Self-incompatibility alleles in Esatern European and Asian almond ( Prunus dulcis ) genotypes : a preliminary study

Almond [Prunus dulcis (Mill.) D.A. Webb. syn. P. amygdalus Batsch] is a member of Rosaceae familiy, Prunoideae subfamily. The Rosaceae family is one of the most important plant families in the temperate zone. This family includes economically important species (e.g. apple, apricot, plum, sweet and sour cherries, almond, strawberry and rose). Almond is commercially grown worldwide for its nuts. The putative origin of almond is in the arid mountainous regions of Central Asia (Grassely, 1976; Arús et al., 2009). Several wild species grow in the areas ranging from Tianshan through Afghanistan into Iran and Iraq (Grasselly, 1976; Kester & Gradziel, 1996). Almond shows gametophytic self-incompatibility (de Nettancourt, 2001), which is governed by the highly polymorphic, multiallelic S-locus. In pistils, the S-locus encodes for an S-ribonuclease (S-RNase) enzyme, which degrades RNA in self-pollen tubes and stops their growing (McClure et al., 1989). Almond is a typically outbreeding species, which has dramatic consequences on the genetic diversity of both wild growing and cultivated accessions (Szikriszt et al., 2011), similarly to other fruit species (Halász et al., 2011; Hegedûs et al., 2011). The S-genotypes have been identifi ed for many USA, Spanish, Italian and French almond cultivars (López et al., 2006). Cross-incompatibility, that means the mutual failure in the fruit set, is a well-known phenomenon in almond. Until now, more than forty self-incompatibility (SI) RNase alleles have been identifi ed (López et al., 2006; Ortega et al, 2006; Boskovic et al., 2007; Halász et al., 2008, 2010; Kodad et al, 2008). One allele, S f , allows for self-compatibility in its inactive form, while its active form is functional (Fernández i Martí et al., 2010). The application of DNA-based molecular markers allows for the early selection of the common S-alleles (Martínez-Gómez et al., 2007; Szikriszt et al., 2011). Rapid and reliable S-genotyping protocols were reported based on the PCR amplifi cation of the fi rst and the second intron regions in the S-RNase gene using degenerate primers and fragment size determination (Ortega et al., 2005). This work is the fi rst information from a comprehensive study to determine the S-genotype of some Eastern European almond cultivars and some Turkish wild growing seedlings to characterize their S-allele pool.


Introduction
Almond [Prunus dulcis (Mill.)D.A. Webb.syn.P. amygdalus Batsch] is a member of Rosaceae familiy, Prunoideae subfamily.The Rosaceae family is one of the most important plant families in the temperate zone.This family includes economically important species (e.g.apple, apricot, plum, sweet and sour cherries, almond, strawberry and rose).Almond is commercially grown worldwide for its nuts.The putative origin of almond is in the arid mountainous regions of Central Asia (Grassely, 1976;Arús et al., 2009).Several wild species grow in the areas ranging from Tianshan through Afghanistan into Iran and Iraq (Grasselly, 1976;Kester & Gradziel, 1996).
Almond shows gametophytic self-incompatibility (de Nettancourt, 2001), which is governed by the highly polymorphic, multiallelic S-locus.In pistils, the S-locus encodes for an S-ribonuclease (S-RNase) enzyme, which degrades RNA in self-pollen tubes and stops their growing (McClure et al., 1989).
Almond is a typically outbreeding species, which has dramatic consequences on the genetic diversity of both wild growing and cultivated accessions (Szikriszt et al., 2011), similarly to other fruit species (Halász et al., 2011;Hegedûs et al., 2011).The S-genotypes have been identifi ed for many USA, Spanish, Italian and French almond cultivars (López et al., 2006).
Cross-incompatibility, that means the mutual failure in the fruit set, is a well-known phenomenon in almond.Until now, more than forty self-incompatibility (SI) RNase alleles have been identifi ed (López et al., 2006;Ortega et al, 2006;Boskovic et al., 2007;Halász et al., 2008Halász et al., , 2010;;Kodad et al, 2008).One allele, S f , allows for self-compatibility in its inactive form, while its active form is functional (Fernández i Martí et al., 2010).
The application of DNA-based molecular markers allows for the early selection of the common S-alleles (Martínez-Gómez et al., 2007;Szikriszt et al., 2011).Rapid and reliable S-genotyping protocols were reported based on the PCR amplifi cation of the fi rst and the second intron regions in the S-RNase gene using degenerate primers and fragment size determination (Ortega et al., 2005).This work is the fi rst information from a comprehensive study to determine the S-genotype of some Eastern European almond cultivars and some Turkish wild growing seedlings to characterize their S-allele pool.

