Density-weighted Algorithms for Similarity Computation and Cluster Tree Construction in the RAPD Analysis of Natural Cordyceps sinensis

Abstract

Objective: The goal of this study was to develop and validate density-weighted arithmetic methods for the analysis of the dynamic maturational changes in random amplified polymorphic DNA (RAPD) polymorphisms in Cordyceps sinensis containing multiple fungi. Methods: Ten random primers were used for PCR amplification to monitor changes in the RAPD molecular marker polymorphisms in caterpillar body, stroma and ascocarp portion samples of C. sinensis collected at different stages of maturation. We compared (1) the density-unweighted Nei-Li similarity equation and new similarity equations considering the densities of DNA bands and (2) the density-unweighted and weighted arithmetic methods for cluster analysis. Results: The algorithm using the Nei-Li similarity equation did not account for the differences in the density of the matched randomly amplified amplicons, whereas the new similarity equations were capable of integrating different amplicon densities into the similarity computation. With improvements in the similarity computation and cluster construction, the new methods revealed dynamic maturational changes in the RAPD polymorphisms of molecular markers in C. sinensis caterpillar bodies and stromata. The polymorphism analysis revealed similarities of 74%-88% between the RAPD polymorphisms of the ascocarp portion versus premature and mature C. sinensis stromata, but low similarities of<70% versus the caterpillar bodies and maturing stromata. Apparent dissimilarities (similarity <66%) were also found between Hirsutella sinensis and C. sinensis samples. Conclusions: These new algorithmic methods represent an advance in similarity computations when comparing polymorphisms of RAPD molecular markers in C. sinensis and provide accurate analytic tools to capture the molecular information related to the dynamic Am. J. Biomed. Sci. 2014, 6(2), 82-104; doi: 10.5099/aj140200082 © 2014 by NWPII. All rights reserved 83 changes in differential expression of C. sinensis intrinsic fungi during maturation. The apparent dissimilarity in RAPD polymorphisms between H. sinensis and C. sinensis suggests differences of mycological background and challenges the hypothesis of the anamorph-teleomorph connection between H. sinensis and Ophiocordyceps sinensis.

Suitability and Advantages of Abundance-Weighted Algorithms for the Holistic Comparison of Sample Systems

“…Among these methodologies, RAPD molecular marker polymorphism analysis is the most frequently used technique for comparing overall similarities or dissimilarities (genetic distances) and exploring the phylogenetic (cluster) relationship between the test systems [2,29,[32][33][34][35][36][37][38][39][40][41], although it has been suggested that ISSR may be more sensitive than RAPD [42][43][44] and costly metagenomic approaches may demonstrate advantages in qualitative studies of microbial genetic diversity and molecular ecology [45][46][47]. Among the key factors in study design for molecular marker polymorphism comparisons, the importance of unbiased selection of a plurality of random primers for random amplification of the genomic DNA templates isolated from the examined systems has been addressed [1][2][3][4]40,41]. The use of only a few random primers without reporting the objectivity and representativeness of the selection could lead to bias in the data analysis and thus biased conclusions [1][2][3][4]40,41,48].…”

“…The ZUNIX equation (2) defines similarity as the total density of all common parts present in the samples of comparison divided by the total density of all bands across the samples. This algorithm is mathematically general with no specific prerequisites and governs all conditions [40] in contrast to the abundance-unweighted Nei-Li equation (1) [49], which narrows the specific cases under the strict prerequisites (a) and (b) described above. The second ZUNIX equation (3) …”

“…This algorithm is mathematically general with no specific prerequisites and governs all conditions [40] in contrast to the abundance-unweighted Nei-Li equation (1) [49], which narrows the specific cases under the strict prerequisites (a) and (b) described above. The second ZUNIX equation (3) Extending further, the third ZUNIX equation (4) The abundance-weighted ZUNIX equations (2) (3) (4) define similarity as the total density (abundance) of all common parts present in the matched DNA bands of the samples being compared divided by the total density (abundance) of all bands across the samples [40]. The ZUNIX equations are mathematically general, with no specific prerequisites, and govern all conditions including the special cases under the strict prerequisites set forth by the Nei-Li equation (1) [49].…”

“…This large phylogenetic distance and dissimilarities (0.546-0.686) between H. sinensis and the stroma, caterpillar body and ascocarp samples of C. sinensis determined using the abundance-weighted algorithms is inconsistent with the sole O. sinensis anamorph hypothesis for H. sinensis. The types of algorithms and software should be carefully considered when designing RAPD, ISSR, SSCP and other holistic profile comparison studies, although both fully and semi-quantitative abundance-weighted clustering algorithms can be used in general for C. sinensis molecular and proteomic polymorphism studies [40,41,52].The mathematically general, abundance-weighted algorithms have also been employed in a proteomatic polymorphism study [52], in which the proteins of C. sinensis insect-fungi complex and of mycelial cultures of H. sinensis and Paecilomyces hepiali were extracted and separated through SELDI-TOP mass spectrometry. In this study, Dong et al [52] analyzed more than 1,900 protein bands, computed the holistic similarities between the samples and constructed a phylogenetic tree.…”

“…These macrocosmic molecular techniques for holistic comparisons include AFLP (Amplified Fragment Length Polymorphism), CAPS (Cleaved Amplified Polymorphic Sequence), DAF (DNA Amplified Fingerprints), ISSR (InterSimple Sequence Repeat), RAPD (Random Amplified Polymorphic DNA), RFLP (Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Regions), SSCP (single-strand conformation polymorphism) and SSR (Simple Sequence Repeat) [1][2][3][4]30,31]. Among these methodologies, RAPD molecular marker polymorphism analysis is the most frequently used technique for comparing overall similarities or dissimilarities (genetic distances) and exploring the phylogenetic (cluster) relationship between the test systems [2,29,[32][33][34][35][36][37][38][39][40][41], although it has been suggested that ISSR may be more sensitive than RAPD [42][43][44] and costly metagenomic approaches may demonstrate advantages in qualitative studies of microbial genetic diversity and molecular ecology [45][46][47]. Among the key factors in study design for molecular marker polymorphism comparisons, the importance of unbiased selection of a plurality of random primers for random amplification of the genomic DNA templates isolated from the examined systems has been addressed [1][2][3][4]40,41].…”