糯性胚乳小麦的选育

在自然界,大多数禾谷类作物,如玉米、水稻、高梁、粟、黍子等,都存在糯性类型.但是由于普通小麦是异源六倍体作物,在它的染色体7AS、4AS和7DS 上各有一个基因,即Wx-A1、Wx-B1 和Wx-D1控制Wx蛋白(淀粉粒束缚淀粉合成酶),Wx蛋白与直链淀粉的合成有关.当三个位点基因全部缺失或不表达时不能合成直链淀粉,只有支链淀粉、自然界不存在三个Wx基因同时缺失的小麦.所以迄今为止尚未发现自然界存在的糯性小麦.小麦面粉中直链淀粉含量是决定面条品质的一个重要因素.本研究以"两个部分糯性"材料作亲本,并结合花粉染色、籽粒剖面染色、SDS-PAGE电泳等手段,在杂种后代中选出了若干糯性小麦品系,定名为"农大糯麦"系列.     

Grain Quality of Emmer Wheat Derived Synthetic Hexaploid Wheats

The wild diploid goat grass (Aegilops tauschii Cosson), and the cultivated tetraploid emmer wheat (Triticum turgidum L. subsp. dicoccon (Schrank) Thell.) may be important sources of genetic diversity for improving hexaploid bread wheat (Triticum aestivum L.). Through interspecific hybridization of emmer wheat and Ae. tauschii, followed by chromosome doubling, it is possible to produce homozygous synthetic hexaploid wheat. Fifty-eight such synthetic hexaploids were evaluated for grain quality parameters: grain weight, length, and plumpness, grain hardness, total protein content, and protein quality (SDS-Sedimentation volume, SDS-S). Most synthetics showed semi-hard to hard grain texture. Results showed significant genetic variation among the synthetic hexaploids for protein content, SDS-S values, and grain weight and plumpness. Quality measurement values of synthetic hexaploids were regressed on corresponding values of the emmer wheat parents. With this offspring-parent regression, protein content and SDS-S values explained 8.7 and 28.8%, respectively, of the variation among synthetics, indicating a significant contribution from the emmer wheat parents for these traits. The synthetic hexaploids, in general, had significantly higher protein content (15.5%, on average) and longer grains than ‘Seri M82’, the bread wheat control (13.1% protein content). Synthetics with SDS-S values and grain weights higher than those of ‘Seri M82’ were also identified. Protein content among synthetics showed significantly negative correlations with grain weight and plumpness, but no correlation with SDS-S values. Despite these negative correlations, 10 superior synthetic hexaploid wheats, derived from nine different emmer wheat parents and with above average levels of protein content, SDS-S values, and either grain weight or plumpness, were identified. This study shows that genetic variation for quality in tetraploid emmer wheat can be transferred to synthetic hexaploid wheats and combined with plump grains and high grain weight, to be used for bread wheat breeding.     

Paste Properties of Modified Starches from Partial Waxy Wheats

Properties of modified starches from partial waxy wheats have not been examined. Protease digestion of cracked kernels of three hard winter wheats varying in amylose content led to 82–85% recovery of starch, whereas kneading of the flour-water doughs gave 75–83% recovery. All starches had a protein content of <0.3% and ash content of <0.01%. Granule size distributions showed that starch from Ike kernels contained 86% A-type granules with a peak size of ≈18μm, and Karl-92 starch contained 77% A-type granules with a peak size of ≈16μm. The A-type granules (82%) from Rio Blanco starch were intermediate in size. The amylose content of Karl-92 starch, determined by concanavalin-A precipitation of amylpectin, was 28%, which was 17% higher than that of Ike starch (23%). The amylose content of Rio Blanco starch was 26%. The lipid content of Karl-92 starch, determined as fatty acid methyl esters, also was 18% higher than that of Ike starch (601 vs. 488 mg/100 g of starch, respectively). Wheat starches were modified with hydroxypropyl (HP) groups to low (1.5–2.5%) and medium (≈4.0%) levels, and the HP starches were cross-linked with phosphoryl chloride at levels of 0.003–0.075%. Pasting curves (amylograms) showed that Ike starch substituted with a low level of HP and optimally cross-linked with 0.025% phosphoryl chloride (starch basis) had a greater paste consistency than low HP cross-linked Karl-92, and Rio Blanco starches. At 4% HP and optimum cross-linking (0.003% phosphoryl chloride), the paste consistencies of the modified starches were nearly the same. The clarity of unmodified Ike starch paste was higher than that of Karl-92 or Rio Blanco starch pastes, and the clarity of all three pastes decreased as cross-linking was increased. Unmodified Ike starch formed a stronger gel than unmodified Karl-92 and Rio Blanco starches, but gel properties largely converged as the starches were modified.     

