An Automated Near‐Infrared System for Selecting Individual Kernels Based on Specific Quality Characteristics

An automated sorting system was developed that nondestructively measured quality characteristics of individual kernels using near‐infrared (NIR) spectra. This single‐kernel NIR system was applied to sorting wheat (Triticum aestivum L.) kernels by protein content and hardness, and proso millet (Panicum miliaceum L.) into amylose‐bearing and amylose‐free fractions. Single wheat kernels with high protein content could be sorted from pure lines so that the high‐protein content portion was 3.1 percentage points higher than the portion with the low‐protein kernels. Likewise, single wheat kernels with specific hardness indices could be removed from pure lines such that the hardness index in the sorted samples was 29.4 hardness units higher than the soft kernels. The system was able to increase the waxy, or amylose‐free, millet kernels in segregating samples from 94% in the unsorted samples to 98% in the sorted samples. The portion of waxy millet kernels in segregating samples was increased from 32% in the unsorted samples to 55% after sorting. Thus, this technology can be used to enrich the desirable class within segregating populations in breeding programs, to increase the purity of heterogeneous advanced or released lines, or to measure the distribution of quality within samples during the marketing process.     

Single Kernel Near‐Infrared Analysis of Tetraploid (Durum) Wheat for Classification of the Waxy Condition

Plant breeding programs are active worldwide in the development of waxy hexaploid (Triticum aestivum L.) and tetraploid (T. turgidum L. var. durum) wheats. Conventional breeding practices will produce waxy cultivars adapted to their intended geographical region that confer unique end use characteristics. Essential to waxy wheat development, a means to rapidly and, ideally, nondestructively identify the waxy condition is needed for point‐of‐sale use. The study described herein evaluated the effectiveness of near‐infrared (NIR) reflectance single‐kernel spectroscopy for classification of durum wheat into its four possible waxy alleles: wild type, waxy, and the two intermediate states in which a null allele occurs at either of the two homologous genes (Wx‐1A and Wx‐1B) that encodes for the production of the enzyme granule bound starch synthase (GBSS) that controls amylose synthesis. Two years of breeders' samples (2003 and 2004), corresponding to 47 unique lines subdivided about equally into the four GBSS genotypes, were scanned in reflectance (1,000–1,700 nm) on an individual kernel basis. Linear discriminant analysis models were developed using the best set of four wavelengths, best four wavelength differences, and best four principal components. Each model consistently demonstrated the high ability (typically >95% of the time) to classify the fully waxy genotype. However, correct classification among the three other genotypes (wild type, wx‐A1 null, and wx‐B1 null) was generally not possible.     

Quality of Spaghetti Made from Full and Partial Waxy Durum Wheat

The waxy character is achieved in durum wheat (Triticum turgidum L. var. durum) when the granule‐bound starch synthase activity is eliminated. The result is a crop that produces kernels with no amylose in the starch. The presence of two Waxy loci in tetraploid wheat permits the production of two partial waxy wheat genotypes. Advanced full and partial waxy durum wheat genotypes were used to study the effect of waxy null alleles on pasta quality. Semolina from full and partial waxy durum wheats was processed into spaghetti with a semicommercial‐scale extruder, and pasta quality was evaluated. Cooked waxy pasta was softer and exhibited more cooking loss than pasta made from traditional durum cultivars. These features were attributed to lower setback of waxy starch as measured with the Rapid Visco Analyser. High cooking loss may be due to the lack of amylose‐protein interaction, preventing the formation of a strong protein network and permitting exudates to escape. Waxy pasta cooked faster but was less resistant to overcooking than normal pasta. Partial waxy pasta properties were similar to results obtained from wild‐type pasta. This indicates that the presence of a single pair of functional waxy genes in durum wheat was sufficient to generate durum grain with normal properties for pasta production. Waxy durum wheat is not suitable for pasta production because of its softening effect. However, this property may offer an advantage in other applications.     

