Strawberry GenBank-derived and Genomic Simple Sequence Repeat (SSR) Markers and Their Utility with Strawberry, Blackberry, and Red and Black Raspberry

Abstract

<jats:p>Although simple sequence repeat (SSR) markers have been developed for species in the closely related genera <jats:italic>Fragaria</jats:italic> L. (strawberry) and <jats:italic>Rubus</jats:italic> L. (raspberry and blackberry), the number of SSRs available is insufficient for genetic mapping. Our objective was to use and compare multiple approaches for developing additional SSRs for <jats:italic>Fragaria</jats:italic> and <jats:italic>Rubus</jats:italic>. The approaches included: the development of SSRs from GenBank sequences from species of varied relatedness to <jats:italic>Fragaria</jats:italic> and <jats:italic>Rubus</jats:italic> and identified with two different data-mining methods (BLAST and SSRIT); the evaluation of some previously published SSRs designed from related species; and the development of SSRs from a genomic library made from <jats:italic>F</jats:italic>. ×<jats:italic>ananassa</jats:italic> Duschene ex Rozier `Earliglow'. When an SSR was developed from a known gene sequence, the location of the repeat in the gene was determined to evaluate the effect on amplification and polymorphism detection. Cross-generic amplification between closely related <jats:italic>Fragaria</jats:italic> and <jats:italic>Rubus</jats:italic> as well as transference from species of varied relatedness to <jats:italic>Fragaria</jats:italic> and <jats:italic>Rubus</jats:italic> also was evaluated and indicated limited transference within the subfamily Rosoideae. However, development of SSRs for <jats:italic>Fragaria</jats:italic> and <jats:italic>Rubus</jats:italic> from <jats:italic>Rosa</jats:italic> L. (rose) and Rosaceae genera outside Rosoideae was not efficient enough to be practical for new map development. SSRIT was superior to BLAST for identifying GenBank sequences containing repeats. SSRs developed from repeats found in either the 5′UTR (80% polymorphic) or 3′UTR (85% polymorphic) were most likely to detect polymorphisms, compared with those developed from coding regions (30%). SSRs developed from the genomic library were only slightly superior to GenBank-derived SSRs in their ability to detect polymorphisms.</jats:p>

Construction of black (Rubus occidentalis) and red (R. idaeus) raspberry linkage maps and their comparison to the genomes of strawberry, apple, and peach

“…Of these, 136 (14.5%) mapped, 505 (54%) were monomorphic, and 177 (18%) had complex HRM curves. The percentage of monomorphic markers in our population is similar to what was reported by Lewers et al (2005) (40-83%) and Zorrilla-Fontanesi et al (2011) (34-60%) and suggests that the cultivars and selections most often used for mapping studies of Rubus may have high gene homozygosity. Our marker design technique focused on the discovery of de novo SNP using HRM to detect sequence polymorphisms, rather than amplification of SSR repeats.…”

“…However, the variation in heterozygosity for some markers found in this study does not appear to influence greatly the ability to compare the linkage maps, as the overall alignment of anchor loci agree among the mapping studies. The recent studies on marker transferability by Lewers et al (2005), Zorrilla-Fontanesi et al (2011), and our own results, demonstrate the difficulty of identifying polymorphic loci in Rubus, and also illustrate that the amplification of a PCR product does not necessarily make a marker useful for map construction. In general, amplification of polymorphic loci in Rubus using PCR primer pairs designed from Fragaria sequence was more successful if the sequence was from EST (8-13%, and a total of 18 markers) than if the sequence was from genomic SSR (5-6%, and a total of 4 markers) (Table 4).…”

“…The mapping of gene-based markers from Prunus G6 on to Rubus LG2 demonstrated by Woodhead et al (2010), and the more efficient inter-generic transferability of EST-SSR relative to genomic SSR markers (Lewers et al 2005;Zorrilla-Fontanesi et al 2011) suggests that orthologous markers are key to successful comparative genomic analyses in Rosaceae.…”

“…Lewers et al (2005) assessed the transferability of GenBank-derived Fragaria EST-SSR to be 32% to blackberry, and 20% to raspberry. In the same study, the transferability of primers from GenBank-derived Fragaria genomic SSR was lower, at 26% in blackberry, and 18% in raspberry.…”

“…Members of the sub-family Rosoideae, including Fragaria (strawberry) and Rubus (raspberry and blackberry), have the same base chromosome number of x = 7, similar fruit form, and are closely related based on chloroplast and nuclear DNA phylogenies (Potter et al 2007), as well as morphological data (Eriksson et al 2003). These lines of evidence suggest colinearity between the Fragaria and Rubus genomes; however, a lack of transferable markers has made genomic comparisons between these genera unachievable to date (Lewers et al 2005).…”