US 20120017291A1 (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2012/0017291 A1 SUPERAK et a]. (43) Pub. Date: Jan. 19, 2012 (54) SQUASH LEAF CURL VIRUS (SLCV) C12Q 1/68 (2006.01) RESISTANCE IN CUCURBITS A01H 5/06 (2006.01) C07H 21/04 (2006.01) (75) Inventors: Ted SUPERAK, Davis, CA (US); A01H 5/10 (2006.01) Julie Fauve, Nimes (FR); Eric C12N 5/04 (2006.01) Lionneton, Bouchemaine (FR) (52) US. Cl. ....... .. 800/260; 800/298; 800/310; 435/410; (73) Assignee: Vilmorin & Cie, Paris (FR) 536/23.1; 435/611 21 A 1.N .: 12/836 888 ( ) pp 0 ’ (57) ABSTRACT (22) Filed: Jul‘ 15’ 2010 The invention provides a neW Severe Leaf Curl Virus (SLCV) _ _ _ _ resistant gene slc-2 in cucurbit plants and plants comprising Pubhcatlon Classl?catlon the slc-2 gene. The invention also provides molecular mark (51) Int, Cl, ers linked to slc-2 gene. The invention further provides meth A01H 1/02 (200601) ods of breeding to produce plants that are resistant to SLCV, A01 H 5/00 (200601) and the resistant plants produced by such methods. Patent Application Publication Jan. 19, 2012 Sheet 2 0f 2 US 2012/0017291 Al was:2% 00m.fmum95mm;2%5:82.2226.lll-v2_#0 I xNewAll.8E‘III‘III.35536x 5.323 053050WEE82.0:25>$3128 m|=z AwVhQHWN-WUU>HZQF2USE-WQ-PCQ-uNUOTUZ-JE{t K Ew??om35.596355> oibocwmmAmwmuwamE?wmmwh 22mEQEQQEQQ08-8: as.gm6.3 N@552 E2202". US 2012/0017291A1 Jan. 19, 2012 SQUASH LEAF CURL VIRUS (SLCV) alternative form of resistance to SLCV. The resistance of the RESISTANCE IN CUCURBITS present invention is imparted by the neWly discovered reces sive gene slc-2. TECHNICAL FIELD SUMMARY [0001] The application relates to a novel Squash Leaf Curl [0007] The present invention provides cucurbit seeds con Virus (SLCV) resistance gene identi?ed in Cucurbila taining one or tWo copies of a recessive gene for resistance to moschala plants; non-C. moschala cucurbit plants, including geminiviruses. The recessive resistance gene is designated as inbred plants and hybrid plants, having said resistance gene; slc-2. In one embodiment, the geminivirus is Squash Leaf molecular markers associated With the resistance gene; meth Curl Virus (SLCV). In one embodiment, said cucurbit seed is ods of developing plants With resistance to SLCV and plants a squash seed. In one embodiment, the SLCV resistant gene developed by such methods. slc-2 is present in the cucurbit seeds deposited as NCIMB Accession No. 41728. BACKGROUND [0008] The present invention provides a non-Cucurbila moshala cucurbit seeds Whose genome contains at least one [0002] Cucurbitaceae is a plant family commonly knoWn as copy of a recessive allele of a slc-2 gene coding for resistance melons, gourds or cucurbits and includes crops like cucum to Severe Leaf Curl Virus (SLCV), said at least one allele bers, squashes (including pumpkins), luffas, melons and being present in the seeds deposited as NCIMB Accession Watermelons. According to Food and Agriculture Organiza No. 41728. In one embodiment, the genome of the non-C. tion (FAO, 2002), World production of Watermelon exceeded moshala cucurbit seeds contain tWo copies of the recessive 80 million tons; cucumber exceeded 36 million tons; melon alleles of the slc-2 gene. The present invention also provides exceeded 22 million tons; and squash exceeded 17 million plants, explants, scions, cuttings, seeds, fruit, rootstock, pol tons. len, ovules, and/or plant parts produced by groWing said non-Cucurbila moshala cucurbit seeds. In some embodi [0003] A variety of pathogens (virus, fungi, bacteria, nema ments, the cucurbit plants are squash plants, including geneti todes, and insects) affect the productivity of cucurbits (Blan cally related squash populations comprising such squash chard et al., 1994A color atlas of cucurbit diseases, NeWYork: plants. The present invention also provides tissue cultures of Manson Publishing/ John Wiley; Zitter et al., 1996 Compen regenerable cells of the non-C. moshala cucurbit plants of the dium of cucurbit diseases, St Paul, Minn., APS Press). Cucur present invention, Wherein the regenerable cells are derived bits are susceptible to many viruses and virus resistance is therefore of major agricultural importance (Provvidenti, from embryos, protoplasts, meristematic cells, callus, pollen, 1993, Resistance to viral disease of cucurbits, In Kyle, M. M., leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, ed. Resistance to viral diseases ofvegelables, Portland, Oreg., ?oWers, cotyledons, and/or hypocotyls. Timber Press, 1993: 8-43). [0009] The present invention also provides methods for producing a cucurbit seed that can groW into a cucurbit plant [0004] The taxonomic family Geminiviridae includes some comprising at least one recessive allele of a slc-2 gene, said of the most important plant viruses causing severe diseases in methods comprising groWing non-Cucurbila moshala cucur agricultural, ornamental and horticultural crops. During the bit seeds Whose genome contains at least one copy of a reces last three decades numerous