Diversity Suppression‐Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

Abstract

Genomic DNA polymorphisms are very useful for tracing genetic traits and studying biological diversity among species. Here, we present a method we call the "diversity suppression-subtractive hybridization array" for effectively profiling genomic DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization (SSH) to establish a subtracted gDNA library, from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomic DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphic fragments of pairwise comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array is a highly effective platform for profiling genomic DNA polymorphisms and dendrograms.

Development and use of molecular markers: past and present

“…In recent years, DArT has assumed the status of highly reliable, robust and useful markers for analysis of genetic diversity, as well as genetic mapping using linkage or association studies both in a variety of model and non-model crops such as Eucalyptus Restriction digestion, ligation of adapters, end sequencing and sequence analysis Baird et al (2008) Specific-locus amplified fragment sequencing (SLAF-seq) Deep sequencing, Sequence alignment Sun et al (2013) pearl millet (Supriya et al, 2011), carrot (Grzebellus et al, 2014), barley (Lex et al, 2014), sugarcane (Aitken et al, 2014). Subtracted diversity array (SDA; Li et al, 2006) is an advancement over DArT incorporating the benefits of subtractive suppression hybridization (SSH) based microarray, in order to enrich the resulting data with polymorphic genomic DNA sequences. Thus, making it is more relevant genotyping method for gene tagging and molecular mapping.…”

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

“…The alternative SSH involves the pooling of gDNA representations and single subtraction instead of making multiple pair-wise subtractions between the species as proposed by other subtraction techniques [17]. Multiple pair-wise subtractions could be costly and time consuming; for example, a total of four subtractions had to be performed in order to fingerprint only six Dendrobium species [18]. Furthermore, SDA could potentially be a superior technique for assessing the genetic relationships among Echinacea species since it does not require previous DNA sequence information and has shown to be capable of differentiating closely-related species of the same genus [19], [20].…”

First report of fingerprinting dried herbal products using a subtractive diversity array

“…Moreover, some PCR-based methods like microsatellites and sequence characterized amplified regions (SCAR) require prior sequence information. Therefore, these techniques may not be suitable for fingerprinting herbal species with poor genomic resources [13,14].…”

“…The oligonucleotide microarrays used in these studies required prior sequence information for probe and target synthesis. Alternatively, to fingerprint species with no sequence information, sequence-independent arrays like Suppression Subtractive Hybridization (SSH)-based arrays [13,17] and Diversity Array Technology (DArT™, [18]) were developed.…”

“…Instead of making pairwise subtractions between the species to be fingerprinted [13,17], they pooled genomic DNA from 49 representative angiosperm species and subtracted it from pooled genomic DNA of five representative non-angiosperm species to extract angiosperm-specific DNA fragments. The (SDA) successfully differentiated species from the six main clades of angiosperms [19], and correctly clustered nine flowering plants at the family level [20].…”