Analysis Of Plant Height Difference And PCR Fragments Of SCAR Primers About Male Sterile Mutant Induced By Space Flight In Maize

1996, a male sterile mutant controlled by one single recessive gene was screened in the offspring of chuan Dan No.9 hybrid. Previous analysis by DNA-AFLP has been conducted, polymorphism fragments were got between male sterile and male fertile. The polymorphism fragment of DNA-AFLP has been converted into SCAR marker, PCR product sequencing and clone sequencing were used in this experiment. At the same time, according to the preview observation, the plant height of male sterile always accompanied with lower plant height compared to male fertile plant. So, the difference analysis of plant height between male sterile and male fertile were also conducted in this study.The main results are as follows:1. PCR was made among 5 plants of male fertile and 5 plants of male sterile, respectively, with 5 pairs of SCAR primers, there were no length polymorphism fragments among male fertile and male sterile plants with agarose gel electrophoresis. Target fragments re-attain, clone sequencing and PCR product sequencing were applied at the same time.5 plants of male sterile and 5 plants of male fertile were sequenced with 3 clones each plants.5 plants of male sterile and 5 plants of male fertile were sent for direct sequencing with 2 PCR products each plants.14 and 13 effective sequences were got for male fertile and male sterile respectively by clone sequencing.10 effective sequences were got for male fertile and male sterile respectively by PCR product sequencing.2. sequence analysis.there are some single base's differences within 3 clones of per plant and 2 PCR products of per plant in the sequences.Strategy was adopted to adjust the sequences according to the principle of the more frequency,the more possible.Then analyze the differences between male sterile and male fertile with the adjusted sequence.After that few single base's differences between male sterile and male fertile still can be screened. These difference may be related with fertility phynotype. Program FGENESH was used to forecast the introns and the open reading frame for the objective sequence.3. Sequencing analysis through NCBI blast showed that sequences was homology as a cloning AF466646.1, target sequence is in a coding region of AF466646.1, this coding region is produced gene Z195D10.19,it is presumed retrotransposons, Blastp with AA sequences in NCBI,found the most similar protein is prpol. Clone AF466646.1can be translated into prpol. So, the target fragment's function may be related to retrotransposons.4.Analysis.Looking for restriction site in the sequences of clone sequencing,found that 2 single base's differences lead to 3 restriction sites changed in clone sequencing,and 1 single base's differences lead to 1 restriction sites changed in PCR product sequencing. There is the possibility that primers and enzyme could be used together as a CAPS marker with which.male sterile plants and male fertile plants of maize which origined from space flight could be distinguished to some extent.5. Plant height, internode number, internode length, tassel panicle length of plants in segregation populations were surveyed both at Yunnan and Sichuan experiment plots. Fertility were observed during adults periods.By statistic analysis,we found that the difference of Plant height, internode number, internode length and tassel panicle length of plants between male sterile and male fertile were significant, it were lower for male sterile than that of male fertile.This results indicated that the difference of plant height between male sterile and male fertile which origined from space flight in maize may be due to the difference of internode number, internode length., tassel panicle length.