Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers

Abstract

PCR-based identification of all 13 known self-incompatibility (S) alleles of sweet cherry is reported. Two pairs of consensus primers were designed from our previously published cDNA sequences of S(1) to S(6) S-RNases, the stylar components of self-incompatibility, to reveal length variation of the first and the second introns. With the exception of the first intron of S(13), these also amplified S(7) to S(14) and an allele previously referred to as S(x), which we now label S(16). The genomic PCR products were cloned and sequenced. The partial sequence of S(11) matched that of S(7) and the alleles were shown to have the same functional specificity. Allele-specific primers were designed for S(7) to S(16), so that allele-specific primers are now available for all 13 S alleles of cherry (S(8), S(11) and S(15) are duplicates). These can be used to distinguish between S alleles with introns of similar size and to confirm genotypes determined with consensus primers. The reliability of the PCR with allele-specific primers was improved by the inclusion of an internal control. The use of the consensus and allele-specific primers was demonstrated by resolving conflicting genotypes that have been published recently and by determining genotypes of 18 new cherry cultivars. Two new groups are proposed, Group XXIII (S(3) S(16)), comprising 'Rodmersham Seedling' and 'Strawberry Heart', and Group XXIV (S(6) S(12)), comprising 'Aida' and 'Flamentiner'. Four new self-compatibility genotypes, S(3) S(3)', S(4)' S(6), S(4)' S(9) and S(4)' S(13), were found. The potential use of the consensus primers to reveal incompatibility alleles in other cherry species is also demonstrated.

Molecular and quantitative signatures of biparental inbreeding depression in the self-incompatible tree species Prunus avium

“…Fragments were run on a Megabace genetic analyser (Amersham Biosciences) using the Megabace ET 550-R size standard (GE Healthcare). When amplification of the SFB intron was not successful and the S-allele could not be identified, we also amplified the second intron of the S-RNase gene (Sonneveld et al, 2003;Vaughan et al, 2008). We used 60 ng DNA, 2.5 ml 10Â Buffer, 2 mM MgCl 2 , 0.2 mM dNTP, 0.2 mM of each forward and reverse primer, 1.25 ml W-1 buffer (Invitrogen, Darmstadt, Germany) and 1.25 U Taq polymerase for a total reaction volume of 25 ml.…”

Identification and characterization of S-RNase genes and S-genotypes in Prunus and Malus species

“…The Malus S-RNase gene contains a single intron, located within the HV region (Heng et al 2008b;Long et al 2010), whereas, the Prunus S-RNase contains two introns. The first intron is located between a signal peptide and the C1 region, while the second intron is located within the HV region (Sonneveld et al 2003;Wu et al 2009). Moreover, the length of this second intron shows higher levels of polymorphisms when compared with that of the first intron, and this has been widely used to discriminate S-RNase alleles among Prunus species (Heng et al 2008a;Zhang et al 2008).…”

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).…”

“…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.…”

“…The cloning and sequencing of PCR amplified products is used to determine the S genotype by sequence comparisons (Janssens et al 1995;Richman and Kohn 1996;Verdoodt et al 1998;Ishimizu et al 1999;Tamura et al 2000;Ma and Oliveira 2001). To confirm a presumptive new S-allele that shows a new band pattern or to determine structural differences with previous alleles, S-alleles can be partially amplified by using consensus primers (Sonneveld et al 2003;De Cuyper et al 2005). Previous comparisons of cultivated almond with closely related species has demonstrated genetic variation among S-alleles (Rahemi et al 2010(Rahemi et al , 2012a including differences in S-allele amplification (Rahemi et al 2014).…”