Developing molecular genetic markers for grape breeding; using polymerase chain reaction procedures

Stenospermocarpic seedlessness in grapes has been discussed in detail by PEARSON (1932) and STOUT (1936), and recently by RAMMING et al. (1990), SPIEGEL-RoY et al. (1990), and LEDBE"ITER and BURGOS (1993). A classification of seeded vs. seedless individuals is influenced and biased also by several extraneous factors such as berry firrnness. Although few genetic factors are assumed to be invoJved (SPIEGEL-ROY 1979; LEDBETTER and RAMMING 1989), it is hard to overlook the quantitative nature of seed development and its response to environmental effects. We compared several characters of seedlessness in an Early-Muscat x Flame-Seedless progeny. The progeny segregates as to the seedlessness subtraits, as weil as to berry colour and muscat flavor. For the present study we analysed seven variables representing traits of seedlessness: mean fresh weight of one seed, total fresh weight of seeds per berry; seed contents evaluated by perceptibility (SPtEGEL-RoY 1979); four seed size categories visually evaluated; degree of hardness of the seed coat; degree of development of the endosperm and degree of development of the embryo (STRIEM et al. 1992). Excluding seeded offsprings at an early stage would be of great value to the breeder concerned with a program · aimed at seedlessness. This will be facilitated by establishing a linkage between this trait and reliable markers. Identification of linkage between DNA markers and Joci controlling quantitative traits (QTLs) is rather complicated, requiring a ! arge number of polymorphic markers (BECKMANN and SoLLER 1986). Linear Model Analysis has been applied to quantify the effects of QTL-associated bands. A new genetic assay to detect nucleotide sequence polymorphisms by polymerase chain reaction (PCR) procedures has been developed (WELSH and McCLELLAND 1990; WtLLIAMS et al. 1990). Using the technique for producing molecular genetic markers, more than 160 different lOmer primers (Operon kits A, B, C, E, F, G, H, J, and other primers) were tested by us, some of which were highly polymorphic. Band patterns of two primers, successfully differentiated between 24 different V. vinifera grape cultivars examined (STRIEM et al., unpublished, 1992), confirming the suitability of this technique to uncover genetic variability among grape cultivars which was also demonstrated by others (GoGoRCENA et al. 1993; BüseHER et al. 1993; JEAN JAQUES et a/. 1993). Results and discussion: 110 of the primers tested, gave a distinct band pattern. 17 primers distinguished between two closely related cultivars, one is a mutant of the other (Sultanina vs. Seeded Sultanina). Bandsharing ratio of 96.1 %was found in this pair, compared to 78.5% bandsharing ratio between two nonrelated cultivars (Early-Muscat vs. Flame-Seedless). 78 primers which uncovered polymorphism between Early-Muscat and Flame-Seedless were tested with 82 individuals of the progeny resulting from the cross between these cultivars. Three traits which segregate in the progeny were investigated: seedlessness, muscat flavor and berry color. More than 400 polymorphic markers were identified. 12 markers were statistically analysed, using SAS procedures. Significant correlations with several of the seven subtraits of seedlessness were obtained, mainly with the quantitative ones. For example: primer OP-E10, band