Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi.
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 L16(45) design was employed to investigate the effects of five factors (Mg2+, 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 conditions for SCoT-PCR in R. kamoji included 1.5 mM Mg2+, 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 L16(45) 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.
Department of Animal Science, Southwest University, Rongchang, Chongqing Province, China.
B
B
Zeng
Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Chengdu, Sichuan, China.
H D
HD
Yan
Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Chengdu, Sichuan, China.
L K
LK
Huang
Animal Husbandry and Veterinary Research Institute of Guizhou Province, Guizhou, Guiyang, China 1007795901@qq.com.
Y C
YC
Wang
Guizhou Institute of Prataculture, Guizhou, Dushan, China.
J H
JH
Wu
Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Chengdu, Sichuan, China.
X
X
Huang
Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Chengdu, Sichuan, China.
A L
AL
Zhang
Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Chengdu, Sichuan, China.
C R
CR
Wang
Guizhou Institute of Prataculture, Guizhou, Dushan, China.
Q
Q
Mu
D018387:Codon, Initiator / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D014644:Genetic Variation; D006109:Poaceae / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D018387:Codon, Initiator; D017931:DNA Primers