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Authenticity and Purity Identification of Corn (Zea mays) Variety Using DHPLC |
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Abstract Denaturing high-performance liquid chromatography (DHPLC) could be used to test the single nucleotide polymorphism (SNP) and Insertion/Deletion (InDel) polymorphism markers, which would be applied for the rapid identification of diverse hybrid maize. In order to test the feasibility and reliability of DHPLC for molecular marker genotyping in maize (Zea mays), the DHPLC analysis on 10 hybrids and their parents were conducted. In this study, based on the information in maize genome database, two DNA fragments harboring multiple and informative SNPs were screened. PCR products of the two amplicons from all maize varieties were analyzed by DHPLC and DNA sequencing. We first analyzed the PCR product from C7-2 at the temperate predicted by WaveMarker software to obtain a homoduplex elution profile for the two fragments. The experimental results showed the software-predicted temperature was only suitable for the analysis of homoduplex DNA for AmpⅡ. By analyzing the PCR product from C7-2 in 0.5 ℃ decrements over the range of predicted temperature for AmpⅠ, the optimal condition appropriate for the analysis of homoduplex DNA and other genotypes was obtained. Our results showed under the optimal separation condition, the two screened DNA fragments were polymorphic across hybrids with distinct DHPLC elution profiles produced. AmpⅠexhibited 3 different DHPLC elution profiles and AmpⅡproduced 7 types of elution profiles. Different DHPLC profiles could be detected and distinguished easily due to their significant differences in peak number and retention time. For each of the two DNA fragments, some samples showed nearly identical profiles, which had the same peak number and similar retention times. The sequencing results showed that the samples exhibiting nearly identical elution profiles harbor common sequence variants, and each sequence variant presented a unique elution profile for each of the two DNA fragments. It confirmed that the distinct DHPLC patterns corresponded to different DNA sequences. A set of distinct characteristic profiles in two DNA fragments could differentiated between 7 hybrids with at least a single profile pattern difference, except three varieties (C4, C87 and 6047×618) which had identical profiles. We also found that because hybrids displayed a characteristic pattern of DHPLC elution profile resulting from parental variation in allelic nucleotide sequence, the DHPLC pattern of the hybrid maize was in accordance with the results obtained by DHPLC analysis of a mixed sample of the two parents. We proposed it was practical to test genetic purity of F1 hybrid seeds by comparing their DHPLC elution profiles with that of a mixed sample of their two parents. Due to the advantages of high resolution, high through-put, simplicity and safety, DHPLC would be a useful technology for rapid identification of hybrids and seed genetic purity test.
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Received: 19 May 2015
Published: 23 November 2015
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