Construction and validation of a prototype microarray for efficient and high-throughput genotyping of angiosperms.
Until recently, the identification of plants relied on conventional techniques, such as morphological, anatomical and chemical profiling, that are often inefficient or unfeasible in certain situations. Extensive literature exists describing the use of polymerase chain reaction (PCR) DNA-based identification techniques, which offer a reliable platform, but their broad application is often limited by a low throughput. However, hybridization-based microarray technology represents a rapid and high-throughput tool for genotype identification at a molecular level. Using an innovative technique, a 'Subtracted Diversity Array' (SDA) of 376 features was constructed from a pooled genomic DNA library of 49 angiosperm species, from which pooled non-angiosperm genomic DNA was subtracted. Although not the first use of a subtraction technique for genotyping, the SDA method was superior in accuracy, sensitivity and efficiency, and showed high-throughput capacity and broad application. The SDA technique was validated for potential genotyping use, and the results indicated a successful subtraction of non-angiosperm DNA. Statistical analysis of the polymorphic features from the pilot study enabled the establishment of accurate phylogenetic relationships, confirming the potential use of the SDA technique for genotyping. Further, the technique substantially enriched the presence of polymorphic sequences; 68% were polymorphic when using the array to differentiate six angiosperm clades (Asterids, Rosids, Caryophyllids, Ranunculids, Monocots and Eumagnoliids). The 'proof of concept' experiments demonstrate the potential of establishing a highly informative, reliable and high-throughput microarray-based technique for novel application to sequence independent genotyping of major angiosperm clades.
Jayasinghe
Ruchira
R
Division of Chinese Medicine, School of Health Sciences, RMIT University, Building 223, Level 1, Plenty Road, PO Box 71, Bundoora, Vic. 3083, Australia.
Kong
Stephan
S
Coram
Tristan E
TE
Kaganovitch
Janna
J
Xue
Charlie C L
CC
Li
Chun G
CG
Pang
Edwin C K
EC
D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D018745:Genome, Plant; D005838:Genotype; D019684:Magnoliopsida / Q000145:classification / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*