Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes

Abstract

The work aimed to develop a reliable and convenient PCR approach for determining incompatibility S genotypes in almond. Initially, genomic DNAs of 24 accessions of known S genotype were amplified with novel consensus primers flanking the first and second introns of the S-RNase gene. The PCR products separated on agarose showed length polymorphisms and correlated well with the reference alleles S 1 -S 23 and S f . In addition, to improve discrimination between alleles of similar sizes, the same sets of primers but fluorescently labelled were used, and the products sized on an automated sequencer. These fluorescent primers were particularly informative in the case of the first intron, variation in the length of which has not been used previously for S genotyping in almond. Some reference alleles showed the same patterns with first and second intron primers, and others showed a microsatellite-like trace. Subsequently, the S genotypes of 26 cultivars not genotyped previously and of four of uncertain genotype were determined. An allele described in Australian work as putative S 10 was shown to be a ÔnewÕ allele and ascribed to S 24 and evidence of five more ÔnewÕ S alleles was found, for which the labels S 25 -S 29 are proposed. This PCR approach should be useful for genotyping in other Prunus crops.Most cultivars of almond [Prunus dulcis (Mill.) D.A. Webb] are self-incompatible and some are cross-incompatible (Tufts and Philp 1922). The incompatibility system is of the gametophytic type and controlled by a multi-allelic S locus (Gagnard 1954). To obtain good yields at least two cross-compatible cultivars of coincident flowering time should be planted together.Initial studies conducted to determine incompatibility genotypes in almond cultivars used controlled crosses. CrossaRaynaud and Grasselly (1985) proposed the existence of six different self-incompatibility alleles (S 1 , S 2 , S 3 , S 4 , S 7 and S 8 ) and the self-compatible allele S f in various European cultivars. Later, Kester et al. (1994) assigned four S alleles (S a , S b , S c and S d ) to explain the incompatibility groups of some American cultivars studied.Finding that almond incompatibility S alleles code for stylar proteins with ribonuclease activity that can be separated electrophoretically by isoelectric focusing (IEF) and non-equilibrium pH gradient electrofocusing (NEPHGE), Bosˇkovic´et al. (1997) determined the S genotype of 29 almond cultivars. This study corroborated the results obtained previously by Crossa-Raynaud andGrasselly (1985) andKester et al. (1994), and identified S 1 with S b , numbered S a as S 5 , and proposed two more alleles, S 6 and S 9 . In subsequent studies, the alleles S 10 (Bosˇkovic´et al. 1999), and S 11 and S 12 (Bosˇkovic´et al. 1998) were indicated and two more cultivars genotyped. Recently, 35 more cultivars (mostly of American origin) were genotyped with the same techniques and the alleles S 13

Phylogenetic relationships among the first and second introns of selectedPrunus S-RNase genes

“…sequences were used for PCR amplification in accordance with previous reports (Sonneveld et al 2003;Ortega et al 2005;Rahemi et al 2010). The PaConsI-FD forward and EM-Pc1ConsRD reverse primers were designed using the SP of cherry S-RNases (Sonneveld et al 2003) and the first conserved region flanking the first intron, respectively (Ortega et al 2005).…”

“…Total genomic DNA was extracted from dried leaves using CTAB protocol (Doyle and Doyle 1987) with some modification (Ortega et al 2005;Rahemi et al 2012b). DNA quantity and quality were determined by spectrophotometry and agarose gel electrophoresis.…”

“…The PaConsI-FD forward and EM-Pc1ConsRD reverse primers were designed using the SP of cherry S-RNases (Sonneveld et al 2003) and the first conserved region flanking the first intron, respectively (Ortega et al 2005). The EMPc2ConsFD and EM-PC3ConsRD primers were designed based on the second and third conserved regions (Ushijima et al 1998;Sutherland et al 2004) flanking the second intron, which is variable among genotypes.…”

“…Variation in the size of PCR amplification products of the first intron (Ortega et al 2005), the second intron (Tamura et al 2000;Channuntapipat et al 2001), or both introns can be used to identify S-alleles (Sutherland et al 2004). Degenerate primers designed using conserved sequences can be used to amplify alleles that have previously been identified or alleles that are thought to be new (De Cuyper et al 2005).…”

“…Molecular characterization of S-allele identity of crossing parents allows rapid prediction of incompatibility reaction. Currently, more than 50 S-alleles have been identified in almond (Ortega et al 2005(Ortega et al , 2006Boskovic et al 2007;Kodad et al 2008a;Mousavi et al 2011) using different molecular-based methods.…”