Comparison of Waxy vs. Nonwaxy Wheats in Fuel Ethanol Fermentation

Fermentation performance of eight waxy, seven nonwaxy soft, and 15 nonwaxy hard wheat cultivars was compared in a laboratory dry‐grind procedure. With nitrogen supplements in the mash, the range of ethanol yields was 368–447 L/ton. Nonwaxy soft wheat had an average ethanol yield of 433 L/ton, higher than nonwaxy hard and waxy wheat. Conversion efficiencies were 91.3–96.2%. Despite having higher levels of free sugars in grain, waxy wheat had higher conversion efficiency than nonwaxy wheat. Although there was huge variation in the protein content between nonwaxy hard and soft wheat, no difference in conversion efficiency was observed. Waxy cultivars had extremely low peak viscosity during liquefaction. Novel mashing properties of waxy cultivars were related to unique pasting properties of starch granules. With nitrogen supplementation, waxy wheat had a faster fermentation rate than nonwaxy wheat. Fermentation rates for waxy cultivars without nitrogen supplementation and nonwaxy cultivars with nitrogen supplementation were comparable. Ethanol yield was highly related to both total starch and protein content, but total starch was a better predictor of ethanol yield. There were strong negative relationships between total starch content of grain and both yield and protein content of distillers dried grains with solubles (DDGS).     

Physicochemical and Structural Characteristics of Flours and Starches from Waxy and Nonwaxy Wheats

A waxy spring wheat (Triticum aestivum L.) genotype was fractionated into flour and starch by roller and wet‐milling, respectively. The resultant flour and starch were evaluated for end‐use properties and compared with their counterparts from hard and soft wheats and with commercial waxy and nonwaxy corn (Zea mays L.) starches. The waxy wheat flour had exceptionally high levels of water absorption and peak viscosity compared with hard or soft wheat flour. The flour formed an intermediate‐strength dough that developed rapidly and was relatively susceptible to mixing. Analysis by differential scanning calorimetry and X‐ray diffractometry showed waxy wheat starch had higher gelatinization temperatures, a greater degree of crystallization, and an absence of an amylose‐lipid complex compared with nonwaxy wheat. Waxy wheat and corn starches showed greater refrigeration and freeze‐thaw stabilities than did nonwaxy starches as demonstrated by syneresis tests. They were also similar in pasting properties, but waxy wheat starch required lower temperature and enthalpy to gelatinize. The results show analogies between waxy wheat and waxy corn starches, but waxy wheat flour was distinct from hard or soft wheat flour in pasting and mixing properties.     

Measurement of Blend Concentrations of Conventional and Waxy Hard Wheats Using NIR Spectroscopy

Breeding development of waxy (amylose‐free) hard wheat lines adapted to the North American climate has been underway for more than a decade, with releases of competitive varieties imminent. Because of required identity preservation and a possible premium value placed on waxy lots, a rapid and accurate method is desired to identify and quantify the mixing of conventional wheat with waxy wheat, a condition that might occur at harvest or any point downstream. Our previous work demonstrated that lines pure with waxy starch can be identified from nonwaxy lines by use of near‐infrared (NIR) spectroscopy applied either on a whole kernel or ground meal basis. However, mixture quantification by NIR techniques has not been examined until now. Using hard winter wheat grown in two seasons (2011 and 2012) and at two locations (Nebraska and Arizona), a series of mixtures ranging in proportion (conventional/waxy) percentage by weight, from 0:100 to 100:0, were formed from nine pairs of waxy and nonwaxy varieties or lines, with year and location being consistent within a pair. Twenty‐nine mixtures (0, 1, 2, 3, 4, 5, 10, 15, …, 85, 90, 95, 96, 97, 98, 99, and 100%) were formed for each pair. Partial least squares regression models were developed by using eight of the nine pairs, with model validation accomplished by using the pair excluded. This procedure was repeated for each pair. The results indicate that, regardless of sample format or spectral pretreatment, the optimal models typically produce coefficients of determination in excess of 0.98, with standard errors of 4–7%, thus demonstrating the feasibility of the use of the NIR technique to predict the mixture level to within 10% by weight.     

