«) United States US 201200424081 «2 Patent Application Publication 10 Pub. No.: US 2012/0042408 Al Mercier et al. (S4)_ PLANTS PRODUCING 2N GAMETES OR APOMELOTIC GAMETES (76) Inventors: Raphael Mercier. Fontenay-lo-Fleury (FR); Isabelle Laurence Cromer, Clamart (FR) 21) Apple, 13/143,530 (22) PCT Fie San. 6, 2010 (86) PCTNo, PCTABIOOISS §371 00), (2),(4) Date: Sep. 16,2011 0) Foreign Application Priority Data Jan.7,2009 (EP) 092900109 (4) Pub. Date: Feb. 16, 2012 (1) Iat.Cl 01H 5/00 (2006.01), AOLIE 1/06 (2006.01), CBN 1582 (2006.01), (52) US.Cl 800/276; 800/278; 435/320.1: 00/298 on ABSTRACT Te iavention relates to plants wherein the protein OSDI. involved in the transition from meiosis [to meiosis I is inactive, These plants produce Second Division Restitution (SDR) 2n gametes. The invention further relates to plats ‘wherein the inaetivation of SSDI is combined with the inac- tivationofagene involved ia meiotic recombination inplants, and of a gene involved inthe monopolar orientation ofthe kinetochores during meiosis, These plants produce apome= fie gametes. These plants are useful in plant breedingPatent Application Publication Feb. 16, 2012 Sheet 1 of 7 US 2012/0042408 AI Wild type meiosis = osdt ~~ mies am Th 4 oA) i) 1)-Ga —Ga~-4) Wl EE i) i Atspo1t-1 2a wre ill ca Atspott-t/Atrec8 meiosis ae i ae EM me Mine (Atspot-1/Atrectlosdt) meiosis Figure 1Patent Application Publication Feb. 16, 2012 Sheet 2 of 7 Us 2012/0042408 AI Figure 2Patent Application Publication Feb. 16, 2012 Sheet 3 of 7 Us 2012/0042408 AI Figure 3Patent Application Publication Feb. 16, 2012 Sheet 4 of 7 Us 2012/0042408 AI Figure 4Patent Application Publication Feb. 16, 2012 Sheet 5 of 7 Us 2012/0042408 AI « é 2 Ca ri cr Figure 5Patent Application Publication Feb. 16, 2012 Sheet 6 of 7 Us 2012/0042408 AI Figure 6Patent Application Publication Feb. 16, 2012 Sheet 7 of 7 US 2012/0042408 AI Figure 7US 2012/0042408 AI PLANTS PRODUCING 2N GAMETES OR APOMELOTIC GAMETES [0001] The invention ceates to plants that produce 2n See- ‘ond Division Restitution (SDR) gametes, and to plants that produce apomeiotc gametes, and to their use in plant breed ing. [0002] 2n gametes (also known as diplogametes) are ‘aametes having the somatic chromosome number rather than the gametophytic chromosome number. They have been showin tobe useful forthe genetic improvement of several crops (For review, ef. fr instance RAMANNA & JACOB- SEN, Fuphyica 133, 3-18, 2008). In particular the prod tionofdiplogametesallow emsses between plants of iferent ploidy level, Jor instance crosses between tetraploid crop plans and thir diploid wild relatives, in oder to use their ‘genetic diversity in plant breeding programs. [0003] ‘The formation of 2n gametes results from anomalies uring meiosis (or review ef. VEILLEUX, Plant Breeding Reviews 3, 252-288, 1985, r BRETAGNOLLE & THOMP- ‘SON, New Phytologist 129, 1-22, 1995), [0004] In normal meiosis, chromosomes first duplicate, resulting in pairs of sister chromatids. This round of repli ‘ion is followed by two rounds of division, known as meiosis, [and meiosis I. During meiosis homologous chromosomes recombine and are separated into two cells, each of them comprising one entre haploid content of chromosomes. In meiosis IT the two cells esuting from meiosis I farther vide, andthe sister chromatids segregate. Thesporesresult- ‘ng from this division are thus haploid and carry recombined szeneti information, [005] |The abnormalities leading to 2n gametes formation include inparicular abnormal cytokinesis, the skip ofthe first ‘or second meiotic division, or abnormal spindle geometry (ar eview ef. VEILLEUX, Plant Breeding Reviews 3, 252- 288, 1985, or BRETAGNOLLE & THOMPSON, New Phy {ologist 129, 1-22, 1995). These abnormalities lead to dfer- cnt classes of unredueed gametes, Foriasiance, failure of the fist meiotic division results in Fist Division Resttation (FDR) gametes, while failure ofthe second meiotic division results in Second Division Restitution (SDR) gametes. 006] Although aumerous mutants able to produce 2n ‘gametes have been reported in various plant species only one ‘gene implicated in the fommation of 2a pollen bas been iden- ‘ied and characterized atthe molecular evel until now. The inactivation ofthis pee, designated AtPSI (Tor Arabidopsis ‘thaliana parallel spindles), generates diploid male spores, avin rise to viable diploid pollea grains and to spontaneous ‘rploid plants in the progeny. This gene and its use for pro ducing 2a pollen are disclosed in European Patent application (08490672, filed on Jul 8, 2008, and in the publication of DERFURTH et al (PLoS Genet 2008 November; 4(1): 1000274. Epub 2008 Now. 28). [0007] Theinventors have now identified in the model plant Arabidopsis thaliana, another gene implicated in the Forma tion of 2n gametes in plants, The inventors have found tht ‘inactivation ofthis gene results ia the skipping ofthe second meiotic division. This generates diploid male and female spores, giving rise to viable diploid male and female gamete, Which are SDR gametes. This gene will be hereinate ds nated OSDI, for omission of second division. The sequence ofthe OSDI gene of Arabidopsis thaliana is available in the TTAIR database under the accession number AigS7860, orin Feb. 16, 2012 the GenBank database under the accession number [NM_1IS648. This gene encodes a protein of 243 aa (Cen unk NP_191345), whose sequence is also represented in the enclosed soquence listing as SEQ ID NO: I. [0008] The OSDI gene of rabidepsis thaliana hes been previously depicted as “UVI4-Like” gene (UVIEL), in a publication of HASE etal. (Plant J 46.317-26, 2006), whieh escrbes its paralogue, named UVI4, According to HASE et al, UVIG aets a a suppressor of endo-reduplication and is rcessary for maintaining the mitotie stte whereas OSDI (UVL4L) does nat appeae to be requite for his proces. In ‘contest as shown herein, OSDI appears necessary fo allow- ing the transition fom meiosis ho meiosis I 10009] The inventors have also identified in vice (Oryza sativa) anortbologafthe OSD1 gene of Arabidopsis thaliana, ‘Thesequenceo!the OSD1 gene of Oryza sativaisavailable in the OryGenes or TAIR databases unde the scoession number (0302g37850. It encodes a prtein of 234 a, whose sequence {represented inthe enclosed sequence listing as SEQIDNO: 35. The OSDI proteins of Arabidopsis thaliana and Oryza sativa have 23.6% identity and 35% similarity over the whole eng oftheir sequences [0010] ‘The invention thus provides a method for obtaining plant producing Second Division Restitution 2n gametes, ‘wherein said method comprises the ikibition in aid plant of 4 protein hereinafter designated «s OSD] protein, wherein said protein has atleast 20%, and by order of increasing preference, at least 25,30, 35, 40, 45, $0, $5, 60, 65, 70,75, 80, 85, 90, 95 or 98% sequence idenity, or af least 29%, and by order of increasing preference, at leas 35, 40, 45,50, 55, 60,65, 0,75, 80, 85, 90,95 oF 98% sequence similarity with the AIOSD1 protein of SEQ ID NO: 1 or with the OSOSD1 protein of SEQ ID NO: 35. [0011] Unless otherwise specified, the protein sequence ‘dent and similarity values provided herein are calculated cover the whole length of the Sequences, using the BLASTP. program under defult parameters, or the Needleman-Wan- sch global aligameat algorithm (EMBOSS pairwise align ‘ment Needle tool under default parameters). Similarity eal- culations are performed) using the scoring matrix BLOSUM®2, [0012] The SDR 2n gametes produced according tothe invention are useful in all the usual applications of 2n gametes, for instance for producing polypoids plants, or to allow crosses between plans of differeat ploidy level. They ‘can also be usefilia methods of genetic mapping, fr instance the method of “Reverse prozeny mapping” disclosed in US. Pateat Application 20080057583. 