Construction Of The SSR Reaction System And Application Research In Maize Breeding With The Heavy Ion Irradiation

With the heavy ion irradition is used inducing crop genetic variation, compared to traditional new approaches of physical mutagenesis, having the characteristics of the less physiological damage, wide mutation spectrum, higher mutation frequency, a certain repeatability and directivity. So recently, it has been widely appiled in rice breeding, rapeseed breeding, wheat breeding ect. SSR molecular marker does not specially need design primers, simple operation, it has been studied on diversity of crop germplasm resources and the identification in hybrid, issubjected universal attention by researchers. In this study selection 37 maize (Zea mays L.) material by treating with heavy ion radiation 12C6+, establishment SSR molecular marker reaction system, analysis of diversity of crop germplasm resources and genetic variation,control field biology phenotypic effects, exploration relationship among SSR molecular marke, heavy ion irradiation and mutative traies, provide theoretical basis in reasonable selection maize hybrids, improve utilization level of heterosis. Main results as follows:(1) Using maize as material, construction stable SSR reaction system,1cycle of prede-natureation at 94℃for 3 min; followed by 30 cycles of denaturation at 94℃30 s, annealing at 50~58℃30 s, and extension at 72℃for 1 min; the final cycle had a 5 min extension at 72℃. The PCR mixtures 25 ul contained 2.5 ul 10×buffer(Mg2+ free), 2.0 ul dNTP(2.5 Mm each),0.4 ul Taqpolymerase(2.5 U/ul),2.0 ul Mg2+(25 mM),2.0 ul each primer. Amplified the genomic DNA of 37 maize by SSR, 18 primes produced 56 polymorphic amplified fragments. The Molecular weight was concentrated between 100~350 bp. The average number of alleles per SSR locus was 3.11 with range from 2 to 6.(2) With the basis for genetic divergence preparation excellent bybridized combination. The cluster analysis showed that material could be classified into 4 distinct clusters. The firt class is F30, on the molecular level, it has the better specificity. The second class including E10, E20, E30, E40, E50, Eck. The third class A10, A15, A20, A30, Ack, C30, D20, D25, D30, B25, Dck, F10, F20, F50, Fck and C20, C25, Cck, D10, D15, F40. The fourth class including A25,C10,C15 and B10,B15,B20,Bck,B30,C5.(3) Plant character and characteristic of spikes on the quantitative character exhibits different by heavy ion irradiation. With increase irradiation dose, the plant of seedling emergence rate and seedling rate decrease. So the more irradiation dose, the greater rate decrease 12.0 %~34.6 %; M3 generation, seeding emergence decrease 5.6 %~47.2 %, seeding rate decrease 1.91 %~42.0 %. Suitable irratiation dosage of 12C6+ irradited maize seed is 20~25 Gy. M2 generation, the more irradiatong dose, the lower plant height and ear heigh, but leaf number above the ear and total leaf number have no obvious difference. With the change of irradiation dose, change color of anthers, increase ear rows, granulosity change into hard grain type, increase grain weight, increase varieties of rust resistancered-leaf diease, producing the positive variation.(4) Irradiation dose is 30 Gy in maize inbred lines 478 on the molecular level become a kind, plant height and ear heigh are lower than irradiation dose is 10 Gy, 20 Gy, 40 Gy, 50 Gy and it has obvious side variation, mutil-spike and longer ligule etc. Irradiation dose is 10 Gy, 20 Gy, 40 Gy, 50 Gy in maize inbred lines 478 and other materials cluster one class, showed morphological, grain type and grain weight have changed, panicle type and ear rows increase, plant height and ear heigh lower the whole immane. Maize inbred line 308 through a field experiment, the consistent with plant type but plant height and ear heigh have differences. The forth type, basically, agronomic characters is consistent: the higher plant height and ear heigh, occuring white axis, changes of grain. On the molecular level becoming a kind of maize inbred line, finding a certain similarity on the campestral agronomic characters. The results showed that SSR molecular marker can be used to analyse mutagenic material of radioactive breeding.