Significance of Starch Properties and Quantity on Sponge Cake Volume

We evaluated the qualitative and quantitative effects of wheat starch on sponge cake (SC) baking quality. Twenty wheat flours, including soft white and club wheat of normal, partial waxy, and waxy endosperm, as well as hard wheat, were tested for amylose content, pasting properties, and SC baking quality. Starches isolated from wheat flours of normal, single‐null partial waxy, double‐null partial waxy, and waxy endosperm were also tested for pasting properties and baked into SC. Double‐null partial waxy and waxy wheat flours produced SC with volume of 828–895 mL, whereas volume of SC baked from normal and single‐null partial waxy wheat flours ranged from 1,093 to 1,335 mL. The amylose content of soft white and club wheat flour was positively related to the volume of SC (r = 0.790, P < 0.001). Pasting temperature, peak viscosity, final viscosity, breakdown, and setback also showed significant relationships with SC volume. Normal and waxy starch blends having amylose contents of 25, 20, 15, and 10% produced SCs with volume of 1,570, 1,435, 1,385, and 1,185 mL, respectively. At least 70 g of starch or at least 75% starch in 100 g of starch–gluten blend in replacement of 100 g of wheat flour in the SC baking formula was needed to produce SC having the maximum volume potential. Starch properties including amylose content and pasting properties as well as proportion of starch evidently play significant roles in SC baking quality of wheat flour.     

Characteristics of Noodles and Bread Prepared from Double‐Null Partial Waxy Wheat

Double‐null partial waxy wheat (Triticum aestivum L.) flours were used for isolation of starch and preparation of white salted noodles and pan bread. Starch characteristics, textural properties of cooked noodles, and staling properties of bread during storage were determined and compared with those of wheat flours with regular amylose content. Starches isolated from double‐null partial waxy wheat flours contained 15.4–18.9% amylose and exhibited higher peak viscosity than starches of single‐null partial waxy and regular wheat flours, which contained 22.7–25.8% amylose. Despite higher protein content, double‐null partial waxy wheat flours, produced softer, more cohesive and less adhesive noodles than soft white wheat flours. With incorporation of partial waxy prime starches, noodles produced from reconstituted soft white wheat flours became softer, less adhesive, and more cohesive, indicating that partial waxy starches of low amylose content are responsible for the improvement of cooked white salted noodle texture. Partial waxy wheat flours with >15.1% protein produced bread of larger loaf volume and softer bread crumb even after storage than did the hard red spring wheat flour of 15.3% protein. Regardless of whether malt was used, bread baked from double‐null partial waxy wheat flours exhibited a slower firming rate during storage than bread baked from HRS wheat flour.     

Characteristics of French Bread Baked from Wheat Flours of Reduced Starch Amylose Content

Wheat genotypes of wild type, partial waxy, and waxy starch were used to determine the influence of starch amylose content on French bread making quality of wheat flour. Starch amylose content and protein content of flours were 25.0–25.4% and 14.3–16.9% for wild type; 21.2 and 14.9% for single null partial waxy; 15.4–17.1% and 13.2–17.6% for double null partial waxy; and 1.8 and 19.3% for waxy starch, respectively. Wheat flours of double null partial waxy starch produced smaller or comparable loaf volume of bread than wheat flours of wild type and single null partial waxy starch. Waxy wheat flour, despite its high protein content, generally produced smaller volume of bread with highly porous, glutinous, and weak crumb than wheat flours of wild type and partial waxy starch. French bread baked from a flour of double null partial waxy starch using the sponge-and-dough method maintained greater crumb moisture content for 24 hr and softer crumb texture for 48 hr of storage compared with bread baked from a flour of wild type starch. In French bread baked using the straight-dough method, double null partial waxy wheat flours with protein content >14.3% exhibited comparable or greater moisture content of bread crumb during 48 hr of storage than wheat flours of wild type starch. While the crumb firmness of bread stored for 48 hr was >11.4 N in wheat flours of wild type starch, it was <10.6 N in single or double null partial waxy flours. Wheat flours of reduced starch amylose content could be desirable for production of French bread with better retained crumb moisture and softness during storage.     