N‐Terminal Amino Acid Sequence Analysis of Endosperm Proteins in Japanese Hexaploid Wheat

Semidry electroblotting is convenient and allows a rapid and efficient protein transfer from two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) gels onto sequencer stable supports for protein microsequence analysis in a gas‐phase sequencer. Using this technique, I determined the amino acid sequences of the endosperm proteins in Japanese hexaploid commercial wheats (Triticum aestivum). Based on sequence determination of the Japanese hexaploid wheats, the endosperm protein could be easily characterized. Wheat endosperm protein, extracted in the presence of 2‐mercaptoethanol and SDS, fractionated into many protein polypeptides using 2D‐PAGE under dissociating conditions. These components were grouped into HMW glutenin subunits, α‐, β‐ or γ‐gliadins, and novel protein polypeptides by using the N‐terminal amino acid sequences. The novel endosperm protein polypeptides were detected, and two new types of N‐terminal amino acid sequences have been found for protein poly‐peptides. These polypeptides have much faster electrophoresis mobility during 2D‐PAGE and are therefore probably a much smaller size than any other peptides of endosperm protein groups found in hexaploid wheat. Ten protein polypeptides have been purified from cultivars of Japanese wheat. Some differences in the contents of amino acids for four protein polypeptide spots were apparent in Japanese wheat.     

Observations on the Quality Characteristics of Waxy (Amylose‐Free) Winter Wheats

Previous investigations have suggested waxy (amylose‐free) wheats (Triticum aestivum L.) possess weak gluten properties and may not be suitable for commercial gluten extraction. This limitation could prevent the use of waxy wheat as a source of unique starch, because gluten is a by‐product of the wheat starch purification process. Fifty waxy wheat lines were used to determine the extent to which gluten protein and other grain quality related traits might vary and, consequently, allow the development of waxy wheat with acceptable gluten properties. Among the waxy lines, significant variation was observed for all measured quality traits with the exception of flour protein concentration. No waxy entries statistically equaled the highest ranking nonwaxy entry for grain volume weight, falling number, flour yield, or mixograph mix time. No waxy lines numerically exceeded or equaled the mean of the nonwaxy controls for falling number, flour yield, or mixograph mix time. For grain and flour protein related variables, however, many waxy lines were identified well within the range of acceptability, relative to the nonwaxy controls used in this study. Approximately 50% of the waxy lines did not differ from the highest ranking nonwaxy cultivar for grain and flour protein concentrations. Forty‐three (86%) of the tested waxy lines were not sig‐nificantly different from the nonwaxy line with the highest mixograph mixing tolerance, 22/50 (44%) of the waxy wheat lines did not differ from the highest ranking nonwaxy line in gluten index scores, and 17/50 (34%) did not differ from the highest ranking nonwaxy line in extracted wet gluten. All waxy experimental lines produced gluten via Glutomatic washing. The quality of the gluten, as measured both by mixograph and gluten index, varied widely among the waxy lines tested. These observations suggest that weak gluten is not a natural consequence of the waxy trait, and waxy cultivars with acceptable gluten properties can be developed.     

Starch Characteristics of Waxy and Nonwaxy Tetraploid (Triticum turgidum L. var. durum) Wheats

Manufacture of pasta products is paramount for durum wheat (Triticum turgidum L. var. durum). The recent development of waxy durum wheat containing starch with essentially 100% amylopectin may provide new food processing applications and present opportunities for value‐added crop production. This investigation was conducted to determine differences in some chemical and functional properties of waxy durum starch. Starch was isolated from two waxy endosperm lines and four nonwaxy cultivars of durum wheat. One of the waxy lines (WX‐1) was a full waxy durum wheat whereas the other line (WX‐0) was heterogeneous, producing both waxy and nonwaxy seed. Effects on starch swelling, solubility, pasting, gelatinization, and retrogradation were examined. The full waxy starch had four times more swelling power than the nonwaxy durum starches at 95°C, and was also more soluble at three of the four temperatures used. Starch pasting occurred earlier and peak viscosities were greater for starches from both waxy lines than for the nonwaxy starches, but their slurries were less stable with continued stirring and heating. Greater energy was required to melt gelatinized waxy starch gels, but no differences were found in either refrigerated storage or freeze‐thaw retrogradation, as determined by differential scanning calorimetry. The results of this investigation showed some significant differences in the starch properties of the waxy durum wheat lines compared to the nonwaxy durum wheats.     

