Bread wheat (cv. Chinese Spring)/Ae. geniculata (TA2899) chromosome addition lines 1U^g, 2U^g, 3U^g, 4U^g, 5U^g, 6U^g, 7U^g, 1M^g, 2M^g, 3M^g, 5M^g, 6M^g, and 7M^g were kindly provided by Dr. Bernd Friebe (Kansas State University, Manhattan, KS, United States) and maintained by the Cereal Genebank, Martonvásár, while a set of bread wheat (line Mv9kr1)/Ae. biuncialis (MvGB642) chromosome addition lines 1U^b, 1U^b6U^b, 3U^b, 2M^b, 3M^b, and 7M^b were produced in Martonvásár (Molnár-Láng et al., 2002; Schneider et al., 2005). The recessive crossability allele kr1 was transferred from bread wheat cv. Chinese Spring (CS) into bread wheat cv. Martonvásári 9 (Mv9) by backcrossing the Mv9 × CS hybrids with Mv9. The Mv9kr1 line carries recessive crossability alleles Kr1 and Kr2, but the genotype is 93.75% Mv9 (Molnár-Láng et al., 1996). Ae. geniculata or Ae. biuncialis accessions were provided by the Cereal Genebank, Martonvásár (MvGB), by the Wheat Genetics Resource Center, Kansas State University, United States (TA) and by the Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany (AE) and maintained by the Cereal Genebank, Martonvásár.

Four grams of seed from each sample was milled using a Retsch Mixer Mill MM 200 ball mill to produce wholemeal samples, which were immediately cooled and stored at -20°C until compositional analysis.

No studies have yet been reported on the composition of DF in Aegilops species with the U and M genomes, but previous results on HMW glutenin subunit alleles showed wide variation within these species (Ahmadpoor et al., 2014; Dai et al., 2015; Medouri et al., 2015; Wang et al., 2015; Garg et al., 2016). In order to determine the extent of variation in the amounts of protein, β-glucan, TOT-pentosan, and WE-pentosan in Ae. geniculata and Ae. biuncialis, five genebank accessions of each species were studied. The Ae. geniculata and Ae. biuncialis accessions had significantly lower TKW than the two wheat genotypes (cv. Chinese Spring and line Mv9kr1) used in the present study (Figure 1A), with values ranging between 17.4 and 22.1 g (accessions AE754/90 and AE274/80, respectively) in Ae. biuncialis, and between 9.3 and 17.0 g (accessions AE1311/00 and AE839/91, respectively) in Ae. geniculata.

The β-glucan and protein content were significantly higher in all Aegilops accessions than in the wheat genotypes (Figure 1B). Within Ae. biuncialis and Ae. geniculata, the β-glucan content varied between ∼30 and 50 mg/g irrespective of the TKW of the accessions. Interestingly, the protein content was more variable among the Ae. biuncialis accessions (∼22–37%) than in Ae. geniculata genotypes (∼26%), where differences were not observed for this parameter. The amounts of TOT-pentosan and WE-pentosan also varied (∼30–60 and ∼8–14 mg/g, respectively), but with the exception of one accession (MvGB376) were similar to or above those in wheat (Figure 1C).

The parental Ae. geniculata TA2899 and Ae. biuncialis MvGB642 genotypes have significantly lower (<50%) TKW than the parental wheat genotypes cv. Chinese Spring and cv. Mv9kr1 (Figure 2A). In the Chinese Spring × Ae. geniculata combination, chromosome addition lines 1U^g, 4-5-6U^g, 1M^g, and 7M^g showed significantly higher TKW than the wheat parent, while addition lines containing chromosomes 2U^g, 2M^g, and 5M^g exhibited significantly lower TKW. Ae. biuncialis chromosomes had no effect on this parameter. In parallel with the lower TKW values, the Aegilops accessions had higher protein content relative to wheat (Figure 2B), which can be attributed to the “yield dilution” effect, i.e., the high grain weight of wheat, resulting from the increased starch content and the consequent relative decrease in grain storage proteins. The addition of chromosomes 2U^g, 4U^g, 5U^g, 7U^g, 2M^g, 5M^g, and 7M^g of Ae. geniculata significantly increased the protein content of wheat cv. Chinese Spring, while the addition of Ae. biuncialis chromosomes 3U^b, 2M^b, 3M^b, and 7M^b significantly increased that of wheat line Mv9kr1 (Figure 2B).

