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2025年4月4日 星期五
  2016, Vol. 24 Issue (9): 1364-1373    
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
高通量检测花生油酸含量相关基因AhFAD2等位变异的方法
徐平丽1,唐桂英2,付春3,刘玮2,鲁成凯3,姜言生3,刘皓3,单雷2
1. 山东省农业科学院生物技术研究中心
2. 山东省农业科学院生物技术研究中心/山东省作物遗传改良与生理生态重点实验室
3. 山东省潍坊市农业科学院
Methods for High-throughput Detecting the Allelic Variation of AhFAD2 Gene Related with Oleic Acid Content in Peanut (Arachis hypogaea)
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摘要 花生(Arachis hypogaea)Δ12脂肪酸去饱和酶基因(Δ12 fatty acid desaturase, AhFAD2A和AhFAD2B)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AhFAD2A 448位G/A差异和AhFAD2B 442位A碱基的插入/缺失(insertion-deletion, InDels)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)、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、TaqMan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TaqMan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AhFAD2B 442 nt A/-InDel差异结果的一致率高达96.7%;而针对AhFAD2A 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、TaqMan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。
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徐平丽
唐桂英
付春
刘玮
鲁成凯
姜言生
刘皓
单雷
关键词 花生??12脂肪酸去饱和酶基因(AhFAD2)等位变异油酸检测方法花生    
Abstract:Cultivated peanut (Arachis hypogaea) is one of the important oil crops, and the planting area stabilize at about 4.7 million hectares in China in recent years. In general, the oleic acid content of kernels is about 36%~67% in cultivars currently grown in China, and the ratio of oleic acid to linoleic acid (O/L) ranges from 0.78 to 3.5. Increasing the O/L of peanut cultivars significantly improves the nutritional quality and prolongs the shelf-life, and has become one of the major goals of researchers in the area of plant lipid modification. Peanut ??12 fatty acid desaturase genes(AhFAD2A and AhFAD2B) are the key genes controlling the content of oleic acid and O/L in the peanut kernels, and the mutations at both AhFAD2A and AhFAD2B loci exist in the high oleic acid peanut varieties. In view of the G/A variation in AhFAD2A 448 nt and the A base insertion-deletion (InDel) variation in AhFAD2B 442 nt in the low-oleate and high-oleate peanut germplasm, several molecular markers and detection methods including cleaved amplified polymorphic sequence (CAPS), allele-specific PCR(AS-PCR), and so on, have been developed and applied. In this study, the primers, probes and detection protocol of the TaqMan q-PCR for high-throughput detection of the two SNP allelic variations were developed, and a number of samples including some varieties and individual plants from the backcross group BC2F1 and BC1F2 were detected. It was found that this method was highly efficient and accurate, and the detection results were in accord with the sequencing results. Furthermore, in order to confirm the results of genotype discrimination, their oleic acid and linoleic acid contents were detected by gas chromatography or near infrared reflectance spectroscopy. Kompetitive allele specific PCR (KASP) was a homogeneous, fluorescent, endpoint genotyping technology, which offered the simplest, most cost-effective and flexible way to determine both SNP and insertion/deletion genotypes. An efficient KASP method for detecting these two allelic variations was also developed in this study. Comparison with the method of TaqMan probe, the KASP was more economic due to applying the universal fluorescence-labeled probe. Using the TaqMan qPCR method and the KASP method, the genotypes of AhFAD2A and AhFAD2B gene in 14 peanut varieties and the seeds of the backcross group BC2F1 and BC1F2 were identified. The consistency rates of the two methods for G/A SNP and A/-InDel were 71.7% and 96.7%, respectively. The lower accuracy for G/A SNP by KASP results from the interfering of the G at 448 nt of AhFAD2B and the most similarity of sequences between AhFAD2A and AhFAD2B. Based on the different fluorescent probes for G/A or A/-InDel allele variation, these two genotyping assays could distinguish three different genotypes (the homozygote, heterozygote of wild-type and mutant-type) in each reaction. In this paper, the advantages and disadvantages of different methods including CAPS, AS-PCR, sequencing, TaqMan probe and KASP were compared. Among these methods, TaqMan probe and KASP with the obvious detection accuracy and stability were high-throughput detection methods, and need a higher requirement for the instruments, such as the q-PCR equipment or laboratory of the government chemist (LGC) SNPline XL PCR equipment. Finally, the application prospect of the new high-throughput method was also discussed. These high-throughput methods will greatly improve the accuracy and efficiency of selecting for high oleic acid and effectively accelerate the breeding course of peanut varieties with the target traits.
Key wordsPeanut Δ12 fatty acid desaturase gene (AhFAD2)    Allelic variation    Oleic acid    Detection method    Peanut
收稿日期: 2016-01-06      出版日期: 2016-07-22
基金资助:调控FAD2不同剪接体参与膜脂和储藏脂合成的机制研究
通讯作者: 单雷     E-mail: shlei1025@sina.com
引用本文:   
徐平丽 唐桂英 付春 刘玮 鲁成凯 姜言生 刘皓 单雷. 高通量检测花生油酸含量相关基因AhFAD2等位变异的方法[J]. , 2016, 24(9): 1364-1373.
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http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I9/1364
 
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