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Isolation and Analysis of B-chromosome Specific cDNA-AFLP Markers in Maize
Chang, Stephen Kuo-Hu
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In the present research, maize inbred lines (L289 and W23) with and without B chromosomes, and the TB-10L38 translocation inbred line were used to isolate B-chromosome specific cDNA-AFLP markers by using cDNA-AFLP analysis. A total of 44 B-chromosome specific cDNA-AFLP markers were isolated from L289 inbred lines with and without B chromosomes, and 39 of which were cloned successfully. By using W23 inbred lines with and without B chromosomes and the TB-10L38 translocation, 8 B-chromosome specific cDNA-AFLP markers were isolated, and then 6 of which were cloned and sequenced. Sequence analysis showed that 4 out of 45 cDNA-AFLP markers had homology with B-chromosome related sequence, 28 markers had homology with sequence of different gene, mRNA, or BAC clone sequences in maize, and 13 markers showed no homology with any published sequence. Southern hybridization analysis indicated that 10 markers showed B-specificity or dosage effect signals, 20 markers had identical signals in both genomic DNA with and without B chromosomes and the remaining 15 markers showed no signal. Therefore, sequences of 2 markers with B-chromosome related sequence, 3 markers with B-specificity or dosage effect signals, and 3 markers randomly chosen were used to design primers for RT-PCR and SCAR-PCR analysis. Results of RT-PCR showed that 4 markers could be amplified from RNA of L289 with and without B chromosomes indicated that these markers had transcriptional activity. SCAR-PCR results showed that these 4 markers could be amplified from DNAs of L289 with and without B chromosomes, indicating these 4 markers were existed in genomic DNA and had the ability for expression.
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