Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L

Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted 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 using bulked segregant analysis. DNA samples from 6 trees each were pooled into four 'bulks', one for each of the following: S1 S2, S1 S26, S2 S12, and S12 S26. 'Super bulks' of 12 trees each for S1, S2, S12 and S26 were then created for each allele by combining the appropriate 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 allele S2 (OPI07(750) and S1 (OPJ14(1700). OPJ14(1700) was identified in 13 of 14 S1 individuals of the cross OSU23 X VR6 used in bulking and yielded a false positive in 1 non-S1 individual. This same marker was not effective outside the original cross, identifying 4 of 5 S1 progeny in another cross, 'Willamette' X VR6-28 ('Will' X VR6), but yielded false positives in 4 of 9 non-S1 individuals from the cross 'Casina' X VR6-28 ('Cas' X VR6). OPI07(750) served as an excellent mark for the S2 allele and was linked closely to this allele identifying 12 of 13 S2 individuals in the OSU23 X VR6 population with no false positives. OPI07(750) was found in 4 of 4 S2 individuals from 'Will' X VR and 7 of 7 S2 individuals of 'Cas' X VR6 with no false positives, as well as 10 of 10 S2 individuals of the cross OSU 296.082 (S1S8) X VR8-32 (S2S26), with only 1 false positive individual out of 21 progeny. OPI07(750) was also present in 5 of 5 cultivars carrying the S2 allele, with no false-positive bands in non-S2 cultivars, and correctly identified all but 2 S2 individuals in 57 additional selections in the breeding program. In the OSU23 X VR6 population the recombination rate between the marker OPJ14(1700) and the S1 allele was 7.6% and between the OPI07(750) marker and the S2 allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S1 and S2 markers were successfully cloned and 18 bp primers yielded the sole OPJ14(1700) product, while 24-bp primers yielded OPI07(750) as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined.