Bread wheat milling behavior: effects of genetic and environmental factors,and modeling using grain mechanical resistance traits

Key message

Genetic (Pinb-D1 alleles) and environment (through vitreousness) have important effects on bread wheat milling behavior. SKCS optimal values corresponding to soft vitreous or hard mealy grains were defined to obtain the highest total flour yield.

Abstract

Near-isogenic lines of bread wheat that differ in hardness, due to distinct puroindoline-b alleles (the wild type, Pinb-D1a, or the mutated forms, Pinb-D1b or Pinb-D1d), were grown in different environments and under two nitrogen fertilization levels, to study genetic and environmental effects on milling behavior. Milling tests used a prototype mill, equipped with two break steps, one sizing step, and two reduction steps, and this enabled 21 individual or aggregated milling fractions to be collected. Four current grain characters, thousand grain weight, test weight, grain diameter, and protein content, were measured, and three characters known to influence grain mechanical resistance, NIRS hardness, SKCS hardness index, and grain vitreousness (a character affecting the grain mechanical behavior but generally not studied). As expected, the wild type or mutated forms of Pinb-D1 alleles led to contrasted milling behavior: soft genotypes produced high quantities of break flour and low quantities of reduction flour, whereas reverse quantities were observed for hard genotypes. This different milling behavior had only a moderate influence on total flour production. NIRS hardness and vitreousness were, respectively, the most important and the second most important grain characters to explain milling behavior. However, contrary to NIRS hardness, vitreousness was only involved in endosperm reduction and not in the separation between the starchy endosperm and the outer layers. The highest flour yields were obtained for SKCS values comprised between 30 and 50, which corresponded either to soft vitreous or hard mealy grains. Prediction equations were defined and showed a good accuracy estimating break and reduction flours portions, but should be used more cautiously for total flour.

     

Expression of the gamma-zein protein of maize in seeds of transgenic barley: effects on grain composition and properties

A cDNA clone encoding the gamma-zein protein of maize was expressed in developing grain of barley using the starchy endosperm cell-specific promoter from the wheat Glu-1D-1 (HMW subunit 1Dx5) gene. Seven transgenic lines were recovered from 226 bombarded immature embryos, of which two were sterile and four tetraploid, while five were shown to express the gamma-zein protein based on western blotting. Southern blot analysis showed the presence of between about three and twelve transgene insertions. Detailed comparative studies of five null and five homozygous transformed sub-lines from transgenic line A showed that gamma-zein accounted for over 4% of the total prolamin fraction, corresponding to about 1.9% of the total grain N. Comparison of the proteins present in the gel protein fraction demonstrated that the gamma-zein was incorporated into polymers, as in maize. However, there was no effect on grain hardness measured using the Perten Single Kernel Characterisation System or on the vitreousness measured by visual inspection. This contrasts with the situation in maize where a clear association with vitreousness has been reported.     

Expression of wild-type pinB sequence in transgenic wheat complements a hard phenotype

Wheat grain hardness is a major factor in the wheat end-product quality. Grain hardness in wheat affects such parameters as milling yield, starch damage and baking properties. A single locus determines whether wheat is hard or soft textured. This locus, termed Hardness ( Ha), resides on the short arm of chromosome 5D. Sequence alterations in the tryptophan-rich proteins puroindoline a and b (PINA and PINB) are inseparably linked to hard textured grain, but their role in endosperm texture has been controversial. Here, we show that the pinB-D1b alteration, common in hard textured wheats, can be complemented by the expression of wild-type pinB-D1a in transformed plants. Transgenic wheat seeds expressing wild-type pinB were soft in phenotype, having greatly increased friabilin levels, and greatly decreased kernel hardness and damaged starch. These results indicate that the pinB-D1b alteration is most likely the causative Ha mutation in the majority of hard wheats.     