Quality Characteristics of Waxy Hexaploid Wheat (Triticum aestivum L.): Properties of Starch Gelatinization and Retrogradation

The viscoelastic properties and molecular structure of the starch isolated from waxy (amylose-free) hexaploid wheat (WHW) (Triticum aestivum L.) were examined. WHW starch generally had lower gelatinization onset temperature, peak viscosity, and setback than the starch isolated from normal hexaploid wheat (NHW). Differential scanning calorimetry (DSC) showed that WHW starch had higher transition temperatures (T_o, T_p, and T_c) and enthalpy (ΔH) than NHW starch. However, when compared on the basis of amylopectin (AP) content, ΔH of WHW starch was almost statistically identical to that of its parental varieties. Typical A-type X-ray diffraction patterns were observed for the starches of WHW and its parental varieties. Somewhat higher crystallinity was indicated for WHW starch. WHW starch was also characterized by having greater retrogradation resistance. The high-performance size-exclusion chromatography (HPSEC) of amylopectin showed that each amylopectin yielded two fractions after debranching. Although WHW amylopectin had somewhat long B chains, little difference was observed in the ratio of Fr.III/ Fr.II between WHW and its parental varieties.     

Evaluation of Waxy Grain Sorghum for Ethanol Production

The objective of this research was to investigate the fermentation performance of waxy grain sorghum for ethanol production. Twenty‐five waxy grain sorghum varieties were evaluated with a laboratory dry‐grind procedure. Total starch and amylose contents were measured following colorimetric procedures. Total starch and amylose contents ranged from 65.4 to 76.3% and from 5.5 to 7.3%, respectively. Fermentation efficiencies were in the range of 86.0–92.2%, corresponding to ethanol yields of 2.61–3.03 gallons/bushel. The advantages of using waxy sorghums for ethanol production include easier gelatinization and low viscosity during liquefaction, higher starch and protein digestibility, higher free amino nitrogen (FAN) content, and shorter fermentation times. The results showed a strong linear relationship between FAN content and fermentation rate. Fermentation rate increased as FAN content increased, especially during the first 30 hr of fermentation (R² = 0.90). Total starch content in distillers dried grains with solubles (DDGS) was less than 1% for all waxy varieties.     

Ultrastructure of Developing Hard and Soft Red Winter Wheats After Air- and Freeze-Drying and Its Relationship to Endosperm Texture

A transmission electron microscopic study was conducted on air- and freeze-dried developing wheat to determine the effects of drying on the structure of the starchy endosperm. Field-grown hard red winter wheat (Karl) and soft red winter wheat (Clark) were harvested at 15, 18, 21, 23, 25, 28, and 35 days after flowering (DAF). Wheat was dried by either air-drying in the spike at 28°C or freeze-drying following freezing in liquid nitrogen. Dried wheat was prepared for microscopy. Fresh samples of Karl and Clark were also harvested on the same days and prepared immediately for microscopy. The method of drying greatly affected cellular ultrastructure. The most pronounced change upon air-drying of developing samples was disappearance of individual protein bodies and conversion of the cytoplasm into a matrix-like material similar in appearance to storage protein matrix found in mature wheat endosperm. Freeze-dried wheats maintained nearly natural ultrastructure but exhibited various amounts of freeze damage. Conversion of protein bodies to a matrix was not observed in freeze-dried samples. The results suggest that hardness develops as a result of endosperm senescence rather than accumulation of particular grain components. Senescence may cause changes in the starch granule surface such that surrounding components bind tightly in hard wheats, whereas the binding is weaker in soft wheats. Therefore, the surface of starch granules might be more important than components the starch granules bind to in determining hardness.     

