Articles
RAPD MARKERS DEVELOPED FOR IDENTIFYING MALE STERILITY IN TAGETES ERECTA
Article number
855_43
Pages
289 – 294
Language
English
Abstract
Various experimental protocols for RAPD analysis of genes related to fertility in Tagetes erecta were studied and an optimum procedure was developed for setting up a stable system.
The CTAB and SDS method were compared to extract DNA from different tissues of Tagetes erecta. The quality of DNA extracted was evaluated by spectrophotometry and PCR. Results showed that total DNA extracted from young leaves by CTAB was suitable for the analysis of RAPD. The concentration of template DNA, MgCl2, buffer and dNTPs were examined.
Amplifications were then carried out in a 20 μl volume containing 5 μl diluted template DNA, 2 μl 10× PCR buffer, 2.5 mmol MgCl2, 0.3 mmol dNTP, 0.3 μmol primer and 1.50 U Taq DNA polymerase.
PCR were operated under the following program: 5 min at 94°C before amplification, 40 cycles of 94°C for 30 s, 37°C 1 min, 72°C 1 min, followed by one cycle of 72°C for 1 min.
By using this system, 100 sterile and fertile plants of W205, a two type male sterile line in Tagetes erecta were tested.
Two hundred eighty eight bands were obtained using 64 RAPD primers, among which one band of 980 bp derived from the primer G-02(5-GGCACTGAGG-3) was cosegregating with fertile plants.
This RAPD marker named as G-02-980 could be used to identify male and fertile seedlings.
The CTAB and SDS method were compared to extract DNA from different tissues of Tagetes erecta. The quality of DNA extracted was evaluated by spectrophotometry and PCR. Results showed that total DNA extracted from young leaves by CTAB was suitable for the analysis of RAPD. The concentration of template DNA, MgCl2, buffer and dNTPs were examined.
Amplifications were then carried out in a 20 μl volume containing 5 μl diluted template DNA, 2 μl 10× PCR buffer, 2.5 mmol MgCl2, 0.3 mmol dNTP, 0.3 μmol primer and 1.50 U Taq DNA polymerase.
PCR were operated under the following program: 5 min at 94°C before amplification, 40 cycles of 94°C for 30 s, 37°C 1 min, 72°C 1 min, followed by one cycle of 72°C for 1 min.
By using this system, 100 sterile and fertile plants of W205, a two type male sterile line in Tagetes erecta were tested.
Two hundred eighty eight bands were obtained using 64 RAPD primers, among which one band of 980 bp derived from the primer G-02(5-GGCACTGAGG-3) was cosegregating with fertile plants.
This RAPD marker named as G-02-980 could be used to identify male and fertile seedlings.
Publication
Authors
Zhigang Wang, Dongsheng Yin, Ping Wang, Jingyun Zhao, Baitao Pan, Huihua Zhang
Keywords
Tagetes erecta, male sterility, RAPD, molecular marker
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