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. Author manuscript; available in PMC: 2016 Apr 22.
Published in final edited form as: J Plant Regist. 2014 Aug 25;8(3):334–338. doi: 10.3198/jpr2014.02.0007crg

Registration of Durum Wheat Germplasm Lines with Combined Mutations in SBEIIa and SBEIIb Genes Conferring Increased Amylose and Resistant Starch

Brittany Hazard 1, Xiaoqin Zhang 1, Mahmoudreza Naemeh 1, Jorge Dubcovsky 1,*
PMCID: PMC4840879  NIHMSID: NIHMS754769  PMID: 27110322

Abstract

Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations in the linked SBEIIa and SBEIIb paralogs were combined on chromosomes 2A (SBEIIa/b-A; Reg. No. GP-968, PI 670159), 2B (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and on both chromosomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160) in the tetraploid wheat cultivar Kronos, a semidwarf durum wheat cultivar that has high yield potential and excellent pasta quality. These three double and quadruple SBEII-mutant lines were compared with a control sib line with no SBEII mutations in two field locations in California. The SBEIIa/b-AB line with four mutations showed dramatic increases in amylose (average 66%) and resistant starch (average 753%) relative to the control. However, the SBEIIa/b-AB line also showed an average 7% decrease in total starch and an 8% decrease in kernel weight. The release by the University of California–Davis of the durum wheat germplasm combining four SBEIIa and SBEIIb mutations will accelerate the deployment of these mutations in durum wheat breeding programs and the development of durum wheat varieties with increased resistant starch.

Wheat (Triticum ssp.) is an important cereal crop that contributed over 651 million t of food worldwide in 2012 and is a valuable source of carbohydrates, amino acids, vitamins and minerals (FAO, 2012). Durum wheat [T. turgidum L. subsp. durum (Desf.) Husn.] production has been increasing globally since the 1950s and has currently reached about 33 million t per year (Ma et al., 2013). Durum wheat is most useful for producing pasta, couscous, and flatbreads because of its unique quality aspects, including hardness, high protein content, and high gluten strength (Ma et al., 2013). The demand for food products rich in fiber is growing among consumers as a result of increased awareness of the associated health benefits, such as reduced risk of type II diabetes, obesity, cardiovascular diseases, and cancers of the colon (Yong-Cheng and Maningat, 2013; Sestili et al., 2014). High levels of amylose in the starch of wheat and other cereals are associated with increased resistant starch, an important component of dietary fiber. Resistant starch is defined as the undigested portion of starch that passes through the small intestine and is ultimately fermented in the large intestine by the gut microflora (Englyst et al., 1992). The portion of the starch that is resistant to enzymatic digestion is thought to originate in long amylose glucan chains that associate and form complexes that function similar to other dietary fibers. Foods with resistant starch have reduced glycemic indices, which are important in obesity and diabetes prevention. The fermentation of resistant starch in the large intestine produces short chain fatty acids that provide additional health benefits by lowering the pH of the intestinal environment, which reduces the proliferation of pathogenic bacteria and absorption of cytotoxic compounds (Sestili et al., 2014). Along with improved nutritional properties and health benefits, moderate increases in the proportion of amylose in wheat have been associated with improved qualities in food products such as improved texture in baked goods and increased firmness in pasta (Waring, 2005; Soh et al., 2006).

Starch-based foods comprise more than 50% of the human diet. Thus, improving the starch composition in food products by increasing the amount of amylose and resistant starch has the potential to provide consumers with the associated health benefits. Because of this, increasing amylose and resistant starch in wheat grain is a valuable objective for both durum and common wheat (T. aestivum) breeding programs. Starch branching enzymes play a major role in determining the proportion of amylose and amylopectin in the wheat grain, and downregulation or mutations of the STARCH BRANCHING ENZYME II (SBEII) genes result in increased amylose and resistant starch in the grain (Regina et al., 2006; Sestili et al., 2010; Botticella et al., 2011; Hazard et al., 2012; Slade et al., 2012). We previously obtained and described ethyl methane sulfonate (EMS) mutants for the paralogous genes SBEIIa and SBEIIb from a TILLING (targeted induced local lesions in genomes) population of the tetraploid durum wheat cultivar Kronos (Uauy et al., 2009; Hazard et al., 2012). We also identified recombinant lines that combined SBEIIa and SBEIIb mutations on a single chromosome, both in the A and B genome copies of these genes (homoeologs), but only the single SBEIIa mutants were characterized for amylose and resistant starch content in the previous study (Hazard et al., 2012). In this study, we register three mutant durum wheat germplasm lines developed at the University of California, Davis that carry linked SBEIIa and SBEIIb mutations in coupling in the A genome (SBEIIa/b-A; Reg. No. GP-968, PI 670159), in the B genome (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and in both genomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160). We also determined the effect of these mutations on amylose and resistant starch content in the grain, on total starch content, and on kernel weight (KW) relative to a sib control line with no SBEII mutations.

