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The Role of Puroindoline A and B Individually and in Combination on Grain Hardness and Starch Association

Dep. of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT, 59717-3150

View larger version (87K): [in this window] [in a new window]   Fig. 1. Cleaved amplified polymorphism sequence (CAPS) test used to distinguish between the Pinb-D1e (Canadian Red [CR]) and Pinb-D1b (Hi-Line) alleles at Ha locus in segregating populations among CR crosses. BstNI does not digest the Pinb-D1e PCR product (469 bp) from CR. Pinb-D1b PCR product from Hi-Line is cut once yielding 234- and 235-bp products. Lanes 1 and 2 represent progeny lines possessing the Pinb-D1e allele while lanes 5 and 6 represent progeny lines carrying the Pinb-D1b allele. Lanes 3 and 4 are heterozygous for the Pinb-D1e and Pinb-D1b allele. M denotes a 100-bp ladder marker (Promega, Madison, WI) that goes up to 900 bp.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Scatter plots of F3–derived F4 progeny vs. F3 single plant grain hardness for segregating progeny from crosses of: (A) McNeal (McN) (Pina-D1b/Pinb-D1a) x Hi-Line Ha locus homozygous lines with added Pina or Pinb and the McN Ha locus. Pina-D1b denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles and lacking a transgene, Pina-D1b (PINA+) denotes genotypes possessing the McN Pina-D1b/Pinb-D1a alleles with added Pina transgene and Pina-D1b (PINB+) denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles with added Pinb transgene; (B) Canadian Red (CR) (Pina-D1a/Pinb-D1e) x Hi-Line Ha locus homozygous lines with added Pina or Pinb and the CR Ha locus. Pina-D1b denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles and lacking the transgene, Pina-D1b (PINA+) denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles with added Pina transgene, and Pina-D1b (PINB+) denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles with added Pinb transgene.

View larger version (117K): [in this window] [in a new window]   Fig. 3. SDS-PAGE gel of total TX-114 extractable proteins levels from PIN null and transgenic overexpressing lines. Heron (Pina-D1a/Pinb-D1b) is a soft wheat control while Canadian Red, PINB null (Pina-D1a/Pinb-D1e) and McNeal, PINA null (Pina-D1b/Pinb-D1a) are the hard wheat controls. TX-114 extractable puroindoline protein levels were performed on three randomly selected lines per group as shown. The position of PINA and PINB is marked.

View larger version (94K): [in this window] [in a new window]   Fig. 4. SDS-PAGE gel of starch-associated puroindolines (PINA and PINB). Total puroindolines were extracted from water-washed starch granules of control and soft and intermediate-textured PIN overexpressing lines. Heron (Pina-D1a/Pinb-D1a) is a soft wheat control while Canadian Red, PINB null (Pina-D1a/Pinb-D1e) and McNeal, PINA null (Pina-D1b/Pinb-D1a) are the hard wheat controls. Starch-associated proteins (PINA or PINB) were extracted from three randomly selected lines per group as noted. The position of PINA and PINB is marked.