Effects of vitreousness and particle size of maize grain on ruminal and intestinal in sacco degradation of dry matter,starch and nitrogen

We assessed the effects of vitreousness and particle size of maize grain on ruminal and intestinal in sacco degradation of dry matter, starch and nitrogen. Six maize grain (Zea mays) genotypes characterized by differing vitreousness (proportion of vitreous in total endosperm) were ground (3-mm screen; Gr, ground particles, mean particle size (MPS): 526 μm) and cracked with a roller mill using two gap width settings (CS, cracked small particles, MPS: 1360 μm; CL, cracked large particles, MPS: 2380 μm). The ruminal and intestinal in sacco degradation of dry matter, starch and nitrogen was measured on three dry Holstein cows, fitted with rumen, proximal duodenum and terminal ileum cannulas, fed maize silage ad libitum twice daily. The ruminal starch degradability and intestinal digestibility differed among genotypes (P<0.001) and decreased as particle size increased (P<0.001). For the same particle size, starch ruminal degradability decreased (P<0.05) and intestinal digestibility decreased (P<0.002) with vitreousness. Particle size and vitreousness of maize grain are efficient factors for manipulating the amount of starch escaping rumen degradation, but may be limiting for the amount of starch digested in the small intestine.     

Relationships between kernel vitreousness and dry matter degradability for diverse corn germplasm: I. Development of near-infrared reflectance spectroscopy calibrations

Negative correlations between corn vitreousness and ruminal dry matter and starch degradabilities have been widely reported. To measure corn vitreousness and density more rapidly, Correa et al. [Correa, C.E.S., Shaver, R.D., Pereira, M.N., Lauer, J.G., Kohn, K., 2002. Relationship between corn vitreousness and ruminal in-situ starch degradability. J. Dairy Sci. 85, 3008–3012] initiated the development of near-infrared reflectance spectroscopy (NIRS) calibrations from 47 samples derived from 14 US and five Brazilian commercial hybrids. In this study, we generated more data to add to these NIRS calibrations with the objective of making them more robust. We also evaluated the potential of using Stenvert hardness measurements for NIR calibrations. Thirty-three diverse corn germplasm sources were grown at University of Wisconsin West Madison Research Station. These included a wide range of endosperm characteristics from opaque 2 (o2) types to densely packed flint types, and a number of intermediates. Harvest was at 1/2 milkline and black-layer maturity stages. Dried kernels from middle portions of ears from 12 selected inbreds, four each from low (0–30%), medium (30–70%), and high (70–100%) vitreousness classifications were used to determine vitreousness by manual dissection and density by water displacement using a pycnometer. Hardness was determined on all 33 inbreds on a 20 g sample using a Stenvert micro hammer-cutter mill with 2 mm screen size and 3600 rpm to measure time to collect ground sample to a set receptacle height (T); total column height (CH); and height ratio of coarse to fine (C/F) particles. The NIRS equations were selected on the basis of high R²-values (0.90, 0.92, 0.85, and 0.85) and low SEC (4.85, 0.01, 1.39, and 0.19) and SECV (6.04, 0.02, 1.79, and 0.25), for vitroueness, density, T and CH factors, respectively. Calibrations for vitreousness and density were regarded as the best prediction models compared to stenvert hardness measurements as determined by their RPD values (3.73 and 2.50, respectively). These results show that NIRS can be used as a screening tool in large-scale breeding trials to develop corn hybrids of desired endosperm properties for improved ruminal degradabilities.     

The role of chromosomes from homeologous group 5 in regulation of grain hardness and protein content in substitution lines of common wheat cultivar Saratovskaya 29

Genetic regulation of grain hardness and protein content in intervarietal substitution lines for chromosomes of homeologous group 5 was examined. Common wheat cultivar Saratovskaya 29 with high bread-backing properties served as the recipient. Donors of chromosomes 5A and 5D were 18 cultivars with variable traits examined, including high-protein cultivars (Atlas 66 and Diamant 2), and soft-grain cultivars (Ul’yanovka and Chinese Spring). Analysis of substitution lines pointed to a substantial effect of chromosome 5D on the regulation of both traits. It was demonstrated that as a result of intervarietal substitution for chromosome 5D from donor cultivars Ul’yanovka and Chinese Spring, the endosperm softness was increased compared to the recipient cultivar Saratovskaya 29. Substitution lines Saratovskaya 29/Atlas 66 5D and Saratovskaya 29/Diamant 2 5D were characterized by high grain protein content, as well as by high endosperm hardness. In addition, the line Saratovskaya 29/Novosibirskaya 67 5D, characterized by grain hardness higher than in Saratovskaya 29, was isolated. In the lines with intervarietal substitution of chromosome 5A, grain protein content was found to be lower than in recipient cultivar Saratovskaya 29.     