10013] The inventors have furuer found that by combining the inactivation of OSDI., with the insetivation of two other tenes, one (SPOI-1} which encodes a protein necessary for ficient meiotic recombination in plats, and whose ini tion eliminates recombination and pairing (GRELON etal, Embo4, 20, 589-600, 2001), andanother (REC8, At2g47980) which encodes a protein necessary forthe monopolar aren tation ofthe kinetochores during meiosis (CHELYSHEVA et al, J Coll Sei, 118, 4621-32, 2008), and whose inhibition ‘modifies chromatid sepregation, resulted in @ genotype in ‘which meiosis is totlly replaced by mitosis without affecting stibsoquent sexual processes. This genotype willbe called horeilter MiMe for “mitosis instead of meiosis, This replacement of meiosis by mitosis results in apomeioticUS 2012/0042408 AI gametes, retaining all the paren's genetic information (BICKNELL & KOLTUNOM, Plant Cel 16 Sop S22 45,2008) [W014] FIG. 1 provides a schematic comparison been the mecbannms of ites, nomal mon, meiosis in he ond mat, meiosis ina mat fcking SPOLT-1asivity (AxpolL-1y, meiosis in double mutant lacking. both ‘SPOLI-1 and RECS activity (Atspol 1-1/Atrec8), and meio- sis inthe MiMe mutant, [0015] | During mitosis in diploid cells, chromosomes rep- Tate and ser chromatids segregate to generate dager call tht sre ip and genetical entice Yo the inal cell, During normal nicoss, two rounds of chromosome Searegition ella single round of eliation, At dvson on, homologous chromosomes recombine nd are separ Meiosis emore similar to mitosis resin ns ist bution of sister cromatis, The obtains! spores are thus plc and cary recombined. gens infomation In the cosa nrutant (this study) meiosis II is skipped giving rise to. diploid spores and SDR gametes wih eeombined genetic infomation (W016). The A‘poll-1 mutem vodeoes an unbalanced fit dvson lowed bys ssond dvnon lang oonbal- anced spores and sterility [W017] The Apoll-1/AtwcS double mutant wdexgoes titot-ike division isteadof anormal stmeaicvson, followed by an unbalanced second vison lang ounbal- anced sors and sty [W018] Inthe taple. os/Aspol1-1Atec® mutant (Mie) he presence fhe Apo snd tes matations Jens foams fit meiotic dvvion and the presence of the oxdl tation prevents the second mai dvsion from occuring Ths inci i voplaed by a mittee dixon, The obtained spores and pimetss ate genetically dential tthe inal el [WI9] The spomeiote gametes produced by the MiMe ‘nt can be hn These way asthe SDR 2n games, for prodcing polyploid pln o for crossing plants of diferent ploidy level. Tey are also of intrest or the pro duction of sonst plans plants which rable to frm seods fromthe maternal es ofthe ovale esting in progeny tht are genetic clones of the maternal parent ‘Athogh it exist in over 400 species of angiosperms, very few emp pies ar apomicic and atenplsto nto this tr by crossing be fle (SAVIDAN, The Floweting of ‘Agomis: From Mechanisms o Genetic Engineering 201; SPILLANE etal, Sexual Plant Reproduction, 1, 2001, [0020] A further object of the present invention is thus a ‘method fr obtaining plant prodcing pomeotc gametes, Ahern sid method comprises the inion insaid plant of the following pons {0021} a) an OSD1 protein as defined above; [0022] b) @ protein involved in initiation of meiotic recombinstion in plants, sid protein being selected among [0023] i)a prin henner designed as SPOUI-L proein,whecin si rtein hast es an by ‘ner ofincensing preference, t es 45,50, 55,60, 6,70, 75,80, 85, 90,95 or 8% sequence enti oF atlas and by onder afinereing referee, st Teast, 65,7, 95, 0, 85,90, 95 of 98% sequence silat with thSPOI 1 proteincfSEQIDNO:2. {0024} iija protein hereinafter designated as SPO1-2 (i, wherein sid etn has eat 0% and by Feb. 16, 2012 ‘onder of increasing preference, atlas 45, 50,55, 0, 65,70, 75,80, 85,90, 95 or NY sequence identity, ot atleast 6%, and by order finereasing preference at least, 6S, 70, 75, 80, 85, 90,95 or 98% sequence similar with theSPOI 1-2 protein SEQ IDNO: 3, 10025] i) 2 proein hereinafter designated as PRD protein, wherein sid protein hs at least 25%, and by ‘nde of increasing preference, east 30, 35,40 45, 50,55, 60,65, 70.75, 80,85, 9,950 98% sequence identi oat least 35%, and by order of ineresing preference at as, 4, 45,50, 55, 60, 65,70, 75,80, 85, 0,98 o 98% sequence similarity with the PRDT protein of SEQID NO: & {0026} iv)» protein hereinafter designated as PAIR protein wherein sid poten has at east 3%, and by ‘onder of increasing preference, atleast 38, 40,45, 50, $5, 60,65, 70, 75,80, 85, 90,95 or 98% sequence ident oat least 40%, and by oder of ineresing preference a eas, 4, $0, 55, 60, 65, 70 75,80, 85, 50, 95 oF 98% sequence similarity with the PAIRT protein af SEQID NO: 5; 10027] c) a protein heriniter designated as Rec pro- ‘ein, wherein sud protein st east 40%, and by oder of increasing preference, at last 45,50, $5, 60, 65.70, 75,80, 85, 90, 98 oF SBM sequence ident, rat east 45%, and by one of increasing preference, lat 50, $5, 60, 68, 10, 75, 80, 85, 90, 95 or 98% sequence Similarity wi the Re protein of SEQ ID NO: 6 {0028} SEQ ID NO: 2 represents the saguence of the SPOI-1 proein of Arabidopsis thaliana. This sequence is ako available in the Swissprotdataase under the accession numer QUMAA2, 10029] SEQ 1D NO: 3 represents the sequence of the SPOIL? protein of Arabidopsis thaliana. This sequence is ko salable in the SwissProt database under the accession umber QOMAL {0030} SEQID NO: 4 represents the sequence ofthe PRD pein of trabidepsis thaliana, This sequence is als availe able in the GenBank database under the accession number ‘ABQI2682. [0031] SEQIDNO: rpresentsthe sequence ofthe PAIR protein of Arabidopsis thaliana. This sequence i als avai able in the GenBank database under the accession number NP_I71675. [0032] SEQ ID NO: 6 repeesents the sequence of the Rec’ protein of traidopsis thaliana, This sequence is also avaie able in the GenBank database under the accession number NP_i96168 {0033} The SPO11-1, SPOIT-2, PRDI, PAIR, and Recs cits are conserved in higher plants, monocotydns as ‘well as dicoyledons. By way of non-fiitatve examples of ctbologs of SPOI-1, SPOL-2, PRDI, PAIR and ResS pics of Arabidopsis thaliona in monocotyledonous plants, one can cite the Ora sativa SPOLI-I, SPOI2, RDI, PAIRI, and Rec peteins. The sequence ofthe Ore saliva SPOIL protein avaiable in GenBusk unde the accession number AAPOR363: the sequence ofthe Sativa SPOL-2 protein is avaiable in GenBank unde the accession number NP_OOIOSIOZ7: the sequence of the (Ora sativa PRD\ pricinis availabe in GenBank under the accession number EAZ30811 the sequence of the Ore Stiva PAIR] prtcin is salable ia SwissProt under theUS 2012/0042408 AI accession number Q7SRY2; the sequence ofthe Oryza sriva Ree® protein i available in GenBank under the accession number AAQTSO8S [0034] The inhibition of the sbove mentioned OSDI, SPOII-, SPOII-2, PRDI, PAIR, or Ree proteins can be tained either by abolishing, blocking, or decreasing thei fame, or advantageously, by preventing or downsezula- {ng the expression ofthe coresponding genes. {0035} By way ofexampl, inhibition of said prosincanbe obtained by mutagenesis ofthe coresponding gene or ofits promoter, and section of the mutants having partially or totaly los the activity of id proen. Fr instanee, 3 mut tion within the coding sequence can indice depending onthe satu of the mutton, the expression ofan native prot, cr of & potin with impoirel atv in the same way, a ‘tation within the promoter sequence can noe a lack of expression of ai protein, odacease thereof [0036] _ Mutagenesis ean