Under field conditions, the 6U^g, 3M^g, 6M^g, and 7M^b additions exhibited the most stable TKW similar to wheat, while the TKW of all the other lines were decreased by adding the Aegilops chromosomes (Supplementary Figure S2a). It is probable that the very low TKW resulted the significantly higher protein contents of Aegilops than wheat (with the exception of 2M^b and 3M^b), with 1U^b, 1U/6U^b, 2U^g, 3U, 4U^g, 2M^g, and both 7M having the greatest effects (Supplementary Figure S2b).

The good processing quality of wheat is related to the high content of polymeric glutenins relative to gliadins (Glu/Gli) and to the high ratio of UPP (UPP%). Ae. geniculata accession TA2899 had a higher Glu/Gli ratio and UPP% than cv. Chinese Spring, whereas these two parameters were lower in Ae. biuncialis MvGB642 than in Mv9kr1 (Figures 2C,D). It should be noted that the model wheat genotype cv. Chinese Spring, which has poor processing quality properties, exhibited lower Glu/Gli ratio and UPP% than line Mv9kr1 whose breadmaking quality parameters are good. This is why the effect of Aegilops chromosomes on the quality parameters of wheat was more pronounced in cv. Chinese Spring than in Mv9kr1. In agreement with this, the added chromosomes 1U^g and 1M^g significantly increased the proportion of polymeric glutenin proteins (higher Glu/Gli ratio and UPP%) in the Chinese Spring background, which can be expected to result in improved processing quality (Figures 2C,D). The addition of chromosomes 3U^g, 3M^g, and 5M^g also increased the Glu/Gli ratio, but did not affect the UPP%. In the case of Ae. biuncialis, chromosomes 1U^b and 1U^b/6U^b again had the greatest effects on the protein composition and the proportion of glutenin polymers (Figures 2C,D), but these were negative, with the proportion of monomeric gliadins increasing instead of the glutenins.

The β-glucan content expressed in mg/g dry weight of wholemeal (measured using the commercial kit for total β-glucan content) was about fivefold higher in Ae. geniculata TA2899 than in cv. Chinese Spring and 2.5-fold higher in Ae. biuncialis MvGB642 than in line Mv9kr1 (Figure 3A). Chromosome addition lines 5U^g, 7U^g, and 7M^g, which had similar or higher TKW than wheat, had a positive effect on β-glucan content in the Chinese Spring genetic background. In the case of Mv9kr1 × Ae. biuncialis addition lines, significantly higher β-glucan content was also observed in the 7M^b addition line.

Aegilops chromosomes 5U^g, 7U^g, 7M^g, and 7M^b were able to increase the β-glucan content of wheat wholemeal under field conditions, which fully support the results of the glass house experiments (Supplementary Figure S2c). Moreover, the significant effect of chromosome 1U^b was also shown in the field experiment.

The content of total AX (measured as TOT-pentosan) was similar in Ae. geniculata and cv. Chinese Spring and lower in Ae. biuncialis than in line Mv9kr1 (Figure 3B). The effects of chromosome additions were generally not significant, but small increases were observed with the addition of chromosomes 5U^g and 7U^g, which increased the total AX content of wheat (measured as TOT-pentosan in mg/g dry weight of wholemeal), while in the Mv9kr1 genetic background only chromosome 1U^b gave a higher value than the parental wheat genotype (Figure 3B). No significant differences were observed in the contents of TOT-pentosan in the parental lines and the addition lines in the field experiment (Supplementary Figure S2d).

The water-extractable fraction of total pentosans (WE-pentosan) was higher in the Aegilops parents in comparison with the corresponding wheat genotypes (Figure 3C). At the level of single chromosomes, significantly higher WE-pentosan content was observed in chromosome addition lines 3U^g, 4U^g, 5U^g, 6U^g, 7U^g and 5M^g and 7M^g, the highest positive effect being exerted by the group five chromosomes of Ae. geniculata. In the case of Ae. biuncialis, chromosome addition lines 2M^b and 7M^b showed the highest level of WE-pentosan, exceeding those of all the other genotypes investigated (wheat–Ae. geniculata and wheat–Ae. biuncialis additions). The results of the glasshouse experiment were supported by the field experiment. Similar to the glasshouse experiments, chromosome additions 5U^g and 5M^g showed the highest WE-pentosan contents among the Chinese Spring–Ae. geniculata addition lines, although their values were not significantly different from the wheat parent, while the Ae. biuncialis 7M^b chromosome addition had significantly higher WE-pentosan content than the parental wheat Mv9kr1 (Supplementary Figure S2e).

