Short communication. Cloning and sequencing of partial genomic DNA fragments corresponding to the S11 and S12 alleles of the Spanish almond cultivar ‘Marcona’

This paper reports the cloning and partial genomic DNA sequencing of two S-RNases (S11 and S12) of almond. DNA from the Spanish almond cultivar Marcona, the most highly appreciated in Spain, was amplified by PCR using the primer pair AS1II and AmyC5R designed for the conserved regions of almond S-alleles. The cloned and sequenced S11 allele of cv. Marcona appears to be identical to that of cvs. Rumbeta and Bertina (which have been previously cloned and sequenced). This is the first time, however, that S12 has been sequenced in almond. The amplified and cloned S12 allele fragment possessed 1080 bp corresponding to the second intron and 506 bp corresponding to the second and third exons. The product of PCR-specific amplification showed that cv. Marcona and the Portuguese cultivar Pestaneta carry the same S12 allele. Knowledge of the sequences of these alleles will be helpful in the design of specific primers for S11 and S12, and could be of use when employing antisense techniques in genetic engineering projects. Additional key words: PCR, Prunus amygdalus Batsch, self-incompatibility, stylar ribonucleases.

Knowledge of the allelic composition of almond cultivars is important in orchard design; it helps to ensure cross-pollination and good crop yields. It is also important for choosing parental plants when performing crosses. Traditionally, the alleles carried have been determined by test crosses in the field (Crossa-Raynaud and Grasselly, 1985), a time-consuming technique, the results of which are strongly influenced by the environment. Recently, it has been shown that the selfincompatibility S-alleles of almond (Bos v ković et al., 2003) code for specific glycoproteins in the style tissue, as in other Rosaceae (Sassa et al., 1996). These possess ribonuclease activity that inhibits pollen tube growth.
The development of new molecular techniques, such as the use of genomic DNA in PCR analysis (Tamura et al., 2000), has dramatically advanced the identification of S-alleles in almond. However, the primers developed by Tamura et al. (2000) do not always distinguish alleles with a similar number of nucleotides, such as S 5 and S 25 (López et al., 2004), or S 3 and S f (Sánchez-Pérez et al., 2004). Allele sequencing is therefore often advisable for identifying almond S-alleles.
The allelic composition of the Spanish almond cultivar Marcona (S 11 S 12 ), the most highly appreciated in Spain, was established using stylar RNases by Bos v ković et al. (2003). However, the allelic sequences remained unknown. The aim of this work was to establish the DNA genomic sequence of the S 11 and S 12 alleles in this cultivar.
The PCR reactions were very eff icient in the identification of almost all the S-alleles of the cultivars reported in this study (S 1 , S 3 , S 6 , S 11 , S 12 , S 13 , S 23 and S 25 ) (Fig. 1). The PCR-amplified fragments of genomic DNA of cv. Marcona were of about 700 bp for S 11 and 1600 bp for S 12 , in agreement with that reported by Sánchez-Pérez et al. (2004).
Genomic DNA was inserted into the 3.9 kb vector pCR ® 2.1 using the TA Cloning ® Kit (Invitrogen) and confirmed both by restriction enzyme digestion with EcoRI and by PCR using the same primers. The positive clones were isolated and the plasmid DNAs purified using the QIAquick PCR Purification Kit (Qiagen). DNA sequence data were analysed using the BLAST (NCBI) program (Altschul et al., 1997) and the Clustal W program (DNAStar Inc.). The nucleotide sequence data was deposited in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers DQ437650 for S 11 and DQ437651 for S 12 .
Sequencing included the second and third exons of the S 11 and S 12 alleles of cv. Marcona as well as the second intron between these two exons (Fig 2). The partial nucleotide sequence for cv. Marcona S 11 was determined to be of 686 bp after sequencing. The intron size, determined by matching the partial exon sequence with those of S 1 , S 3 , S 6 and S 23 (Fig. 2), was 194 bp. The partial exon sequence of the S 11 -gene amplified in this study was 100% identical to that reported for cvs. Bertina (GenBank AF487915) and Rumbeta (GenBank AF510415).
The S 11 allele band amplified from cv. Marcona was of the same size as the S 11 from cv. Bertina (Fig. 1), and after sequencing was found to show 100% homology 200 100 S11 S11 S25 S23 S13 S12 S12 S3 S6 S1 S1  Figure 2. Sequence alignment of alleles S 1 and S 3 from cv. Ferragnès (acc. nos. AF 149039 and AF 510417, respectively), S 6 and S 23 from cv. Ramillete (AF 510419 and AF 454002, respectively), and S 11 and S 12 from cv. Marcona (this work). The front exons start at 1-158 bp, the partial introns start at 159-871 bp, and back exons start at 872-1,196 bp. The AS1II and AMYC5R primer regions are underlined. Arrows indicate the primer orientation. ᮣ ᮣ with S 11 of cvs. Bertina and Rumbeta. These cultivars therefore share the same S 11 allele, although its origin is unknown (Felipe, 2000). The presence of S 11 in these cultivars, all of which originated in eastern Spain, suggests they have a common ancestor.