DNA extraction and genomic PCR with consensus primers
Genomic DNA was extracted from fully expanded young leaves using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).DNA concentration and purifi cation parameters were measured using a NanoDrop ND-1000 Spectrophotometer (Bio-Science, Budapest, Hungary).
PCR was conducted according to Sutherland et al. ( 2004) using the consensus degenerate primer pairs of the EM-PC2consFD and EM-PC3consRD for the amplifi cation of the second intron region of the S-RNase gene.To amplify the fi rst intron of the S-RNase gene, the fl uorescently labelled ( 6- FAM ) PaConsI-F primer (Sonneveld et al., 2003) was used in a combination with the EM-PC1consRD primer (Ortega et al., 2005).To identify the S 31 -allele, an allele-specifi c primer pair was used according to Halász et al. (2008).
Approximately 20-80 ng of genomic DNA was used for PCR amplifi cation in a 25μl reaction volume, containing 10× DreamTaq™ Green Buffer (Fermentas, Szeged, Hungary) which contains KCl and (NH 4 ) 2 SO 4 at a ratio optimized for robust performance of DreamTaq™ DNA Polymerase in PCR with fi nal concentrations of 4,5 mM MgCl2, 0.2mM of dNTPs 0.2μM of the adequate primers and 0.06 U of DreamTaq DNA polymerase (Fermentas).PCR was carried out in a PTC 200 Thermocycler (MJ Research, Budapest, Hungary) according to Sonneveld et al. (2003).
The second intron PCR products were separated by electrophoresis in 1,2 % TAE or TBE agarose gels for 2 h at 100V and DNA bands were visualized by the ethidium bromide staining.Fragment lengths were estimated by comparison with the 1 kb DNA ladder (Promega, Madison, USA).To determine the exact size of the S-RNase fi rst intron region fragments under 500 bp, the fl uorescently labelled products were run in an automated sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Budapest, Hungary).
Plasmid DNA was isolated with the E.Z.N.A. Plasmid Miniprep Kit I. (Omega Bio-tek Incl., USA) and sequenced in an automated sequencer, ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Budapest, Hungary).For each fragment, the nucleotid sequences of two clones were determined in both directions with sequencing primer M13.DNA and deduced amino acid sequences were compared using BLASTN and TBLASTN at NCBI and CLUSTAL W program (Thompson et al., 1994) and the aligned sequences were edited with BioEdit v. 7.0.9.0.

Results and discussion
The consensus primers for the fi rst and the second introns of the S-RNase gene was used for the S-genotype determination of all samples, and the sizes and sequences of the PCR products were compared with those previously published (Ortega et al., 2005;Boskovic et al., 2007;Kodad et al., 2008;Halász et al., 2008Halász et al., , 2010)).To confi rm the S-genotypes, fl uorescently labelled PCR products of the fi rst intron fragments were sized on automated sequencer.
The regions of Bademli and Akdamar Island are two distinct places of almond natural occurrence in Turkey.Figure 2 and Figure 3 show the second intron PCR analysis of the investigated Turkish almonds.Trees growing wild around Bademli village in Turkey showed greater genetic diversity than those originated on the Akdamar Island (located in lake Van).The two regions locate approx.300 km apart.The Akdamar germplasm showed relatively few S-alleles presenting only a limited number of genotypes.In conclusion, many trees had identical genotypes, which might be associated with the geographical isolation of the germplasm.Some of the previously described 45 S-RNase alleles have been also detected in these regions.More interestingly, homology searches revealed that Turkish almonds carried some P. webbii alleles indicating hybridization between the two cultivars and massive introgression events.
The most important concliusion of our study is that the exclusive application of the consensus PCR primers amplifying the fi rst and second intron regions was not suitable to the reliable discrimination of the S-RNase alleles in a germplasm of great genetic diversity.Since several alleles from other species like P. webbii also occurred and some of those alleles may have similar intron lengths as P. dulcis S-RNases, the identifi cation of the alleles seems to be challenging.Therefore, new strategies (allele-specifi c primers) will be inevitable to improve reliability in such cases.
The great genetic diversity is due to the fact that selfcompatible accessions (at least seedlings carrying the S f ) were not found in the Hungarian and Turkish germplasm.Self-compatibility in almond is a relatively new trait of limited occurrence and hence it has not been enough time to operate for inducing genetic erosion like in apricot (Halász et al., 2010b).Interestingly, the genetic constitution of the almond germplasm seemed to be quite different according to the regions investigated and the geographic isolation was clarifi ed to have strong effects on genetic variability.Our results supply long-awaited information on almond S-allele diversity from regions between the main cultivation centres (the Mediterranean Europe and California) and the centre of origin of this species.

A
total of 19 Eastern European and 70 Turkish almond accessions were used in the experiments including 39 Self-incompatibility alleles in Esatern European and Asian almond (Prunus dulcis) genotypes: a preliminary study sampled trees growing wild on the Akdamar Island and 31 trees occurring around the village Bademli (Figure 1).The local Hungarian cultivars were obtained from the orchard of the Corvinus University of Budapest, Department of Genetics and Plant Breeding in Szigetcsép, Hungary.

Figure 1 .
Figure 1.Map of Turkey, the origin of Turkish almond accessions (Bademli and Akdamar Island) are signed with small almond symbols