be perfomes for instance by tae ced deletion ofthe coding sequence or ofthe promoter of te gone encoding sid pron o ofa potion thereof, or by targeted insertion ofan exogenous Sequence within said cod- ing sequence or said promoter. It ca also be performed by inducing random mutation, for instance through EMS ‘mutggeness or rand insertional mutagenesis followed by Screening ofthe mutants within the desired gene Metbods for high throughput mutagenesis and screning are avaiable in teat By way of example, one can mention TILLING (Tar ating Induced Local Lesions IN Genomes, described by McCallum ea, 200 [0037] Among the mutations within the OSDI gene those resulting in the ability to produce SDR 2n gametes can be ‘dented onthe bss of the phnorypie charters of he plans which are homozygous fr this mutation: these plants an formatleast preferably at ast 10% mare referbly at east 20%, stl more prefebly atleast S0%, and up to 100% of dyads asa pret of meoss {0038} Among themmtatons withina gen encoding po- ‘ein avolved in nitation of meote recombination in plans, such the SPO +1 gene or the SPOL-2, PRD, or PAIR gene those sei for obianing a plant producing apomiivic gametes can be identified on the basis of the phenotypic characteristics ofthe plants which are homozygous foe this ‘lato, inpaiculr the presence of unvaents instead oF ‘bivalents at meiosis [and te sterity othe plant {0039} Among the mutants hviag @ mutation within the RECS gene, thse useful for obtaining plant producing apomeioic gametes can be idenifed on the bass of the ‘henotpie characteristics ofthe plants which are homo7y- ou for this mutation, in particular chromosome Iragmenta- tion at meiosis, and sterityof the plant {0040] According aprefered embodiment ote method othe invention fr obtaining a plant able to produce SDR 2 zanees, sid method comprise: {0041} 2) providing a plant having & mutton within an allele ofthe OSDI gene resting inthe inhibition of he protein encodes by this allele said plan beng heterozygous for ths mutation: [0042] b) self etlizing said plant of step a) ia order to bina plant homozygous for si mottion [0043] According to prefered embodiment oftte method ofthe invention for obtaining a plant able to proloe pom oe gametes, sid method comprise: Feb. 16, 2012 {0044} 2) providing 2 plant having @ mutton within an allele of the OSDI gene resulting inthe inhibition of the protein encoded by this allel, sid plant being heterozygous for this tation; [0045] 5) providing plant having « mutton within aa allele ofa gene selected among the SPOLI-1, SPOI-2 PRDI, orPAIR gene resulting the nbibition ofthe prot encod by sai ale, sit plant being heterozygous fr this mutation: (0046) c) providing a plant heving a mutation within an allele of the RECS gene resting in the inhibition of the prtcin encoded by sid alee said plant beng heterozygous forthis mutation; {0047]e)erssngte plants of steps) b)ande) in onerto obtana plant having a mutation within anallele ofthe OSD1 gene, anuttion within alleleofa gene seleted among the SPOII-I, $PO11-2, PRDI, or PAIR] gene nd» mation ‘itn an allele ofthe RECS gen, si plat being hsterozy- ous foreach mutation; {0048} 1)selfferilizingtheplntofsepe)inondertoobtain 2 plant homozygous forthe mutation within the OSD1 gene, forthe mutation within the gene selected amoag the SPOLI- 1, SPO1-2, PRDI, of PAIRI gen, and for tbe mutation ‘within the RECS gene. {0049} _Altematvely, te inhibition of the target protein is obtained by silencing ofthe conesponding gene, Methods or gene silencing in plants are knows in themselves inthe ar Forinstance, one can mention by antisense inhibition or co- suppression, as described way ofexample in U.P. Nos, 510,068 and 5,288,323. tisalso possible to use ribozymes ‘areting the mRNA of said poten. {0050} Prefered methods ae those wherein gene silencing Js induced by means of RNA interference (RNA), sing a silenging RNA tasting the gone to be silenced. Various ‘methods an DNA const or dfvery of silencing RNAS ar available i the at [0051] _A*slencng RNA"isherein defined esasmall RNA that can sence age gene in a soquence-specitic manner bye paring to complementary mRNA molecules Sle {ng RNAs inlude in paricular small interfering RNAS (iR- [NA and mieroRNAS (miRNA). {0052} Intay, DNA construe for delivering silencing RNA ina plant included a frgment of 300 bp or more (aen- erally 300800 bp alinough shorter soquenes my sometine Jnkce efficient silencing of the eDNA of the target gene, under transcriptional conto of @ promoter active in sid plant. Curent, the more widely use silencing RNA eon- Strvets are those tht ean proce fipin RNA. (BpRNA) transcrip. Intheseconsirts the frgment ofthe target gene ‘sinversely repeated, with generally a sacer region between the epeat (for review ef WATSON ct al, 2008) One can aso use atl microRNAs (amiRNAS) directed against te gene to be silenced (Jor review about the design and aplication of sieacing RNAs including nparcularamik- 'NAs,inplantsef-forinstaee OSSOWSKI etal (Plant, 83, 674.90, 2008, {00S3] The present invention provides tools for silencing one or mare tet peoe() sleted among OSDI, SPO! +L SPOII-2, PRDI, PAIRI, and RECS, including i particular expression castes for bpRNA or miRNA targeting said ase).US 2012/0042408 AI {W954] An exresson caste ofthe mention my com ise for instance [W155] promt anton ns pst cel: [0056] on ortnore DNA constracts)o£200t 1000p. prefebly f 3001 900bp cach comprising fragment Of a eDNA of a target gene selected among OSDI, SPOI-1, SPO12, PRD, PAIR and RECR,o ofits onpemenary or having at eax 98% identi, ad by onde of neresing preference a st 9%, 97%, 98%, 0F 99% identity wih sai agent aid DNA consict (s) being placed under transcriptional control of said promoter {0987} According 19 prefered embodiment ofthe inven- tion, an expression cnet for pRNA comprises [OSE] a promote fictional ina plant cel {WiS9] one o more fxn DNS coasts) capable, when tanseribed of forming hip RNA targeting» fone selected among OSDI, SPOIL, SPOIT2, PRDI,PAIRI, and RECS: {0060] ssiGDNA construc) being plod under enserp- tinal contol of aid promoter {Wv61] Gencraly sid hin DNA constr comprises) Ast DNA sequence of 200 1000p, perch 03000 Seb. constingata trizmentofaeDNA ofthe tet gene, or having a ent 95% ten, and By oner of increasing preference ates 96%, 97% 9% or 9M entity with sid fragment i second DNA sore that the complem tary of si fit DNA, sid ir and secon segoenes eng in opposite onenttons andi) a space sequence Separating ‘sidfistand second sequen, ih thatthe fistand second DDNA senses re capable when ransrbe, of forming singe doublestanded RNA moll. The pacer canbe rund ment of DNA, However, prefembly. one wil we an intron which is spliceable by the target plant cell, tssizeis erally 00 to 2000 melts in ng, [0062] According to anther refered embodiment ofthe invention an expression case foranamiRNA conri {63} a promoter finetonl in plant el [0068] one or more DNA constet(s) capable, when wransriba of forming an amiRNA targeting a gene Selected among OSDI, SPOIT-1, SPOTL2, PRDI, PAIRI, and RECS; {0068} said DNA conseuc) being placatuntertansrp fiona contol of id promoter (W066) Advanigsoinly, an expression cassete of the invention comprises DNA consi ating the OSDI gene. According to paricserly prefered embodiment i omprisesa DNA construct artigo OSD1 gone, aDNA onstucttagtinga gee selected among SPOL-1, SP011- 2: RDI, and PAIR anda DNA constr trsting RECS. [W067] large oie of promoters suitable for expeesion of etemlogos genes in plans is avilable inthe at {0068) They canbe obtained fr instance rom plants pant vinses, of bacteria such ss Agrobacterium. They include onsttsive promot, is. promoter which re asin thos sis and cells