Establishment And Correlative Analysis Of DNA Fingerprinting Of Partial Maize Hybrids And Their Parents In Guizhou

One hundred and twenty nine maize materials including 48 inbred lines and 81 hybrids, which widely used or being extended in Guizhou province in China,were analyzed by using SSR (Simple Sequence Repeat) technic.A highly effective,accurate and reliable technical system SSR molecular marker was set up for germplasm identification.The DNA fingerprints database of above inbred lines and hybrids was constructed.The main results were summarized as follows:1.Through comparing several methods of DNA-extracting,SSR-PCR reaction system,SSR-PCR amplification procedures,non-denaturing and denaturing polyacrylamide gel and silver staining,a protocol for SSR marker technique in maize was as follows:(1) SDS or CTAB method should be chosen for maize DNA extraction.(2) The SSR reaction system was 20μL PCR reaction consisted of 1×PCR buffer,2.0 mM Mg²⁺,0.15mM dNTPs,1U Taq polymerase,0.3μM of each primer,and 60 g of DNA.(3) The PCR amplification procedure was 93℃for 1 min;30 cycles of 93℃for 1 min,60℃for 2min,and 72℃for 2min;and a terminal extension step at 72℃for 5 min.(4) It was appropriate to use 8%non-denaturing polyacrylamide gel or 6%denaturing polyacrylamide gel to separate SSR products.(5) The silver staining procedure was:washing two times with distilled water;staining with 0.2%silver nitrate for 10min;washing two times with distilled water; visualizing with 3%NaOH and 0.5%formaldehyde.By our test,the above protocol for SSR marker analysis in maize was economical and effective.2.It was feasible to use SSR technique to construct corn fingerprinting.The SSR analysis of above maize hybrid varieties and their parent materials were conducted.The 20 pairs of maize SSR primers screened from 100 pairs of SSR primers by Beijing Academy of Agriculture and Forestry were used to establish a DNA fingerprinting of these hybrids and their parents.What's more,the 129 Corn materials can be discriminated from each other by only 10 pairs of primers used in the fingerprint.Therefore,the fingerprinting can be extended for use.3.It was feasible to use SSR technique to identify authenticity of maize hybrids and inbred lines. Twenty pairs of polymorphic primers were used to analyze maize hybrids and inbreds,their amplified products have 4～7 bands generally,each with molecular weight in the range of 100～400bp.The number of alleles detected at each site is 5.3 averagely,polymorphism information 0.611,marking index coefficient 3.34.The DNA fingerprinting constructed by the 20 pairs of SSR primers can be used to identify the reality of hybrids and inbred lines in maize.4.A Two-PCR-set was consisted of two pairs of SSR primers chosen combinatorially from the 20 pairs of SSR primers used in the fingerprint.A Three-PCR-set was actualized after increasing one pairs of primers to the better double combination.In the same way,sets of Four,Five,and Six-PCR were achieved.The PCR amplified results of the Two,Three,Four,Five,and Six-PCR-set were accorded with the results of single PCR.