We evaluated the potential of microsatellite markers for use in Citrus genome analysis. Microsatellite loci were identified by screening enriched and nonenriched libraries developed from `Washington Navel' Citrus. Microsatellite-containing clones were sequenced and 26 specific PCR primers were selected for cross-species amplification and identification of cultivars/clones in Citrus. After an enrichment procedure, on average 69.9% of clones contained dinucleotide repeats (CA)n and (CT)n, in contrast to <25% of the clones that were identified as positive in hybridization screening of a nonenriched library. A library enriched for trinucleotide (CTT)n contained <15% of the clones with (CTT)n repeats. Repeat length for most of the dinucleotide microsatellites was in the range of 10 to 30 units. We observed that enrichment procedure pulled out more of the (CA)n repeats than (CT)n repeats from the Citrus genome. All microsatellites were polymorphic except one. No correlation was observed between the number of alleles and the number of microsatellite repeats. In total, 118 putative alleles were detected using 26 primer pairs. The number of putative alleles per primer pair ranged from one to nine with an average of 4.5. Microsatellite markers discriminated sweet oranges [Citrus sinensis (L.) osb], mandarin (Citrus reticulata Blanco), grapefruit (Citrus paradisi Macf.), lemon [Citrus limon (L.) Burm.f.], and citrange (hybrids of trifoliate orange and sweet orange), at the species level, but individual cultivars/clones within sweet oranges, mandarins and grapefruit known to have evolved by somatic mutation remained undistinguishable. Since these microsatellite markers were conserved within different Citrus species, they could be used for linkage mapping, evolutionary and taxonomic study in Citrus.
Riaz Ahmad, Darush Struss and Stephen M. Southwick
Darush Struss, Riaz Ahmad, Stephen M. Southwick and Manuela Boritzki
Simple sequence repeats (SSRs) and amplified fragment-length polymorphisms (AFLPs) were used to evaluate sweet cherry (Prunus avium L.) cultivars using quality DNA extracted from fruit flesh and leaves. SSR markers were developed from a phage library using genomic DNA of the sweet cherry cultivar Valerij Tschkalov. Microsatellite containing clones were sequenced and 15 specific PCR primers were selected for identification of cultivars in sweet cherry and for cross-species amplification in Prunus. In total, 48 alleles were detected by 15 SSR primer pairs, with an average of 3.2 putative alleles per primer combination. The number of putative alleles ranged from one to five in the tested cherry cultivars. Forty polymorphic fragments were scored in the tested cherry cultivars by 15 SSRs. All sweet cherry cultivars were identified by SSRs from their unique fingerprints. We also demonstrated that the technique of using DNA from fruit flesh for analysis can be used to maintain product purity in the market place by comparing DNA fingerprints from 12 samples of `Bing' fruit collected from different grocery stores in the United States to that of a standard `Bing' cultivar. Results indicated that, with one exception, all `Bing'samples were similar to the standard. Amplification of more than 80% of the sweet cherry primer pairs in plum (P. salicina), apricot (P. armeniaca) and peach (P. persica L.) showed a congeneric relationship within Prunus species. A total of 63 (21%) polymorphic fragments were recorded in 15 sweet cherry cultivars using four EcoRI-MseI AFLP primer combinations. AFLP markers generated unique fingerprints for all sweet cherry cultivars. SSRs and AFLP polymorphic fragments were used to calculate a similarity matrix and to perform UPGMA cluster analysis. Most of the cultivars were grouped according to their pedigree. The SSR and AFLP molecular markers can be used for the grouping and identification of sweet cherry cultivars as a complement to pomological studies. The new SSRs developed here could be used in cherry as well as in other Prunus species for linkage mapping, evolutionary and taxonomic study.
Claudio Cantini, Amy F. Iezzoni, Warren F. Lamboy, Manuela Boritzki and Darush Struss
The U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS) tetraploid cherry (Prunus L. sp.) collection at Geneva, N.Y., contains ≈75 accessions of sour cherry (P. cerasus L.), ground cherry (P. fruticosa Pall.), and their hybrids. Accurate and unambiguous identification of these accessions is essential for germplasm preservation and use. Simple sequence repeats (SSRs) are currently the markers of choice for germplasm fingerprinting because they characteristically display high levels of polymorphism. Recently SSR primer pairs from sweet cherry (P. avium L.), sour cherry, and peach [(P. persica L. Batsch (Peach Group)] have been reported. Ten SSR primer pairs were tested on 59 tetraploid cherry accessions to determine if they could differentiate among the accessions. Scorable SSR fragments were produced with all primer-accession combinations. The cherry accessions exhibited high levels of polymorphism with 4 to 16 different putative alleles amplified per primer pair. Most of the putative alleles were rare with frequencies <0.05. Heterozygosity values ranged from 0.679 to 1.00, while gene diversity values ranged from 0.655 to 0.906. The primer pairs differentiated all but two of the 59 cherry accessions. Based upon the ability of the SSR data to differentiate the cherry accessions and the high level of gene diversity, we propose that all the tetraploid cherry accessions in the USDA/ARS collection be fingerprinted to provide a mechanism to verify the identity of the individual accessions. The fingerprinting data are available on the World Wide Web (http://www.ars-grin.gov/gen/cherry.html) so that other curators and scientists working with cherry can verify identities and novel types in their collections and contribute to a global database.