论文部分内容阅读
限制性位点扩增多态性(RSAP)是一种新型分子标记技术。为获得适用于芝麻的RSAP-PCR的反应体系,本研究通过L16(45)正交设计与单因素试验相结合的方法,对PCR反应体系中模板DNA、Mg2+、d NTPs、引物和Taq DNA聚合酶等影响因素进行了试验优化。结果表明,芝麻RSAP-PCR的最佳反应体系为:在15μL反应体系中,30 ng模板DNA、2.0 mmol/L的Mg2+、0.3 mmol/L的d NTPs、0.6μmol/L的引物和1.0 U的Taq DNA聚合酶。利用17个芝麻品种对该体系进行了稳定性验证和多态性检测,结果表明,该反应体系稳定、可靠,29对随机引物组合共扩增DNA条带454条,每对引物扩增8~21条,平均15.66条,多态性比例为0%~56.25%,平均25.77%。利用普通芝麻与有限生长习性芝麻构建的F2分离群体对RSAP标记进行了初步遗传验证,结果表明,F2群体植株中出现了亲本位点的分离。该RSAP-PCR反应体系的建立与优化为芝麻遗传多样性分析、连锁图谱构建和分子标记辅助育种提供了新技术。
Restriction site-amplified polymorphism (RSAP) is a novel molecular marker technique. In order to obtain the RSAP-PCR reaction system suitable for sesame, the objective of this study was to polymerize template DNA, Mg2 +, dNTPs, primers and Taq DNA in a PCR reaction system by a combination of L16 (45) orthogonal design and single factor experiments Enzyme and other factors were tested and optimized. The results showed that the best reaction system of RSAP-PCR was as follows: 30 ng template DNA, 2.0 mmol / L Mg2 +, 0.3 mmol / L dNTPs, 0.6 μmol / L primer and 1.0 U Taq DNA polymerase. The stability and reliability of this system were tested with 17 sesame cultivars. The results showed that the reaction system was stable and reliable. A total of 454 DNA bands were amplified by 29 pairs of random primers. Each pair of primers amplified 8 ~ 21, an average of 15.66, the proportion of polymorphisms was 0% ~ 56.25%, an average of 25.77%. The RSAP markers were preliminary verified by F2 segregation population of common sesame and limited growth habit of sesame. The results showed that there was a segregation of parental loci in F2 population. The establishment and optimization of the RSAP-PCR reaction system provided new technologies for genetic diversity analysis, linkage map construction and molecular marker-assisted breeding of sesame.