Craig F. Morris

A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch-surface friabilin

Abstract The quantitative level of friabilin 15-kDa protein present on the surface of water-washed starch is highly correlated with wheat grain softness. Friabilin is composed primarily, if not exclusively, of the proteins puroindoline a and b. The transcript levels of these two proteins are similar among hard and soft wheat varieties, and the expression of both is controlled by the short arm of chromosome 5D, also the chromosomal location of the Hardness gene. We report here a glycine to serine sequence change in puroindoline b associated with hard grain texture. This amino acid change results from a single nucleotide mutation and resides in a region thought to be important for the lipid-binding properties of puroindolines. No recombination was observed between the serine puroindoline-b mutation, hard grain texture and low levels of starch surface friabilin among a set of 83 homozygous 5D recombinant lines derived from the soft-textured variety ‘Chinese Spring’ and the substitution line ‘Chinese Spring’ containing the 5D chromosome of the hard-textured variety ‘Cheyenne’. The sequence change reported here may adversely affect the lipid-binding properties of puroindoline-b and so effect hard grain texture. The results suggest that grain hardness results from puroindoline-b functionality such that the Hardness gene is a direct manifestation of puroindoline structure. We are suggesting the tentative molecular marker loci designations of Pinb-D1a and Pinb-D1b for the glycine and serine puroindoline-b types, respectively.

A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe

Abstract Endosperm hardness in wheat (Triticum aestivum L.) is determined by one major genetic factor, the Hardness (Ha) gene on the short arm of chromosome 5D. Grain hardness has previously been reported to result from either a failure to express puroindoline a (Pina–D1b) or a glycine to serine mutation at position 46 in puroindoline b (Pinb–D1b). In this study, which involves a large survey of 343 wheat genotypes of mostly Northern European origin, we report two new mutations in puroindoline b associated with hard endosperm. These were characterized as involving a leucine to proline change at position 60 (Pinb–D1c), and a tryptophan to arginine change at position 44 (Pinb–D1d), respectively. While the former seems to be widely distributed in germplasm around the world, the latter was only found in three winter wheats from Sweden and Netherlands. As discussed in the paper, the three known mutations in puroindoline b can be considered ”loss-of-function” mutations (i.e. soft to hard), and structural analysis may serve to predict that their dramatic effect on wheat grain texture is a result of reduced lipid–binding ability.

Molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses

The hardness or texture of cereal grains is a primary determinant of their technological and processing quality. Among members of the Triticeae, most notably wheat, much of the variation in texture is controlled by a single locus comprised of the Puroindoline a, Puroindoline b and Grain Softness Protein-1 (Gsp-1) genes. Puroindolines confer the three major texture classes of soft and hard common wheat and the very hard durum wheat. The protein products of these genes interact with lipids and are associated with the surface of isolated starch (as a protein fraction known as ‘friabilin’). During the past ten years a great diversity of alleles of both Puroindoline genes have been discovered and significant advances made in understanding the relationship between the gene presence/absence, sequence polymorphism and texture of cereal grains. Efforts have also focussed on Puroindoline and Gsp-1 genes in diploid progenitors, other Triticeae grasses and synthetic wheats in order to understand the evolution of this gene family and find potentially useful variants. The puroindoline homologues in other cereals such as rye and barley are also receiving attention. This work summarises new developments in molecular genetics of puroindolines in wheat and related Triticeae grasses, and the related genes in other cereals.

Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars

Kernel hardness that is conditioned by puroindoline genes has a profound effect on milling, baking and end-use quality of bread wheat. In this study, 219 landraces and 166 historical cultivars from China and 12 introduced wheats were investigated for their kernel hardness and puroindoline alleles, using molecular and biochemical markers. The results indicated that frequencies of soft, mixed and hard genotypes were 42.7, 24.3, and 33.0%, respectively, in Chinese landraces and 45.2, 13.9, and 40.9% in historical cultivars. The frequencies of PINA null, Pinb-D1b and Pinb-D1p genotypes were 43.8, 12.3, and 39.7%, respectively, in hard wheat of landraces, while 48.5, 36.8, and 14.7%, respectively, in historical hard wheats. A new Pinb-D1 allele, designated Pinb-D1t, was identified in two landraces, Guangtouxianmai and Hongmai from the Guizhou province, with the characterization of a glycine to arginine substitution at position 47 in the coding region of Pinb gene. Surprisingly, a new Pina-D1 allele, designated Pina-D1m, was detected in the landrace Hongheshang, from the Jiangsu province, with the characterization of a proline to serine substitution at position 35 in the coding region of Pina gene; it was the first novel mutation found in bread wheat, resulting in a hard endosperm with PINA expression. Among the PINA null genotypes, an allele designed as Pina-D1l, was detected in five landraces with a cytosine deletion at position 265 in Pina locus; while another novel Pina-D1 allele, designed as Pina-D1n, was identified in six landraces, with the characterization of an amino acid change from tryptophan-43 to a ‘stop’ codon in the coding region of Pina gene. The study of puroindoline polymorphism in Chinese wheat germplasm could provide useful information for the further understanding of the molecular basis of kernel hardness in bread wheat.

Assessing genotypic softness in single wheat kernels using starch granule-associated friabilin as a biochemical marker

SummaryThe end-use quality of wheat (Triticum aestivum L.) is determined in large part by the texture of the grain (soft or hard). Endosperm texture is currently determined by several empirical methods. These methods are limited because the use bulk grain lots, as opposed to individual kernels; assess phenotypic, as opposed to genotypic hardness; require a quantity of grain greater than that generally available in the early generations of wheat breeding programs, and are destructive. Recent approaches that use single kernels address the problems associated with bulk grain lots, but suffer the other limitations of providing only the phenotype and being destructive. An objective method for determining the texture genotype of single kernels of wheat was developed using starch granule-associated friabilin, a family of closely related 15 kDa proteins, as a biochemical marker. The occurrence of friabilin on water-washed wheat starch granules is apparently unaffected by the environment and is perfectly correlated (no exceptions) with grain softness. The technique presented here can detect friabilin on as little as 0.2 mg of starch and provides a 250-fold improvement in friabilin detection compared to previous methods. The method is capable of correctly assessing the genotype of F1 heterozygotes from hard x soft and soft x hard crosses. Further, the method uses only a portion of the endosperm from the kernel and therefore accommodates embryo propagation and high molecular weight glutenin subunit characterization. This single kernel method also facilitates the genetic characterization of mixed, bulk grain lots.

Influence of Cultivar and Environment on Water-Soluble and Water-Insoluble Arabinoxylans in Soft Wheat

ABSTRACT Arabinoxylans are hydrophilic nonstarch polysaccharides found in wheat grain as minor constituents. Arabinoxylans can associate with large amounts of water through hydrogen bonding and can form oxidative gels. These properties are important factors in end-use quality of wheat. The objective of this study was to delineate the influence of wheat cultivar and growing environment on variation in water-soluble (WS-AX), waterinsoluble (WI-AX), and total (TO-AX) arabinoxylan contents of flour and whole grain meal. This study included seven spring and 20 winter soft white wheat cultivars grown in 10 and 12 environments, respectively (each evenly split over two crop years). Univariate analysis of variance (ANOVA) and multivariate analysis of variance with canonical analysis (MANOVA) was used to evaluate sources of variation. Variation in arabinoxylan contents and absolute amounts (xylose equivalents) among the two cultivar sample sets (spring and winter) was similar, and both cultivar and environment were...

Occurrence of puroindoline alleles in chinese winter wheats

ABSTRACT Grain hardness is one of the most important characters that determine the end-use quality of bread wheat (Triticum aestivum L.). Mutations in genes encoding either puroindoline a (Pina) or b (Pinb) have been associated with hard grain texture, i.e., Pina null at Pina-D1 or seven mutations at Pinb-D1. In this study, the diversity of puroindoline alleles in 251 Chinese winter wheat cultivars and advanced lines from four major autumn-planted wheat regions were investigated. Among the examined cultivars, 79 were classified as soft, while 53 were mixed in hardness, and 119 were uniformly hard. Of these hard winter wheats, three of the seven reported mutation types were observed, with Pina-D1a/Pinb-D1b being the dominant type for hard texture; 91 genotypes carried this allele. Sixteen genotypes had the Pina-D1b allele, and two genotypes had the Pinb-D1d allele. A new mutation, designated as Pinb-D1p, was detected in 10 hard genotypes, with a single nucleotide (A) deletion corresponding to position 42 i...