in under most enviromental cont tions, aswell a Gatuespece or cellapectc promters which are active only or mainly in certain tissues or certain Cell ype, and indcble promoters tht ate activated by rlysitl ce chemical simi, soc a those resin fen emit nto [0969] Non-imtaive examples of eonsiatve promoters that are commonly used in plant cells are the catlfower Feb. 16, 2012 ‘mosaic virus (CaMV) 388 promoter, the Nos promoter, the rbisco promoter, the Cassaya vein Mosaic Virus (CsVMV) promoter [0070] | Onuan or tissue specific promotes that ean be used ‘nthe present iveation include in particular promoters able to confer meosis-assoviated expression, such as the DMC promoter (KLIMYUK & JONES, Plat J, 11, 1-14, 1997), ‘one can also use aay of the endogenous promoters of the ‘genes OSDI, SPOII-1, SPOI-2, PRDL, PATRI, or RECS. [0071] The DNA constructs ofthe invention generally also include a transcriptional terminator (for instance the 358 transcriptional terminator, or the nopaline synthase (Nos) ‘wanserptionalteratinato) 10072] The invention also includes rocombinant vostors ‘containing a chimeric DNA construct of the iaventio, Clas sicaly, said recombinant vectors also inchude one ar more ‘marker genes, which allow fr selection of transformed host 10073] The selection of suitable vectors and the methods for inserting DNA constrets therein are well known to pet= sons of onlinary skil in the art. The choice of the vector depends onthe intended hos and on te intended method of transfomation of sid host. variety of methods for genotc ‘wansfomiation of plant cells or plants are available inthe art. for many plant species, dcotyledons or monocotyledons. By ‘way of non-imitative examples, one can mention virus medi ated transformation, transformation by microinjection, by electroporation, miroprojetile mediated transformation, Agrobacterium mesisted transformation, and the like [0074] Theiaveaton also provides ast cell comprising 2 recombinant DNA constrict of the vention. Seid host cell ean bea prokaryotic cel, or nstance an Agrobacterium cell, ‘ora eukaryotic eel, for instancea plant cell genetically tans forme by @ DNA construe ofthe invention. The construct ‘may be transiently expressed it can also be incorporated in a stable extrachromosomal replicon, or ntegated inthe chro= [0075] According to preferred embodiment ofthe method ‘ofthe invention for providing plant able to produce SDR 2a szametes, ssid plant is a ansgenie plant, and said method ‘comprises 10076] 9) wansforming atleast one plat cel with a vector containing a DNA constuet of the invention targeting the OSDI gene; 0077). ) cultivating said transformed plant cell in onder regenerate a plant having i is genome a transgene contin ing said DNA coastroct 10078] Acoording to prefered embodimentof the method ‘ofthe invention fr obtaining a plant abe to produce apome- ‘oft gametes ssid plat sa transgenic plant, and sid method. comprises: [0079] 2) wansforming at leat one plat cell with a vector containing a DNA construct ofthe invention targeting the (OSD1 gene, a vector containing a DNA construct of the ‘vention targeting a gene selected among SPOL-1, SPOL- 2, PRDI, and PAIRI, and a vector containing a DNA eon- struct of the invention targeting the RECS gene; [080] bj culvating suid transformed plant cell ia onlerto regenerate plant having inits genome transgenes containing sid DNA constructs, [081] According to another preferred emabodimeat othe ‘method ofthe invention for obtaining a plant able to produce apomeiotc gametes suid plant is transgenic plant, and aid method comprises:US 2012/0042408 AI [0082] _) transforming at least one plant cell wi a vector containing a DNA construct ofthe invention targeting the (OSDI gene, « DNA construct of the invention targeting a _geneselected among SPO1I-1, SPON-2, PRDI. and PATRI, and a vector containing « DNA construct of the invention targeting the RECS gene; [0083] -) cultivating ssid transformed plant cell in onder regenerate a plant having i ts genome a transgene contin ing said DNA constructs [0084] _The invention also encompasses plants abe to pro- dduce SDR 2n gametes or apomeiotc gametes, obtainable by the methods ofthe invention, [085] This includes in particular plans comprising: {0086 a mutation within the OSDI gene, wherein the SDI protein is inhibited as a result ofthis mutation, and {0087} "a mutation within gene selected among SPOL- 1,SPO1I-2, PRDI,orPAIRI gene, wherein the SPO 1, SPOII-2, PRDI, of PAIRI protein encoded by said ene is inhibited asa result ofthis mutation: and [0088] "2 mutation within the RECS gene, wherein the Rex8 procin is inhibited asa result ofthis mutation. [0089] -Thisalso includes plants sentially transformed by ‘one or more DNA constructs) of the invention. Preferably, suid plants are transgenic plants, wherein sad coastrict is contained in a transgene integrated in the plant genome, so that its passed oato successive plant generations 0090] -Theexpression ofa chimeric DNA construct taet- ing the OSDI gene, resulting in a down regulation of the ‘OSDI protein, provides to said transgene plat the ability to produce 2n SDR gametes. The co-expression ofa chimeric DNA construct targeting the OSDI gene, a chimeric DNA. ‘construct targeting a gene selected among SPOL-1, SPON- 2,PRDI and PAIR anda chimeric DNA constrct targeting the RECS gone, resis in a dawn rgulaton ofthe proteins encoded by these thee genes and provides o said transgenic plant the ability to produce apomeiotc gametes [0091] ‘The invention aso encompasses a method for pro- ducigg SDR 2n gametes, wherein said method comprises cultivating plan’ obtaisableby method of the iaveationsnd recovering the gametes produced by said plant. Preferably suid gametes comprises at least 10%, more preferably at east 20%, and by order of increasing preference, atleast 30%, 4076, 0%, 6%, 70%, 80%, or 9O% of viable 2n gametes. Feb. 16, 2012 {W092} The vention slo encompasses mstod for ro ducing spomeoi yantes, wherein sid tod comprises calvatingaplntobsnabiebyamethodoftheimentionand ‘recovering the gametes produced by said plant. Preferably: ‘sid gates comprises at es 1%, more prefer test 20% and by order of inereasing preference, at est 3%, 406,540 4%, 70%, 80%, oF of vite aposigc ames [0093] The present invention aples oa broad range of ‘monocot or dicted plants of pronomal interest. By ‘ay ofaonlimtaveexamplesonecanmestion ptt, He, ‘whet, maz, tomato, cucimbes,llt, sar cane, sweet Poiao, manic, clover, saybean,ry-grs, banana, melo, ‘ateronciton of epament plants suchas os, is, tus and nacssus [0994] Frepning and oter oct and advantages ofthe invention wil become more sparet from the following detailed desernion and accompanying drovings. Isobe ndestooghorsever tht his foregoing detailed escrintions exemplary ony an isnot restive ofthe invention EXAMPLES Experimental Procedures lant Material and Growth Conditions [0095] trabidopsi plats were cultivated ws desribed ia VIGNARD et al, (PLoS Genet, 3, 1894-906, 2007). For germination assays and cytometry experiments Arabidopsis ‘Were cultivated in vitro on Arabidopsis medium (ESTELLE. & SOMERVILLE, Mol. Gen. Genet, 206, 200-06, 1987) a 21° C. with a 16 h dayi® b night photoperiod and 70% haygromety. Genetic Analysis. 