Early expression of grain hardness in the developing wheat endosperm.

Seeds from near-isogenic hard and soft wheat lines were harvested at regular intervals from 5 days post-anthesis to maturity and examined for hardness using the single kernel characterisation system (SKCS). SKCS analysis revealed that hard and soft lines could be distinguished from 15 days post-anthesis (dpa). This trend continued until maturity where the difference between the hard and soft lines was most marked. SKCS could not be applied to the small 5- and 10-dpa wheat kernels. Fresh developing endosperm material was examined using light microscopy and no visible differences between the cultivars were detected. When air-dried material was examined using scanning electron microscopy (SEM) differences between soft and hard lines were visible from as early as 5 dpa. Accumulation of puroindoline a and puroindoline b was investigated in developing seeds using both Western blotting and ELISA. Low levels of puroindoline a could be detected in the soft cultivar from 10 dpa, reaching a maximum at 32 dpa. In the hard cultivar, puroindoline a levels were negligible throughout grain development. Puroindoline b accumulates in both the soft and hard cultivars from 15 dpa, but overall contents were higher in the soft cultivar. These findings indicate that endosperm hardness is expressed very early in developing grain when few starch granules and storage proteins were deposited in the endosperm cells. Further, the near-isogenic soft and hard Heron lines could be differentiated by SEM at a stage in development when the accumulation of puroindolines could not be detected by the methods used in this study.

Mapping QTLs for grain hardness and puroindoline content in wheat ( Triticum aestivum L.).

Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.