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花生(Arachis hypogaea)Δ12脂肪酸去饱和酶基因(Δ12fatty acid desaturase,Ah FAD2A和Ah FAD2B)是决定花生籽粒油酸含量和高油酸亚油酸比值(oleic acid/linoleic acid,O/L)的关键基因,高油酸花生品种中这2个基因均发生突变。针对普通油酸花生和高油酸花生Ah FAD2A 448位G/A差异和Ah FAD2B 442位A碱基的插入/缺失(insertion-deletion,In Dels)等位差异,已开发了包括酶切扩增多态性序列法(cleaved amplified polymorphic sequence,CAPS)和等位基因特异PCR(allele-specific PCR,AS-PCR)等多种检测的标记和检测方法。本研究针对上述2个SNP位点,开发了可进行高通量检测的实时定量PCR引物、Taq Man探针和检测技术,该方法检测效率和准确率均很高。本研究还开发了更为经济和高效的竞争性等位基因特异性PCR(kompetitive allele specific PCR,KASP)引物和检测方法。利用开发的Taq Man探针检测方法和KASP方法检测了14个花生品种和高油酸选育回交组合BC2F1和BC1F2群体部分种子的基因型,并比较了两种方法检测结果的一致率,发现这两种方法检测Ah FAD2B 442 nt A/-In Del差异结果的一致率高达96.7%;而针对Ah FAD2A 448位G/A差异位点检测一致率仅为71.7%。本研究还比较了目前常用的CAPS、AS-PCR、测序法、Taq Man探针法及KASP方法的优缺点,对相关方法的应用前景进行了讨论。本研究涉及的高通量检测方法可有效地辅助高油酸品种的选育,极大地提高了目标性状选择的准确性和效率。
The Δ12 fatty acid desaturase (Ah FAD2A and Ah FAD2B) genes of peanut (Arachis hypogaea) are the key determinants of oleic acid content and oleic acid / linoleic acid (O / L) Genes, high oleic acid peanut varieties in these two genes were mutated. In view of the allelic variation of the 448-position G / A difference between Ah-FAD2A and Ah-FAD2B 442-position A bases in common oleic and high-oleic peanuts and the insertion-deletion (In Dels) (CAPS), allele-specific PCR (AS-PCR) and other markers and detection methods. In this study, real-time PCR primers, TaqMan probes and detection techniques for high-throughput detection were developed for the above two SNP sites. The detection efficiency and accuracy of the method are high. This study also developed a more economical and efficient kampetitive allele specific PCR (KASP) primer and detection method. The genotypes of partial seeds of BC2F1 and BC1F2 populations from 14 peanut varieties and high oleic breeding backcross combinations were detected by Taq Man probe assay and KASP method. The agreement between the two methods was also compared. The coincidence rate of Ah FAD2B 442 nt A / -In Del difference between these two methods was as high as 96.7%, while that of Ah FAD2A 448 G / A difference site was only 71.7%. This study also compared the current advantages and disadvantages of CAPS, AS-PCR, sequencing, TaqMan probe and KASP methods, and discussed the application prospects of the methods. The high-throughput detection methods involved in this study can effectively assist breeding of high-oleic varieties and greatly improve the accuracy and efficiency of target trait selection.