Using the Hexaploid Nature of Wheat To Create Variability in Starch

Jan 25, 2016 - The corrected version published February 3, 2016. ... end-product quality, since bread made from type 5-5 flour showed a 3 day lag in s...
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Using the Hexaploid Nature of Wheat To Create Variability in Starch Characteristics Takayuki Inokuma,† Patricia Vrinten,‡ Tomoya Shimbata,† Ai Sunohara,† Hiroyuki Ito,§ Mika Saito,§ Yoshinori Taniguchi,§ and Toshiki Nakamura*,§ †

Nippon Flour Mills Company, Limited, Atsugi, Kanagawa 243-0041, Japan Bioriginal Food & Science Corporation, Saskatoon, Saskatchewan S7J 0R1, Canada § Tohoku National Agriculture Research Center, Morioka, Iwate 020-0198, Japan ‡

S Supporting Information *

ABSTRACT: In hexaploid crops, such as bread wheat, it should be possible to fine-tune phenotypic traits by identifying wildtype and null genes from each of the three genomes and combining them in a calculated manner. Here, we demonstrate this with gene combinations for two starch synthesis genes, SSIIa and GBSSI. Lines with inactive copies of both enzymes show a very dramatic change in phenotype, so to create intermediate phenotypes, we used marker-assisted selection to develop near-isogenic lines (NILs) carrying homozygous combinations of null alleles. For both genes, gene dosage effects follow the order B > D ≥ A; therefore, we completed detailed analysis of starch characteristics for NIL 3-3, which is null for the B-genome copy of the SSIIa and GBSSI genes, and NIL 5-5, which has null mutations in the B- and D-genome-encoded copies of both of these genes. The effects of the combinations on phenotypic traits followed the order expected on the basis of genotype, with NIL 5-5 showing the largest differences from the wild type, while NIL 3-3 characteristics were intermediate between NIL 5-5 and the wild type. Differences among genotypes were significant for many starch characteristics, including percent amylose, chain length distribution, gelatinization temperature, retrogradation, and pasting properties, and these differences appeared to translate into improvements in end-product quality, since bread made from type 5-5 flour showed a 3 day lag in staling. KEYWORDS: hexaploid, wheat, starch, variability, near-isogenic line, SSIIa, GBSSI

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INTRODUCTION Mutations in plant genes have played an important role in crop domestication1 and have also been important in the development of traits that increase crop value, such as seed retention, flowering time, disease resistance, and plant height. In large part, selection for these mutations was based on phenotype. In the case of null mutations, phenotypic effects are more likely to be observed in diploid plants. However, wheat is an allohexaploid plant with three copies of each gene derived from homoeologous chromosomes, and for the majority of genes, all three copies are expressed.2 This provides a buffering effect because the presence of a single null mutation, which results in the lack of a single protein isoform, is normally masked by the presence of the proteins produced by the two remaining wild-type genes. Thus, mutations cannot easily be observed or selected for in wheat unless they show a dominant effect over the wild-type genes. Although this means that mutations in hexaploid crops, such as wheat, are harder to identify, the presence of three genes encoding three proteins also provides an unusual advantage; not only fully null but “partial” null lines, with elimination of one or two homoeologous genes, can be created. Different combinations of homozygous null and wild-type alleles can provide a level of variation in a trait of interest, a hypothesis that has been solidly demonstrated with the genes involved in starch synthesis. For example, granule-bound starch synthase I (GBSSI) is largely responsible for amylose synthesis in endosperm tissue,3 and waxy wheat lines with null mutations in all three GBSSI genes © XXXX American Chemical Society

(GBSSI-A1, GBSSI-B1, and GBSSI-D1) produce endosperm starch that is essentially amylose-free.4 However, lines missing one or two proteins show a small but significant reduction in endosperm amylose content. Both gene dosage effects and variation in effects among the three genes are observed; lines missing two proteins show a greater reduction in amylose than lines missing a single protein, and among lines missing a single GBSSI, the strongest reduction in amylose content is seen in GBSSI-B1 null lines.5−7 The small reduction in amylose content due to the absence of GBSSI-B is sufficient to alter the viscoelasticity of noodles, and most lines used for making Asian salted noodles lack GBSSI-B or both GBSSI-A and GBSSIB.8−10 Comparable results have been observed with lines carrying mutations in the SSIIa gene, which is involved in amylopectin synthesis in endosperm starch. Although a number of enzymes are required for amylopectin synthesis, the starch synthase SSIIa, which elongates the short side chains of amylopectin, appears to play a pivotal role. Lines with null mutations in all three SSIIa genes (SSIIa-A1, SSIIa-B1, and SSIIa-D1) produce seed starch with a higher proportion of amylose, and the structure of amylopectin in these lines is also modified in that a higher proportion of short branch chains are observed.11 As Received: October 21, 2015 Revised: December 17, 2015 Accepted: January 4, 2016

A

DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. GBSSI and SSIIa Dosage in Wheat Linesa GBSSI (A B D) SSIIa (A B D) a

type 1-1 (wild type)

type 8-1 (waxy)

type 1-8 (high amylose)

type 3-3

type 5-5

+++ +++

−−− +++

+++ −−−

+−+ +−+

+−− +−−

Types 1-1, 3-3, and 5-5 correspond to NILs used in this study. D1, GBSSI-B1, and GBSSI-D1) were selected from the F2−F4 generations. To evaluate the recovery of the recurrent parent genotype in 1-1, 33, and 5-5 NILs, an analysis was performed using a set of 123 simple sequence repeat (SSR) markers that were distributed across all chromosomes and were selected on the basis of their ability to detect polymorphisms between the recurrent parent and the original donor lines for the HA and waxy NILs. The polymerase chain reaction (PCR) conditions were as follows: each 12.5 μL reaction included 25 μg of template DNA, 1× Ex Taq buffer, 0.2 mM deoxynucleotide (dNTP) (each), 1.5 mM MgCl2, 3% dimethyl sulfoxide (DMSO), 1 M betaine, 0.5 μM each primer, and 0.04 unit/μL Takara Ex Taq (Takara, Osaka, Japan). The PCR cycle consisted of an initial 5 min denaturation at 98 °C, followed by 40 cycles of 98 °C for 30 s, 55 or 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min. The annealing temperature was selected on the basis of melting temperatures of primers. PCR products were analyzed by a CEQ8000 DNA sequencer (Beckman Courter) or the QIAxcel electrophoresis system (QIAGEN). Plants were grown in a greenhouse using controlled conditions (25 °C day/17 °C night temperature; 60% relative humidity) under natural light, and seeds were harvested at maturity. Field-grown seeds of NILs 1-1 and 5-5 from two growing seasons were used for analysis of starch characteristics and bread quality tests. Samples grown in the 2013−2014 season are referred to as 2013 samples, and samples grown in the 2014−2015 season are referred to as 2014 samples. A randomized block design with three replications was used for field tests. Seed Weight, Seed Diameter, Seed Hardness, and Moisture, Ash, Protein, and Starch Contents. Thousand kernel weight (TKW) measurements were based on the mean of five 100 kernel weight measurements. The seed moisture content was determined using the American Association of Cereal Chemists (AACC) approved method 44-15A.20 Whole grain was ground into flour using a Retsch ZM200 centrifuge mill (Retsch GmbH, Germany) with a mesh of 0.75 mm using a centrifuge speed of 10 000 rpm. The ash content was measured by the AACC approved method 08-02.20 The grain protein content was based on 13.5% moisture and was determined by near-infrared reflectance spectroscopy using an Infratec 1241 Grain Analyzer (FOSS). The starch content was measured using the Total Starch Assay Kit (Megazyme) according to the manufacturer's protocol. Starch Extraction and Amylose Content. Starch was extracted as described by Hayakawa et al.21 The amylose content was determined using an Amylose/Amylopectin Kit (Megazyme) as previously described.13 Microscope Observations. Starch granules from mature seed were stained with iodine and potassium iodide solution (0.2% KI and 0.04% I2) and observed under an optical microscope at a magnification of 400×. Scanning electron microscopy (SEM) analysis was conducted with a VE-8800 SEM (Keyence, Osaka, Japan) under the same conditions reported previously.16 SEM images were taken at a magnification of 1200×. Chain Length Distribution. The chain length distribution was determined as previously described.16 Essentially, starch was gelatinized by suspending 1 mg of starch granules in 50 μL of DMSO and heating in a boiling water bath for 20 min. An isoamylase treatment was performed by combining 20% starch in DMSO, 10 mM sodium acetate buffer (pH 4.0), and 10 units/mL isoamylase or denatured isoamylase and incubating overnight at 37 °C. A 10 μL aliquot of the reaction was dried using a vacuum centrifuge, and the resulting pellet was dissolved in 2 μL of sodium cyanoborohydride and 8-

with GBSSI, gene dosage and gene-specific effects are observed in partial SSIIa mutants, including effects on amylopectin structure and starch gelatinization and pasting properties.12,13 Wheat lines with null mutations in all SSIIa and GBSSI genes is known as Sweet Wheat (SW), because seeds of these lines have high levels of sugars, particularly maltose.14,15 The starch characteristics of SW are also dramatically different from wildtype starch characteristics, and mature SW seed shows a shrunken phenotype.14,16 By combining null and wild-type alleles for both the GBSSI and SSIIa genes, it is possible to create 64 homozygous genotypes, which would be expected to show a range of starch characteristics unlikely to be observed by chance in a breeding program. The dosage effects of GBSSI and SSIIa enzymes both follow the order B > D ≥ A, and for both enzymes, the effect of two inactive proteins is stronger than the effect from a single inactive protein.7,13 Therefore, of the 64 possible genotypes, we chose to focus on type 3-3, which has null mutations in the Bgenome-derived genes for both GBSSI and SSIIa, and type 5-5, which carries null mutations in both the B- and D-genomederived genes of GBSSI and SSIIa (Table 1). Genotype 3-3 should theoretically show the strongest phenotype among genotypes that are missing a single protein of each type, and type 5-5 the strongest among the lines that carry two null mutations for each enzyme, while type 5-5 should show stronger differences from wild type than type 3-3. In agreement with this, prototype 3-3 and 5-5 lines isolated from initial crosses between the waxy and high-amylose lines used to produce SW14 showed significant differences from wild type in terms of starch characteristics (our unpublished data). However, the genetic backgrounds of the prototype lines were not uniform; therefore, here, we used near-isogenic lines (NILs) to confirm differences between types 3-3, 5-5, and wild type. Analytical results indicated that very clear and statistically significant differences in starch characteristics occurred between the mutant and wild-type NILs, with NIL 5-5 consistently showing stronger deviations from wild type than NIL 3-3. On the basis of this example, the potential for producing desirable variation in wheat quality characteristics using combinations of null and wild-type homeologous genes will be discussed.

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MATERIALS AND METHODS

Plant Materials. To clearly demonstrate the variation in starch characteristics arising from the combinations of null and wild-type alleles of the GBSSI and SSIIa genes investigated here, background effects were minimized by the generation of NILs. Near-isogenic waxy (Wx) (BC5F1) lines, with null alleles for all GBSSI genes, and nearisogenic high-amylose (HA) (BC6F1) lines, with null alleles for all SSIIa genes, were developed using marker-assisted selection with markers described previously.17−19 MD-B004, a breeder’s line that carries wild-type alleles for all GBSSI and SSIIa genes, was used as the recurrent parent in NIL development. After crossing the Wx and HA NIL lines, NIL 1-1 (homozygous wild-type alleles for all SSIIa and GBSSI genes), NIL 3-3 (homozygous null alleles for SSIIa-B1 and GBSSI-B1), and NIL 5-5 (homozygous null alleles for SSIIa-B1, SSIIaB

DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry aminopyrene-1,3,6-trisulfonate (APTS, Beckman Coulter), incubated in the dark for 4 h, and diluted with 46 μL of water prior to analysis. Samples were analyzed using a PA800 Proteome Lab (Beckman Coulter) equipped with a N−CHO coated capillary, 50 μm (inner diameter) × 50.2 cm, using N-linked carbohydrate separation gel buffer (Beckman Coulter) and a 488 nm laser module detector (excitation, 488 nm; emission, 520 nm). The separation conditions were as follows: wash with distilled water, 20.0 psi for 3.0 min; buffer, 20.0 psi for 3.0 min; sample injection, 0.5 psi for 10 s; and separation, 30.0 kV for 35 min. The peak areas in the range of degree of polymerization (DP) 2−45 were calculated using the 32 Karat version 8.0 software package. Differential Scanning Calorimetry (DSC) Analysis. DSC analysis was conducted using a DSC-60A automated DSC instrument (Shimazu, Kyoto, Japan) essentially as described previously.13 Samples were loaded onto the DSC pan, and water was added to achieve a 33% (w/w) starch solution. The thermal conditions were as follows: heating from 20 to 120 °C at 5 °C/min, 1 min hold at 120 °C, followed by cooling from 120 to 30 °C at −10 °C/min. To determine the degree of retrogradation, the DSC cell was left at 4 °C for 14 days and a second measurement was taken using the same thermal conditions. The percent retrogradation was calculated using the ratio of the enthalpy of retrogradation to the enthalpy of gelatinization. Rapid Visco Analyzer (RVA) Measurements. The pasting profile was established using a RVA according to the AACC method 76-21 using profile STD3.20 For RVA measurements, starch or 60% extracted flour was suspended in a 1 mM AgNO3 solution to prevent amylase activity during the analysis.22 Turbidity Measurements. Changes in turbidity were measured as described by Shimbata et al.13 After heating, starch was cooled to room temperature and stored at 4 °C. Absorbance was measured after 1, 2, 3, 7, 10, and 14 days. The absorbance reading obtained immediately after cooling to room temperature was used as a baseline and was subtracted from the readings obtained after storage. Bread-Making Quality Tests. For bread-making quality tests, field-grown seeds of NILs 1-1 and 5-5 planted in 2013 and 2014 were milled to 60% extraction using a Buhler experimental mill (model MLU-202, Uzwil, Switzerland). Baking tests were performed by the sponge−dough method.23 Bread firmness was analyzed 1 and 3 days after baking according to the AACC approved method 74-0920 using a texture analyzer (TA XTplus, Stable Micro Systems, Godalming, U.K.). To ensure that the protein profiles for the major proteins influencing bread-making quality were identical in NILs, sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) analysis of seed proteins extracted from flour was performed according to Payne et al.24 In addition, PCR evaluations of Glu-B1, Glu-D1, GluA3, Glu-B3, Glu-D3, Pina-D1, and Pinb-D1 loci were performed as described above. Statistical Analysis. Analysis of variance (ANOVA) and Scheffe’s test were conducted using SPSS software.

Figure 1. Appearance of seed and starch granules. The appearance of NIL 3-3 and 5-5 seeds is indistinguishable from wild-type seed (NIL 11). Starch granules from all genotypes stain red−brown with iodine solution, indicating the presence of amylose. Analysis of starch granules by electron microscopy (EM) indicates that starch granules from NIL 3-3 and 5-5 seeds are normally shaped and have the expected large A-type and smaller B-type starch granules.

