Genetic variability for the Waxy genes in Argentinean hexaploid wheats

VANZETTI L.; PLUGER L.; RODRIGUEZ-QUIJANO M.; CARRILLO J. M.; DUBCOVSKY J.; HELGUERA M.

Mar del Plata

Conferencia; 7th Internacional Wheat Conference; 2005

The major component of wheat flour is starch, a polymer composed of two carbohydrates: amylose and amylopectin. Waxy (Wx) protein, which is produced by waxy genes, is a granule-bound starch synthase (GBSSI) responsible for amylose synthesis in starch. Because common wheat is allohexaploid (AABBDD genomes) it has three different Wx isoproteins – Wx-A1, Wx-B1 and Wx-D1 –. Wheat with one or two non-functional (null) Wx genes produces starch with significant lower levels of amylose (partial Wx starch). Partial Wx starch is a desirable trait in the development of wheat suitable for noodle quality. The objective of this study was to characterize the genetic variability of the Wx-A1, Wx-B1 and Wx-D1 loci in 86 Argentinean wheat cultivars using molecular markers. Wx-A1: 78% of cultivars showed WxA1a (normal) allele and 22% showed Wx-A1b (null) allele when using A-1 primers. When these cultivars were tested with primers Wx-A1-specific that include A-1 primers region, all of them showed PCR products of similar size. Sequence comparison of PCR products obtained from cultivars previously scored as Wx-A1a and Wx-A1b showed two silent one-point-mutations in Wx-A1b alleles, one in the annealing site of the reverse primer A-1. As A-1 primers amplify simultaneously Wx-A1, Wx-B1 and Wx-D1 genes, this mismatch could be enough to favor Wx-B1 and Wx-D1 amplification against Wx-A1. This is not a real Wx-A1b (null) allele, so we propose to name it Wx-A1f. In line with these arguments, waxy protein patterns in cultivars scored as Wx-A1a and Wx-A1f were identical.  Wx-B1: using A-1 primers 73% of tested cultivars showed Wx-B1a allele and 27% Wx-B1b (null). However, a second combination of primers Wx-B1-specific showed again 73% of tested cultivars as Wx-B1a, but 19% was Wx-B1b (absence of amplification) and 8% showed a larger fragment designated Wx-B1e. Sequence comparison of Wx-B1 alleles showed two amino acids differences in the coding region of Wx-B1e (Ser to Aspa and Arg to Met) and a 34bp-insertion in an intron that could explain its different size. The effect of the amino acid mutations on  Wx-B1e function has not been evaluated yet, but SDS-PAGE analysis in cultivars scored as Wx-B1e have shown a waxy protein that migrate slightly slower than the Wx-B1a allele. As expected cultivars scored by PCR as Wx-B1b, showed lack of the Wx-B1 protein (null allele). Wx-D1: using A-1 primers 100% of tested cultivars showed a Wx-D1a allele. Similar data was obtained using primers Wx-D1-specific.