096] Plants were genotyped by PCR (80 eyeles of 30s at 94° C., 305 at 6° C. and I min at 72°C.) using two primer pairs, For each genotype the primer par is shown in Table 1 ‘ad the primer pair specifi tothe insertion is shown in Table n TABLE 1 rinses tor #ild-type allele ‘POCIS307U (S'CGTCACTCTOCCCARGAAAG 3°) (SBQ ID 20: 7) eta6207L (S'OGcRAAGERACCCTECTAEG 3°) (59 1D MO 6) orzudetu (s'ccaatarrerTorsxctes 3°) (680 1D mo: 9) ‘orzideiL (S'ccxaarteeTaarmeacere 3°) (859 19 Wo: 10] seopent-1-3 ARTCGGTORGTCAGGTTTEAG 3°} (SED ID HO: a2) aetea7aL, (5! cenTaaRTEAMAGCGRTEAG 3°} (69 1D MO. 221, 1tenc0270 (S'cTeREATECACGGTECTECC 3°) (880 1D WO: 13) enco27L (S'coaaaMMARGaGRARGGTTC 3°) (859 1D Wo: 14)US 2012/0042408 AI Feb. 16, 2012 6 TABLE IT Priners for mutant allele cart pensom Bos-2n (S'TCCGTECCOTTTICGTTNTTTACS"} (RQ oom-2 oraieen0 bast (5"CCGDCCCGCAAOTIARATATED'| [88 1D HO: 16) peepowia-3 neaer7aL bgatez (S' aerercrtccermcernnere 3°) (S89 EBtoail (5 TAOCRICTGAATTICATAACCARTCTCGATACRCS| (SEQ 1D 10: 18) 097] Genetic markers used to genotype the oxdl-I(No- OyosdI-2(Ler)*Col-) FI population and_osd1-1(No-Dy spol-I(Col0)lrec8(Col-0) triple mutantsLer FI popula ‘ion relisted in Table IL. The PCR conditions were 40 cycles 488 nm and 405 am with AlexaFhior488 and DAPI espoc- Lively. Arabidopsis eenome sizes were measured as described in MARIE & BROWN, (Biol Cell, 78, 1-S1, 1993) using tomato Lycopersicon esculentum ev “Montfavet as the stan (of 305 at 94° C., 305 at Tm and 30s at 72°C. dard (2C=1.99 pg, % GC-40.0%) TABLE 11 a Chron. Position pb Primer + (G89 1D NO.) Primer 2 (68) ID DO) ia 1 as8a74a3 GRACERGEROGETE (18) ‘GICARACCAGTTCRATER(201 reins 128374008 craceronAArTeToaRaAc(2t! ancatcacasrrenaartee(22) veat2 2 asasaseo TaRMGxGTCccTOoTAAKG(27) oTTUTTorTOTOOCATT(26) Ccapors_10355 4 «1044800. AccenrTTooTGANECTAAC(20) _ cagckareTecgeTITETCe( 30) eate-a 4 naseen04 naraaasarcoscrerana(a:} cH@MAACRAATCOCATTA(321 pats 5 cesosa2 grerrencaaorrrmacraa(aa}_ ckatcrssaaaconasarasaara 24) [0098] These markers were amplified (40 cycles of 30 at Example 1 94° C., 308 at $8° C. and 30s at 72° C.) with the indicated primers and observed after migration om 3% agarose gel [0099] CAPSK4 10385 wasobserved after Boos IV Hpall ‘double digestion, The wa primer pats specifi forthe osdl-L ‘and osdl-2 insertion borders Were used asa marker on chro mosome 3. Cytology and Flow Cytometry: [0100] Final meiotic produets were observed as desribed in AZUME eta, (Embo J, 21, 3081-95, 2002) and viewed ‘with a conventional light microscope with a 40x dry objec- tive, Chromosomes spreads and observations were caried ‘out using the technique described in MERCIER et a, (Bio chimie, 83, 1023-28, 2001). The DNA iluorescence of sper- matic pollen nuclei was quantified using open LAB 40.4 software. For each nucleus the surrounding Background was, calculated and subtracted fom the global fuorescenceof the rucleus. Meiotc spindles were observed according 1 the protocol described in MERCIER etal. (Genes Dev, 15, 1859- ‘7, 2001) except that the DNA was counterstained with DAPI. Observations were made using an SP2 Leica confocal microscope, mages were aequied with a63x wate objective in xyz and 3D reooastructions were made using Leica soft- ‘ware, Projections are shown. Cells were imaged at excitation Production of Diploid Gametes by os Mutants [0101] Asa part of an expression profiling sereen for mei- ‘otic genes, using the Expression Angee tool (TOUFIGHI et al, Plant J 43, 153-63, 2005) with the AIGenExpress tissue sol (SCHMID ctal., Nat Genet, 37, $01-6,2005),At3g57860 ‘was elected asa good candidate det its co-regulation with several knowa meiotic genes. ABg57860 corresponds tothe UVId-Like gene (UVI-L) which was briefly deserted ina snd of its paralogue, the UVI4 gene (HASE etal, Pant J, 46, 317-26, 2006). Due to its roe in meiosis (see below) we renamed the At3p57860 gene OSDI, for omission of second division, The OSD and UVId proteins axe conserved ‘throughout the plant kingdom but do ot contain any obvious conserved Known functional domains. No homologues were ‘dentified outside the plant kingdom. [0102] _ We investigated the role of the OSDI gene by iso- lating and characterising vo mutans. Tae osdl-1 (stl 5307) and the o1-2 (GT21481) Ds insertional mutans are inthe "Nooseen (No-O) and Landsberg (Ler) backgrounds, respec tively, and in both cases the insertion sin the second exon of the OSDI gone [0103] | Theintronéexon stricture of the OSDI geneand the location ofthe two diferent Ds insertions re shown in FIG 2.TheOSDI gene contains 3 exons and introns aadencodesUS 2012/0042408 AI 4 protein of 243 amino acids. The positions of the two Ds insertions are indicated by triangles, [0104] FIG. 3 represents meiosis in wildtype plans and FIG. 4 represents meiosis in os mutants [0105] Legend of FIG. 3: (A) Pachytene, Homologous chromosomes are fll synapsod.(B) Diakiness. Five pairs ‘of homologous chromosomes (bivalent), linked by chias- ‘mata, are observe. (C) Metaphase I. The five bivalent are aligned on the metaphase plate. (D) Anaphase I. The bomolo- ‘gous chromosomes are separated. (E) Telophase 1. (F) ‘Metaphase Il. The pairs of sister chromatids aljgn on the ‘metaphase plates (3) Anaphase I. The sister chromatids are separated. (H and 1) Telophase I. Four haploid spores are formed (trad) Scale Bar-10 um [0106] Legend of FIG. 4: (A and B) Male meiotic products stained with toluidine blue. (A) wildtypetetrad.(B) A dyad inthe osd-1 mutant. (C to D) Male meiosis in os is incis- tinguishable from wild type uatil telophase I (compare to FIG. 3, butno figures charictersic ofa second division were ‘observed. (C)pactytene (D) dakiness. (E) metaphase |. (F) ‘Anaphase I. (G) Telophase 1, (11) Metaphase I of female ‘ncicsis in os [0107] In both independent osdl mutans the products of ‘male meiosis were dyads (osdl-I: 7147714 osdl-2:334/334) instead ofttrds (FIGS. 44 and B), Complementation tests ‘between osdl-1 and osdl-2 confirmed that these mutations are allelic (sdl-osdl-2: 369 dyads’369), and thus demon- strated thatthe observed dyads are due to disruption ofthe (OSDI gene, Osd] mutants did not show any somatic devel- ‘opmental defets, male and female gametophyte lethality or redced ferility (wildtype 38=11 seeds, osd 9526) [0108] Next, we measured ploidy levels among the off springof diploid os mutans. Among selfed progeny, ttra- ploid (84%) and triploid (16%), but no diploid plants were Found (osdl-I: 256; osdl-2: 1-24). When mutant pollen ‘was used to fertilise a wildtype plant all he resulting prog ny were triploid (osdl-1: 2-75). When mutant ovules were Ferilised with wild type pollen grains we iolated 12% dip- oid and 88% triploid plants (0-25). This demonstrated that the osdl mutants produce high levels of male (100%) and female (-85%) diploid spores, which result in funcional aamotes. [0109] To unravel the mechanisms leading to dyad produe- tion in osd, we investigated chromosome behaviour during ‘meiosis, Both male and female meiosis | were indstingush- able from wildtype (compare FIG. 4 with FIG. 3). Notbly, chiasma, the cytological manifestation of crossovers, and bivalents Were observed, However, we were unable to find ‘any meiosis I figures (among >S00 male meioeytes from prophase to spore formation), strongly suggesting that dyad production is due to an absence of the sevond meu sion, If this second division does not take place then feeroqjort at ofsomeres wil be bet ia te diploid sgamotes because of sistr cheomatids co-segregation and homologues separation during the fist division, Because of ‘recombination, any loci which are not inked to centromeres will segregate, We ested ourassumption by taking advantage ofthe two diferet genetic backgrounds ofthe os -1 (No-0) and osd1-2 mutants (Ler). plants bearing the two muta tions—mutant for osdl and heterozygous for any No-O/Ler polymorphisms—were crossed as male or female oa third senetic background, Columbia (Col), Karytyping and szenolyping ofthe obtained plats for tcimorphie molecular ‘markers provided direct information onthe genetic make-up Feb. 16, 2012 of pollen grains and female gametophytes produced by the ‘mutant, All the diploid gametes tested had the predicted ‘genetic characteristics. They were systematically homozy- ‘gous at centromeres and segregating —because of recombi ‘ation —at other loci (0-48 for male diploid gametes and for female diploid gametes) These results confirmed that the absence of a second meiotic division is indeed the ‘cause of 2n gametes production nos. This mechanism also ‘implies that unbalanced chromosome segregation st meiosis T would give rise to unbalanced dyads in osdl this was confined by analysing a double Atspol-1osdl-I mutant (ata not shown. [0110] Due to an absence ofthe second meitie division, ‘sd mutants produce high frequencies of viable diploid male and female ametophytes, which generate after fecundation, able tetraploid plants. However, tis phenomenon differs from apomeiosis in thatthe produced gametes are genetically cffereat from the mothe plant Example 2 Production of Apomeiotic Gamete by Triple os Alc®!Atspol 1-1 Mutants [0111] _Indouble Atspol1-1/Atec8 mutants the fist mei- otc division is replaced by 2 micotic.ike division, followed. by an unbalanced second division which leads to unbalanced spores and sterility (CHELYSHEVA eta, J Cell Sei, 118, 4621-32, 2008), 0112] "We generated osdl/Atre8/Atspol-1_ mutans Plants heterozygous for both Apo! -1 and Atree8 mutations ‘wereobtaned by crossing plants heterozygous foreach muta- tion, and were erossed by a plant heterozygous for os “Triple heterozygous plants identified were sell-fertlized and plants homozygous for the three mutations were analy ed [0113] | Observation ofchromosome behaviour during mle ‘and female meiosis ofthese mutants is shown in EG. 8 [0114] Legend of FIG. 5: (A) Male metaphase I (B) Male ‘anaplase 1 The vignette shows 2 dyad ia MiMe, (C) Female ‘metaphase I (D) Female anaphase I, Scale bar=10 un, [0115] These observations revealed a mitotilke division: 0 univaleas aligned on the metaphase plate and sister chro- ‘matid separated af anaphse (FIG. 5). [0116] The Atspoll-1 and Atrec8 mutations lead to 2 rmitotc-ike fist meiotic division andthe osll mutation pre- vents the second meiotic division from taking place. This sul in replacement of meiosis by a mitotc-like division, and in spomeiosis, [0117] We called this wenorype MiMe for “mitosis instead ‘of meiosis". MiMe plans generate dyads (408/408) and are Ferile (2526 seeds per fruit). The osdl mutation therefore suppressed the sterility phenotype ofthe AtspolI-1/Atree8 double mutant, [0118]. Theselfed progeny of MiMe plants were systemati- cally tetaploid (a-24) and backerosses between diploid IMiMe plats ad wildtype plants generated tpl plants regardless of whether male (a=24) or female (9-67) MiMe gametes were used, showing that this mitotic-ike division tives rise wo fiaetional diploid gametes. All the gametes {male sn female), tested similary as described above, 95> ‘ematicallyetaned the mother plant heterozygosity fr every ‘genetic marker tested and were thus genetically ieatial to the mother plant. These results confirm that MiMe plants ‘undergo « mitoti-ike division instead ofa normal meiotic vision, without alfecting subsequeat sexual processesUS 2012/0042408 AI 0119] When meiosis is replaced by mitosis ploidy is expected to double with each generation. This was observed in MiMe plant, as shown in FIG. 6 [0120] Legend of FIG. 6 Left column: mitotic metaphase, scale bar=10 pm. Right columns: the coresponding four ‘weeks old plans, (scale har-2 em) and flowers (scale har= am) [0121] In subsequent generations, we obtained tetraploid (AN, 20 chromosomes, n=26) and octoplid (SN, 40 chromo somes, 1-33). Example 3 Identification of a Rice Onholog atthe Arabidopsis (SDI Gene 0122] The Orisa sativa genome contains wo OSDUUVIE ‘homologue candidates (Os02g37850 and Os04g39670). We isolated two T-DNA insertion mutants in one of this putative Domologue (0302937850). The two lines, AMBAI2 and AMO were genotyped by PCR to select homozygotes. In ‘oth ines we observe spontaneous tetraploid plants among the offspring of diploid mutant plants, suggestive ofthe pro Feb. 16, 2012 400) and observed the production of 100% of dyads instead of tetrad, a illustrated by FIG. 7, 0123] Legend of FIG. 7: A: Tetrad of spores ia wildtype; : Dyad of spores in AMBI2. [0124] This phenotype is identical tthe Arabidopsis osd ‘mut To unravel the mechanisms leading to dyad produc tion in AMBAI2 homozygote mutants, we investigated chro ‘mosome behavior during. meiosis. Meiosis I was indstin- guishable from wild ype. Notably, chiasmata,theeytological ‘manifestation of crossovers, and bivalents were observed However, we were unable to find any meiosis I figures, strongly suggesting that 2N spores produetia is due to aa absence of the second meiotic division, lke in Arabidopsis ‘os. Altogether, these results show that O302g37880 isthe functional homologue of Arabidopsis OSDL and therefore called it OsOSDI-OSDI and OsOSDI proteins have 23.6% ‘dentty and 35% similarity onan aljgnment that covers the ‘whole length ofthe sequences (EMBOSS paiewise alignment Needle too) SEQUENCE LISTING 60> MMBER OF 5B 1D HOS: 35, 20> $60 10 Ho 1 00> sequEHCE: 1 Wot Pao Glu Ala Axg Aep Arg Thr Ola Arg Pro Val Aap Tyr Ser thr Te Phe Ala Aon Ag Arg Ang Hie Gly Tle teu tat Aap G14 Pro Aap Sex Aug Leu Sex Leu Tle Glu Ser Pro Val Aen Pro Aap Tle Gly sax "he oy Ghy Mr Gly Gly Le Vek Aap Gly Han me Thx Te Tp Ag ro Gly An Gly Arg Gly Gly His Tar Fro Phe Arg lau Pro Gin Gly & Ey = ® ‘ang Gli Aan Nee Fro Te Val Thy Ala Arg Arg Oly Arg Gly Sty oy) les Le Pro Ser Mp Tyr ro ARG TRE Pro Lau Ag Aap The Th His Ne Val Ag Ala Te cle Seg Arg Arg Gly Ala Gly The Oly Gly Aap Asp Gly Arg Val Te Glu Ze Pro The tie Arg Gin Val Gly Val teu Gls Ser Pro Val Fro Leu Gar Gly la Hie Lye Cys Ser Hat Val he Pro Gly Pro Ser Val Gly Ohe Lye Arg Ser Gye Pro Pro Ser Thr Ala lye Wal Gin tye Hat Leu Cee Aap Ie Thr Lye Glu Te Ala ote oieUS 2012/0042408 AI Feb. 16, 2012 -continued Coy Ala oy Phe Tle Thr Pro Glu ye tye Lew Leu Aen Ser The Aap tye Val lu tye Tle Val Not Ala Gly Tle Gin Lys Leu tye Ser The <210> $29 10 Wo 2 SRDS ORGAIISH: Arabidopase thaliana ‘4oo> segomce: 2 Net Glu Gly ye Phe Ala Tie Ser Glu Ser The Aen Leu Leu Gln 2g 1e Lye Asp fhe The Gin ser Yel Val Vel Asp Lau Ala Glu Gly Ang Ser Pro Lye Te Sex 11e Aan Gin Phe Axg Aen Tye Cys Het Aan Pro eu Ata Asp oye Leu ce aay cin ctu ne ‘Phe Thr Leu lye Lye Glu Pro Gin Thr Tyr aay Te Aep vet Leu La ‘sg Val Leu Leu THe Val Gin Gin Leu Leu Gin Glu Aen Ary Hie Ae Ser bye Arg Aap Te Tyr Tye Net ste Pro Ser Ala Ser Te Yel Aep Atg Ala Ie Ghy Aap Tle Cye Tle on ‘Ser Axg tye Ban Lew Aen Val Val Ser Val Gly Ban Gly Lew Val Met hy Top Leu Iye Phe Arg Glu Ala Gly Arg Lye Phe Ap Oye Leu Aan Ser Leu Ron The Ala Tyr Pxo Val Pro Val Leu Val Glu Glu vat Glu ‘ep Te val Sex Leu Ala Glu Tyr Te teu Vat Yal Glu Lye clu Tar Val Phe Gin Arg Leu Ata Aan Asp Met Phe Oye Lys Thr Aun Arg cys He Val Ie Thr Gly Arg Gay Tye Pro Aep Vol Ser Thr Arg Arg Phe ‘Ley Rog Leu Lau Met Ghu Uys Lew Ble Leu Pro Val Hla Cye Leu Val ‘ep Ove Aap Pro Tyr Gly Phe Glu Te Leu Ale The Tye Rey Phe Gly Ser Met Gln Net Ala Tyr Agp Tle Glu ser Leu Arg Ala Pro Aap Net lye Tap Leu Gly Ala Phe exo Sex Agp Sex Giu Val Tyx Ser Val P10 28 380 28 lye Gin ye Les Lew Pro Leu The Glu GLa Aap Lye Lye Ary The GIUS 2012/0042408 AI 10 -continued Feb. 16, 2012 aa ce teu su arg oye cos the wee 210» 559 10 90 > SUD Grama arebiaopese ‘e4oo> segomce: 3 tet Glu Glu sar ser oly mie oe ae an ala an aa Fro 3s wa ne ne xan sor Phe vat neg uu wpe 30 pep va aia sor ay wy ne the nis Arg vat atu ye ety mor thr aa oy ty teu 2 Ha a ser Aep tou ye oly ne he Lye Ser wal Bp oye sag Val Thr ap Tye Phe va Ala tow fer aug oxy cay ala val ay Aep bow ye ne ne sap arg Vuk Wy Po A he Pro ep lew ala Te cs ala rye ne val 8 ne Poe ne