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Effect of Multiple Copies of Puroindoline Genes on Grain Softness

^a Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS 66506
^b Dep. of Plant Sci., Montana State Univ., Bozeman, MT 59106

View larger version (60K): [in a new window]   Fig. 1. Pina and Pinb gene loci number in different lines at the F3 generation determined by restriction fragment length polymorphism analysis. ‘Chinese Spring’ (CS) is homozygous at the 5D locus and has two copies. Other lines have four or six copies except lines 4 and 196, which have five copies each as they were heterozygous at the 5A locus as indicated by reduced 32P signal and their segregation in the F4 generation (data not shown).

View larger version (81K): [in a new window]   Fig. 2. Puroindoline expression analysis. RNA was extracted from 20-d post anthesis seeds. Membranes were probed with Pina and Pinb. Glutenin levels were used to normalize loading differences. ‘Chinese Spring’ (CS) was loaded in concentrations from 0.5x (one copy) to 4x (eight copies) as a reference. The adjusted values indicate a graphical representation of the expression levels compared with CS standards. The numbers in parentheses indicate puroindoline copy number.

View larger version (53K): [in a new window]   Fig. 3. Starch surface protein levels, though not normalized, show a clear increase in starch surface protein levels that can be seen in lines 6, 12, 4, and 127, as indicated by the 15Mr (relative mobility) fragment. Starch surface protein is absent in the durum cultivar Langdon (L), but reappears at similar levels to ‘Chinese Spring’ (CS) in the Langdon 5D(5B) substitution line L5D(5B) that contains the 5D chromosome of CS. The numbers in parentheses indicate puroindoline copy number.

View larger version (25K): [in a new window]   Fig. 4. Friabilin proteins from ‘Chinese Spring’ (CS) and the lines with multiple copies of Pina and Pinb genes. The friabilin levels indicated by the upper Pina and lower Pinb bands show an increased concentration of friabilin in the lines 12, 30, and 284, each with three copies of the puroindoline genes. Lines 4 and 127 with two copies produce less protein, but are slightly more concentrated than the 1x (two copy) CS standard. Line 196 with two copies shows increased friabilin levels above that of other two-copy lines. The numbers in parentheses indicate puroindoline copy number.