Evaluation of Nebraska Waxy Sorghum Hybrids for Ethanol Production

To evaluate the ethanol production performance of waxy sorghum hybrids and the effects of location and harvest year on ethanol yield, samples of four waxy sorghum hybrids collected from two Nebraska locations (Mead and Lincoln) in both 2009 and 2010 were tested for ethanol production in a dry‐grind process. No significant difference (P = 0.216) in starch contents was observed among the four hybrids, but starch contents of the hybrids were significantly affected by growth location (P = 0.0001) and harvest year (P = 0.0258). Location, hybrid, and harvest year all had significant effects on ethanol fermentation efficiency in the dry‐grind process. Lincoln sorghum samples showed higher (P = 0.022) ethanol fermentation efficiency (90.4%) than did Mead sorghum samples (90.0%). Sorghums harvested in 2010 had higher (P < 0.001) ethanol fermentation efficiency (91.1%) than those harvested in 2009 (89.3%). The 2009 sorghum flours had more amylose‐lipid complexes than the 2010 samples did, and amylose‐lipid complexes as previously reported had adverse effects on ethanol fermentation. Residual starch contents in distillers dried grains with solubles (DDGS) were significantly affected by hybrid and harvest year (P < 0.0001), but we observed no difference in protein content in DDGS from the four hybrids.     

Extrusion of Regular and Waxy Barley Flours for Production of Expanded Cereals

Grains of two regular and two waxy barley cultivars were milled into break and reduction stream flours using a wheat milling mill, granulated to facilitate feeding and flow through the barrel, and extruded to form expanded products using a modified laboratory single‐screw extruder. As moisture content of barley granules decreased from 21 to 17%, the expansion index of extrudates increased from 1.81 to 2.68, while apparent modulus of compression work (AMCW) decreased from 17.1 × 10⁴ to 7.8 × 10⁴ N/m². Break stream flours of both regular and waxy barley produced extrudates with higher expansion index (2.72–3.02), higher water absorption index (WAI), and lower AMCW than extrudates from reduction stream flours. Extrudates produced from regular barley had generally higher expansion and lower density than those produced from waxy barley. The specific mechanical energy (SME) was greater during extrusion of regular than of waxy barley. Barrel temperatures of 130, 150, and 170°C for the feeding, compression, and metering sections, respectively, resulted in higher SME, higher expansion index, lower water absorption index and lower AMCW of extrudates compared with a constant extruder barrel temperature of 160°C. Increased screw speed generally resulted in larger expansion index and increased WAI of extrudates. With increased feed rate from 89 to 96 g/min, the expansion index of extrudates decreased from 3.20 to 2.78 in regular barley and 3.23 to 2.72 in waxy barley, and harder extrudates were produced.     

Quality of Flours from Waxy and Nonwaxy Barley for Production of Baked Products

One nonwaxy (covered) and two waxy (hull-less) barleys, whole grain and commercially abraded, were milled to break flour, reduction flour, and the bran fraction with a roller mill under optimized conditions. The flour yield range was 55.3–61.8% in whole grain and increased by 9–11% by abrasion before milling. Break flours contained the highest starch content (≤85.8%) independent of type of barley and abrasion level. Reduction flours contained less starch, but more protein, ash, free lipids, and total β-glucans than break flours. The bran fraction contained the highest content of ash, free lipids, protein, and total β-glucans but the lowest content of starch. Break flours milled from whole grain contained 82–91% particles <106 μm, and reduction flours contained ≈80% particles <106 μm. Abrasion significantly increased the amount of particles <38 μm in break and reduction flours in both types of barley. Viscosity of hot paste prepared with barley flour or bran at 8% concentration was strongly affected by barley type and abrasion level. In cv. Waxbar, the viscosity in bran fractions increased from 428 to 1,770 BU, and in break flours viscosity increased from 408 to 725 BU due to abrasion. Sugar snap cookies made from nonwaxy barley had larger diameter than cookies prepared from waxy barley. Cookies made from break flours were larger than those made from reduction flours, independent of type of barley. Quick bread baked from nonwaxy barley had a loaf volume similar to that of wheat bread, whereas waxy barley bread had a smaller loaf volume. Replacement of 20% of wheat flour by both waxy and nonwaxy barley flour or bran did not significantly affect the loaf volume but did decrease the hardness of quick bread crumb.     

