Subtracted Diversity Array Identifies Novel Molecular Markers Including Retrotransposons for Fingerprinting Echinacea Species

Abstract

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.

Application of subtracted gDNA microarray-assisted Bulked Segregant Analysis for rapid discovery of molecular markers associated with day-neutrality in strawberry (Fragaria x ananassa)

“…This subtraction efficiency is comparable to the first prototype angiosperm SDA (97.0%) and Asterids-specific SDA (99.6%)24. The high subtraction efficiency in this study was achieved using 1:60 tester:driver ratio as recently described for the Echinacea -SDA6 that enabled 97% efficiency compared to the Salvia -SDA where 1:30 tester:driver ratio produced 88% efficiency5. Therefore, we are confident that all common DNA sequences between strawberries and non-angiosperms were eliminated, and the resulting FDP is significantly enriched with angiosperm- and/or strawberry-specific DNA sequences.…”

“…Firstly, genomic representations were prepared by pooling equal quantities of DNA extracted from five strawberry genotypes and nine non-angiosperm species (Table 5) into tester and driver pools, respectively to a final amount of 4 μg and fragmented overnight with 5 units of Alu I and Hae III (NEB, Ipswich, MA) in a 100 μL digestion mixture. DNA subtraction was performed using PCR-Select TM cDNA Subtraction kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions but the tester:driver ratio was increased to 1:606. Microarray probe preparation and printing was performed according to Jayasinghe et al 2.…”

“…We subsequently refined the SDA technique to efficiently eliminate common sequences between two DNA pools, and achieved a subtraction efficiency of 97.0%4. We also successfully extended the use of this technique for narrow subtraction at clade (Asterids-specific SDA) and genera ( Salvia - and Echinacea -specific SDA) levels and achieved high subtraction efficiency between 88 to 99.6%456. The SDA technique is not only capable of fingerprinting a wide range of plants, but more importantly, identifying novel species and family-specific sequences, and markers associated with accumulation of bioactive compounds456.…”

Identification and validation of FaP1D7, a putative marker associated with the biosynthesis of methyl butanoate in cultivated strawberry (Fragaria x ananassa)

“…We combined the application of FDP with Bulked Segregant Analysis (BSA) 25 , a rapid method for detecting DNA markers linked to any specific gene in the genome. Taking advantage of the discriminatory power of SDA for genotyping closely related species within the same genus 26 – 28 , we have successfully identified a putative marker, FaP2E11 possibly involved in controlling day-neutrality in octoploid strawberry 24 . Here, we extended the utility of the subtracted gDNA microarray-assisted BSA for the identification of polymorphic markers linked to the loci determining strawberry aroma.…”