Hordoindolines are associated with a major endosperm-texture QTL in barley (Hordeum vulgare).

Endosperm texture has a tremendous impact on the end-use quality of wheat (Triticum aestivum L.). Cultivars of barley (Hordeum vulgare L.), a close relative of wheat, also vary measurably in grain hardness. However, in contrast to wheat, little is known about the genetic control of barley grain hardness. Puroindolines are endosperm-specific proteins found in wheat and its relatives. In wheat, puroindoline sequence variation controls the majority of wheat grain texture variation. Hordoindolines, the puroindoline homologs of barley, have been identified and mapped. Recently, substantial allelic variation was found for hordoindolines among commercial barley cultivars. Our objective was to determine the influence of hordoindoline allelic variation upon grain hardness and dry matter digestibility in the 'Steptoe' x 'Morex' mapping population. This population is segregating for hordoindoline allele type, which was measured by a HinA/HinB/Gsp composite marker. One-hundred and fifty lines of the 'Steptoe' x 'Morex' population were grown in a replicated field trial. Grain hardness was estimated by near-infrared reflectance (NIR) and measured using the single kernel characterization system (SKCS). Variation attributable to the HinA/HinB/Gsp locus averaged 5.7 SKCS hardness units (SKCS U). QTL analysis revealed the presence of several areas of the genome associated with grain hardness. The largest QTL mapped to the HinA/HinB/Gsp region on the short arm of chomosome 7 (5H). This QTL explains 22% of the SKCS hardness difference observed in this study. The results indicate that the Hardness locus is present in barley and implicates the hordoindolines in endosperm texture control.     

Seed-specific expression of the wheat puroindoline genes improves maize wet milling yields

The texture of maize ( Zea mays L.) seeds is important to seed processing properties, and soft dent maize is preferred for both wet-milling and livestock feed applications. The puroindoline genes ( Pina and Pinb ) are the functional components of the wheat ( Triticum aestivum L.) Hardness locus and together function to create soft grain texture in wheat. The PINs (PINA and PINB) are believed to act by binding to lipids on the surface of starch granules, preventing tight adhesion between starch granules and the surrounding protein matrix during seed maturation. Here, maize kernel structure and wet milling properties were successfully modified by the endosperm-specific expression of wheat Pins ( Pina and Pinb ). Pins were introduced into maize under the control of a maize γ- Zein promoter. Three Pina/Pinb expression positive transgenic lines were evaluated over two growing seasons. Textural analysis of the maize seeds indicated that the expression of PINs decreased adhesion between starch and protein matrix and reduced maize grain hardness significantly. Reduction in pressure required to fracture kernels ranged from 15.65% to 36.86% compared with control seeds. Further, the PINs transgenic maize seeds had increased levels of extractable starch as characterized by a small scale wet milling method. Starch yield was increased by 4.86% on average without negatively impacting starch purity. The development of softer maize hybrids with higher starch extractability would be of value to maize processors.     

Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize

The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.     

Chromosomal loci associated with endosperm hardness in a malting barley cross

A breeding objective for the malting barley industry is to produce lines with softer, plumper grain containing moderate protein content (9–12%) as they are more likely to imbibe water readily and contain more starch per grain, which in turn produces higher levels of malt extract. In a malting barley mapping population, ‘Arapiles’ × ‘Franklin’, the most significant and robust quantitative trait locus (QTL) for endosperm hardness was observed on the short arm of chromosome 1H, across three environments over two growing seasons. This accounted for 22.6% (Horsham 2000), 26.8% (Esperance 2001), and 12.0% (Tarranyurk 2001) of the genetic variance and significantly increased endosperm hardness by 2.06–3.03 SKCS hardness units. Interestingly, Arapiles and Franklin do not vary in Ha locus alleles. Therefore, this region, near the centromere on chromosome 1H, may be of great importance when aiming to manipulate endosperm hardness and malting quality. Interestingly, this region, close to the centromere on chromosome 1H, in our study, aligns with the region of the genome that includes the HvCslF9 and the HvGlb1 genes. Potentially, one or both of these genes could be considered to be candidate genes that influence endosperm hardness in the barley grain. Additional QTLs for endosperm hardness were detected on chromosomes 2H, 3H, 6H and 7H, confirming that the hardness trait in barley is complex and multigenic, similar to many malting quality traits of interest.     

