Tucson, Arizona

University of Arizona

Npi402 and ncsu1 are identical ; inra1 (tmp) maps upstream of the b promoter --Stam, M, Lavin, T and Chandler, VL The analysis of intragenic recombinants showed that b is transcribed, 5ý 3, from the telomere towards the centromere and that the sequences required for paramutation of the b gene map upstream of the b coding region (Patterson et al., Genetics 140:1389, 1995). To further define the sequences involved in paramutation we wanted to identify recombination events between the B (paramutagenic) and B-P (non-paramutagenic) alleles further upstream of the b gene. To simplify the screen for recombinants we needed to convert RFLP markers tightly linked and upstream of b into a PCR assay to detect sequence length polymorphisms (SLPs) linked to the different b alleles. The RFLP markers npi287A, umc61, ncsu1 and npi402, which map 1.1 cM upstream of b on chromosome 2S (www.agron.missouri.edu/images/bnlc2.jpg), were sequenced. Two markers, ncsu1 and npi402, were found to have exactly the same sequence and therefore are the same RFLP marker. Primers designed to amplify the genomic sequences corresponding to npi287A, umc61, ncsu1/npi402 did not reveal any SLP between B and B-P. No further experiments were performed with these markers.

The map UMC 98 2 showed inra1 (tmp) mapped to 1.2 cM 3 of umc61; inra1 was not mapped relative to b, however. Southern analysis using DNA from B, B-P and three B-P//B recombinant alleles (recombinations verified by PFGE analysis) showed that all three B-P//B alleles had the B-P inra1 RFLP, while B had another sized fragment (not shown). This suggested that inra1 is located upstream of b and could be used to look for a PCR SLP between B and B-P. Tmp is an mRNA for a transmembrane protein that is highly conserved. We hypothesized that SLPs might be present within the introns of the tmp alleles linked to B and B-P. To predict the intron positions in the tmp gene, the protein sequence was used to search the database for homologous genes. The genomic DNA sequence of the homologous Arabidopsis thaliana water channel-like protein (EMBL accession nr. CAA20461) contained three introns. Protein sequence alignment between the translated maize tmp cDNA and the Arabidopsis protein predicted the intron positions in the maize gene. Primers surrounding the predicted third intron gave rise to different sized PCR products for the tmp alleles linked to B and B-P. Sequence analysis of both PCR products identified multiple deletions and insertions in the tmp intron 3 between the alleles linked to B and B-P. Primers were designed that only amplified tmp sequences linked to B-P. This enabled us to screen for recombination events upstream of the b promoter. The B allele (colorless seeds due to B promoter proximal region), flanked with the homozygous recessive phenotypic markers gl2 (19 cM 5 of b) and wt (11 cM 3 of b), was combined with the B-P allele (purple seeds), flanked with the wild-type alleles of gl2 and wt. The F1 was crossed to gl2 B-I wt plants (colorless seeds); colorless seeds were planted and screened for a recombination event upstream of b using the gl2 marker. 1861 seedlings wild-type for the marker gl2, were tested for a recombination event near to b using the B-P specific primers on pooled DNA samples. 18 recombinant alleles (0.97% of 1861 seedlings) were identified using PCR and verified by Southern blot analysis. All 18 alleles have wt as the 3 marker, consistent with a single recombination event between tmp and b. These results show that the tmp allele maps 0.18 cM upstream of b. This was calculated by multiplying the 19 cM between gl2 and b by the % of recombinants obtained within this region.
 
 


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