Novel ssIIa Alleles Produce Specific Seed Amylose Levels in Hexaploid Wheat

Novel starch synthase II (ssIIa ) alleles were created in a soft wheat (Triticum aestivum L.) via ethyl methanesulfonate mutagenesis and combined by crossing to create four unique ssIIa triple mutant haplotypes. A range of starch amylose content was obtained from the ssIIa triple mutant lines (35.6–46.8%), with moderate increases in amylose content found in lines carrying two ssIIa null alleles and one partially functional ssIIa allele. Seeds from all ssIIa triple mutants had significantly higher amylose and protein contents but also lower starch content, kernel weight, and flour swelling power compared with their wild‐type sister lines. Seed starch amylose content was negatively correlated with individual kernel weight (r = –0.54), starch content (r = –0.85), and flour swelling power (r = –0.91) but positively correlated with grain protein (r = 0.78), demonstrating that unique ssIIa triple mutant null combinations confer defined levels of seed starch amylose, protein content, and kernel size. The ssIIa mutant lines also had a hard grain texture (≈86 single kernel characterization system units), whereas all parental material and wild‐type sister lines had soft grain texture (≈35). This change in grain texture was independent of Ha locus genotype, because all lines carried a functional (soft type) Ha locus. The ssIIa alleles and allelic combinations reported here demonstrate the ability to create defined levels of seed starch, amylose, and protein.     

Grain Quality Characteristics and Milling Performance of Full and Partial Waxy Durum Lines

Mutation of the gene coding for the granule bound starch synthase (waxy protein) leads to reduced amylose content in cereal endosperm. Durum wheat (Triticum turgidum L. var. durum) has one waxy locus in each of its two genomes. Full waxy durum wheat is produced when both genomes carry the waxy null alleles. When only one locus is mutated, partial waxy durum wheat is obtained. Partial and full waxy near‐isogenic lines of durum wheat developed by a breeding program were analyzed as to their quality characteristics. Amylose was largely eliminated in full waxy lines; however, no reduction in amylose content was detected in partial waxy lines. The waxy mutation did not affect grain yield, kernel size, or kernel hardness. Full waxy durum lines had higher kernel ash content, α‐amylase activity, and a unique nonvitreous kernel appearance. Protein quality, as evaluated by SDS microsedimentation value, gluten index, and wet gluten was slightly lower in the full waxy lines than in the other genotypes. However, comparisons with current cultivars indicated that protein quality of all derived lines remained in the range of strong gluten cultivars. Semolina yield was lowered by the waxy mutations due to lower friability that resulted in less complete separation of the endosperm from the bran. Waxy semolina was more sensitive to mechanical damage during milling, but modified tempering and milling conditions may limit the damage. Overall, quality characteristics of waxy durum grain were satisfactory and suitable for application testing.     

Effect of Variation in Amylose Content and Puroindoline Composition on Bread Quality in a Hard Spring Wheat Population

Milling and breadbaking quality of hard‐textured wheat may be influenced by alternative alleles at the Wx loci controlling percent amylose in the endosperm, and the puroindoline (pin) loci controlling grain hardness. For this experiment, we developed recombinant inbred lines (RIL) from a cross between Choteau spring wheat cultivar and experimental line MTHW9904. Choteau has the PinB‐D1b mutation conferring grain hardness and the Wx‐B1a allele at the Wx‐B1 locus conferring wild‐type amylose content. MTHW9904 has the PinA‐D1b allele conferring grain hardness and the Wx‐B1b allele conferring lower amylose content, causing a partial waxy phenotype. RIL with the PinB‐D1b mutation (n = 49) had significantly softer kernels, higher break flour yield, and higher loaf volume than lines with the PinA‐D1b mutation (n = 38). Lines with partial waxy phenotype due to Wx‐B1b (n = 43) had significantly lower kernel weight, lower amylose content, and higher flour swelling power than lines with wild‐type starch due to Wx‐B1a (n = 51). These results provide additional evidence for the positive effect of PinB‐D1b on bread quality in hard wheats, while genotype at Wx‐B1 was generally neutral for bread quality in this population. Interactions between the Pin and Wx loci were minimal.     

