利用AFLP技术对谷子抗锈基因进行分子标记
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摘要
谷子锈病是对谷子产量有严重影响的主要病害之一,培育和利用谷子抗锈品种是当前控制其危害的一种最为经济有效的措施。寻找与谷子抗锈基因连锁的分子标记,构建遗传连锁图,对于抗谷子抗锈病基因的分离和利用具有重要意义。
本研究对谷子高抗锈病品种十里香和高感锈病品种豫谷1号及其杂交后代F2代分离群体进行AFLP分子标记分析。取得以下主要结果:
1.采用改良的CTAB法获得高质量的谷子基因组DNA,并用限制性内切酶MseⅠ和EcoRⅠ双酶切谷子基因组DNA。应用AFLP分子标记技术对谷子F2代分离群体进行分析。随机选择270对AFLP引物组合对抗锈亲本,感锈亲本以及F2代分离群体的抗锈基因池和感锈基因池进行分析,发现有92对引物组合能揭示出多态性。
2.对筛选出的92对引物组合多次重复性验证F2代单株,检测结果显示引物组合E-AAC/M-ATA,E-AAC/M-CGT和E-AC/M-CC能够在两亲本和F2代分离群体间扩增出稳定的多态性条带。此3对引物组合均能在父本十里香和抗锈基因池中扩增出目的条带,条带长度分别为188bp,212bp和176bp,而母本和感锈基因池中没有相应条带。经MapMaker 3.0软件分析,AFLP分子标记E-AAC/M-ATA,E-AAC/M-CGT和E-AC/M-CC与抗锈病基因Rusi1连锁,遗传距离分别为9.2 cM,9.8 cM和12.4 cM。应用Mapdraw软件构建了这3个AFLP分子标记和谷子抗锈病基因Rusi1的遗传连锁图谱。
3.优化了AFLP分子标记技术在谷子抗锈病基因研究中的反应体系。根据相关文献报道的AFLP分子标记技术在其它作物中的应用情况,结合谷子基因组DNA的的特点,对影响最终检测结果的相关因素进行了详尽研究,对AFLP分子标记技术进行了相关改良,使其能够最有效地应用于谷子抗锈病基因的研究中,构建了一套适合于谷子抗锈病基因研究的AFLP分子标记技术体系。
Millet rust is one of the most infectious fungus which affects yield of foxtail millet (Setaria italica (L.) Beauv). Therefore, breeding and using herbicide resistant varieties in agriculture production seems to be one of the most effective and economical methods to control Uromyces setariae-italicae (Diet) Yoshino so as to improve yield. According to this, scanning the linkage molecular markers and construct the genetic linkage map for millet rust resistance gene should be very important to isolate and use this resistant gene. It must be of great value for promoting the herbicide resistance breeding in foxtail millet.
In this study, the parents, Shilixiang which shows high resitance to foxtail millet rust, Yugu1 which is highly susceptible to foxtail millet rust and their F2 progeny were used as experimental materials for the AFLP analysis of millet rust resistant gene.
1. Modified CTAB method was used to extract DNA of foxtail millet, which meets AFLP requirements in high quantity. And then two restriction enzymes MseI and EcoRI were used to deal with the DNA of foxtail millet. AFLP analysis was conducted in the two parents and their F2 progeny. 92 out of the 270 pairs of randomly selected primers amplified the polymorphic bands.
2. Three pairs (E-AAC/M-ATA,E-AAC/M-CGT and E-AC/M-CC) of 92 pairs which amplified the polymorphic bands, showed the same discriminating results in replications. The primers E-AAC/M-ATA,E-AAC/M-CGT and E-AC/M-CC produced fragments with the length of 188bp, 212bp and 176bp linked to rust resistant gene Rusi1 with the genetic distance of 9.2 cM, 9.8 cM and 12.4 cM. And then the three AFLP molecular markers were used to construct a genetic linkage map for millet rust resistance gene. The AFLP markers and the genetic linkage map would be facilitated the selection on millet rust resistant gene.
3. Modified AFLP molecular marker technique was used in scanning the linkage molecular markers and construct the genetic linkage map for millet rust resistance. According to the already published documents which reported the use of AFLP molecular marker technique in other plants and the features of the DNA of foxtail millet, the factors that eventually affected the results in scanning the linkage molecular markers for millet rust resistance were discussed in details. And finally, a set of modified AFLP molecular marker technique was established, which is of great use in scanning the linkage molecular markers and construct the genetic linkage map for millet rust resistance.
