Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi

Abstract

ABSTRACT. Roegneria kamoji Ohwi is an excellent forage grass due to its high feeding value and high resistance to some biotic and abiotic stresses. However, the start codon targeted (SCoT) polymorphism has not been conducted on R. kamoji. In this study, an orthogonal L 16 (4 5 ) design was employed to investigate the effects of five factors (Mg 2+ , dNTPs, Taq DNA polymerase, primer, and template DNA) on the polymerase chain reaction (PCR) to determine the optimal SCoT-PCR system for R. kamoji. The results showed that the most suitable , 0.15 mM dNTPs, 1.0 U Taq DNA polymerase, 0.4 pM primer, and 40 ng template DNA. SCoT primers 39 and 41 were used to verify the stability of the optimal reaction system, and amplification bands obtained from diverse samples were found to be clear, rich, and stable in polymorphisms, indicating that this reaction system can be used for SCoT-PCR analysis of R. kamoji. We have developed a simple and rapid way to study the mutual effects of factors and to obtain positive results through the use of an orthogonal design L 16 (4 5 ) to optimize the SCoT-PCR system. This method may provide basic information for molecular marker-assisted breeding and analyses of genetic diversity in R. kamoji.

Development of a high specificity typing method for the detection of herpes simplex virus

“…Referring to others’ work ( Zeng et al, 2015 ; Zeng et al, 2016 ; Tang et al, 2018 ; Mou et al, 2019 ; Gong et al, 2021 ), the gradient experiments for single factor and multilevel orthogonal design were integrated and results from the two experiments were analyzed to find the best PCR reaction system ( Table 3 ).…”

Species identification of polygonati rhizoma in China by both morphological and molecular marker methods

“…The fresh rhizomes were identified based on their morphological characteristics and cultivated to score and analyze morphological characters. The ISSR [36] and SCoT [32,37] markers were developed and implemented for the identification of the specimens. Finally, the identification results by both morphological characteristics and molecular markers schemes were compared.…”