The ratio of TOT-pentosan (mainly AX) to β-glucan reflects the composition of non-starch cell wall polysaccharides. In the present experiment, the parental wheat genotypes had TOT-pentosan to β-glucan ratios of 4.5 and 5.5 (in cv. Chinese Spring and line Mv9kr1, respectively). In contrast, TOT-pentosan to β-glucan ratios of only 1.0 and 1.5 were determined for the Ae. geniculata TA2899 and Ae. biuncialis MvGB642 (Figure 4A), which was attributed to the high β-glucan content of the Aegilops genotypes (Figure 3A). In Chinese Spring–Ae. geniculata chromosome additions, this ratio was higher in lines containing chromosomes 1U^g, 2U^g, 3U^g, 6U^g, and 1M^g, 2M^g, and 5M^g than in the wheat parent, while the chromosome addition line 7U^g showed a lower value for this parameter. Within the set of Ae. biuncialis additions, chromosomes 2M^b and 7M^b significantly decreased the ratio of TOT-pentosan to β-glucan relative to wheat. The ratio of water-extractable to unextractable pentosans (WE/WU pentosan) indicates the relative amounts of the two pentosan fractions, which have different health-promoting effects. The higher WE/WU pentosan ratio showed that the two Aegilops accessions have higher proportions of water-extractable pentosans than the wheat parents (Figure 4B). A relatively higher amount of water-extractable pentosans was also detected in the wheat–Aegilops addition lines 5M^g, 2M^b, and 7M^b (Figure 2C).

Differences in the structure of β-glucan were determined after digestion with lichenase. Lichenase releases glucooligosaccharides (GOS) with degrees of polymerization (DP) of up to 10, with DP3 and DP4 GOS being the major forms. The values for TOTAL-GOS were between 1.6- and 1.3-fold higher in Ae. geniculata TA2899 and Ae. biuncialis MvGB642, respectively, than in wheat (Figure 5A). The ratio of DP3:DP4 glucan units, which represents the ratio of β-(1–3) to β-(1–4) bonds in the polymer, was significantly lower in both Aegilops species than in wheat (Figure 5B). The effect of chromosomes 7U^g and 7M^g on the TOTAL-GOS content, determined using HPAEC (Figure 5A) was similar to that of these chromosomes on the β-glucan content of wheat, determined with a Megazyme kit (Figure 3A). The addition of chromosome 5U reduced the ratio of DP3:DP4 glucan units released by lichenase from TOTAL-GOS. In addition, chromosomes 6U^g and 3U^b had decreasing effects on the DP3:DP4 ratio (Figure 5B), reflecting a higher ratio of larger polymers.

Differences in the structure of AX were determined after digestion with endoxylanase. AX molecules have a backbone of xylose residues which may be substituted with arabinose. They vary in structure, in terms of both the proportion and distribution of unsubstituted, monosubstituted, and disubstituted xylose residues. Digestion with a specific endoxylanase releases AXOS which can be separated and quantified by HPAEC, giving “fingerprints” for the samples. As the structures of the separated AXOS have been determined (Ordaz-Ortiz et al., 2005), the peak areas can also be used to compare the proportion of AXOS containing unsubstituted, monosubstituted, and disubstituted xylose residues. This comparison showed that TOTAL-AXOS was ∼50% lower in both Ae. geniculata and Ae. biuncialis than in wheat (Figure 5C). The proportion of substituted xylose residues was also significantly lower in both parental Aegilops accessions than in the wheat genotypes (Figures 5D–F,H). In addition, the ratio of monosubstituted to disubstituted xylose residues was almost twice as high in Ae. geniculata than in wheat or Ae. biuncialis (Figure 5G). The ratio of unsubstituted AXOS to total AXOS (Figures 5C,D) was increased by the addition of chromosomes 5M^g, 1U^b, 1U^b6U^b, 3M^b, and 7M^b, with monosubstituted AXOS being affected by chromosomes 2U^g, 3-5U^g, 2M, and 6M^g (Figure 5E) and disubstituted AXOS by chromosomes 4U^g, 6M^g, and 2M^b (Figure 5F). The ratio of monosubstituted to disubstituted AXOS was increased by the addition of chromosomes 2U^g, 4-6U^g, 2M, 3M^b, and 7M^b (Figure 5G).

In order to identify Aegilops homologs of the key genes responsible for the biosynthesis of storage proteins, β-glucan, or AX, a comparison was made between wheat or barley and the Aegilops genomes. As complete sequences of the U- and M-genomes of Ae. geniculata and Ae. biuncialis are not available, chromosome survey sequences of the Ae. umbellulata genomes were used for comparative analysis (see text footnote 3). A BLASTn search on the cDNA sequences of the key genes responsible for wheat grain storage proteins (Table 1 and Supplementary Table 1) showed that most of the investigated genes (HMW glutenins, LMW glutenins, γ-gliadins) were assigned to the same homeologous group chromosomes (group 1) in the U-genome of Ae. umbellulata as in bread wheat (Table 1 and Supplementary Table 1). The α-gliadin genes, which are assigned to group 6 chromosomes in bread wheat, were located on the 1U and 3U chromosomes of Aegilops. As for the genes involved in β-glucan biosynthesis (OsCslF1-F2, HvCslF3-4, HvCslF6-10, HvCslF12-13, HvCslH1), the Aegilops homologs were again assigned to the same homeologous group chromosomes (group 1, 2, 5, and 7) as in bread wheat (Table 2 and Supplementary Table 2). This was also true for most of the genes responsible for AX biosynthesis, which were assigned to group 4 or 7 chromosomes (TaGT43 family), group 3 chromosomes (TaGT47 family), and group 2 chromosomes (TaGT75 family) (Table 2 and Supplementary Table 3). However, some Aegilops homologs were found on different chromosomes than the wheat genes. For example, gene TaGT47-12 was assigned to the group 3 chromosomes of hexaploid wheat, while its Aegilops homolog was found on chromosome 6U. Differences in chromosomal assignments were also found for genes HvCslF11, TaGT61-1, TaGT61-2, and TaGT75-4 (Table 2 and Supplementary Tables 2, 3).

The addition of the group 1 chromosomes of Ae. geniculata (1U^g and 1M^g) to bread wheat was found to increase the proportion of insoluble glutenins relative to total glutenins (%UPP). These chromosomes also increased the ratio of glutenins to gliadins (Glu/Gli) (Figures 2C,D). These results are in line with the observations of Garg et al. (2016), who used the same set of Chinese Spring–Ae. geniculata addition lines and found that chromosome 1M^g exhibited greater dough strength than the parental wheat. Wheat gluten protein has been studied in great detail for more than a half century, with the first genetic studies dating back to the 1960s. It has been established that three major groups of wheat gluten proteins (LMW subunits of glutenin, ω-gliadins, and γ-gliadins) are encoded by genes on the short arms of the group 1 chromosomes of all three genomes of wheat (A, B, and D), while the HMW subunits of glutenin are encoded by genes on the long arms of the same chromosomes. A further group of gluten proteins, the α-gliadins, are encoded by genes on the short arms of the group 6 chromosome (Payne, 1987; Shewry et al., 2003a,b, 2009).

Wild homologs of the gluten protein genes were detected on the 1U chromosome sequence contigs of Ae. umbellulata (Table 1 and Supplementary Table 1), consistently with previous studies on Ae. geniculata (Medouri et al., 2015). A recent comparative analysis of individual flow-sorted chromosomes of Ae. umbellulata, Ae. comosa, and wheat indicated that chromosomes 1U and 1M are syntenic with the group 1 chromosomes of hexaploid wheat at the macro level (Molnár et al., 2016), suggesting that the 1M^g chromosome of Ae. geniculata may also contain wild alleles of gluten protein genes.

