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
Riaz Ahmad, Louise Ferguson and Stephen M. Southwick
A genomic DNA library enriched for dinucleotide (CT)n and (CA)n and trinucleotide (CTT)n microsatellite motifs has been developed from `Kerman' pistachio (Pistacia vera L.). The enrichment method based on magnetic or biotin capture of repetitive sequences from restricted genomic DNA revealed an abundance of simple sequence repeats (SSRs) in the pistachio genome which were used for marker development. After an enrichment protocol, about 64% of the clones contained (CT)n repeats while 59% contained (CA)n for CT and CA enriched libraries, respectively. In the (CT)n enriched library, compound sequences were 45% while for (CA)n it was 13.5%. In both dinucleotide enriched libraries, about 80% of the clones having microsatellites have a repeat length in the range of 10 to 30 units. A library enriched for trinucleotide (CTT)n contained <19% of the clones with (CTT)n repeats. Of the clones that contained microsatellites, 62% had sufficient flanking sequence for primer design. An initial set of 25 pairs of primers was designed, out of which 14 pairs amplified cleanly and produced an easily interpretable PCR product in the commercially important American, Iranian, Turkish, and Syrian pistachio cultivars. The efficient DNA extraction method developed for pistachio kernels and shells (roasted and nonroasted) yielded DNA of sufficient quality to use PCR to create DNA fingerprints. In total, 46 alleles were identified by 14 primer pairs and a dendrogram was constructed on the basis of that information. The SSR markers distinguished most of the tested cultivars from their unique DNA fingerprint. An UPGMA cluster analysis placed most of the Iranian samples in one group while the Syrian samples were the most diverse and did not constitute a single distinct group. The maximum number of cultivar specific markers were found in `Kerman'(4), the current industry standard in the United States, and the Syrian cultivar Jalab (5). The technique of using extracted DNA from pistachio kernal or shell coupled with the appropriate marker system developed here, can be used for analyses and measurement of trueness to type.
Riaz Ahmad, Dan Potter and Stephen M. Southwick
Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) molecular markers were evaluated for detecting intraspecific variation in 38 commercially important peach and nectarine (Prunus persica) cultivars. Out of the 20 SSR primer pairs 17 were previously developed in sweet cherry and three in peach. The number of putative alleles revealed by SSR primer pairs ranged from one to five showing a low level of genetic variability among these cultivars. The average number of alleles per locus was 2.2. About 76% of cherry primers produced amplification products in peach and nectarine, showing a congeneric relationship within Prunus species. Only nine cultivars out of the 38 cultivars could be uniquely identified by the SSR markers. For SRAP, the number of fragments produced was highly variable, ranging from 10 to 33 with an average of 21.8 per primer combination. Ten primer combinations resulted in 49 polymorphic fragments in this closely related set of peaches and nectarines. Thirty out of the 38 peach and nectarine cultivars were identified by unique SRAP fingerprints. UPGMA Cluster analysis based on the SSR and SRAP polymorphic fragments was performed; the relationships inferred are discussed with reference to the pomological characteristics and pedigree of these cultivars. The results indicated that SSR and SRAP markers can be used to distinguish the genetically very close peach and nectarine cultivars as a complement to traditional pomological studies. However, for fingerprinting, SRAP markers appear to be much more effective, quicker and less expensive to develop than are SSR markers.
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.
Riaz Ahmad, Miki Okada, Jeffrey L. Firestone, Chris R. Mallek and Marie Jasieniuk
We isolated and characterized microsatellite loci in the ornamental pampas grass Cortaderia selloana (Schult. & Schult. f.) Asch. & Graebn. for purposes of identifying cultivars and assessing genetic relationships among cultivars. Small insert genomic libraries were enriched for dinucleotide (CT)n and (CA)n repeats. Ninety clones were sequenced of which 76% contained at least one microsatellite with a basic motif greater than six repeat units. Nine primer pairs amplified 10 polymorphic and putatively disomic loci, and were used to genotype 88 individuals representing 17 named cultivars and four selections. In total, 93 alleles were detected with a maximum of two to 19 per locus. Effective number of alleles varied from 1.3 to 9.5. Observed heterozygosity ranged from 0.07 to 0.81. The 10 microsatellite loci distinguished the majority of pampas grass cultivars. An unweighted pair group method with arithmetic mean (UPGMA) cluster analysis, based on proportion of shared alleles among individuals, revealed groups of cultivars corresponding to origin and morphological characteristics. With few exceptions, individuals of a single cultivar clustered together with moderate to strong bootstrap support (greater than 50%). Interestingly, `Pumila' from Europe and the United States formed separate clusters indicating independent origins. A large, diverse cluster with low bootstrap support consisted of selections and cultivars sold as seed, rather than potted or bare-root clonal plants. Primers designed for C. selloana amplified microsatellite loci in other Cortaderia Stapf species concordant with phylogenetic relationships among the species. Cross-amplification was 100% in C. jubata (Lemoine ex Carrière) Stapf; 77% in C. pilosa (d'Urv.) Hack. and C. rudiuscula Stapf; 66% in C. fulvida (Buch.) Zotov; and 55% in C. richardii (Endl.) Zotov and C. toetoe Zotov.