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Reduced Grain Cadmium Concentration

Contributed by Curtis Pozniak, Krystalee Wiebe and John M. Clarke

Cadmium (Cd) is a toxic metal that is naturally present in trace quantities in almost all soils. Cadmium-contaminated foods are the dominant source of human exposure to environmental Cd, with cereals and vegetables contributing the majority of dietary Cd. Recently the Codex Alimentarius Commission of FAO/WHO has set a level for Cd in wheat grain of 0.2 mg kg-1 (CODEX STAN 193-1995, 2009). Some durum wheat (Triticum turgidum L. var durum) varieties have the genetic potential to accumulate Cd in grain to levels that exceed the Codex standard. Genetic variation for grain Cd accumulation exists in durum (1), and breeding for low grain Cd concentration is a target of durum wheat breeding programs globally.

In durum wheat, Cd accumulation is governed by the major gene that has been designated as Cdu1 (1), which has been localized to chromosome 5BL (2, 3). Marker assisted selection is preferred for selecting breeding lines expressing low Cd as measuring grain Cd is laborious and expensive relative to PCR-based assays. A SCAR marker, designated as ScOpc20 (2) has been used effectively to develop low Cd durum wheat varieties (4), but it is a dominant marker that is linked in repulsion to the low Cd phenotype, and thus has limited application in backcross breeding programs (2). Wiebe et al. (3) identified an EST-derived marker (XBF474090) which co-segregated with variation in grain Cd concentrations. This marker has since been converted to a co-dominant CAPS marker designated as usw47. This marker detects two alleles of usw47 following digestion of PCR amplicons with Hpy188 I. When evaluated on a set of 96 diverse durum wheat cultivars and breeding lines, usw47 successfully classified all lines into either high or low Cd accumulators (Wiebe et al, in preparation). Thus, usw47 will have broad application for selection of the low Cd phenotype in durum wheat.

usw47

usw47 is a co-dominant CAPS marker. The PCR amplification product is digested with restriction enzyme Hpy188 I.

Primer sequences
usw47-F  5'- GCT AGG ACT TGA TTC ATT GAT -3'
usw47-R  5'- AGT GAT CTA AAC GTT CTT ATA -3'

Final concentrations of the reagents used in the PCR amplification

  • 1X Genscript buffer
  • 1.5 mM MgCl2
  • 0.2 mM dNTPs
  • 0.4 µM of each primer
  • 1.25 U of Genscript Taq DNA polymerase
  • 100 ng of genomic DNA
PCR conditions
  • Denaturing step: 95°C, 5 min
  • Amplification step (35 cycles)
    • 95°C, 30 sec
    • 55°C, 30 sec
    • 72°C, 60 sec
  • Extension step: 10 minutes at 72°C.

Volumes of reagents used in the enzymatic digestion (per reaction)

  • PCR product, 4µL
  • NEB Hpy188 I (10U/µL), 0.25µL
  • NEB buffer 4, 1.5µL
  • H2O (sterile), 9.25µL
Enzymatic digestion
  • 37°C for 1 hour
  • 65°C for 20 min
  • Hold 10°C
Expected products

After digestion with the Hpy188 I, products were separated on 2% agarose gels.

CAPS marker usw47
Alleles of usw47 : Kofa, Commander and Langdon are high Cd accumulators. W9262, Strongfield, and CDC Verona are lines with lower contents of grain Cd. The red and blue lines shows the high-Cd and low-Cd alleles, respectively.

References

1. Inheritance of cadmium concentration in five durum wheat crosses. Clarke JM, Leisle D, Kopytko GL. In: Crop Science, 1997, 37:1722-1726. [Journal Link]

2. Chromosomal location of the cadmium uptake gene (Cdu1) in durum wheat. Knox RE, Pozniak CJ, Clarke FR, Clarke JM, Houshmand S, Singh AK. In: Genome, 2009, 52:741-747. DOI:10.1139/G09-042

3.Targeted mapping of Cdu1, a major locus regulating grain cadmium concentration in durum wheat (Triticum turgidum L. var durum). Wiebe K, Harris NS, Faris JD, Clarke JM, Knox RE, Taylor GJ, Pozniak CJ. In: TAG Theoretical and Applied Genetics, 2010,121:1047-1058. DOI:10.1007/s00122-010-1370-1

4. Forty-Six Years of Genetic Progress in Canadian Durum Wheat Cultivars. Clarke JM, Clarke FR, Pozniak CJ. In: Canadian Journal of Plant Science, in press.

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

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