Molecular Technique Of Examination For Hybrid Seed Purity Of Maize

The genuineness and purity test of seed is important on maize (Zea mays L. ) production.With the development of maize breeding and seed trade, the protection of breeder's right isgraduaIIy recognized. DNA fingerPrinting approach can be used to identify the distinctness andunifOrmity of the variety rapidly and exactly, and could have a significant sense on hybrid seedpurity test and new variety protection. This paper established a moIecular technique of testingfOr maize seed purity based on SSR markers, which included DNA molecular detectingapproach, core primers used in PCR (Polymerase Chain Reaction), DNA fingerprintingdatabase of elite maize hybrids and 'biometricaI anaIysis method. With the rapid DNAextracting procedure frorn single kernel, the molecular technique could be effectiveIy expIoitedfor hybrid seed purity test of maize. The results were summarized as follows1. The molecular technique was established based on SSR markers, which included PCRsystem, polyacrylamide gel electrophoresis, silver staining method, the biometrical analysis ofband profile, etc.2. Sixty-six out of 98 pairs of SSR primers were selected, which gave clear, stable,repeated, polymorphic amplification profiles in the sample of 21 inbred lines. 66 primersproduced 25l poIymorphic aIIeIes. The average number of alleIes per SSR loci was 3.80 with arange from 2 to 9. The size of fragments ranged from 63 to 502bp. Tl1e Polymorpl1icInformation Content (PIC) for the SSR loci varied from 0.l49 to 0.800 with an average of0.5 l5. The experiment showed that 7 primers possessing a higher level of polymorphism couIdbe used to identify the 2l inbreds.3. The experiment established DNA fingerprinting database of 94 eIite maize inbred linesused predominantly in China. 66 primer8 produced 295 aIIeIes with the average of 4.47 per lociand a range from 2 to 19. The size of fragments varied from 63 to 502bp, and the PIC valueranged from 0.040 to 0.875 with an average of 0.5l6.4. Twenty-nine out of 66 pairs of primers were further selected, which gave very cIear,stable, repeated, polymorphic amplification profiles. 29 primers produced l l6 aIIeles among 26inbreds ranged from 2 to 6 with the average of 4.00. The size of alleIes ranged from 63 to502be, and the PlC value for the SSR Ioci varied from 0.038 to 0.767 with an average of 0.545,5. The resuIts showed that there were four ampIified profiles between the hybrid and itsparental lines, incIuding maIe, female and hybrids, of which the parents were the main ones.6. The rapid DNA extracting procedure from single kemel was established incIudingquickIy miIIing embryo, centrifugeIizing-milled liquid. absorbing top clear liquid, etc.7. The resuIts showed that l3 hybrids couId be distinguished by SSR fingeI'Printingpatterns amplified by 2 to 3 primers and statistical anaIysis, informing that the DNA detectingtechnique established could be effectiveIy expIoited for hybrid seed purity test of maize.