to ys ys arg ota as ay me ag ag val oe vat ma oe ne np au an oe ma aw mie ne ome Arg tyr ala xa ne ay ala ne xa Phe bye ag oe ng ng oe Phe va bs et Fro ln t3p ys Phe olu The is val tye He Phe aw Phe va Mo ay one as Phe Phe val ala 3 ag ne an ye ne ne vm va ay we neg aa ne a ihe pe ser uP 2 ya ye ay an ay ne ag mp ne ag aw an ag sop ma on va ne aw te a me ay ayUS 2012/0042408 AI Feb. 16, 2012 ul -continued Ley Rog GLY Aap Aap Leu Aan Leu Te Pro Giu Glu Sex Leu Val Pro eu Lys to lye Aap Ser Gin Tle Ala Lye Ser Leu Leu Ser Ser Lye fly bye Arg Ala Gla Tae Glu Ala Les tye Gye lie Gly Ty Ren hE ‘eu Gly Lye Tyr Te ata Thr Lye The Val Gin oly Wye Tyr Te ‘ sug 30 10 4 2135 ORGRTISH Arabidopose thaliana soos segumten: « Co Ser Net Ala Asp Ser Aan Hi Gin Ser Leu Ser Pro Pro cya Ala ‘Aen Gly His Arg Ser Thr Te ser Leu Arg Aap Asp Gln Gly Oly The Phe cye Leu Ie cys Phe Ser Aan Leu Val Ser Agp Pro Reg Ile Pro ‘moe Val Hse Val Ger Tye Ala Lau ite Gin Leu ser Tle Ala He ser ‘Ala Tle Gin Te Wet Asp Not The Ser teu Leu cys Ser Val Olu Glu ‘Ser Ser te Gly Gla Asp Phe Vel Glu Arg He Ser Agp Gln Leu ger ‘Phe Gly al Leu Wat Ser Gye Glu Asn Ile tap te Amn Ser Hee Tle rg Aep ya Gls Ala Leu Val Gye Gin Ue Val lu Gly Lew Gin tea Pro Ser Glu Glu Te Arg Gly Glu Te Leu Phe Ala Leu Tyr ya Phe ‘Ser Ala Leu Gln Phe Thr Giu Gin Aen Val Asp Gly Tle Glu Val Law Ser Leu Leu Gye Fro Lye eu Lew Gye Ue Ser Leu Glu Ala Leu Ala ‘ye Thr Gln Arg Aep Asp Val Arg Leu Aen Gye Yal Ala Leu Leu ThUS 2012/0042408 AI 12 -continued Feb. 16, 2012 ow ne as ow ow ow va ow Aen ye ae vat val val oe ne aw ne ain ain ae ain val ay ag ne a ay ne 8 ne ay ain ihe ae vs aw aa ap ay oe an ue ow T! aa ne me an les ne aa sie ae va we aw ap va ay a ala ™ aa oa as aw ow ser me oye he tye ap cvs ie sar Phe oe nie oe me ws val an oy ys ma ag me 520 nop aa ne Phe aan a3 ep Rep Aap bap 3 ap ma ne ne a mie ma va va mie aw ay ag ser as a an as ay ne ap ala va au va sh aw ne aw ne thr aw ne Phe Ma ay ma val aap eg ne me ma ain te an sie we 1 oe aa aa ne 8 ae ite pla es a Rep Aap Lye aa ne 380 aa aw 20 a ap aw ow an ala ap ne oe ne ow ye ove ye va, ne va, va, om Phe va ma me ye neg ay an Phe. me ie ma ieUS 2012/0042408 AI Feb. 16, 2012 13 -continued ‘Ala Ser Leu Glu Gin Tyr Ile Tie Leu Aen Lye The Ser Leu Te Cys ‘Ais Te ser Aap ger Pro Ala Leu Lau Aen Leu Val Amn Lew ye Gly eu cye Arg Ser Leu Gin Aan Glu Arg Tyr Gin tle ser tyr Ser Leu Glu Ala elu Arg He We Phe ie Les Lew Aon Glu Bye Glu tep Aap. eu Gly Ser Tle Aim Te Hie Lew Glu ser Leu Lye Typ Lew Phe Gin Gin Glu ser Tle Ser Lye Ser Leu Tle tyr Gin Te Gln Lys Te ser ‘arg Ren Zon Leu Tle Gly Aan Glu Val Ble Aen al Tye Gly ep Gly ‘ep Aen Tyr Ala Ala Thr Geu teu Val Aen Leu Leu The Gin teu Ala Glu bye lu Glu Gin Gtu Aon Aap Val The Ser Te Leu Aen Leu Met ‘hen Gly He Gly Ser Val Vat Hie Arg Leu Val Ser Gly Phe Ser Aan Ser Ser Leu Gly Thr Ser Phe Lye Tar Leu Leu Leu Leu Val Phe Aan Te Leu The Ser Val Gin Pro Ale Val Leu Met Tle Aap Glu Sex Tep {ye Ale Val Sex Tle Lye Leu Leu Aen Phe Leu Ser Leu Rey Rep Thr ‘Mla Te tye Gln Aen Hie Glu Asp tet Yel Val Tie Gly Tle teu ser ‘eu Val Leu Tye ile Ser Sex Aap Gly Ale Leu Val Glu Ala Ser Arg ‘hen Te Val Sex Aon Sor Tyr Leu Val Sex Ala Te Aen Thr Val Val ‘ep Vai Ala Cyo Ser Lyo Gly Pro Ala Leu Thr Gln cye Gin Asp Glu ‘Mor Aen Te Gly Glu Ala Leu Ala Phe The Leu Lew Leu tyr Phe Phe ‘Ser Leu Avg Ser Lau Gln Te Val Law Ala Gly Ala Val Aep Tip Gln Ala Phe Phe Gly Thr Ser Te Sex Leu Glu Thr Leu Pro Vad Val oye Te Tyr Cye tie Aan Leu cye Arg teu Net le Phe Gly, bia Pro in Te lye teu ie Ma Sex tyr cya Lew Leu Glu La 108s xeco 1085 ‘eu Thr ly Lou Sex Glu Gln Val Aap THe bye Lye Gla Gln LewUS 2012/0042408 AI 4 -continued Feb. 16, 2012 ex arg tou cos Lex boa fin ai cys ‘tr arg ola ye ae ay ser ser oe as sep ow ain ain va au Arg He 20> 540 10 10 5 S213 oReRIISM, axaptanpose 400» sequmce: § top The Mia es Pro ala Pro val ain val ser Fro Wet tye Ret an Tle Aen Gye Ala he fre Po Aah ed Ag tg Ser teu Arg ser Gln Gin Ser cin cin Ser Gin Arg Gly Cys Gly Gly Phe ep tu tou leu Te Asn Asp cin lex Ser tou Uys ye Val ser ser oe aa as oe a va up om ure sep ts cs Pre cain vee Phe ser noe tye ala tu Rep Met ols yr Gea Tar val arg clu Lew et tye eu Aep Arg fn ly Tae Arg val Ala Thx en tos Arg Thr val ys ser ne fin ala ser in ely ro sr Gin ser ser cin ols ro tle aan val bes oly ser ala yr Arg he Te Ala cla lye Sor Ala. ser Ser the ais not ais Phe pep ou Me in Gia Gla the Arg gin ser val ain oan ser ne arg Aap wis uveUS 2012/0042408 AI Feb. 16, 2012 15 -continued ‘sg Glu Asp Sex Gln Leu Val Ala Ser Arg Ser Ser Ser Gly Leu ser arg tog ep Ser Ser Ala Ser Iie Gly Glu Ser Lys Ser Gin te ser Phe Gly Met tet Leu hep Sar Ie Gin Ger Aap te Net Gla Aa Aan ‘sg Gly The Ie Gla Val Phe Lew Glu The Glu Aeg Tle Gln Gin tye ‘Leu Thr Leu Gl Agp Thr Ser Leu Gin Gin Leu Arg Lye Glu Gin Aa ‘ep Ser Lys Ala Ser Leu Asp Gly Gly Val Lye Phe Tle Leu Glu Glu ‘Phe Ser Lya Aap Fro fon Gin Glu Lye Leu Gin Lys le Lew cin Met ‘eu Thr The Te Pro Glu Gin Val Glu Mr ala Lau Gla Lye te Gin arg ctu ‘arg Glu Te Gla Val Lew Ala Ser leu Arg The Pro Glu Pro Arg Vel Arg Val Pro The Ala Pro Gin Val liye Ala tya Glu Aen Leu Oxo Giu Gin Arg Gly Gln Ala Ala ty2 Val eu Thr Ser Lau Lye bet Pro Glu Pro Arg Vel Gln Val Pro ALA Ala Pro Gin Ala lye Gla Aen Phe Pro Glu Gin Axy Gly Pro Val Aa Lye Ser Aen See Phe Cye Aen The The Leu Lye The Lyw Gln Pro Gln Phe ‘ro Arg Aen Pro Aon Aep Ala Ser Ale Arg Ala Val Lys Pro Tye Lat Ser Pro Lya Te Gin Val Gly oye Tep lye Thr Val Lys Pro Glu tye Sex Aon the yo Lye Arg Ala Tar Arg tye Pro val Lye Ser clu ser ‘Tar Azg Thr Gln Fhe Giu Gin cye Ser Vat Val Te Aop Ser Asp cu (cu Rep Te Aep cly Gy Phe Ser ye Leu Tie Aen Glu Aen The Arg iy Thr Aen Phe Glu Tep Aap Ala Glu lye Glu The Glu Ary Te Law ‘ng Thr Ala Arg Arg Thr Lys Arg lye Phe Gly Aen Pro Te Tle Tle <210> 849 10 30 6 Su later. 627 S12) ORGRTEH, Arabtaopese thaLtaneUS 2012/0042408 AI Feb. 16, 2012 16 -continued ‘Ho0> segumice: « Net Phe Tye Sex His Gin Leu Leu Ale Axg Lye Ale Pro Leu Gly Gin He Tp Met Ala Ala Thr Geu ia Ale Lye Te Aan Azg Lye lye Lew ‘ep Lye Leu Aap Te Ie Gin Tie cys Glu Giu He Leu Aen Pro ser 1e val tye Glu arg tye Phe Rep Aep Val Ren tag ‘Phe Leu Val Glu THe Aen Gly Ala Tep Arg The Lys Ser Val Pro Aap Pro Thr Leu Leu Pro Lye Gly ye Tar He Ale Arg Lys Glu Ra Val ‘Arg Aen Val Peo Lye Phe Gly Aan Tyr Wet Aap Phe Gln Gln Thr Phe Gal Aep Leu Gly Gln Gin Phe Hie Gin ALa Aap Ale GIM Asn Te The ‘Leu Phe Glu Tye ile Gly Ser Phe Gin The Aan Aen Glu Thr tye Aap) ‘ng Phe Glu Atg Phe Aep Ile Glu Gly Aep Aap Glu Te Gln Met an Ser Aen Pro Arg Glu Gly Ale Glu Ile Pro Thr Thr Leu Tle Pro sex Pro Pro Arg ile Kl hap Tle Pro Glu Gly Val Aan Pro Thr Ser Pro Gen Rog Gln Glu Gin in Glu Aan Arg Arg Aap Gly Phe Ala Glu cin Net Giu Glu Gln Aen Te Pro Rep Lys Glu Giu His Aep Arg Pro Gin Pro Ala ya iyo Azg Ala Arg uo Tar Ala Thr Ser Ala Wet Asp TYE Gu Gin thr Tle Te Ate Gly ie Val Tyr Gin ser Typ Leu Gin Pap. ‘The Ger Asp Tle Leu yo Arg Gly Glu lye Reg Lys Val Reg chy Thr Te Arg Pro Aap Met Glu Ser Phe Wye Rog Ale Aan Wet Pro Pro The Cn Leu Phe Glu Lye Rep Sex Ser Tyr Pro Pro Gln Leu Tyr Gin Lau ‘up Ser Lye Aan Thx Gln Val Lew Gln Thr Sex ser sex Glu cer Ag fie Pro Aap Lau Aug Ala Glu Gin Ser Pro Gly Phe Val Gin Glu Axg 30 8 380 Net Hle Asn Hie He Gin hr Aap lle le Giu Arg Sex Aep Thr sexUS 2012/0042408 AI 7 -continued Feb. 16, 2012 ser ain uy tye oy ala val ala cy noe ‘the Fro ma aa ser aw ome ala a a ag aw <210> 630 10 10.7 au m1 xa va aan ue ser an aw on np val ‘00> segomce: 7 cgtenctote oceaagaaag <210> $49 10 10 6 S20: ona INpowatran, pea peiner ‘4oo> segue: @ ae 20> 549 10 10 9 peg gectgctatg ATURE. ‘4o0> segue: 9 ma aw Gu the teu arg The Val arg Wet wer ala oly, Als Rep He Aan Gs tle