Endosperm Protein Quality and Kernel Modification in the Quality Protein Maize Inbred Lines

Twenty-two selected quality protein maize (QPM) lines, including 13 lines developed in India (DMRQPM series) and nine lines released by CIMMYT, Mexico (CML series), were evaluated for their endosperm protein content and quality, besides kernel modification in terms of vitreousness. Endosperm protein contents in 13QPMlines were on par or better than that of the normal maize ‘checks’ (Trishulata and Parkash). The QPM endosperm proteins showed significantly higher % tryptophan as well as EF-1α (a multifunctional protein with a positive and highly significant correlation with lysine content in the endosperm) contents, in comparison with the normal maize genotypes. Evaluation of kernel modification revealed considerable scope for accumulation of endosperm modifiers in some of the QPM lines. Positive and highly significant correlation was revealed between tryptophan and EF-1α contents in the endosperm proteins, whereas the correlations between the quality parameters with kernel modification in the QPM genotypes were found to be non-significant. The study led to the identification of some promising QPM lines, such as DMRQPM-37, DMRQPM-44, CML176, CML142 and CML149, which could be effectively deployed in the QPM breeding programmes.     

Additive and Non-additive Genetic Correlations. II. Application to Data Obtained from a 12 × 12 Diallel Cross

The proposed genetic correlation analysis, that involves the partitioning of the overall genetic correlation into an additive and a non-additive component, has been applied to data obtained from a diallel experiment involving 12 white modified opaque-2 maize inbred lines. The correlation analysis provided an insight into possible indirect selection strategies for the improvement of inferior kernel quality traits associated with the opaque-2 gene. Direct selection for high yield and low vitreousness rating would provide an efficient selection strategy for the development of high-yielding modified opaque-2 maize hybrids with desirable endosperm traits. It was concluded that it is not necessary to conduct the density, hardness and breakability determinations.     

The detection of QTLs in barley associated with endosperm hardness, grain density, grain size and malting quality using rapid phenotyping tools

Using a barley mapping population, ‘Vlamingh’ × ‘Buloke’ (V × B), whole grain analyses were undertaken for physical seed traits and malting quality. Grain density and size were predicted by digital image analysis (DIA), while malt extract and protein content were predicted using near infrared (NIR) analysis. Validation of DIA and NIR algorithms confirmed that data for QTL analysis was highly correlated (R ² > 0.82), with high RPD values (the ratio of the standard error of prediction to the standard deviation, 2.31–9.06). Endosperm hardness was measured on this mapping population using the single kernel characterisation system. Grain density and endosperm hardness were significantly inter-correlated in all three environments (r > 0.22, P < 0.001); however, other grain components were found to interact with the traits. QTL for these traits were also found on different genomic regions, for example, grain density QTLs were found on chromosomes 2H and 6H, whereas endosperm hardness QTLs were found on 1H, 5H, and 7H. In this study, the majority of the genomic regions associated with grain texture were also coincident with QTLs for grain size, yield, flowering date and/or plant development genes. This study highlights the complexity of genomic regions associated with the variation of endosperm hardness and grain density, and their relationships with grain size traits, agronomic-related traits, and plant development loci.     

Overexpression of Puroindoline a gene in transgenic durum wheat (Triticum turgidum ssp. durum) leads to a medium–hard kernel texture