Physicochemical and Thermal Characteristics of Starch Isolated from a Waxy Wheat Genotype Exhibiting Partial Expression of Wx Proteins

A unique wheat genotype carrying waxy‐type allelic composition at the Wx loci, Gunji‐1, was developed, and its starch properties were evaluated in comparison to parental waxy and wild‐type wheat varieties. Gunji‐1 was null in all three of the Wx genes but exhibited a lower level of Wx proteins than the wild‐type. Starch amylose content and cold water retention capacity were 10.1 and 70.5% for Gunji‐1, 4.2 and 76.6% for waxy, and 27.9 and 65.0% for wild‐type, respectively. No significant differences were observed in microstructure, granule size distribution, and X‐ray diffractograms of the starch granules isolated from Gunji‐1 compared with those of waxy and wild‐type wheat varieties. Starch pasting peak, breakdown, and setback viscosities and peak temperature of Gunji‐1 were intermediate between waxy and wild‐type wheat. In starch gel hardness, Gunji‐1 (1.1 N) was more similar to waxy wheat (0.5 N) than to the wild‐type variety (17.6 N). Swelling power, swelling volume, paste transmittance during storage, and gelatinization enthalpy of Gunji‐1 were lower than those of waxy wheat but greater than those of wild‐type wheat. Retrogradation of starch stored for one week at 4°C expressed with DSC endothermic enthalpy was absent in the waxy wheat variety, whereas Gunji‐1 exhibited both retrogradation of amylopectin and amylose‐lipid complex melting similar to the wild‐type parent, even though enthalpies of Gunji‐1 were much smaller than the wild‐type parent.     

Significance of Amylose Content of Wheat Starch on Processing and Textural Properties of Instant Noodles

The effect of amylose content of starch on processing and textural properties of instant noodles was determined using waxy, partial waxy, and regular wheat flours and reconstituted flours with starches of various amylose content (3.0–26.5). Optimum water absorption of instant noodle dough increased with the decrease of amylose content. Instant noodles prepared from waxy and reconstituted wheat flours with ≤12.4% amylose content exhibited thicker strands and higher free lipids content than wheat flours with ≥17.1% amylose content. Instant noodles of ≤12.4% amylose content of starch exhibited numerous bubbles on the surface and stuck together during frying. Lightness of instant noodles increased from 77.3 to 81.4 with the increase of amylose content of starch in reconstituted flours. Cooking time of instant noodles was 4.0–8.0 min in wheat flours and 6.0–12.0 min in reconstituted flours, and constantly increased with the increase in amylose content of starch. Hardness of cooked instant noodles positively correlated with amylose content of starch. Reconstituted flours with ≤12.4% amylose content of starch were higher in cohesiveness than those of wheat flours of wild‐type and partial waxy starches and reconstituted flours with ≥17.1% amylose content. Instant fried noodles prepared from double null partial waxy wheat flour exhibited shorter cooking time, softer texture, and higher fat absorption (1.2%) but similar color and appearance compared with noodles prepared from wheat flour of wild‐type starch.     

Biochemical Characterization of the Wheat Waxy A Protein and Its Effect on Starch Properties

Granule bound starch synthase1 (GBSS1) is a key enzyme in amylose biosynthesis and is encoded by the A, B and D GBSS1 wx loci in wheat. Wheat lines with mutations at the three GBSS1 loci have been identified. We have characterized and compared the grain starch of CDCW6 wheat line (null B and D for GBSS1) with PI235238 (null A and B for GBSS1), waxy (null A, B and D for GBSS1), and AC Reed (wild type wheat) grain starches. The grain starch of waxy, CDCW6, PI235238, and AC Reed lines contained ≈0, 12, 23, and 25% amylose (w/w), respectively. Waxy, partially waxy, and wild wheat grain starches showed significant differences in onset and peak transition temperatures as determined by differential scanning calorimetric analysis. Grain starches extracted from waxy, CDCW6, and PI235238 also had higher enthalpy of gelatinization values than did wild wheat starch. X-ray diffraction analysis revealed the highest crystallinity for starch extracted from waxy wheat, followed by CDCW6. The starch produced from the CDCW6 line may find special food and industrial applications because of its relatively low amylose concentration.     

