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.
[1] Henry RJ.Plant Genotyping: The DNA Fingerprinting of Plants[M]. Wallingford: CABI, 2001.
[2] Phillips RL, Vasil IK.DNA-based Markers in Plants[M]. Dordrecht: Kluwer Academic Publishers, 2001.
[3] Flint-Garcia SA, Thuillet AC, Yu J, et al.Maize association population: a high-resolution platform for quantitative trait locus dissection[J][J].The Plant Journal, 2005, 44:1054-1064
[4]Simko I, Haynes KG, Ewing EE, et al.Mapping genes for resistance to Verticillium albo-atrum in tetraploid and diploid potato populations using haplotype association tests and genetic linkage analysis[J].Molecular Genetics and Genomics, 2004, 271(5):522-531
[5]Szalma SJ, Buckler ES, Snook ME, et al.Association analysis of candidate genes for maysin and chlorogenic acid accumulation in maize silks[J].Theoretical and Applied Genetics, 2005, 110(7):1324-1333
[6] Diego Lijavetzky, José Antonio Cabezas, Ana Ibá?ez, et al.High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology[J].BMC Genomics, 2007, (8):424-
[7] José A Cabezas, Javier Ibá?ez, Diego Lijavetzky, et al.A 48 SNP set for grapevine cultivar identification[J].BMC Plant Biology, 2011, (11):153-
[8] Tenaillon MI, Sawkins MC, Long AD, Gaut RL, Doebley JF, Gaut BS.Patterns of DNA sequence polymorphism along chromosome 1 of maize (Zea mays ssp. mays L.) [J].Proc Natl Acad Sci USA, 2001, 98:9161-9166
[9] Ching A, Caldwell KS, Jung M, Dolan M, Smith OS, Tingey S, Morgante M, Rafalski AJ.SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines[J]., 2002, 3:19-
[10] Vroh Bi I, McMullen MD, Villeda HS, Schroeder S, Gardiner J, Polacco M, Soderlund C, Wing R, Fang Z, Coe EH.Single nucleotide polymorphisms and insertion-deletions for genetic markers and anchoring the maize fingerprint contig physical map[J]., 2006, 46:12-21
[11] Elizabeth Jones, Wen-Chy Chu, Mulu Ayele, Julie Ho, Ed Bruggeman, Ken Yourstone, Antoni Rafalski, Oscar S. Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm[J]. [J].Molecular Breeding, 2009, 24:165-176
[12]Jianbing Yan, Xiaohong Yang, Trushar Shah, et al.High-throughput SNP genotyping with the GoldenGate assay in maize[J].Molecular Breeding, 2010, 25(3):441-451
[13]Jones E S, Sullivan H, Bhattramakki D, et al.A comparison of simple sequence repeat and single nucleotide polymorphism marker technologies for the genotypic analysis of maize(Zea mays L.)[J]. Theor. Appl.Genet., 2007, 115: 361-371.
[14] Wagner T, Stoppa-Lyonnet D, Fleischmann E, et al.Denaturating high-performance liquid chromatography detects reliably BRCA 1 and BRCA 2 mutations[J]. Genomics, 1999, 62:369-376.
[15] Gross E, Arnold N, Goette J, et al.A comparison of BRCA 1 mutation analysis by direct sequencing, SSCP and DHPLC[J]. Hum Genet, 1999, 105: 72-78.
[16] Qiang Li, Li-Yan Li, Sheng-Wen Huang, et al.Rapid genotyping of known mutations and polymorphisms in β-globin gene based on the DHPLC profile patterns of homoduplexes and heteroduplexes[J]. Clinical Biochemistry, 2008, 41:681–687
[17]Hiroshi Takashima, Cornelius F.Boerkoel,James RLupski. Screening for mutations in a genetically heterogeneous disorder: DHPLC versus DNA sequence for mutation detection in multiple genes causing Charcot-Marie-Tooth neuropathy[J].Genetics in Medicine, 2001, 3(5):335-342
[18] Kalliopi Pazaitou-Panayiotou, Athina Kaprara, Leda Sarika, et al.Efficient Testing of the RET Gene by DHPLC Analysis for MEN 2 Syndrome in a Cohort of Patients[J]. Anticancer Research, 2005, 25:2091-2096.
[19]Knud Nairz, Hugo Stocker, Benno Schindelholz, et al.High-resolution SNP mapping by denaturing HPLC[J].PNAS, 2002, 99(16):10575-10580
[20] Raja Kota, Markus Wolf, Wolfgang Michalek, et al.Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.) [J]. Genome, 2001, 44: 523–528
[21] 王关林, 方宏筠.植物基因工程原理和技术[M]. 北京: 科学出版社, 1998:600-601. (Wang G-L，Fang H-J．Principle and technology of plant genetic engineering[M]．Beijing: Science Press, 1998:600-601．
[22]唐立群, 肖层林, 王伟平.分子标记的研究及其应用进展[J].中国农学通报, 2012, 28(12):154-158
[23] Hamblin MT, Warburton ML, Buckler ES.Empirical comparison of simple sequence repeats and single nucleotide polymorphisms in assessment of maize diversity and relatedness[J]. Plos One, 2007, 2:e1367
[24] José A Cabezas, Javier Ibá?ez, Diego Lijavetzky, et al.A 48 SNP set for grapevine cultivar identification[J]. BMC Plant Biology, 2011, 11:153.