Durum wheat is the second-most widely grown wheat species, and is primarily used in the production of pasta and couscous. The grain utilization of durum wheat is partly related to its very hard kernel texture because of the lack of the D genome and consequentially the Puroindoline genes. Our previous study reported the transformation of durum wheat with the Puroindoline a (Pina) gene. Here, we characterized the transgenic durum wheat lines expressing the Pina gene, and studied the effects of PINA on grain texture and other kernel characteristics. SDS-PAGE and Western blotting results demonstrated that starch-bound PINA levels of Pina-overexpressing lines were lower than that of Pina-positive control, common wheat cv. Chinese Spring, suggesting a weak association of PINA protein with starch granules in the absence of Pinb. Grain hardness analysis and flour milling tests indicated that the overexpression of PINA resulted in decreased grain hardness and increased flour yield in transgenic durum wheat lines. The agronomic performance of the transgenic and control lines was also examined and it was found that no significant differences in measured traits were observed between Pina-overexpressing and control lines in the 2-year field trials. Since grain hardness strongly affects milling and end-use qualities, the development of medium–hard-textured durum wheat lines is not only of significance for our knowledge of grain hardness and Puroindolines, but also has practical implications for plant breeders and food technologists for the expansion of utilization of durum wheat.     

QTLs for grain dry milling properties, composition and vitreousness in maize recombinant inbred lines

Vitreousness and kernel hardness are important properties for maize processing and end-product quality. In order to examine the genetic basis of these traits, a recombinant inbred line population resulting from a cross between a flint line (F-2) and a semident line (Io) was used to search for vitreousness and kernel composition QTLs. Vitreousness was measured by image processing from a kernel section, while NIR spectroscopy was used to estimate starch, protein, cellulose, lipid and semolina yield. In addition, thousand-grain weight and grain weight per ear were measured. The MQTL method was used to map the QTLs for the different traits. An additional program allowed for the detection of interaction QTLs between markers. The total number of main-effect and interaction QTLs was similar. The QTLs were not evenly distributed but tended to cluster. Such clusters, mixing main-effect and interaction QTLs, were observed at six positions : on chromosomes 1, 2, 3, 6, 8 and 9. Two of them, on chromosomes 6 and 9, concerned both QTLs for kernel-weight traits and QTLs for kernel-composition traits (protein and cellulose). Technological-trait QTLs (vitreousness or semolina yield) were located less than 16 cM from a protein-content QTL on chromosome 2, and were co-located with lipid- and starch-content QTLs on chromosome 8. The co-location of a vitreousness and a semolina-yield QTL at the telomeric end of the chromosome 2 (Bin 2.02) is likely to be meaningful since measurement of these related traits, made by completely different methods (NIRS vs image processing), yielded very close QTLs. A similar location was previously reported independently for a kernel-friability QTL. Comparing the map location of the numerous loci for known-function genes it was shown that three zein loci were closely linked to QTLs for vitreousness on chromosome 3, for semolina yield and starch on chromosome 4, and for protein, cellulose and grain weight on chromosome 9. Some other candidate genes linked to starch precursor metabolism were also suggested on chromosomes 6 and 8. Received: 27 April 2000 / Accepted: 3 July 2000     

高大山羊草Aegilops longissima puroindoline b基因的克隆与表达分析

籽粒硬度是小麦加工品质的重要影响因素。puroindoline a（Pina）和puroindoline b（Pinb）是控制小麦籽粒硬度的主效基因。根据已报导的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对高大山羊草Aegilops longissima（SS）的基因组DNA和胚乳RNA进行Pinb基因扩增、克隆、序列测定和表达分析,发现了一个新型Pinb等位基因。基因长360bp,编码119个氨基酸残基,对应于麦类作物Puroindoline B（PinB）成熟蛋白的结构区域,具有麦类作物Pinb基因特有的WPTKWWK的色氨酸结构域基因序列和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv．Capitole的Pinb—D1a相比较,其核苷酸和氨基酸同源性分别为93．3％和92．4％。RT—PCR证实了Pinb基因在籽粒胚乳中的表达。研究结果表明,高大山羊草中包含着与小麦差异较大的籽粒硬度控制基因,为栽培小麦品质改良提供了丰富的遗传资源。     

Relationship between structural and biochemical characteristics and texture of corn grains