Materials and Methods

Development of SBEII Germplasm Lines

SBEII mutants were selected from a TILLING population (Uauy et al., 2009) of the Desert durum cultivar Kronos (PI 576168), which was developed by Arizona Plant Breeders Inc. from a male sterile population (selection D03–21). A more detailed description of the identification, selection, and generation of the mutant lines is described in Hazard et al. (2012), and a summary of mutations is provided in Table 1. Mutant lines were backcrossed twice to Kronos to reduce background mutations. In the previous study, we combined the SBEIIa and SBEIIb mutations and selected plants homozygous for the two mutations both in the A (SBEIIa/b-A) and in the B (SBEIIa/b-B) genomes (Hazard et al., 2012). In this study, we intercrossed SBEIIa/b-A and SBEIIa/b-B, self-pollinated the F1 hybrid, and generated a new segregating population. From this segregating population we selected sib lines homozygous for SBEIIa and SBEIIb mutations only in the A genome (SBEIIa/b-A), only in the B genome (SBEIIa/b-B), in both the A and B genomes (SBEIIa/b-AB) and sib lines with no SBEII mutations to be used as controls. Selected lines were increased in the greenhouse for use in phenotyping experiments. Plants carrying the targeted mutations were selected in each generation by sequencing, using the same genome specific primers designed to screen the original TILLING population described in Uauy et al. (2009).

Table 1.

Summary of SBEIIa and SBEIIb mutations. Line is the mutant number in the TILLING population.† Protein coordinates are based on protein sequences from the corresponding genome in Triticum aestivum cultivar Chinese Spring.‡

Gene Genome Line DNA coordinates Protein coordinates and predicted effect
SBEIIa A T4-2179 G401A W220 (stop codon)
SBEIIa B T4-1214 G1347A E296 (splice junction)
SBEIIb A T4-2574 G308A S208 (splice junction plus stop)
SBEIIb B T4-764 C1290T P283L (BLOSUM62 = −3)
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†

DNA coordinates are based on Triticum turgidum cultivar Kronos partial genomic sequence used for TILLING (Uauy et al., 2009).

‡

For SBEIIb-B there was no complete protein available (the P283L coordinate is based on the A and D genome proteins at the same position) (Hazard et al., 2012).

Phenotypic Characterization of SBEII Germplasm Lines

Mutant lines were grown in two field experiments and compared with wild-type sib control lines. The field experiments were grown in the University of California Experimental Field Station in Davis, CA (38°32′ N, 121°46′ W), and the University of California Research and Extension Center in Tulelake (Intermountain Research and Extension Center, 41°57′ N, 121°28′ W). In Davis, sowing occurred in December (winter planting) in a Yolo loam soil (fine-silty, mixed, superactive, nonacid, thermic Mollic Xerofluvents), and the fertilization consisted of a preplanting application of 112 kg ha−1 N and a top-dress application of 67 kg ha−1 N at tillering. In Tulelake, lines were sown in May (spring planting) in a Tulebasin mucky silty clay loam soil (fine, mixed, superactive, mesic Aquandic Endoaquolls) and were fertilized with a preplanting application of 149 kg ha−1 N. Each experiment was set up in a randomized complete block design with six blocks (replicates). For each block, 50 seeds per genotype were sown in 1-m rows (experimental units). Once mature, grains were harvested from each row. For the Davis experiment, KW was estimated from the weight of 100 to 1000 kernels per sample, with the exception of one sample in which 50 kernels were used due to limited seed availability. Five hundred kernels per sample were used to estimate KW in the Tulelake experiment. Whole grain flour was prepared for each sample by grinding in a UDY Cyclone Mill (UDY Corporation) using a 0.5-mm screen, and grain moisture was determined for each sample at the California Wheat Commission Milling and Baking Laboratory using AOAC Official Method 925.10 (AOAC, 2000).

Relative amylose content (amount of amylose as a percentage of total starch) was measured for 25-mg samples of whole-grain flour using the AMYLOSE/AMYLOPECTIN kit developed by Megazyme International (2011a) following the manufacturer instructions. Relative resistant starch content (amount of resistant starch as a percentage of total starch) was measured for 100-mg samples of whole-grain flour using the RESISTANT STARCH kit from Megazyme International (2011b). In this assay, both solubilized and resistant starch were calculated on a dry weight basis using percentage moisture values and following instructions provided in Megazyme International (2011b). Total starch content was measured for 100-mg samples of whole grain flour using the TOTAL STARCH kit developed by Megazyme International (2011c) following the manufacturer’s instructions for the recommended KOH assay format.

To account for potential variation among assays performed at different times, complete sets of samples including all four genotypes were measured in each assay, and sets were then included as a block in the statistical analyses. Data were analyzed first combining results from both locations using a mixed model ANOVA (SAS Institute, 2011), where environment, genotype × environment, and block within environment were included as random factors. Means of the individual mutant lines were compared with the wild-type sib line using the Dunnett test. These mean comparisons are reported separately for each location to show the consistency of the results across environments. The ANOVA assumptions were tested using Shapiro–Wilk’s test for normality of residuals and Levene’s test for homogeneity of variances.

Characteristics

Amylose Content

The ANOVA for relative amylose content combining both locations explained 83% of the variation and showed no significant effects of environment (P = 0.143) or genotype × environment interactions (P = 0.428) but showed significant difference among genotypes (P < 0.005) (Table 2). Mean comparisons of mutant lines against the wild-type sib control using the Dunnett test showed no significant differences for the mutant lines carrying SBEIIa and SBEIIb mutations only in the A or B genomes (Table 3). However, the quadruple SBEIIa/b-AB showed significantly higher levels of amylose than the wild-type sib control in both locations (P < 0.01) (Table 3). The relative increase in amylose content in the SBEIIa/b-AB mutant lines relative to the control was 81% in Tulelake and 51% in Davis (average 66% increase).

Table 2.

Mixed model ANOVAs for combined locations for relative amylose content, relative resistant starch content, total starch content, and kernel weight. Genotypes include SBEIIa/b-A, SBEIIa/b-B, and SBEIIa/b-AB mutants and wild-type sib control lines.

Source of variation Relative amylose content Relative resistant starch content Total starch content Kernel weight
Genotype† (P) 0.005 0.002 0.024 0.059
Environment‡ (P) 0.143 0.027 <0.0001 0.504
Genotype × environment (P) 0.428 0.497 0.874 0.670
Block(environment) (P) 0.726 0.005 0.831 0.001
Proportion of variation explained(R2) 0.832 0.909 0.659 0.626
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†

Error used = genotype × environment.

‡

Error used = MS[block(environment)] + MS(genotype × environment) − MS(error).

Table 3.

Effect of SBEIIa and SBEIIb mutations on relative amylose content, relative resistant starch content (RS), total starch content (TS) and kernel weight (KW) in SBEIIa/b-A, SBEIIa/b-B, and SBEIIa/b-AB mutants and wild-type sib control lines. Untransformed arithmetic means and P values from Dunnett tests are reported separately for the two locations.

Line Amylose P RS P TS P KW P
% % % mg kernel−1
Davis
Control 26.6 ± 1.0 0.3 ± 0.1 68.3 ± 1.3 48.8 ± 1.3
SBEIIa/b-A 27.0 ± 0.7 NS 0.6 ± 0.2 NS 68.5 ± 2.0 NS 47.1 ± 1.7 NS
SBEIIa/b-B 28.6 ± 1.1 NS 0.4 ± 0.1 NS 66.4 ± 1.2 NS 46.7 ± 1.8 NS
SBEIIa/b-AB 40.1 ± 1.3 <0.01 2.9 ± 0.4 <0.01 64.7 ± 2.8 NS 45.8 ± 1.5 NS
Tulelake
Control 30.0 ± 1.4 0.5 ± 0.1 62.9 ± 0.8 49.7 ± 1.0
SBEIIa/b-A 28.3 ± 1.0 NS 0.7 ± 0.1 NS 62.2 ± 1.1 NS 49.2 ± 1.3 NS
SBEIIa/b-B 29.9 ± 1.4 NS 0.7 ± 0.1 NS 60.9 ± 1.5 NS 48.0 ± 1.4 NS
SBEIIa/b-AB 54.4 ± 3.6 <0.01 3.7 ± 0.2 <0.01 57.0 ± 1.4 <0.05 45.2 ± 1.0 <0.01
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Resistant Starch Content

The combined ANOVA for resistant starch showed significant differences among environments (P = 0.027) and genotypes (P = 0.002) but no significant genotype × environment interaction (Table 2). Approximately 91% of the variation in resistant starch is explained by the combined model (Table 2). Similar to results reported above for relative amylose content, differences in relative resistant starch between the mutants and wild-type sib control were significant only for the SBEIIa/b-AB quadruple mutant line (Dunnett test P < 0.01) and were consistent across locations (Table 3). The single-genome mutant lines showed nonsignificant differences when compared to the control (Table 3). On average, the relative resistant starch content of the SBEIIa/b-AB quadruple mutant was 753% higher than the control. The observed differences were higher in Davis (867% increase) than in Tulelake (640% increase) as shown by the significant effect of location (Table 2).

Total Starch Content

The combined ANOVA explained 66% of the variation in total starch content and showed a strong effect of environment (P < 0.0001). The effect of genotype on this trait was smaller than the effect of the environment but was still significant (P = 0.024). Similar trends in the variation in total starch content among genotype were observed in the two locations, resulting in a nonsignificant genotype × environment interaction (Table 2). In both locations, the SBEIIa/b-AB quadruple mutant showed lower values of total starch content than the wild-type sib control, but the differences were significant only in Tulelake (P < 0.05, Table 3). The SBEIIa/b-AB mutant line showed an average 9% decrease relative to the control in Tulelake but only a 5% decrease in Davis. The SBEIIa/b-A and SBEIIa/b-B mutant lines were not significantly different from the control in both locations (Table 3).

Kernel Weight

The combined ANOVA for KW explained 63% of the variation and showed no significant differences among environments, genotypes or their interaction (Table 2). However, the differences among genotypes were close to significant levels (0.059) and when analyzed separately by location, showed significant differences in Tulelake (P = 0.0037) but not in Davis (P = 0.5212). In Tulelake, the SBEIIa/b-AB quadruple mutant showed 9% decrease in KW relative to the control (P < 0.01) (Table 3). Even though the difference was not significant in Davis, the SBEIIa/b-AB mutant lines also showed a 6% reduction in KW relative to the control. The single-genome mutant lines SBEIIa/b-A and SBEIIa/b-B were not significantly different from the wild-type sib control (Table 3).

Discussion

The SBEIIa/b-AB mutant with combined SBEIIa/b linked mutations in both the A and B genome copies of SBEII will be a useful tool for durum wheat breeding programs interested in increasing amylose and resistant starch content. To facilitate the utilization of these mutations in durum breeding programs, we selected a recurrent parent (Kronos) with high yield potential and excellent pasta quality (Jackson, 2011). Here we demonstrate that the quadruple SBEIIa/b-AB mutant has significantly higher levels of amylose (66%) and resistant starch (753%) than the control, while the SBEIIa/b-A and SBEIIa/b-B lines with combined mutations in only one genome did not (Table 3). The increases reported here are also significantly higher than those observed in the SBEIIa-AB mutants reported previously (Hazard et al., 2012) that combine mutations in both genomes but only for the SBEIIa gene (22% increase in amylose content and 115% increase in resistant starch). These results indicate that the SBEIIa and SBEIIb genes have redundant functions and that only simultaneous mutations in all copies of both paralogs result in large increases in amylose and resistant starch.

However, durum wheat breeders need to be aware that these dramatic increases in relative amylose and resistant starch content expected from the SBEIIa/b-AB mutant are also associated with reduction in total starch content (7%) and KW (8%) across both locations. The changes in KW and total starch content are likely a pleiotropic effect of the mutant SBEII alleles, but it is also possible that this effect results from background effects of other mutations that occurred during the original EMS mutagenesis. It is also possible that the negative impact of reductions in KW on grain yield may be compensated in some genotypes by increases in seed number. Finally, there were no significant genotype × environment interactions for the four traits considered here, suggesting that the effects of these mutations are consistent across environments (Table 2). In summary, durum wheat breeders interested in increasing resistant starch using these mutants will need to determine the negative effects of these mutations on total starch and kernel weight in their own genetic backgrounds and environments. We are currently introgressing these four mutations into different durum genetic backgrounds to evaluate their effect on yield and pasta quality in different locations in California. In addition, we are transferring the SBEIIa/b linked mutations into hexaploid wheat and combining them with an available SBEIIa-D genome mutant, to facilitate the future utilization of these mutations in common wheat.

Availability

Seed of the three mutant lines is available from the USDA–ARS National Center for Genetic Resources Preservation (USDA-ARS National Genetic Resources Program, 2014). The seeds for SBEIIa/b-A, SBEIIa/b-B, and SBEIIa/b-AB were generated through a greenhouse increase of the materials harvested from the Davis field experiment and are homozygous for mutations at the SBEIIa and SBEIIb loci.

Acknowledgments

This project was supported by the National Research Initiative Competitive Grant 2011-68002-30029 (Triticeae-CAP) from the USDA National Institute of Food and Agriculture, the Howard Hughes Medical Institute, and the Gordon & Betty Moore Foundation grant number GBMF3031. Brittany Hazard acknowledges the generous support from the Colorado Wheat Research Foundation (CWRF) and ConAgra Foods through a fellowship for her Ph.D. studies.

Abbreviations

EMS

ethyl methane sulfonate

KW

kernel weight

TILLING

targeted induced local lesions in genomes

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