wild-type granules (Figure 1). Starch granules from all three NILs were plump and showed the expected distribution of large, disc-shaped A granules and small, round B granules. Comparison of Starch Characteristics among NILs. Starch granules from types 3-3 and 5-5 stained blue−black with iodine, demonstrating the presence of amylose in endosperm starch (Figure 1). However, significant reductions in amylose were noted in both types 3-3 and 5-5 (Table 2), although neither NIL type 3-3 nor NIL type 5-5 showed major changes in seed weight compared to wild type (Table 2). As would be predicted on the basis of previous studies using lines missing one versus two GBSSI proteins,7,25 type 5-5 had a lower amylose content than type 3-3. Although starch levels were lower in 3-3 than in 5-5, neither genotype differed significantly from wild type. The chain length distribution of amylopectin affects both the physical26 and nutritional27 properties of starches. The SSIIa enzyme plays a role in elongating the short DP 6−11 chains of amylopectin to DP 13−28 chains, and generally, a lack of SSIIa activity results in a relative increase in short branch chains of DP 6−11, with a corresponding decrease in mid-length chains. Although types 3-3 and 5-5 were not significantly different from the wild type in terms of DP 2−5 chains, the proportion of DP 6−10 chains was significantly higher in type 3-3 compared to type 1-1 and the proportion in type 5-5 was significantly higher than that in type 3-3 (Table 3). Type 5-5 had a significantly lower proportion of DP 11−24 chains than type 3-3, which, in turn, had a significantly lower proportion than the wild type (11). NIL types 5-5 and 3-3 follow a similar pattern in their variance from the wild type at each chain length but with lower amplitudes of differences being observed for type 3-3 (Supplementary Figure 1 of the Supporting Information). This demonstrates nicely that a gene dosage effect is occurring, resulting in substantial changes compared to the wild type but not the drastic changes observed by Vrinten et al.16 in the fully null SW (type 8-8) mutant. Gelatinization properties, including retrogradation, can affect the texture and shelf life of baked goods. In terms of gelatinization properties observed by DSC, the intermediate

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RESULTS Evaluation of NILs. Because the gene dosage effect for both genes follows the order B > D ≥ A, the NIL for type 3-3, which has null alleles for the B-genome copy of the SSIIa and GBSSI genes, and the NIL for type 5-5, which has null mutations in the B- and D-genome-encoded copies of both genes, were used for detailed analysis. A total of 123 SSR markers (Supplementary Table 1 of the Supporting Information) and 7 gene-derived markers (described below) were used to test NILs; of these, 123 markers were identical to the recurrent parent, indicating that the genetic backgrounds of NILs were uniform. Comparison of Seed and Starch Granule Appearance among NILs. The appearance of seeds from NILs used in this study were very similar, as shown in Figure 1. Reflecting the normal appearance of seeds from type 3-3 and 5-5 NILs, starch granules from these lines were visually indistinguishable from C

DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. Kernel Weight and Chemical Composition of Seedsa genotype

TKW (g)b

starch (%)c

amylose (%)

protein (%)c

ash (%)c

NIL type 1-1 (wild type) NIL type 3-3 NIL type 5-5

37.8 ± 0.7 a 37.0 ± 0.7 a 38.0 ± 0.7 a

54.5 ± 0.5 a 54.1 ± 0.4 a 54.9 ± 1.5 a

24.1 ± 0.5 a 22.1 ± 0.0 b 18.1 ± 0.6 c

11.5 ± 0.0 ab 12.0 ± 0.4 b 11.2 ± 0.0 a

1.83 ± 0.01 a 1.79 ± 0.01 b 1.77 ± 0.01 c

Values are based on at least three replicates. Values [means ± standard deviation (SD)] followed by the same letter in the same column are not significantly different (p < 0.05). bTKW = thousand kernel weight, indicated on a dry weight basis. cValues are indicated on a 13.5% moisture basis.

a

Table 3. Chain Length Distribution Analysisa chain length distribution genotype

DP 2−5 (%)

DP 6−10 (%)

DP 11−24 (%)

DP >24 (%)

NIL type 1-1 (wild type) NIL type 3-3 NIL type 5-5

0.3 ± 0.1 a 0.4 ± 0.1 a 0.4 ± 0.0 a

13.2 ± 0.4 a 14.4 ± 0.5 b 16.0 ± 0.4 c

64.9 ± 0.4 a 63.8 ± 0.7 b 61.7 ± 0.4 c

21.6 ± 0.9 a 21.5 ± 1.1 a 21.9 ± 0.8 a

Values are based on at least three replicates. Values (means ± SD) followed by the same letter in the same column are not significantly different (p < 0.05). a

