Primers amplifying a range of Prunus S‐alleles

Abstract

Although various consensus polymerase chain reaction (PCR) primers have been reported for identifying Prunus S-alleles, they have been developed from and optimized on a limited set of alleles, which may limit their applicability to a broader allele range. To develop a primer set for use across the genus, degenerate consensus primers were designed from conserved regions of 27 S-RNase sequences available from five Prunus species. The primers were tested in 15 previously genotyped cultivars of cherry, almond and apricot, representing alleles S 1 to S 6 in each crop and also S c in apricot. Comparisons were made with previously published primers tested in the same 15 cultivars under reported reaction conditions. The new primers generated an amplification product for each of the 19 S-alleles whereas those previously available amplified no more than 14. The primers will be useful for genotyping and genetic studies in cultivars and wild populations.

S-Genotype Profiles of Turkish Apricot Germplasm

“…Based on the structure of S-RNase, many pairs of primers have been developed for Prunus species, such as Pru-C2 and PCE-R (Tao et al, 1999a;Yamane et al, 2001), SRc-F and EMPC5consRD, SRc-F and PM-C5 (Vilanova et al, 2005;Sutherland et al, 2004;Habu et al, 2008), ASIII and AmyC5R (Tamura et al, 2000), EM-PC2consFD and ED-PC3cons-RD 70 (Sutherland et al, 2004), PaConsI-F and PaConsI-R, PaConsII-F and PaConsII-R (Sonneveld et al, 2003). Yaegaki et al (2001) first determined S-RNase genotypes using the primer pair Pru-C2 and Pru-C5.…”

“…The determination of the S-genotypes of 236 Turkish and foreign apricot genotypes was carried out using the SRc-F and SRc-R consensus primers (Vilanova et al, 2005) for the first intron and EM-PC2consFD / EM-PC3consRD primers (Sutherland et al, 2004) for the second intron analysis of the S-RNase gene (Table 1). AprFBC8 F and R primers were used for discrimination of SFB C/8 allele (Halász et al, 2007).…”

“…For the second intron, PCR was conducted according to Sutherland et al (2004) using the degenerate primers EMPC2consFD and EM-PC3consRD. For PCR amplification in a 20-mL reaction volume, containing 1X PCR buffer (Thermo) with the final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 0.2 mM of dNTPs, 0.3 mM of each primer, and 1.0 U of Taq DNA polymerase (Thermo).…”

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

“…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. Reactions were done in Eppendorf 5341 Mastercycler † epgradient thermocycler with optimized PCR conditions according with a previous report by Rahemi et al (2010).…”

“…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).…”