Compressive Strength of Wheat Endosperm: Analysis of Endosperm Bricks

The material properties of wheat grain endosperm are central to its processing and end‐use quality. The preparation of geometrically‐defined endosperm specimens free of bran, germ, and pigment strand can facilitate the objective study of endosperm material properties. This study was conducted to characterize the material properties of wheat endosperm from two soft, two hard, and one durum wheat varietal samples. Additionally, each varietal sample was sorted according to vitreous or mealy kernel type. Endosperm ‘bricks’ approximately 0.76 × 2.08 × 1.06 mm were prepared using an abrading (Kernel Sanders, KS) device. Bricks were tested in compression using a texture analyzer (TA.XTPlus). Stress‐strain curves were used to calculate failure strain, failure stress, failure energy, and Young's modulus. Additionally, the effect of brick aging up to one month, and changes in moisture content (freeze drying, oven drying, and equilibration to ≈10.5–11% mc) were studied. Intrakernel variation was assessed by preparing two sibling bricks (one from each cheek) from individual kernels. Failure strain, stress, and energy all had relatively high model R² values (0.68, 0.79, and 0.75, respectively). The ANOVA model R² for Young's modulus was 0.46. All models indicated variety as a highly significant source of variation in brick material properties. The effect of vitreous versus mealy kernel type was not consistent across varietal samples. Brick age and moisture content did not significantly affect brick material properties. Analysis of sibling bricks indicated that the magnitude of intrakernel variation was similar to that observed for individual varietal lots of uniform vitreous or mealy kernel type. Overall, failure strain provided a ranking and mean separation most consistent with kernel texture market class. The results obtained in the present study, although similar to other published reports do not closely agree with them on the material properties of wheat endosperm. Similarly, published results of material properties often differ considerably. The source of these discrepancies are at present unknown, but in some circumstances they may relate to specimen orientation relative to the source kernel, as there was evidence for anisotropic behavior. A companion study compares the variation in kernel texture obtained with the single kernel characterization system (SKCS) with that obtained here using bricks.     

Milling of Regular and Waxy Starch Hull-less Barleys for the Production of Bran and Flour

Five registered cultivars of hull-less barley (HB) with regular or waxy starch were milled in a Quadrumat Jr. mill to obtain whole grain flour; pearled in a Satake mill (cultivar Condor only), and the pearled fractions examined by microscopy to determine true HB bran. The samples were milled after tempering and drying in a Buhler mill to obtain bran and flour yields. Flour color and composition of HB were unaltered on milling in the Quadrumat Jr. mill. Microscopic evidence showed that a 70% pearl yield was devoid of the grain's outer coverings, including the aleurone and subaleurone layers. Therefore, the balance of 30% constitutes true bran in HB. Dry milling (as-is grain moisture) of regular starch HB in the Buhler mill gave 59% total flour and 41% bran (bran + shorts) yields, the comparative values for the waxy starch HB were 42 and 58%. On tempering HB from 9 to 16% grain moisture, the total flour yield decreased in both types of HB but to a lesser extent in the waxy starch HB due to decreases in reduction flour. On drying HB to 5 or 7% moisture, total flour yields increased due to contamination with bran and shorts. The milling study led to the conclusion that HB, at best, be dry-milled and a bran finisher be used to obtain commercial flour extraction rates. Lower total flour yields in the waxy starch HB than in the regular starch HB milled at the same grain moisture levels seemed due to higher β-glucan rather than grain hardness. Waxy starch HB flour had higher mixograph water absorption and water-holding capacity than regular starch HB or soft white wheat flour milled under identical conditions. Roller-milled HB products offer the best potential for entry into the food market.     

Occurrence of Puroindoline Alleles in Chinese Winter Wheats

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 in the amino acid sequence of puroindoline b, involving a lysine (K) to asparagine (N) change, and leading to a shift in the open reading frame (ORF). This deletion disrupts the last part of the tryptophanrich domain, changing it from KWWK to NGGR, which is considered essential for the lipid‐binding activity of this protein, and results in a stop codon corresponding to position Pro‐60 in the amino acid sequence. The characterization of different hardness alleles provides useful information in understanding the mechanism underlying the formation of endosperm hardness while providing breeders the means of manipulating this important trait.