Table 4. Gelatinization and Retrogradation Properties of Starches from NILs as Determined by DSC Analysisa gelatinization peak

amylose−lipid dissociation peak

genotype

Tp (°C)

Tp (°C)

ΔH (J/g)

Tp (°C)

Tp (°C)

ΔH (J/g)

retrogradation (%)

NIL type 1-1 (wild type) NIL type 3-3 NIL type 5-5

60.0 ± 0.1 a 57.2 ± 0.2 b 54.6 ± 0.2 c

60.0 ± 0.1 a 57.2 ± 0.2 b 54.6 ± 0.2 c

10.5 ± 0.4 a 10.7 ± 0.1 a 10.2 ± 0.4 a

93.5 ± 0.3 a 93.2 ± 0.2 a 93.5 ± 0.5 a

98.0 ± 0.3 a 98.0 ± 0.4 a 97.4 ± 0.4 a

1.3 ± 0.1 a 1.3 ± 0.1 a 1.2 ± 0.1 a

51.5 ± 2.6 a 44.2 ± 1.0 b 34.7 ± 2.6 c

Values are based on at least three replicates. Values (means ± SD) followed by the same letter in the same column are not significantly different (p < 0.05). Tp, peak temperature; ΔH, enthalpy change. a

Table 5. Changes in Turbidity over Timea days genotype

0

1

2

3

7

10

14

NIL type 1-1 (wild type) NIL type 3-3 NIL type 5-5

0 0 0

0.054 ± 0.006 a 0.045 ± 0.011 a 0.037 ± 0.004 a

0.081 ± 0.006 a 0.066 ± 0.004 a 0.053 ± 0.011 a

0.086 ± 0.010 a 0.063 ± 0.014 ab 0.050 ± 0.004 b

0.151 ± 0.008 a 0.104 ± 0.014 a 0.084 ± 0.009 b

0.204 ± 0.005 a 0.142 ± 0.014 a 0.104 ± 0.015 b

0.275 ± 0.011 a 0.192 ± 0.019 b 0.128 ± 0.018 c

Values (means ± SD) indicate increased absorbance at A640 nm from 0 day and are based on at least three replicates. Values (means ± SD) followed by the same letter in the same column are not significantly different (p < 0.05).

a

observed (Table 5). The reduced development of turbidity after 14 days of storage in type 5-5 (absorbance value of 0.128 ± 0.018) compared to type 3-3 (absorbance value of 0.192 ± 0.019) or the wild type (absorbance value of 0.275 ± 0.011) is likely related to the downward trend in amylose amount (type 5-5 < type 3-3 < type1-1) along with the corresponding increases in shorter branch chains. Because both the amylose content, which is controlled by the GBSSI genotype, and amylopectin structure, which is influenced by the SSIIa genotype, can affect turbidity, here, we see the effects of a reduction in the amylose content from type 1-1 to type 3-3 to type 5-5, along with a change in the amylopectin structure, particularly an increase in short chains, following the same order. Starch viscosity characteristics, which can be estimated by RVA pasting profiles, influence textural and storage properties of cereal products.33 The RVA profiles for types 1-1, 3-3, and 55 appear similar, because comparable times and temperatures were observed for all parameters (Figure 2). However, significant differences in peak and breakdown viscosity values were observed between types 1-1, 3-3, and 5-5 (Figure 2 and Supplementary Table 2 of the Supporting Information). Here, again, both GBSSI and SSIIa enzymes are predicted to affect this trait, since partial waxy wheat, which lacks GBSSI-B and