Starch waxiness in hexaploid wheat (Triticum aestivum L.) by NIR reflectance spectroscopy

Wheat (Triticum aestivum L.) breeding programs are currently developing varieties that are free of amylose (waxy wheat), as well as genetically intermediate (partial waxy) types. Successful introduction of waxy wheat varieties into commerce is predicated on a rapid methodology at the commodity point of sale that can test for the waxy condition. Near-infrared (NIR) reflectance spectroscopy, one such technology, was applied to a diverse set of hard winter (hexaploid) wheat breeders' lines representing all eight genotypic combinations of alleles at the wx-A1, wx-B1, and wx-D1 loci. These loci encode granule-bound starch synthase, the enzyme responsible for amylose synthesis. Linear discriminant analysis of principal components scores 1-4 was successful in identifying the fully waxy samples at typically greater than 90% accuracy; however, accuracy was reduced for partial and wild-type genotypes. It is suggested that the spectral sensitivity to waxiness is due to (1) the lipid-amylose complex which diminishes with waxiness, (2) physical differences in endosperm that affect light scatter, or (3) changes in starch crystallinity.     

Comparison of Quality‐Related Alleles Among Australian and North American Wheat Classes Exported to Japan

Grain hardness, amylose content, and glutenin subunit composition are critical determinants for end‐use properties of wheat. To improve the end‐use properties of domestic wheats, we studied these traits between the Australian and North American wheat classes exported to Japan in 2009 and 2011 by analyzing the corresponding alleles. Most hard classes had Pina‐D1b or Pinb‐D1b. A partial waxy allele (Wx‐B1b) was found in all Australian Standard White (ASW) seeds in 2009 and two‐thirds of ASW seeds in 2011. All or most American hard wheat seeds had Glu‐D1d. Most U.S. Western White (WW) seeds had a null allele (Glu‐A1c) or alleles that lacked one of the two Glu‐B1 subunits. Most hard red winter (HRW) seeds had Glu‐B3b or Glu‐B3g. Quality characteristics of these classes seemed to be consistent with these results. In addition, we also found new Glu‐1 and Glu‐3 alleles in HRW and WW. These results suggested that although there are variations in its allelic composition from year to year, each class has unique quality‐related alleles corresponding to its end use. We proposed two matrices for classification of starch properties on the basis of Pin and Wx allelic combinations and for classification of gluten strength on the basis of glutenin allelic combinations.     

Determining Vitreous Subclasses of Hard Red Spring Wheat Using Visible/Near‐Infrared Spectroscopy

The percentage of dark hard vitreous (DHV) kernels in hard red spring wheat is an important grading factor that is associated with protein content, kernel hardness, milling properties, and baking quality. The current visual method of determining DHV and non‐DHV (NDHV) wheat kernels is time‐consuming, tedious, and subject to large errors. The objective of this research was to classify DHV and NDHV wheat kernels, including kernels that were checked, cracked, sprouted, or bleached using visible/near‐infrared (Vis/NIR) spectroscopy. Spectra from single DHV and NDHV kernels were collected using a diode‐array NIR spectrometer. The dorsal and crease sides of the kernels were viewed. Three wavelength regions, 500–750 nm, 750–1,700 nm, and 500–1700 nm were compared. Spectra were analyzed by using partial least squares (PLS) regression. Results suggest that the major contributors to classifying DHV and NDHV kernels are light scattering, protein content, kernel hardness, starch content, and kernel color effects on the absorption spectrum. Bleached kernels were the most difficult to classify because of high lightness values. The sample set with bleached kernels yielded lower classification accuracies of 91.1–97.1% compared with 97.5–100% for the sample set without bleached kernels. More than 75% of misclassified kernels were bleached. For sample sets without bleached kernels, the classification models that included the dorsal side gave the highest classification accuracies (99.6–100%) for the testing sample set. Wavelengths in both the Vis/NIR regions or the NIR region alone yielded better classification accuracies than those in the visible region only.     

