Corn Scar Markers For Raw Gene Marker Fragment Sequence Analysis

The appropriate near-isogenic lines (NIL) were constructed to screen RAPD markers for opposite character of maize. The two stable RAPD markers were successfully converted into SCAR markers after they were identified closely link to the two opposite genes through linkage analysis in an F2 population. The SCAR markers closely link to the opposite genes were obtained and the sequences of the SCAR fragments were analyzed. The results showed:1. The genetics investigation indicated there were two different dominant genes between the H14D opposite line and the H53 alternative line we constructed. The two genes are independent and mutually complemental.2. 11 stably polymorphic RAPD primers such as S312, S218, S360 were screened from 167 1 Omer primers between the two near-isogenic lines.3. The linkage of the 11 polymorphic primers was analyzed respectively with RAPD PCR method within an F2 population of 251 individuals. The results revealed the RAPD markers RAPD_S312 and RAPD_S218 were closely linked to the gene OppJ with the genetic distance 3.2cM and 5.2cM respectively, the marker RAPD_S360 was closely linked to the gene Opp₂ with genetic distance 5.2 cM.4. The linkage relationship among 11 RAPD marker was also analyzed: RAPD_S312 and RAPD_S218 belong to one linkage group with genetic distance 8.7cM and they locate in either side of the gene Opp₁.The genomic region between the marker RAPD_S312 and RAPD_S218 includes the complete sequence of the gene Opp_l. The marker RAPD_S360 locates in another linkage group.5. RAPD_S312 and RAPD_S360, the RAPD markers,were successfully converted into SCAR markers named CBJ312,CBJ360 respectively. CBJ312 and CBJ360 were validated in the parent pools and F2 population. The CBJ312 had amplification with template of the positive RAPD_S312 F2 individuals and opposite DNA pool, did not have amplification with the template of the negative RAPD_S312 F2 individuals and alternative DNA pool. So did the SCAR marker CBJ360.6. Through searching databases, the sequence of CBJ312 has high homologous with the mRNA sequence and amino acid sequence of mouse microtube binding protein棗 tau, in 92% homologenity of nucleotide sequence and in 99% homologenity of amino acid sequence in the region between 126th and 375th nucleotide sequence of it. ORF Finder amonline showed it was the most possible that the mentioned region was one of the ORF of CBJ312. CBJ360 had high homologous with rice, human, fruit fly etc. The 578 nucleotides region (between 11th and 588th) of CBJ360 had the same sequence as one PAC clone of the chromosome 1 of Oryza sativa genomic DNA (AP003263.2) (between 61th to 638th). The acquired information facilitate us to study the function of the SCARs fragments. To summarize, this paper makes a great progress in molecular markers linked to dominant opposite genes of maize. Several close RAPD markers for opposite gene were obtained and firstly in domestic converted RAPD markers into SCARs markers. Therefore, the useful SCARs markers were acquired. The results established the foundation of Marker Assisted Selection(MAS) of breeding, the location of the opposite genes and Map-based clone the genes.