genotypes 3-3 and 5-5 were again significantly different from type 1-1 and from each other, with the gelatinization onset temperature (To) and gelatinization peak temperature (Tp) both being lower in the type 5-5 genotype carrying B- and Dnull genes than in NIL type 3-3 carrying only B-null genes (Table 4). Starches with high ratios of long chains reportedly have higher gelatinization temperatures (To and Tp),28 and the reduction in To and Tp seen here corresponds with the increasing proportion of short branch chains (Table 3). Lower starch retrogradation, which is directly related to reduced bread staling,29 has been associated with a reduction in amylose levels.30,31 Amylopectin chain length distributions also affect retrogradation rates, with an increase in the proportion of DP of 6−9 chains, resulting in decreases in retrogradation.26 In agreement with this, type 5-5, which has the lowest amylose levels and the highest proportion of DP of 6−9 chains among the NILs, had a significantly lower percent retrogradation than type 3-3, which, in turn, had a significantly lower value than type 1-1 (Table 4). Similar to retrogradation, changes in turbidity of starch gels over time can be influenced by both the amylose amount and amylopectin chain length.32 Differences in turbidity between types 1-1 and 5-5 were evident by day 3, and after 14 days of storage, significant differences between all three genotypes were D

DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Profiles of NIL 1-1 (wild type), 3-3, and 5-5 starches as measured with a RVA.

GBSSI-D proteins, shows an increased peak viscosity,7 while lines that are wild type for GBSSI but lacking both the SSIIa-B and SSIIa-D proteins show a lower peak viscosity than the wild type.12,13 Thus, in terms of peak viscosity, it appears that the effects from the lack of the GBSSI proteins are over-riding any potential effects from the lack of SSIIa proteins. Starch Characteristics and Bread-Making Quality Analysis of Field-Grown NILs. Analysis of 2 years of fieldgrown samples of NILs 5-5 and 1-1 supported the results described above. Field-grown NIL 1-1 seed was similar to NIL 5-5 seed in terms of TKW and starch amount, with only amylose levels showing significant differences between samples (Supplementary Table 3 of the Supporting Information). No pre-harvest sprouting problems were observed under field conditions, as demonstrated by falling number measurements (Supplementary Table 3 of the Supporting Information). The RVA pasting profiles of NIL 1-1 and 5-5 field-grown samples (Supplementary Figure 2 of the Supporting Information) resembled profiles of samples grown under controlled conditions (Figure 2). Notably, the significantly reduced level of retrogradation in NIL 5-5 compared to NIL 1-1 observed under controlled conditions (Table 4) was also observed in data from 2 years of field-grown samples (Supplementary Table 4 of the Supporting Information). To determine if the reduced retrogradation observed here translated into useful effects in end products, the bread-making quality of NIL 5-5 was compared to that of NIL 1-1 (wild type). Because differences in storage proteins, especially highand low-molecular-weight glutenin subunits and proteins influencing soft- versus hard-grain type, can affect bread-making quality,34 the major glutenin subunits along with puroindoline a and b alleles were compared among NILs. As shown in Supplementary Table 5 of the Supporting Information, genotypes for the glutenin subunits encoded by Glu-A1, GluB1, Glu-D1, Glu-A3, Glu-B3, and Glu-D3 as well for the Pina and Pinb genes were identical between NIL types 1-1 and 5-5. A SDS−PAGE analysis of storage proteins showed identical patterns for other storage proteins as well (Supplementary Figure 3 of the Supporting Information). These data, together with the SSR evaluation results, provide strong evidence that observed effects were due to differences at the GBSSI and SSIIa loci. Typically, crumb firmness increases during bread storage, and mitigating this increase results in a longer shelf life for bakery products.35 As shown in Figure 3, NIL 5-5 showed a lower firmness value 3 days after baking than was observed with NIL

Figure 3. Crumb firmness values of bread samples prepared using flour from type 1-1 and 5-5 NILs. Results of field-grown samples from two growing seasons are shown. Measurements were taken one (D + 1) and three (D + 3) days after baking. Firmness was analyzed with a texture analyzer using the AACC approved method 74-09. The bottom panel shows representative bread slices from NIL 1-1 and 5-5 lines. Mean loaf volumes (LV), weights (W), and specific volumes (SV) for each line are given below the photographs.

1-1 (wild type) after a single day, suggesting that the 5-5 genotype may prove useful in reducing bread staling. Importantly, loaf volumes of bread made from NILs 1-1 and 5-5 were comparable (Figure 3).