Fourier Transform Infrared (FT‐IR) Microspectroscopic Genetic Expression of the Waxy Trait in Isogenic Durum and Common Wheat

The aim of recent breeding activity is to develop new wheat lines that incorporate desirable traits that are advantageous in baking and milling. Waxy wheats differ from wild‐type ones in functionality, end use, and biochemical contents (e.g., amylopectin/amylose ratios and lipid contents). Previous waxy wheat investigations focused only on differences in the carbohydrate and protein fractions. The goals of this work were to apply chemical imaging to discriminate between waxy and wild‐type wheats and to define contrasting lipid profiles that occur in the deliberate alteration of the carbohydrate fraction. Fourier transform infrared (IR) microspectroscopic in situ probing and imaging of kernel sections was applied with the use of high spatial resolution. Mid‐IR provided chemical manifestation of lipid genetic expression in isogenic samples. All waxy wheat specimens contained higher lipid content, mostly in the form of glycolipids. The relative lipid profiles differed among common, durum, and waxy wheats. Isogenic partial waxy cultivars and advanced breeding lines were also examined.     

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.     

Functional Characteristics of Bread Wheat (Triticum aestivum L.) Near‐Isogenic Lines Differing at the Waxy (Wx) Locus

The Waxy (Wx) gene in hexaploid wheat (Triticum aestivum L.) encodes granule‐bound starch synthase (GBSS1), which is involved in the synthesis of amylose, a mostly linear glucan polymer that makes up ∼25% of wheat starch. A null mutation of the Wx gene in each of the three genomes is associated with starch almost entirely consisting of the branched glucan polymer amylopectin (waxy starch), with corresponding changes in functionality. However, the rheological behavior of partially waxy starch remains unclear. The objective of this study was to characterize flour and baking quality in 16 near‐isogenic lines, null at the Wx locus on zero, one, two, or all three genomes, grown in four different environments. Across allelic groups, significant variations in amylose concentrations, flour paste viscosity, loaf structure and texture, dough stability, and proximate variables were observed. Because waxy wheat starch has greater water absorbance and resistance to retrogradation than normal starch, its inclusion in flour blends has been suggested as a means of improving the texture and appearance of bakery products and noodles. The results indicate that wheat encoding <3 functional homeologs of GBSS1 produces starch that has potential in the production of certain food items, such as Asian noodles. However, further research is necessary to determine the optimal amylose‐to‐amylopectin ratio to improve baking quality.     

Genetic variation for waxy proteins and amylose content in Spanish spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.)

Spelt wheat is a neglected crop that could be used in the quality breeding of modern common wheat. One important aspect of this quality is the starch composition which is related to the waxy proteins. A collection of 420 accessions of Spanish spelt wheat was analysed for waxy protein composition by SDS–PAGE. Polymorphism was found in the three waxy proteins, detecting differences both in size and in activity, and a new waxy allele (Wx-D1g) was identified. Seed amylose content was also determined and significant differences were detected among the different allelic combinations. In general, the accessions carrying one or two waxy null alleles showed less amylose content. The variation found could be used to enlarge the genetic pool of common wheat, or to develop lines of spelt with different levels of amylose content.     

Automated Single‐Kernel Sorting to Select for Quality Traits in Wheat Breeding Lines

An automated single kernel near‐infrared system was used to select kernels to enhance the end‐use quality of hard red wheat breeder samples. Twenty breeding populations and advanced lines were sorted for hardness index, protein content, and kernel color. To determine whether the phenotypic sorting was based upon genetic or environmental differences, the progeny of the unsorted control and sorted samples were planted at two locations two years later to determine whether differences in the sorted samples were transmitted to the progeny (e.g., based on genetic differences). The average hardness index of the harvested wheat samples for segregating populations improved significantly by seven hardness units. For the advanced lines, hardness index was not affected by sorting, indicating little genetic variation within these lines. When sorting by protein content, a significant increase from 12.1 to 12.6% was observed at one location. Purity of the red samples was improved from ≈78% (unsorted control) to ≈92% (sorted samples), while the purity of the white samples improved from 22% (control) to ≈62% (sorted samples). Similar positive results were found for sorting red and blue kernel samples. Sorting for kernel hardness, color, and protein content is effective and based upon genetic variation.