Articles
DNA TEMPLATE PREPARATION AND TECHNOLOGY SYSTEM BUILDING FOR AFLP ANALYSIS OF CHINESE CABBAGE (BRASSICA CAMPESTRIS L. SSP. PEKINENSIS)
Article number
944_21
Pages
159 – 167
Language
English
Abstract
In this study, the improved method of CTAB was used to extract genomic DNA from tender leaves of Chinese cabbage (Brassica campestris L. ssp. pekinensis). DNA restriction reaction, ligase reaction, pre-amplification and selective amplification were optimized for establishment of AFLP reaction system and clear DNA fingerprints.
The results indicated: (1) The effects of restriction of DNA, lingation of oligonucleotide adapters as well as amplification should be affected by the quality of genomic DNA. Clear finger map were obtained by the improved CTAB method which is suitable for AFLP analysis on Chinese cabbage. (2) There were 150 ng DNA template, EcoRI 1.25U, MseI 1.25U, 5×Reaction Buffer, in the best 12.5 μl system of restriction at 37°C for 4 h.
The optimum temperature in the lingation system was 20°C for 2.5 h. (3) The clear and stable amplification patterns could be obtained from six Primer pairs (E-AAC/M-CAG, E-AAG/M-CAC, E-ACA/M-CTG, E-ACT/M-CAC, E-ACT/M-CTT, E-ACT/M-CTC). Based on the silver-staining AFLP system, this paper might provide a method for the further study in the field of molecular marker in genetic diversity, new cultivars breeding and genetic relationship in Chinese cabbage.
The results indicated: (1) The effects of restriction of DNA, lingation of oligonucleotide adapters as well as amplification should be affected by the quality of genomic DNA. Clear finger map were obtained by the improved CTAB method which is suitable for AFLP analysis on Chinese cabbage. (2) There were 150 ng DNA template, EcoRI 1.25U, MseI 1.25U, 5×Reaction Buffer, in the best 12.5 μl system of restriction at 37°C for 4 h.
The optimum temperature in the lingation system was 20°C for 2.5 h. (3) The clear and stable amplification patterns could be obtained from six Primer pairs (E-AAC/M-CAG, E-AAG/M-CAC, E-ACA/M-CTG, E-ACT/M-CAC, E-ACT/M-CTT, E-ACT/M-CTC). Based on the silver-staining AFLP system, this paper might provide a method for the further study in the field of molecular marker in genetic diversity, new cultivars breeding and genetic relationship in Chinese cabbage.
Authors
Shuchun Meng, Haiying Zhang, Ling Liu, Yumei Liu
Keywords
Chinese cabbage (Brassica campestris L. ssp. pekinensis), DNA isolation, AFLP, silver-staining
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