Breadmaking quality of selected durum wheat genotypes and its relationship with high molecular weight glutenin subunits allelic variation and gluten protein polymeric composition.

ABSTRACT Twenty-seven durum wheat genotypes originating from different geographical areas, all expressing LMW-2 at Glu-B3, and five bread wheats were evaluated for flour mixing properties, dough physical characteristics, and baking performance. Gluten polymeric composition was studied using size-exclusion HPLC of unreduced flour protein extracts. As a group, durum wheats had poorer baking quality than bread wheats in spite of higher protein and total polymer concentrations. Durum wheats exhibited weaker gluten characteristics, which could generally be attributed to a reduced proportion of SDS-unextractable polymer, and produced less extensible doughs than did bread wheats. However, substantial variation in breadmaking quality attributes was observed among durum genotypes. Better baking performance was generally associated with greater dough extensibility and protein content, but not with gluten strength related parameters. Extensibility did not correlate with gluten strength or SEHPLC parameters. Genotype...

Susceptibility of waxy starch granules to mechanical damage.

ABSTRACT Starch samples isolated from wheat flour that represented four possible waxy states (0, 1, 2, and 3-gene waxy) were subjected to crushing loads under both dry and wet conditions. Calibrated loads of 0.5–20 kg were applied to the starch samples and the percentage of damaged granules was visually determined. Under dry crushing conditions, starches containing amylose (0, 1, and 2-gene waxy) had between 1% (5-kg load) to 3% (15- and 20-kg load) damaged granules, whereas waxy starch (3- gene waxy; <1% amylose) began rupturing at 0.5-kg load (3.5% damaged granules) and had 13% damaged granules when ≥10-kg load was applied. Under wet crushing conditions, normal and partial waxy starch (0, 1, and 2-gene waxy) showed little difference in percentage of damaged granules when compared to the results of dry crushing. Waxy starch (3-gene waxy), however, showed substantially increased numbers of damaged granules: 12% damaged granules at 0.5-kg load, rising to 55% damaged granules at 15-kg load. The results indi...

Molecular characterization of wheat polyphenol oxidase (PPO)

Abstract.It is well-established that the enzyme polyphenol oxidase (PPO) is involved in undesirable browning of noodles, chapattis, middle east flat breads and steamed breads. Methods for measuring PPO activity have been developed, and the variation of PPO activity among wheat (Triticum aestivum L.) cultivars has been well documented. However, there is no report on the identification and characterization of a wheat PPO gene. PCR performed on wheat genomic DNA with oligonucleotide primers designed from conserved copper binding regions of other PPO genes resulted in amplification of a 444-bp DNA fragment. Sequence analysis identified the conserved amino acids of PPO genes indicating that the PCR product was part of the wheat PPO gene. Screening genomic and cDNA libraries using 444- and 760-bp DNA fragments as probes failed to identify a PPO gene based on conserved sequence, even though there were very strong hybridization signals for some isolates. Rapid amplification of cDNA ends (RACE) technique was used as an alternative to obtain the remaining DNA sequences in 5′ and 3′ directions based on the 444-bp partial wheat PPO gene sequence. With the use of ThermoScript Reverse Transcriptase (which functions at higher temperatures) and Advantage-GC cDNA kit, the complete DNA sequence in the 3′ direction was obtained. A similar effort in the 5′ direction resulted in amplification of a truncated 414-bp DNA sequence. Overall, 1,509-bp of putative wheat PPO DNA sequence was obtained. Alignment of deduced amino-acid sequences revealed similarity to the other PPO gene sequences, especially in the conserved copper binding regions. Southern-blot analysis performed with four different restriction enzymes revealed two to four DNA fragments, suggesting a limited number of PPO genes in wheat. Wheat genomic DNA restricted with HindIII and hybridized using a 760-bp wheat PPO probe revealed a clear distinction between wheat cultivars with high and low PPO activities. Northern-blot analysis indicated a transcript size of about 2.0-kb. PPO DNA fragment as well as RNA transcript was observed for the durum cultivar Renville which normally has very low PPO activity. Further study is needed to explain the relationship between PPO activity and the presence of PPO gene (s).