The texture of corn grains is a fundamental characteristic for the industry as well as for grain producers and processors. To further understand the mechanisms involved in grain hardness, contrasting corn cultivars for grain hardness and protein quality were evaluated. The cultivars were Cateto L237/67 (hard endosperm and low protein value), QPM BR 451 (semi-hard endosperm and high protein value); Bolivia-2 (floury endosperm and low protein value), and Opaque-2 (floury endosperm and high protein value). Evaluations were carried out at 10, 20, 30, 40, 50, and 60 days after pollination in developing corn grains and in the mature grain. In developing grains, evaluation consisted in the determination of the area, percentage of starch granules, distribution of starch granules, and protein bodies in the endosperm. In mature corn grains, the parameters evaluated were: density, percentage of total proteins, levels of lysine and tryptophan, and banding pattern of zeins. The results indicate that the higher physical resistance of corn grains from the cultivars analyzed is influenced by the high percentage of total proteins, high synthesis of 27-kDa zeins, presence of protein bodies, and perfect organization of starch granules in the endosperm, independent of their sizes.     

Improving zinc accumulation in cereal endosperm using HvMTP1, a transition metal transporter

Zinc (Zn) is essential for all life forms, including humans. It is estimated that around two billion people are deficient in their Zn intake. Human dietary Zn intake relies heavily on plants, which in many developing countries consists mainly of cereals. The inner part of cereal grain, the endosperm, is the part that is eaten after milling but contains only a quarter of the total grain Zn. Here, we present results demonstrating that endosperm Zn content can be enhanced through expression of a transporter responsible for vacuolar Zn accumulation in cereals. The barley (Hordeum vulgare) vacuolar Zn transporter HvMTP1 was expressed under the control of the endosperm‐specific D‐hordein promoter. Transformed plants exhibited no significant change in growth but had higher total grain Zn concentration, as measured by ICP‐OES, compared to parental controls. Compared with Zn, transformants had smaller increases in concentrations of Cu and Mn but not Fe. Staining grain cross sections with the Zn‐specific stain DTZ revealed a significant enhancement of Zn accumulation in the endosperm of two of three transformed lines, a result confirmed by ICP‐OES in the endosperm of dissected grain. Synchrotron X‐ray fluorescence analysis of longitudinal grain sections demonstrated a redistribution of grain Zn from aleurone to endosperm. We argue that this proof‐of‐principle study provides the basis of a strategy for biofortification of cereal endosperm with Zn.     

Particle size distribution of rice flour affecting the starch enzymatic hydrolysis and hydration properties

Rice flour is becoming very attractive as raw material, but there is lack of information about the influence of particle size on its functional properties and starch digestibility. This study evaluates the degree of dependence of the rice flour functional properties, mainly derived from starch behavior, with the particle size distribution. Hydration properties of flours and gels and starch enzymatic hydrolysis of individual fractions were assessed. Particle size heterogeneity on rice flour significantly affected functional properties and starch features, at room temperature and also after gelatinization; and the extent of that effect was grain type dependent. Particle size heterogeneity on rice flour induces different pattern in starch enzymatic hydrolysis, with the long grain having slower hydrolysis as indicated the rate constant (k). No correlation between starch digestibility and hydration properties or the protein content was observed. It seems that in intact granules interactions with other grain components must be taken into account. Overall, particle size fractionation of rice flour might be advisable for selecting specific physico-chemical properties.     

Creation and functional analysis of new Puroindoline alleles in Triticum aestivum

The Hardness (Ha) locus controls grain texture and affects many end-use properties of wheat (Triticum aestivum L.). The Ha locus is functionally comprised of the Puroindoline a and b genes, Pina and Pinb, respectively. The lack of Pin allelic diversity is a major factor limiting Ha functional analyses and wheat quality improvement. In order to create new Ha alleles, a 630 member M(2) population was produced in the soft white spring cultivar Alpowa using ethylmethane sulfonate mutagenesis. The M(2) population was screened to identify new alleles of Pina and Pinb. Eighteen new Pin alleles, including eight missense alleles, were identified. F(2) populations for four of the new Pin alleles were developed after crossing each back to non-mutant Alpowa. Grain hardness was then measured on F(2:3) seeds and the impact of each allele on grain hardness was quantified. The tested mutations were responsible for between 28 and 94% of the grain hardness variation and seed weight and vigor of all mutation lines was restored among the F(2) populations. Selection of new Pin alleles following direct phenotyping or direct sequencing is a successful approach to identify new Ha alleles useful in improving wheat product quality and understanding Ha locus function.