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DISCUSSION Although developing fully null mutants in hexaploid bread wheat can present challenges, the presence of three copies of each gene also provides the opportunity to create a range of homozygous genotypes carrying zero to three active genes, which can be reflected in a phenotypic gradient. NILs were developed for all 64 of the starch synthase genotypes, and because earlier studies indicated that gene dosage effects of both SSIIa and GBSSI follow the order B > D ≥ A, we chose to focus on types 5-5 and 3-3 in our comparisons to the wild type (type 1-1; Table 1). These NIL lines were carefully compared using SSR markers distributed across all chromosomes to ensure that the observed effects were from the specific SSIIa and GBSSI gene combinations, and this analysis indicated that, except for the GBSSI and SSIIa regions, the genotype of the recurrent parent had largely been recovered. Starch of a prototype 5-5 line selected from crosses with the original SW parents showed similar characteristics (our unpublished data) to those observed in this study, and the more detailed work presented here using NILs allowed us to confirm the accuracy of our original results. Here, we clearly demonstrate that the creation of “partial” null lines for two genes can produce novel and potentially economically useful wheat types that have less severe changes than are found in fully null mutants. One notable aspect of this work was the stepwise gradient observed among the NILs E

DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry caused by varying the dosage levels of two different genes. Results for traits including amylose content, amylopectin chain length, retrogradation, and pasting properties were clear and significant and followed the expected pattern (Tables 2−5). Effects attributable to either GBSSI or SSIIa, such as changes in the amylose content (Table 2), were observable, and changes that resulted from the combined effects of both genes, such as changes in retrogradation (Table 4), were also detected. From a practical point of view, the unique starch characteristics of the type 5-5 line should prove useful for bakery products (Figure 3). Bread staling represents an important source of food wastage, and although the mechanisms of this process are not well understood,29 starch retrogradation is thought to be a major cause of staling.35 Both the amylose content (influenced by GBSSI) and amylopectin chain length (influenced by SSIIa) affect starch retrogradation; therefore, by combining operative and inoperative genes in a calculated manner, we were able to develop a wheat line that produced bread showing reduced staling. Incorporation of the relevant genes into adapted bread-making varieties via marker-assisted selection-based breeding has been initiated in both Japan and Canada. This work also highlights the potential for modifying other quality traits in wheat using combinations of active and inactive genes, particularly for traits where reduced gene expression has a positive effect. The development of “partial” wheat mutants will also be useful for genes that regulate more than one trait or are lethal in the fully null condition; in such cases, combining partial null genotypes can increase the available phenotypic variability while still providing a viable plant. As demonstrated here, using combinations of two or more genes that influence a particular trait can be particularly advantageous; with two genes, 64 homozygous genotypes can be selected, and this increases to 512 genotypes if mutations for three genes are combined, bringing into view a huge amount of hidden variation. The dosage effect of genes in wheat has been of interest to researchers for many years,36,37 but in the past few years, the amount of sequence information available for wheat and related species has greatly increased,38−41 allowing for the development of novel and more efficient screening methods that enable rapid identification of mutations.42−45 These advances greatly increase the practicality of using the approach described here as an auxiliary method for creating a range of variation in quality characteristics, which may lead to increased suitability for particular end uses.

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(Supplementary Table 4), and Glu1, Glu3, and Pin genotypes (Supplementary Table 5) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +81-196433514. E-mail: tnaka@affrc.go.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to the support staff at NARO Tohoku Agricultural Research Center and Nippon Flour Mills Company, Ltd.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05099. Differences in chain length distribution (Supplementary Figure 1), pasting curve of starch from 60% extracted flour from field-grown lines as determined by RVA (Supplementary Figure 2), SDS−PAGE analysis of seed storage proteins (Supplementary Figure 3), genetic backgound of NILs (Supplementary Table 1), pasting properties of starches from NILs as indicated by RVA parameters (Supplementary Table 2), kernel weight and flour characteristics of field-grown NILs (Supplementary Table 3), gelatinization properties of 60% extracted flour from field-grown seed as determined by DSC analysis F

DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b05099 J. Agric. Food Chem. XXXX, XXX, XXX−XXX