A practical method for almond cultivar identification and parental analysis using simple sequence repeat markers

Each primer pair was tested under only one standard set of PCR conditions. More primers pairs might have produced useful results under different PCR conditions. However, our goal was to develop a practical forensic “DNA fingerprinting” method to uniquely characterize all almond cultivars and use this method to confirm the identity of each almond tree in the FPS foundation blocks. Adoption of a single protocol for DNA amplification increases productivity and reduces lab errors.

The goal of this study was to develop a method to uniquely identify all current almond cultivars using automated DNA fragment analysis, to use this method to confirm the identity of the almond cultivars at FPS and to elucidate the relationships of the commercially important cultivars grown in California. This study set represents a very narrow germplasm. Such a limited germplasm is a very good sample set for choosing highly polymorphic markers; however, the resulting data set is not the large, diverse database needed to calculate meaningful allele frequencies for probability analysis.

In addition to allowing an unambiguous identification of almond cultivars, the SSR markers reported here can be used to study cultivar pedigrees. A progeny shares one allele at each locus with each of its parents. This study set of 18 almond cultivars is neither large nor diverse enough to calculate probabilities for parentage analysis. However, a consistent result for both parents and a progeny at all 24 SSR loci provides strong, if not quantifiable, evidence to support the relationship, particularly if it confirms previous reports.

The almond cultivars ‘Aldrich’, ‘Butte’, ‘Carmel’, ‘Monterey’ ‘Norman’, ‘Price’ and ‘Thompson’ have previously been reported to be chance seedling selections probably originating from ‘Nonpareil’ × ‘Mission’ crosses (Asai et al. 1996; Brooks and Olmo 1997). This preliminary characterization was based on early cross-compatibility studies (Kester et al. 1994) where it was shown that most chance-selection cultivars could be grouped into four cross-incompatibility groups (S₁S₇, S₁S₈, S₅S₇, S₅S₈). These cross-incompatibility genotypes were presumed to be the result from natural crosses between the dominant cultivar ‘Nonpareil’ (S₇S₈) and the cultivar ‘Mission’ (S₁S₅) which was the major pollenizer for ‘Nonpareil’ during the early to mid 20th century (Asai et al. 1996; Wood 1925). However, other potential donors of the S₁, S₅ or S₇ allele have now been identified, including ‘Languedoc’ (S₁S₅), ‘Ne Plus Ultra’ (S₁S₇), and ‘Peerless’ (S₁S₆), (Barckley et al. 2006; Lopez et al. 2006) all of which have been reported to be widely planted in California from the late 19th to mid 20th century (Asai et al. 1996; Wood 1925).

The SSR data does, however, support both ‘Fritz’ and ‘Ruby’ as having the cultivar ‘Peerless’ in their lineage since both have the unique alleles 142 at BPPCT039 and 156 at ssrPaCITA12 markers (Table 5) as well as the unique S₆ incompatibility allele. Molecular marker data also support ‘Mission’ as the other parent (Table 5) as does the presence of the S₁ incompatibility allele (Barckley et al. 2006). It is assumed the same null allele at MA034a inherited by ‘Carmel’ is also inherited from ‘Mission’ by both ‘Fritz’ and ‘Ruby’ (Table 6).

Similarly, while the recently released cultivar ‘Kochi’ was discovered as a volunteer seedling near a ‘Drake’ almond orchard (Kochi 2004), the SSR data show that it most likely results from a ‘Peerless’ × ‘Nonpareil’ cross. ‘Kochi’ shares one allele at each locus with both ‘Nonpareil’ and ‘Peerless’, including the unique ‘Peerless’ alleles 142 at BPPCT039 and 156 at ssrPaCITA12. ‘Kochi’ also possesses the unique ‘Peerless’ S₆-allele (Barckley et al. 2006).

Molecular markers can also support published parentage by analyzing only parent/progeny pairs, which will share one SSR allele at each locus. Though this analysis does not show the direction of descent (which is the parent and which the progeny), it can be used in conjunction with other information to support reported pedigrees. In this study marker data for ‘Padre’ support earlier reports of ‘Mission’ being the seed parent. The data are also consistent with ‘Nonpareil’ being one parent of ‘Kanpareil’, ‘Solano’, ‘Sonora’ and ‘Titan’ (Brooks and Olmo 1997). ‘Titan’s’ seed parent is actually known to be ‘Tardy nonpareil’, a late blooming somatic mutant or “bud-sport” of nonpareil [only rarely are differences between somatic mutants observed with SSR data (Riaz et al. 2002)]. S-allele data are also consistent for the reported parentage of ‘Sonora’, ‘Solano’ and ‘Kapareil’. There are no S-allele data for ‘Titan’ which is used primarily as an almond parent in generating almond x peach hybrid rootstocks. Unique molecular marker patterns were also observed for the recent cultivars ‘Sweetheart’ and ‘Winters’, supporting the reported use of novel germplasm to incorporate improved productivity and pest resistance to these cultivars (Gradziel et al. 2007; Martínez-Gómez et al. 2004).