The Puroindoline b-2 Variants are Expressed at Low Levels Relative to the Puroindoline D1 Genes in Wheat Seeds

Grain hardness in wheat is largely controlled by the Hardness locus. This locus contains the Puroindoline a (Pina) and b (Pinb) genes, thought to exist as single-copy genes only on chromosome 5D. However, four additional copies of Pinb have been reported. These new genes termed Pinb-2v1 to Pinb-2v4 map to the group 7 chromosomes of wheat (Triticum aestivum L.). Since PINA and PINB cooperatively bind to starch granules and PINB limits grain softness, we set out to determine Pinb-2 gene variant expression levels relative to Pinb. The tested genotypes included the soft wheat cultivar Alpowa, Chinese Spring, and two Chinese Spring group 7 nullisomic-tetrasomic lines. Deletion of chromosome 7A (and Pinb-2v4) or 7D (and Pinb-2v1) did not impact either the level of total or starch-bound puroindolines, indicating that the expression level of the Pinb-2 genes is not sufficient to modify grain hardness. Due to cross-hybridization, northern blots were not useful in accurately quantifying Pinb-2 transcript levels. Therefore, RNA sequencing was used to accurately measure transcript levels of Pina, Pinb, and each of the four Pinb-2 variants in developing wheat seeds. Relative to Pinb, Pinb-2v1 expression levels averaged 1%, whereas Pinb-2v2 levels ranged up to 7.6%. Pinb-2v3 transcripts were detectable only in Alpowa at 1% of Pinb-D1a, whereas Pinb-2v4 transcripts were undetectable in all genotypes. Given the relative low expression levels of each Pinb-2, it is unlikely that they substantially contribute to grain hardness variation.