sor ser ser ag fig Arg clu Tyr ee Als ep tye Ser Hie Les tye ue ay ow, 2 ser hie ala oe oa ma tea aus aw Phe vat me hee Phe as ayUS 2012/0042408 AI 18 -continued Feb. 16, 2012 cosstotect egtgnctes ‘10> #89 10 10 19 S223) OnHER THFORUTTON: PCR primer soos segumter: 10 ‘<210> $89 30 W011 “aids onenst: areitsciat {22135 OTHER. TnFORMATTON: CR primer soos segumten: 11 sastcgptgeg tengatetcs 9 <210> $89 10 ¥0 12 {21s ORGASM. Ares fseia2 “S213; OTHER THPORBATON: PCR primer soos segmten: 12 4210» #99 10 20 13, {12132 OTHER INFORBATION: PCR primer soos seqomen: 19 <2i0s seq 10 x0 14 ‘00> sagumce: 13 gonaguaseg aganagstts <210s sag 10 x0 15 {22135 OTHER. INFORMATION: CR primer 00> sagumice: 15 ecogtteagt tttagttttt tae 2US 2012/0042408 AI 19 -continued Feb. 16, 2012 [ai0> #89 10 30 16 SHG) oneust. areitsoia1 S20; ona Inpowactan, pea peiner ‘et0o> segue: 16 costecegea agttaaatat 9 aus San ona INpowatran, pea peiner ‘e400> saQ0mNCE: 17 sgetetortce ertectetet ¢ <210> $89 10 10 18 perig Sai ona INpommtron, Bea primer ‘e400> segue: 18 ‘eageatorgs atttoatase caatotogat acae <21o> $39 20 10 18 S213) ongust, axeitsosaa OMNER THPORUETTON: BCR primes 400» s8Q0mNCE: 18 ccarceaceag gete “<2lo> #89 30 10 20 SHG) oneust axeitsoiat S223) OnMER KiroRrToN: eR. primer -et00s saqumer: 20 ‘210> $89 30 10 21, S292 onenst. areitsoiaa Tan oma INpomtTOn, Pca peiner soos segomter: 21 ctgectgasa trgtegaase <210> $89 10 ¥0 22 SEDs ORGASM. areitscia2US 2012/0042408 AI 20 -continued Feb. 16, 2012 {2213s OTHER INFORBATION: PCR primer “00> sagomnce: 22 gecatcaceg exetgattce 210s seq 10 xo 23 {2255 OTHER THFORAATION- PCR primer ‘00> sagumce: 23 agectctee tggtgegeat « <210s sa 10 wo 26 ‘00> segumce: 23 agcctonzea agettagste & <210s 589 10 x0 25 00> sagumce: 25 saceeteagee caateacgtt & <210> $39 1D 10 26 “00> sagomNCE: 26 aggteaagea aaagestaag 9 <210> $89 1D 40 27 S20: ona INpowatran, pea peiner “s4o0> sagomnce: 27 tesaagagte cetegtanig <210> $89 10 10 28 ‘hos PaATORE ‘e4o0> sagUmNCE: 28US 2012/0042408 AI 2 -continued Feb. 16, 2012 geestegetg tageate ‘e21o> #89 10 10 29 S223) OnHER THFORUTTON: PCR primer soos segumter: 29 ‘<210> $89 10 H0 30 “aids onenst: areitsciat {22135 OTHER. TnFORMATTON: CR primer soos sequmter: 30 gagcagttte cacteegtce <<210> $89 10 ¥0 31 {21s ORGASM. Ares fseia2 “S213; OTHER THPORBATON: PCR primer soos segumter: 21 4210» #29 10 10 32 {12132 OTHER INFORBATION: PCR primer soos sagumter: 92 210s 529 10 x0 23 ‘00> sagumee: 32 geeetogges gttttgctas 210s say 10 x0 24 {22135 OTHER. INFORMATION: CR primer 00> sagumice: 33 captctassa gegsgagtst gita 2US 2012/0042408 AI Feb. 16, 2012 2 -continued ‘[a0> #29 10 30 35 SBI: oRouSH: onyes eativa soos sequmter: 35 ‘et Fro Glu Val Seg Aon Gar Gly Gly Arg Ala Ala Leu Ala Aap Pro) Ag Beg Por thr Sor Pro Pro hy Ala Val Ala Val lye Pro Leu Ala Arg Arg Ala Leu Pro Pro Thr Ser Aan liye Glu Aen Yal Pro Pro Ser Trp Ala Val thr Val Arg Ala Thr Fro ly fog Ag Ser P50 Ue Pro Oa Trp Tye ro Arg Ser Fo Law Rg dep te the Mia Vol Glu Arg Lye ser Arg Leu Gly Ser Val Azp Pro Ala Thr Pro Val Gln liye Glu Glu Gly Val ro Gin Ser thr Pro Thr Fro Pro Thr Gin Lye Ala Leu Asp Ala Ala Ala Pro yg Fro ely ser Ts in Ae Yo Ala Ser hr fer Te Ae He tne ‘ls Glu Gly Lys Fro Lye Ala Sex Ser cer Ser ro Sex Aap cys See lye Aen teu bys Arg Ala Pro Uys Ala Ala Gin Pro Ser tye Vat The ie Gla tye Aeg Tar Lew ueu Ser Net Rog 1.A meso for obtaining a plat producing Second Dis 18) an OSDI protein as defined in elaima 1 sion Restitution 2n gametes, wherein sid method comprises b)a protein involved in ntation of meiotic recombination the inition said plant ofa potcin hereinafter designated in pants, std protein being selected among: 4 OSDI protein, wherein suid protein has ot est 20° A). proven reine designated as SPOI-1 protein, sequenceidentiy, oat east 2% sequenesimiariy with the ‘wherein sai protein as teas 40% sequence dene AtOSDI protsin of SEQ ID NO: 1, oF withthe OsOSDI tty, or at least 60% sequence similarity with the pen of SEQ ID NO: 38, SPOI I+ protein of SEQ ID NO: 2; 2.Amethodacconing claim! whecinishibonofthe ia protein herenafer designated as SPOL-2 poi, (OSD! protein s obtained by mutagenesis of the OSDI gene wherein sid protein bas at east 40% sequence iden: cor ofits promoter, andthe nutans having paral or totally iy, or at Least 60% sequence siiaiy with the fost the OSDI protein activity ae selected SPO1-2 protein of SEQ TD NO: 3; 3.Amethodaccontingoclsim I, wherein theinhibitionof i) a protein hereinafter designated as PRD protein, te OSDI pec is obtained by expressing in sid plant a ‘wheccin said protein has at east 25% soquence iden: silencing RNA lant the gene encoding uid protein tity, oat last 3% sequence similarity withthe pl 4. method for obtaining a plant producing apomeiotic protein of SEQID NO: 4; or gametes, wherein said method comprises the iv) protein hereinafter designated as PAIR protein, suid plant ofthe following proteins herein said protein has at least 30% sequence iden-US 2012/0042408 AI tity, or at lest 40% sequence silty with the PAIRI protein of SEQ ID NO- 5: 6) a procin hereinafter designated as Ree8 pron, ‘wherein said proteins al least 4P6 sequence entity, borates 48% ssqueacesimilaty withthe Respatcin ofS5Q IDNO: 6 §.A method according claim 4, whori nhibion oft Jeastoneof the OSDI, SPOI-1, SPOL-2, PRDI, PATRI, Rec protins is obsined by mutagenesis ofthe gee ene {ng sid protein of oo its promote, and the mutants having artall ortoally lost the att of sid protein are sleced ‘A melhod according o claim 4, wherein inhibition of at Jeasion ofthe OSDI, $POM-1,SPO1-2, PRDI, PAIRI or ec proteins blaine by expressing in saidplantasilene- ing RNA targeting the gene encoding said proven. "An expression casetecompasiog promoter fetonal in plant cell; atleast one DNA const selected among: 4) one or more DNA consists) of 200 1000 bp, each comprising @ fragment of a cDNA ofa target gene selected among. OSDI, SPOLI-1, SPOI-2, PRDI, PAIRI and RECE, or ofits complemestay or having at least 95% identity wit sid pment said’ DNA sequence() beng placed under transcriptional contol of said promoter ‘one oF more hapa DNA constr) capable, when ‘canseribd of forming a hain RNA tating 2 gene selected atnong OSDI, SPOI-1, SPOI-2, PRDI, PAIR and RECS: or 6) one of more DNA constr) capable, when tran- sere of fominganamiRNA targeting yneselected among OSDI, SPOLT-L SPO11-2, PRDI, PAIR, and REC: Feb. 16, 2012 said DNA constrot(s) being placed under transcriptional control of said promoter 8, An expression cassette of claim 7 comprising a DNA, ‘construct tangeting the OSDI gene 9. An expression cassette of claim 7, comprising: a DNA. ‘consi targeting the OSDI1 gene, a DNA construct target- ‘ng a gene selected among SPOL-1, SPOI1-2, PRDI, and PAIRI, oa DNA const targeting RECR. 10. A reoombinaat vector comprising an expression cas- sefte of elim 7. 11. A plant producing Second Division Restitution 2a ‘gametes, whichis a transgenic plant containing a transgene ‘comprising an expression cassette of claim 8 12. plant producing apomeiotic gametes, obtainable by the method of claim 4 13. A plant producing apomeiotic gametes, which is @ transgenic plant containing @ transgene comprising an expression casserte of claim 9 14. A method for producing Second Division Restitution 2n gametes, wherein sad method comprises cultivating a plant obtainable by & method of elim 1, and recovering the ‘gametes produced by sid plant. 15. A method for producing apomeiotc gametes, wherein suid method comprises culvating @ plant obtainable by a ‘method of claim 4, and recovering the gametes priced by sid plant 16.4 rosombinaat vector comprising an expression eas- sefte of elim 8 17. A recombinant vector comprising an expression cas- setteofelaia 9