White?y-transmitted Begomovi sive allele of a slc-2 gene coding for resistance to Severe Leaf ruses, a genus of geminivirus, have emerged as devastating Curl Virus (SLCV), crossing the resultant cucurbit plants as pathogens, particularly in the tropics and subtropics, causing the ?rst parent plant With a second parent plant, and harvest huge economic losses and threatening crop production. ing the resultant seed. In one embodiment, such methods Squash Leaf Curl Virus (SLCV), a begomovirus, can cause comprise backcrossing to one of said parent plants for tWo or severe losses in many areas of squash production, but particu more generations. In another embodiment, such methods larly in the major squash groWing areas of Mexico, AriZona, comprise selecting a plant having at least one copy of the California and the Middle East. SLCV Was ?rst observed in recessive allele of the slc-2 gene in each generation. In squash in California during 1977 and 1978 (Flock RA, May another embodiment, the selection comprises molecular heW Del., 1981 Squash leaf curl, a neW disease of cucurbits in marker assisted selection, Wherein the molecular marker California, Plant Dis. 65:75-76) and in cultivated buffalo assisted selection may comprise using any one of the molecu gourd in Arizona at about the same time (Rosemeyer et al., lar markers Whose de?ning primers are listed in Table 8. In 1986 Five viruses isolated from ?eld-groWn buffalo gourd, another embodiment, the second parent plant has one or more Cucurbila foelidissima HBK, a potential crop for semi-arid preferred economically important traits, such as resistance to lands, Plant Dis 70:405-409). one or more biotic and/or abiotic stresses. In yet another [0005] Current methods of preventing and controlling embodiment, such methods comprise selecting a plant having geminivirus include controlling the spread of insect vectors tWo copies of the recessive allele of the slc-2 gene in each that carry the virus, developing transgenic plants expressing generation. the viral coat protein, and using classical breeding methods to [0010] The present invention also provides genetic markers develop plants having natural resistance to the virus. Disease linked With a slc-2 gene. In some embodiments, such genetic resistant plants developed using classical plant breeding offer markers include restriction fragment length polymorphisms an effective, safe, and relatively less expensive method of (RFLPs), isoZyme markers, allele speci?c hybridization controlling many crop diseases. For example, US. Pat. No. (ASH), ampli?ed variable sequences of plant genome, self 7,432,420 describes a squash plant With resistance to SLCV sustained sequence replication, simple sequence repeat due to a dominant resistance gene in the squash genome. (SSR), and ampli?ed fragment length polymorphisms [0006] The present invention provides cucurbit plants, (AFLPs). Some AFLP markers useful in the present invention including squash plants, Which have an important, useful and are de?ned by the primers in Table 8. The present invention US 2012/0017291A1 Jan. 19, 2012 also provides methods of identifying a SLCV resistant cucur Wherein said progeny retains resistance to SLCV. In one bit plant by identifying at least one genetic locus linked With embodiment, at least 5% of said progeny is resistant to SLCV. the genetic markers of the present invention, Wherein such For example, about 5% to about 15%, about 16% to about methods comprise: (i) detecting the presence of at least one 25%, 26% to about 50%, 51% to about 99%, or more of said genetic locus in the plant, Wherein the genetic locus localiZes progeny are resistant to SLCV. to a chromosome interval ?anked on either side by one or [0017] The present invention also provides genetic markers more genetic markers of the present invention; and (ii) iden that are tightly linked to the slc-2 gene. tifying the plant comprising the genetic locus. In some [0018] The present invention also provides a method of embodiments, such methods utiliZe a Whole plant, a plant isolating a nucleic acid sequence designated as slc-2 gene organ, a plant seed, or a plant cell for the identi?cation. In one conferring resistance to SLCV, comprising: a) crossing the embodiment, the methods further comprises constructing a SLCV resistant plant of the present invention as a donor With genetic linkage map of said plant using the genetic maker of a suitable susceptible plant to produce offspring plants as a the present invention. mapping population; b) challenging said offspring plants [0011] The present invention also provides methods of With said virus and determining the resistance in said off introducing a slc-2 resistance gene into a recipient plant com spring population; and c) cloning the slc-2 gene. In one prising crossing a non-Cucurbila moshala cucurbit plant embodiment, said cloning step comprises molecular marker Whose genome contains at least one copy of a recessive allele assisted mapping and cloning.