satellite repeat sequences of P. lindheimeri and P. pallida reaches 94%, while the\naverage sequence identity of centromeric satellite sequences within chromosomes is as\nhigh as 96%. This result suggests the significant conservation of satellite repeat units\nin the centromeric regions of these plant genomes.\nWithin species of the Physaria genus, we identified an enrichment of two classes\nof retrotransposons, Gypsy/CRM and Copia/Ale, in the centromeric regions\n(Fig. 4a and Fig. S9). Specifically, the copy numbers of Gypsy/CRM type\nretrotransposons range from 387 to 577 on each chromosome, while the copy numbers\nof Copia/Ale type retrotransposons range from 147 to 297. Despite P. fendleri having\nthe largest genome among three species, we observed the lowest copy numbers of\nGypsy/CRM and Copia/Ale retrotransposons in its centromeric regions. Subsequently, we\nquantified the proportions of several transposons frequently occurring in the genome.\nWe found that transposons within the centromere region were predominantly composed of\nLTR/Gypsy (85%), followed by LTR/Copia (10%). In contrast, non-centromeric\nregions exhibited similar proportions of LTR/Gypsy (35%) and LTR/Copia (35%),\nindicating distinct transposon distributions between these regions (Response Fig 5).\nResponse Fig 4. The proportion of several major transposons in the genome within\ncentromeric and non-centromeric regions.\nReferences\nBadami, R.C. and Patil, K.B. (1980) Structure and occurrence of unusual fatty acids in minor\nseed oils. Progress in Lipid Research 19, 119-153.\nBarclay, A.S., Gentry, H.S. and Jones, Q. (1962) The search for new industrial crops\nII:Lesquerella (Cruciferae) as a source of new oilseeds. Economic Botany 16, 95-100.\nCarlson, K.D., Chaudhry, A. and Bagby, M.O. (1990) Analysis of oil and meal fromLesquerella\nfendleri seed. Journal of the American Oil Chemists' Society 67, 438-442.\nChen, J., Wang, Z., Tan, K., Huang, W., Shi, J., Li, T., Hu, J., Wang, K., Wang, C., Xin, B.,\nZhao, H., Song, W., Hufford, M.B., Schnable, J.C., Jin, W. and Lai, J. (2023) A\ncomplete telomere-to-telomere assembly of the maize genome. Nature Genetics 55,\n1221-1231.\nCheng, Z., Dong, F., Langdon, T., Ouyang, S., Buell, C.R., Gu, M., Blattner, F.R. and Jiang, J.\n(2002) Functional rice centromeres are marked by a satellite repeat and a centromere-\nspecific retrotransposon. Plant Cell 14, 1691-1704.\nDierig, D.A., Wang, G., McCloskey, W.B., Thorp, K.R., Isbell, T.A., Ray, D.T. and Foster, M.A.\n(2011) Lesquerella: New crop development and commercialization in the U.S.\nIndustrial Crops and Products 34, 1381-1385.\nMiller, J.T., Dong, F., Jackson, S.A., Song, J. and Jiang, J. (1998) Retrotransposon-related DNA\nsequences in the centromeres of grass chromosomes. Genetics 150, 1615-1623.\nMoon, H., Smith, M.A. and Kunst, L. (2001) A condensing enzyme from the seeds of\nLesquerella fendleri that specifically elongates hydroxy fatty acids. Plant Physiol 127,\n1635-1643.\nNaish, M., Alonge, M., Wlodzimierz, P., Tock, A.J., Abramson, B.W., Schmucker, A.,\nMandakova, T., Jamge, B., Lambing, C., Kuo, P., Yelina, N., Hartwick, N., Colt, K.,\nSmith, L.M., Ton, J., Kakutani, T., Martienssen, R.A., Schneeberger, K., Lysak, M.A.,\nBerger, F., Bousios, A., Michael, T.P., Schatz, M.C. and Henderson, I.R. (2021) The\ngenetic and epigenetic landscape of the Arabidopsis centromeres. Science 374,\neabi7489.\nOgunniyi, D.S. (2006) Castor oil: a vital industrial raw material. Bioresource Technology 97,\n1086-1091.\nWolfgruber, T.K., Sharma, A., Schneider, K.L., Albert, P.S., Koo, D.H., Shi, J., Gao, Z., Han,\nF., Lee, H., Xu, R., Allison, J., Birchler, J.A., Jiang, J., Dawe, R.K. and Presting, G.G.\n(2009) Maize centromere structure and evolution: sequence analysis of centromeres 2\nand 5 reveals dynamic Loci shaped primarily by retrotransposons. PLoS Genet 5,\ne1000743.\nWu, Y.V. and Hojilla-Evangelista, M.P. (2005) Lesquerella fendleriprotein fractionation and\ncharacterization. Journal of the American Oil Chemists' Society 82, 53-56."}, {"round": 2, "reviewer_comments": {"reviewer_1": "suggest the revision to be published.", "reviewer_2": "The authors improved the manuscript in some aspects but the key novelty issue remains.", "reviewer_3": "The authors have a good revision on the manuscript according to my comments. Now the manuscript improved a lot. I have no more questions."}, "author_response": "Point-by-point response to REVIEWER’S COMMENTS\nReviewer #1:\nsuggest the revision to be published.\n[Response]: Thank you very much.\nReviewer #2:\nThe authors improved the manuscript in some aspects but the key novelty issue remains.\n[Response]: Thank you for your candid and valuable comment. We fully acknowledge\nthat although the manuscript has been revised in some aspects, the core novelty of our\nwork still needs to be further clarified and strengthened, and that this feedback is crucial\nfor us to better highlight the academic contribution of the study.\nReviewer #3:\nThe authors have a good revision on the manuscript according to my comments. Now\nthe manuscript improved a lot. I have no more questions.\n[Response]: Thank you very much for your positive evaluation and constructive\nsuggestion to publish the manuscript after revision. We highly appreciate your\nrecognition of the core content and academic value of our work, which has greatly\nencouraged us."}]

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