S3 T A A G A A G A C T C A G T T G T T A C A T G A A G T G G T A ---S11 T A C T A A T A C T C A G T T G T T A C A T G A A G T G G T A ---S23 T A A G A A T A C T C A G T T G T T A C A T G A A ---------S6 T A A G A A G A C T C A G T T G T T A C A T G A A G T G G T A ---S1 G C C G A A G A C C C A G T T G T T A C A T G A A G T G G T A ---S12 -----A G A C T C A G T T G T T A C A T G A A G T G G T A T T T
The S 12 band amplified from the genomic DNA of cv. Marcona was of the same size as that of the S 12 band of cv. Pestaneta (Fig. 1). Based on stylar ribonuclease analysis, Certal et al. (2002) assigned the S 12 S 23 allele composition to cv. Pestaneta obtained from the collection belonging to the Direcção Regional de Agricultura de Trás-os-Montes (Portugal). Using the consensus primer pairs PaConsI-F and EM-PC1consR (developed at the East Malling Research Station, UK), Ortega et al. (2005) amplified a band from the genomic DNA of cv. Pestaneta that corresponded to the band size amplified in cv. Marcona. These results, together with those obtained herein, show that the S 12 amplif ied in cv. Marcona appears to be the same as that in cv. Pestaneta.
The partial nucleotide sequence for cv. Marcona S 12 , cloned and sequenced here for the first time, involved 1584 bp (Fig. 2). As with other almond S-RNases, this sequence included the second intron (1080 bp). The partial sequence of the second and third exons was about 504 bp long. When this sequence and those of S 1 , S 3 , S 6 and S 23 were aligned (Fig. 2), the S 11 exons showed 80, 85.5, 84.9 and 82.5% homology with S 1 , S 3 , S 6 and S 23 respectively, while the S 12 exons showed 85.3, 85.1, 86.5 and 83.3% homology respectively. The sequence of the intron/exon splice junction regions followed the GT/AG consensus sequence rule (Thangstad et al., 1993); thus, the sequence adjacent to the splice junctions was highly conserved (Fig. 2).
The nucleotide composition of the partial exons, and of the second intron, for each S-allele showed a higher proportion of A+T nucleotides (69.35% for S 11 and 64.26% for S 12 ) than C+G nucleotides. The partial sequence of the exons contained a higher proportion of C+G nucleotides (44.85% for S 11 and 42.09% for S 12 ) than the introns (30.41% for S 11 and 35.74% for S 12 ). The deduced amino-acid sequence of the S 12 allele (Fig. 3) shows the expected ribonuclease T2 family conserved domain of Rosaceae (Sassa et al., 1996).
Knowledge of the S-allele sequences of cv. Marcona will be helpful in the design of specific primers for S 11 and S 12, and in the identif ication of allelic selfincompatibility groups in Spanish almond cultivars. It may also be useful for modifying the self-incompatible nature of this very important cultivar, for instance via the use of antisense technology in genetic engineering projects.