本研究对谷子高抗锈病品种十里香和高感锈病品种豫谷1号及其杂交后代F2代分离群体进行AFLP分子标记分析。取得以下主要结果:
1.采用改良的CTAB法获得高质量的谷子基因组DNA,并用限制性内切酶MseⅠ和EcoRⅠ双酶切谷子基因组DNA。应用AFLP分子标记技术对谷子F2代分离群体进行分析。随机选择270对AFLP引物组合对抗锈亲本,感锈亲本以及F2代分离群体的抗锈基因池和感锈基因池进行分析,发现有92对引物组合能揭示出多态性。
2.对筛选出的92对引物组合多次重复性验证F2代单株,检测结果显示引物组合E-AAC/M-ATA,E-AAC/M-CGT和E-AC/M-CC能够在两亲本和F2代分离群体间扩增出稳定的多态性条带。此3对引物组合均能在父本十里香和抗锈基因池中扩增出目的条带,条带长度分别为188bp,212bp和176bp,而母本和感锈基因池中没有相应条带。经MapMaker 3.0软件分析,AFLP分子标记E-AAC/M-ATA,E-AAC/M-CGT和E-AC/M-CC与抗锈病基因Rusi1连锁,遗传距离分别为9.2 cM,9.8 cM和12.4 cM。应用Mapdraw软件构建了这3个AFLP分子标记和谷子抗锈病基因Rusi1的遗传连锁图谱。
3.优化了AFLP分子标记技术在谷子抗锈病基因研究中的反应体系。根据相关文献报道的AFLP分子标记技术在其它作物中的应用情况,结合谷子基因组DNA的的特点,对影响最终检测结果的相关因素进行了详尽研究,对AFLP分子标记技术进行了相关改良,使其能够最有效地应用于谷子抗锈病基因的研究中,构建了一套适合于谷子抗锈病基因研究的AFLP分子标记技术体系。
Millet rust is one of the most infectious fungus which affects yield of foxtail millet (Setaria italica (L.) Beauv). Therefore, breeding and using herbicide resistant varieties in agriculture production seems to be one of the most effective and economical methods to control Uromyces setariae-italicae (Diet) Yoshino so as to improve yield. According to this, scanning the linkage molecular markers and construct the genetic linkage map for millet rust resistance gene should be very important to isolate and use this resistant gene. It must be of great value for promoting the herbicide resistance breeding in foxtail millet.
In this study, the parents, Shilixiang which shows high resitance to foxtail millet rust, Yugu1 which is highly susceptible to foxtail millet rust and their F2 progeny were used as experimental materials for the AFLP analysis of millet rust resistant gene.
1. Modified CTAB method was used to extract DNA of foxtail millet, which meets AFLP requirements in high quantity. And then two restriction enzymes MseI and EcoRI were used to deal with the DNA of foxtail millet. AFLP analysis was conducted in the two parents and their F2 progeny. 92 out of the 270 pairs of randomly selected primers amplified the polymorphic bands.
2. Three pairs (E-AAC/M-ATA,E-AAC/M-CGT and E-AC/M-CC) of 92 pairs which amplified the polymorphic bands, showed the same discriminating results in replications. The primers E-AAC/M-ATA,E-AAC/M-CGT and E-AC/M-CC produced fragments with the length of 188bp, 212bp and 176bp linked to rust resistant gene Rusi1 with the genetic distance of 9.2 cM, 9.8 cM and 12.4 cM. And then the three AFLP molecular markers were used to construct a genetic linkage map for millet rust resistance gene. The AFLP markers and the genetic linkage map would be facilitated the selection on millet rust resistant gene.
3. Modified AFLP molecular marker technique was used in scanning the linkage molecular markers and construct the genetic linkage map for millet rust resistance. According to the already published documents which reported the use of AFLP molecular marker technique in other plants and the features of the DNA of foxtail millet, the factors that eventually affected the results in scanning the linkage molecular markers for millet rust resistance were discussed in details. And finally, a set of modified AFLP molecular marker technique was established, which is of great use in scanning the linkage molecular markers and construct the genetic linkage map for millet rust resistance.
引文
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