Quality traits. Waxy mutants

Microsatellite marker for analyzing the allelic variation of the Wx-A1 locus (6)

Sharoflou and Sharp (1999) found a microsatellite sequence near the 3' end of a waxy cDNA sequence and designed PCR primers flanking this region (Accession X57233). The PCR amplification of Chinese Spring DNA produced two bands, one of 204 bp and the other of 265 bp. Using aneuploid stocks the 204 bp product was assigned to chromosome 7D (locus XSun1-7D) and the 265 bp product to chromosome 7A (locus XSun1-7A). This region is particularly useful for analyzing the allelic variation of the Wx-A1 locus (4,6).

Both loci were analyzed in 135 varieties. The XSun1-7A locus is highly polymorphic, with 8 alleles detected. Two varieties that were null for the Wx-A1 gene gave unique fragments of 233 bp. The XSun1-7D locus was not polymorphic in the lines analyzed, but the expected fragment of 204 bp was lacking in varieties null for the Wx-D1 gene.

    Sun1F: 5'-CGC TCC CTG AAG AGA GAA AGA A-3'
    Sun1R: 5'-ATA GGC ACA ACC CCT AAC -3'

1. Denaturing step: 95°C, 3 min
2. 5 cycles of: [94°C 1 min, 58°C 1 min, 72°C 1 min]
3. 25 cycles of: [94°C 1 min, 58°C 50 sec, 72°C 30 sec]
4. Extension step: 72°C 4 min

Final volume: 25 μl

Final concentrations of the products used in the PCR reaction:

• dNTPs: 200 μM each
• MgCl₂: 1.5 mM
• primers: 0.5 μM  each
• Taq DNA polymerase (Advanced Biotechnologies, Epson, UK): 1 unit
• 1X PCR buffer (Advanced Biotechnologies, Epson, UK)
• Genomic DNA: 75 ng

Conditions presented here should be consider only as a starting point of the PCR optimization for individual laboratories.

Amplification products were separated in 3% agarose and ethidium bromide staining or 6% polyacrylamide with silver staining. 
Eight different alleles were detected at the Xsun-7A locus in 135 cultivars. Their sizes were: 219, 233, 260, 271, 275, 285 and 289 bp.
Locus Xsun-7D is not polymorphic. However, it is a recessive marker for null alleles of the Wx-D1 gene, since no amplification was observed for the 3 Wx-D1 null varieties analyzed. For the rest of the varieties the product obtained had a size of 204 bp.