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| Methods for High-throughput Detecting the Allelic Variation of AhFAD2 Gene Related with Oleic Acid Content in Peanut (Arachis hypogaea) |
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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.
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Received: 06 January 2016
Published: 22 July 2016
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