REPORTS BREEDING, CULTIVARS, ROOTSTOCKS, AND GERMPLASM RESOURCES HORTSCIENCE 39(5):943–947. 2004. determined but their presumed gene products were detected by RNase zymogram analysis (Boškovic and Tobutt, 1999). However, so far Update on and Review of the all of the RNases for which the corresponding gene sequences are unknown, except S6 and S8, Incompatibility (S-) Genotypes of have been detected only in a single, generally obsolete cultivar. For some of them, evidence Apple Cultivars has been reported showing that they correspond to known S-alleles, which, if confi rmed, would Wim Broothaerts1 exclude their existence as distinct S-alleles Better3Fruit N.V., Willem de Croylaan 42, B-3001 Leuven, Belgium (Broothaerts, 2003; Matsumoto et al., 2003). Several other new S-alleles that were proposed Ilse Van Nerum and Johan Keulemans by Tobutt’s group may, therefore, also appear Fruitteeltcentrum K.U.Leuven, Willem de Croylaan 42, B-3001 Leuven, to be false if adequately examined. We have, Belgium therefore, decided to limit our investigations to the 16 S-alleles that are characterized at the Additional index words. Malus ×domestica, allele-specifi c PCR, S-allele, self-incompatibility, nucleotide sequence level. Additionally, the S6 pollination and S8-allele were included in our study, as these are characterized both phenotypically and Abstract. Apple cultivars display a self-incompatibility system that restricts self-fertilization at the protein level. While S has been found and fertilization between cultivars bearing identical S-alleles. There has been considerable 6 in two uncommon, local Swiss cultivars, S8 progress in identifi cation of S-alleles in apple in recent years and methods are now avail- has been discovered in a few more common able for the accurate S-genotyping of cultivars. Following a recently revised numerical cultivars (Kobel et al, 1939; Boškovi and identifi cation system for apple S-alleles, we present the fi rst extensive compilation of apple Tobutt, 1999). cultivars with their S-genotypes. This list contains data from our own investigations using Broothaerts (2003) validated the revised S-allele-specifi c PCR methodology, including a number of new data, as well as published conditions for S-allele analysis by analysis of data from various other sources. Eighteen different S-alleles are discriminated, which al- a collection of old apple cultivars that were lowed the determination of the S-genotypes for 150 diploid or triploid European, American, genotyped by Kobel et al. (1939) through ex- and Japanese cultivars. Many of these cultivars are cultivated worldwide for their fruit. tensive cross-pollination studies. Here we have Also included are a number of old, obsolete cultivars and a few nondomestic genotypes. applied the same methodology to assay the S- We observed a wide variation in the frequency of S-alleles in the apple germplasm. Three allele genotypes of a number of generally more S-alleles (S2, S3, and S9) are very common in the cultivars evaluated, presumably as a result common European, American and Japanese of the widespread use of the same breeding parents, and seven alleles are very rare (S4, apple cultivars. Our results were complemented S6, S8, S16, S22, S23, S26). by the inclusion of data from various other sources, resulting in an extensive overview of The production of apples generally requires Several alleles of the S-gene in apple have the current knowledge of the S-genotypes of that pollen be transferred from one cultivar (the been identifi ed and their nucleotide sequences primarily domestic apple cultivars. pollinizer) to the stigmas of the cultivar to be have been determined (Broothaerts, 2003). pollinated (main fruit-bearing cultivar). There, Based on the diversity of nucleotide sequences Experimental Protocols the pollen grains germinate and produce a tube encoding the S-RNase family members, a that enters the stigmatic tissue and elongates method has been developed using allele-spe- The plant material used was primarily through the style towards the ovules where cifi c PCR primers to selectively amplify and derived from the extensive gene bank of the fertilization takes place. During their growth, identify particular S-alleles (Janssens et al., Fruitteeltcentrum K.U.Leuven, with a few the pollen tubes are attacked by abundant 1995). This method was recently re-examined, cultivars from the Brogdale (UK) collection. cytotoxic proteins that enter their cytoplasm. modifi ed, and extended for the identifi cation Unless otherwise indicated, the methods Lethal attack is avoided through the expression of additional alleles (Broothaerts, 2003). At used in our work were described in Broothaerts of specifi c inhibitors of these proteins, allow- the same time, the annotation of S-alleles was (2003). Genomic DNA was isolated from ing pollen tube growth to proceed (Golz et al., revised because of the occurrence of more than leaves of various cultivars, amplifi ed with al- 2001). This mechanism appears to be the way one symbol for some