IDENTIFICATION OF RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS FORSELF-INCOMPATIBILITY ALLELES IN CORYLUS-AVELLANA L

Random amplified polymorphic DNA (RAPD) markers were identified for se lf-incompatibility (SI) alleles that will allow marker-assisted select ion of desired S-alleles in hazelnut (Corylus avellana L.). DNA was ex tracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S1S12) x VR6-28 (S2S26) (OSU23 x VR6) . Screening of 10-base oligonucleotide RAPD primers was performed usin g bulked segregant analysis. DNA samples from 6 trees each were pooled into four 'bulks', one for each of the following: S-1 S-2, S-1 S-26. S-2 S-12, and S-12 S-26 'Super bulks' of 12 trees each for S-1, S-2, S -12, and S-26 were then created for each allele by combining the appro priate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S-2 (OP I07(750)) and S-1 (OPJ14(1700)) OPJ14(1700) was identified in 13 of 14 S-1 individuals of the cross OSU23 x VR6 used in bulking and yielded a false positive in 1 non-S-1 individual. This same marker was not eff ective outside the original cross, identifying 4 of 5 S-1 progeny in a nother cross, 'Willamette' x VR6-28 ('Will' x VR6), but yielded false positives in 4 of 9 non-S-1 individuals from the cross 'Casina' x VR6- 28 ('Cas' x VR6). OPI07(750) served as an excellent marker for the S-2 allele and was linked closely to this allele, identifying 12 of 13 S- 2 individuals in the OSU23 x VR6 population with no false positives. O PI07(750) was found in 4 of 4 S-2 individuals from 'Will' x VR and 7 o f 7 S-2 individuals of 'Cas' x VR6 with no false positives, as well as 10 of 10 S-2 individuals of the cross OSU 296.082 (S1S8) x VR8-32 (S2 S26), with only 1 false positive individual out of 21 progeny. OPI07(7 50) was also present in 5 of 5 cultivars carrying the S-2 allele, with no false-positive bands in non-S-2 cultivars, and correctly identifie d all but 2 S-2 individuals in 57 additional selections in the breedin g program. In the OSU23 x VR6 population, the recombination rate betwe en the marker OPJ14(1700) and the S-1 allele was 7.6% and between the OPI07(750) marker and the S-2 allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of l onger primers (18 or 24 bp) that were used to obtain sequence characte rized amplified regions (SCARs). Both the S-1 and S-2 markers were suc cessfully cloned and 18 bp primers yielded the sole OPJ14(1700) produc t, while 24-bp primers yielded OPI07(750) as well as an additional sma ller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined.