Identification and characterization of genomic DNA sequences of the S‐ribonuclease gene associated with self‐incompatibility allelesS₁toS₅in European pear

Abstract

The stylar products of the S-locus for the gametophytic self-incompatibility (GSI) system in the Rosaceae are ribonucleases (S-RNases). Recently, sequences for 13 pear S-RNase alleles have been published and named following a letter-symbol nomenclature (S a to S d and S h to S p ). To establish the correspondence between these sequences and the self-incompatibility alleles we have described previously (S 1 to S 5 ), we have amplified genomic DNA with consensus primers from the cultivars, ÔWilliamsÕ (S 1 S 2 ), ÔCosciaÕ (S 3 S 4 ), ÔButirra Precoce MorettiniÕ (S 1 S 3 ), ÔSanta Maria MorettiniÕ (S 2 S 3 ) and ÔDoyenne du ComiceÕ (S 4 S 5 ) and identified PCR products specifically associated with each S allele. Cloning and sequencing of the amplification products has revealed that they correspond to European pear sequences already deposited in the database. This allowed us to link S-RNase sequences with S allele phenotypes and to determine a correspondence between the symbol-letter nomenclature used to name S-RNase sequences and the number-based nomenclature used to name S alleles. Based on this result the prediction of new cross-incompatibilities among pear cultivars is discussed. Finally, we propose a unified number-based nomenclature to avoid future confusion denominating S alleles in pear.

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles

“…Sequence identities between new and previously reported alleles varied between 59% (S 14 vs. S 20 ) and 93% (S 5 vs. S 23 ). These values fit within the range of variation (59-92%) observed for S 1 through S 14 , the S-RNases found associated with 14 Salleles as determined by pollination experiments (Sanzol et al 2006;Sanzol and Robbins 2008). Together, these results suggested that the genomic regions characterized in this study correspond to the S-RNases of new S-alleles of European pear.…”

“…The new S-RNases have all the structural features expected for the Maloideae S-RNase, including the conserved and variable regions, and several residues important for the secondary structure and activity of the protein (Ishimizu et al 1996(Ishimizu et al , 1998. Additionally, sequence identities between the new alleles and previously reported S-RNases varied within the same range calculated for S 1 through S 14 , which have distinct recognition specificities (Sanzol et al 2006;Sanzol and Robbins 2008). Pollination tests confirmed that cultivars bearing S 20 or S 21 and having the same S-genotype were cross-incompatible.…”

“…Jules Guyot' (S 1 S 5 ) × 'Bella di Giugno' (S 4 S 20 )], and the S-genotypes assigned to them were in agreement with the inheritance of each allele from one of their progenitors. Moreover, the two cultivars Red Sensation and Rosired and the cultivar Anjou Draft, which are bud mutant derivatives from cultivars with known Salleles, had the same genotypes as those reported for their ancestors 'Williams' (Sanzol et al 2006) and 'Beurré D'Anjou' (Moriya et al 2007), respectively. Among the S-genotypes assigned to diploid cultivars, ten represented new genotypes.…”

“…Leaves for DNA extraction were processed immediately after harvest or kept at −20°C until use. Total genomic DNA was prepared as described (Sanzol et al 2006), following a cetyl trimethylammonium bromide (CTAB) extraction procedure (Hormaza et al 1994). …”

“…In a series of pollination studies, we discriminated between 14 S-alleles (S 1 -S 14 ) and classified cultivars into 15 incompatibility groups based on combinations of these alleles (Sanzol and Herrero 2002;Sanzol and Robbins 2008). The characterization of SRNases in reference cultivars with known S-phenotypes has allowed identification of genomic sequences associated with each S-allele and correlation of the S-phenotypes deduced from test crosses with the S-RNase genotypes inferred from polymerase chain reaction (PCR) analysis (Sanzol et al 2006;Sanzol and Robbins 2008). These results confirmed the utility of the S-RNase gene for molecular S-genotyping of the species.…”