S-alleles in the literature. lele-specifi c primers by PCR, and the fragments separated by agarose gel electrophoresis. For the gametophytic self-incompatibility system The new numbering introduced S16, S19, S22, the unique identifi cation of a few S-alleles (S , operates. The style-encoded toxic proteins are and S23, as a replacement of the former S27a (in 4 S , S , S ), allele-specifi c restriction enzymes the products of the S-gene, which are the S- ‘Baskatong’), S28/Sd (in ‘Delicious’), S27b (in 16 20 22 were employed to digest the amplifi ed products RNases. The pollen-expressed inhibitors of the ‘Alkmene’), and the erroneously assigned “S10” pollen-S gene have not been identifi ed. Both (in ‘Granny Smith’), respectively. Following before gel analysis. Of the 18 S-alleles evalu- genes are part of the polymorphic S-locus. further investigations (S. Matsumoto, personal ated here, only S6 and S8 were not determined by allele-specifi c PCR because their nucleotide If the alleles of the S-gene and of pollen-S communication), it appeared that S19 is a rare, match, i.e. they have the same S-locus speci- but distinct allele, and hence the proposed sequences are unknown. The presence of these two alleles was inferred from the analysis of fi city, an incompatibility reaction is elicited, merger with S28 as suggested by Broothaerts which is manifested by the arrest of pollen (2003) should be corrected. In this paper, we their stylar RNase patterns by Boškovic and Tobutt (1999). Also we did not determine the tube growth somewhere in the style. In such therefore refer to S28 for the S-allele of Deli- presence of the S -allele, which only recently cases, the inhibitors produced in the pollen cious and keep S19 for the S-allele assigned to 25 tube were apparently unable to inactivate the Bohnapfel (Boškovi and Tobutt, 1999) and has been sequenced. stylar S-RNases. sequenced by Matsumoto (unpublished data). Allele-specifi c analysis conditions have been Results described for the detection of 15 S-alleles Received for publication 2 Jan. 2003. Accepted for that operate in the domestic apple germplasm We have assayed a large number of apple publication 23 June 2003. cultivars by allele-specifi c PCR to evaluate (Broothaerts, 2003). An additional allele, S25, 1Current address: Center for the Application of has recently been cloned and sequenced and cross-incompatibility relationships in the Molecular Biology to International Agriculture a PCR/digestion method was suggested for its apple germplasm. Complementing our previ- (CAMBIA), GPO Box 3200, Canberra, ACT 2601, detection (Kitahara and Matsumoto, 2002). The ous report on the S-genotypes of a collection Australia; email [email protected]. sequences of a few other alleles have not been of old cultivars with known incompatibility HORTSCIENCE VOL. 39(5) AUGUST 2004 943 77648-Breed.indd648-Breed.indd 994343 66/22/04/22/04 99:41:23:41:23 AAMM Table 1. S-allele genotypes of apple cultivars. Cultivar S-genotype Referencez Parentage (if known) Adam’s Pearmain S1S3S10 16 Ahrista (scab resistant) S3S10 17 Elstar x TSR15T3 Akane S7S24 10, 11, 13 Jonathan x Worcester Pearmain Akita Gold S2S9 11 Alkmene S5S22 8, 14 Geh. Dr. Oldenburg x Cox O.P. Amanishiki S1S7 11, 13 Ralls Janet x Indo Ambitious S2S9 13 Toko x ? American Summer Pearmain S1S20 11 Arlet S2S7 8, 17 Golden Delicious x Idared Baskatong S16S26 7, 16 y Beni No Mai S7S24 13 Fuji x ? Berner Rosen S1S2 1, 8, 16 Boskoop (=Belle de Boskoop) (3n) S2S3S5 1, 8, 16 Braeburn, Braeburn Hillwell S9S24 7, 8 ? Lady Hamilton x ? Brünnerling S5S7S10 1, 8, 16 Cameo S2S28 13 Charlotte S5S10 8 McIntosh Wijcik x Greensleeves Champagner Reinette S2S4 1, 8, 16 Clivia S5S9 17 Cox Orange Pippin, Queen Cox S5S9 4, 8, 10, 11 Ribston Pippin x ? Danziger Kantapfel S2S7 1, 8, 16 Delbard Jubilé S2S22 14 Golden Delicious x Lunbytrop Delbarestivale, Delcorf S3S10 17 Golden Del. x Stark Jongrimes Delicious, Topred, Starking Delicious S9S28 11, 12, 16 Discovery S10S24 17 Worc Pearmain x Beauty of Bath Ecolette (scab resistant) S3S10 17 Elstar x Prima Elstar, Red Elstar, Elshof S3S5 4, 8 Golden Delicious x Ingrid Marie Empire S10S28 15 McIntosh x Delicious Falstaff S2S5 8 James Grieve x Golden Delicious Fiesta (=Red Pippin) S3S5 4, 8 Cox O.P. x Idared Florina (scab resistant) S3S9 17 Jonathan x 612-1 Fuhong S9S20 11 Fuji S1S9 2, 5, 8, 11, 12, 17 Ralls Janet x Delicious Fukutami S2S9 11 Gala (Galaxy, Regal, Royal) S2S5 4, 6, 8 Kidd’s Orange Red x Golden Del Ginger Gold S3S28 13 Ginrei S3S7 11 Gloster S4S28 16 Glockenapfel x Richared Del. Golden Delicious (Reynders, Smoothee, B) S2S3 2, 3, 6, 8, 11 Grimes Golden x ? Golden Melon S2S7 11 Golden Supreme S3S28 13 Goldrush (scab resistant) S2S28 13, 17 Golden Delicious x Co-op 17 Granny Smith S3S23 16 French crab seedling Greensleeves S2S5 8 James Grieve x Golden Delicious Hacnine (3n) S1S3S9 13 Fuji x Tsugaru Hatsuaki S3S9 11 Himekami S7S9 6, 11 Fuji x Jonathan 3 Hokuto (3n) S1S7S9 2, 13 Fuji x ?Mutsu Holly S9S28 11, 12 Jonathan x Delicious Holsteiner Cox S5S9 17 Cox O.P.