The proportion of dietary fiber components in both Aegilops species was more similar to that in oats, barley, and Brachypodium than to that in wheat, with higher proportions of β-glucan than of AX. There is considerable interest in increasing the content of β-glucan in wheat flour due to its known health benefits (Buttriss and Stokes, 2008; Anderson et al., 2009; Tighe et al., 2010). The addition of chromosome 5U^g or group 7 chromosomes from the U and M genomes of Ae. geniculata and Ae. biuncialis were able to significantly increase the β-glucan content of wheat (Figure 3A) across different growth conditions. The addition of the 1U^g, 7U^g (or 7M^g), and 3M^g chromosomes also increased TOTAL-GOS (Figure 5A). Chromosome 5U was also found to reduce the quantitative ratio of DP3:DP4 glucan units (Figure 5B). Earlier studies indicated that the addition of 0–5% β-glucans to bread flour significantly reduced dough extensibility and loaf volume (Brennan and Cleary, 2007), while the solubility of the fibers was decreased by higher β-glucan levels (Izydorczyk and Dexter, 2008), but increased by a lower DP3:DP4 ratio (Izydorczyk and Dexter, 2008). The two opposite effects probably result in the solubility of β-glucan remaining nearly constant in the Aegilops addition lines, while little changes could be expected in their processing properties.

Cellulose synthase-like (Csl) genes are candidates to encode enzymes that synthetize the backbone of various non-cellulosic cell wall polysaccharides (Doblin et al., 2009). They have been classified into nine gene families, designated CslA to CslJ, of which the CslF, CslH, and CslJ families are restricted to cereals, although the CslJ group is not found in rice (Oryza sativa L.) or Brachypodium distachyon L. (Doblin et al., 2010). Expression in transgenic Arabidopsis thaliana L. plants revealed that the barley CslF and CslH families are probably involved in β-glucan synthesis (Burton et al., 2006; Doblin et al., 2009). Comparative genomic studies have shown that barley has 10 CslF family members; HvCslF3, HvCslF4, HvCslF8, HvCslF10, HvCslF12, and HvCslF13 clustered on chromosome 2H, HvCslF9, located on 1H, HvCslF7, located on 5H and HvCslF6 and HvCslF11, located on 7H (Burton et al., 2008; Schreiber et al., 2014). Among the HvCslF genes of barley, HvCslF6 and HvCslF9 have the highest levels of mRNA transcripts in developing barley endosperms (Burton et al., 2008) and map to loci near the centromeres of chromosomes 7H and 1H, respectively, which are close to quantitative trait loci (QTL) for the β-glucan content of barley grain (Igartua et al., 2002; Molina-Cano et al., 2007; Burton et al., 2008). Taketa et al. (2012) analyzed barley mutants lacking β-glucan and showed that the HvCslF6 gene had a unique role and was the key determinant controlling the biosynthesis of β-glucan, while Nemeth et al. (2010) demonstrated the role of the CslF6 gene in wheat β-glucan synthesis by RNAi suppression in grain of transgenic plants.

The β-glucan content of wheat–Aegilops chromosome addition lines was consistent with the locations of putative β-glucan synthase genes in the U genome of Ae. umbellulata. A sequence similarity search on the barley Cellulose-synthase-like F (CslF) gene family showed that the Aegilops orthologs of the CslF family members were located on the same homeologous group chromosomes as in barley. More precisely, homologs of the CslF6 genes of barley were present on chromosome 7U and those of CslF9 on chromosome 1U, while homologs of other CslF genes were located on chromosomes 2U and 5U (Table 2 and Supplementary Table 2). Previous results (Cseh et al., 2011, 2013) indicated that the chromosome 7H-mediated transfer of HvCslF6 gene from barley cv. Manas to wheat significantly increased the level of β-glucan in the grains. While the increase was statistically significant, it should be noted that the β-glucan level content of the 7H addition line was moderate compared to that of the parental barley genotype (Cseh et al., 2011). An explanation for the moderate increase in the β-glucan level of wheat–barley 7H addition line could be that the barley QTL represented on chromosome 7H is only a part of the genomic regions needed for a more efficient synthesis of β-glucan (Houston et al., 2014; Shu and Rasmussen, 2014) and it is not sufficient to drive higher β-glucan levels. This may also apply to the wheat–Aegilops addition lines 5U^g or 7U^g, 7M^g and 7M^b (and for 1U^b under field conditions) where the increase in the β-glucan amount was also moderate.

A homolog of HvCslF6 gene was also identified on chromosome 7U, which further supports the fact that the addition of group 7 chromosomes from Ae. geniculata and Ae. biuncialis to wheat increases the grain β-glucan level. A putative ortholog of the HvCslF7 gene was detected on chromosome 5U, with an increased β-glucan level in the wheat–Ae. geniculata 5U^g addition line. These results suggest that the variant of HvCslF7 may have a role in grain β-glucan synthesis of Aegilops (Burton et al., 2008).

However, further genome-wide association studies on a diverse population of Ae. geniculata, or Ae. biuncialis would help to identify the chromosomal locations of QTL responsible for high β-glucan levels in grain endosperm. The addition lines contain a whole homeologous chromosome pair from Aegilops and, beside the desirable genomic regions, they also contain many other genes which may affect grain development and composition. The elimination of excess alien chromatin by the production of wheat–Aegilops translocation lines containing desirable QTL and their pyramiding into one genotype could be used to increase β-glucan content.

The observation that the addition of chromosomes 5U^g, 7U^g and 1U^b to wheat increases the total pentosan content, while the addition of chromosomes 3U^g, 4U^g, 5U^g, 6U^g, 7U^g, 5M^g, 7M^g, 2M^b and 7M^b increases the water-soluble pentosan fraction (Figures 3B,C) indicates that goatgrasses are a promising source of genes to increase the AX content of wheat. As well as their health benefits, both water-soluble and unsoluble AX result in higher water absorption, dough development time and loaf volume (Biliaderis et al., 1995; Courtin and Delcour, 2002). Soluble AX contributes to gas bubble formation during baking while insoluble AX destabilizes it (Courtin and Delcour, 2002).

Many efforts have been made to identify genes controlling the biosynthesis of AXs in wheat and several mapping populations have been used to identify QTL for AX in wheat. A major QTL was identified on chromosome 1B (Martinant et al., 1999), which explained 59% of the phenotypic variation in WE-AX content and viscosity (Charmet et al., 2009). Quraishi et al. (2011) identified seven loci (chromosomes 1B, 3A, 3D, 5B, 6B, 7A, and 7B) by association genetics, of which three (chromosomes 1B, 3D, and 6B) corresponded to consensus meta-QTL based on data from seven crosses. These authors also identified candidate genes for the future improvement of grain fiber. Using bioinformatic approaches, Mitchell et al. (2007) identified genes for AX synthesis in glycosyltransferase (GT) families 43, 47, and 61, while Zeng et al. (2010) identified GT75 with a combination of proteomics and transcriptomics analyses. In wheat, most GT genes occur in multiple forms, with three homeoalleles of each form being present on the A, B, and D genomes (Mitchell et al., 2007; Wan et al., 2008; Pellny et al., 2012). RNAi suppression of the expression of these homeoalleles demonstrated that GT43 and GT47 encode subunits of β-1,4-xylan synthase and that GT61 encodes a α-(1,3)-arabinosyltransferase (Anders et al., 2012; Lovegrove et al., 2013). Suppression of either TaGT43_2 or TaGT47 resulted in a 40–50% decrease in total AX but increased Araf residues substitution, with a 50% decrease in cell-wall thickness (Lovegrove et al., 2013). Similarly, the suppression of GT61 (renamed TaXAT1) resulted in a 70–80% decrease in the amount of α-(1,3) linked Araf in the AX of mature starchy endosperm (Anders et al., 2012). Decreases in extract viscosity were observed in all transgenic lines, with greater effects in the TaGT43_2 and TaGT47_2 RNAi lines (located on chromosomes 4ABD and 3ABD of wheat, respectively) than in the TaXAT1 RNAi lines (Freeman et al., 2016). These effects were explained by decreases in the amount and chain length of WE-AX.

Putative orthologs of wheat GT43, GT47 and GT61 and GT75 genes were identified on chromosomes 3U (TaGT47-2B, TaGT47-2D, TaGT47-2A, TaGT47-13), 4U (TaGT43-2B, TaGT43-2A, TaGT61-2), 6U (TaGT47-12, TaGT61-1, TaGT75-4), and 7U (TaGT43-4) in the U genome of Ae. umbellulata, which is consistent with the present observation that the addition of chromosome 7U^g increased total pentosan, while the addition of chromosomes 3U^g, 4U^g, 6U^g, and 7U^g increased WE-pentosan (Figures 3B,C, Table 2, and Supplementary Table 3). It should be noted that the addition of Ae. geniculata chromosome 5U^g increased the total- and WE-pentosan content in wheat even if no orthologs of wheat GT genes were identified on chromosome 5U. However, the increased AX content of the 5U^g addition line is consistent with a recent study by Marcotuli et al. (2015), who used GWAS to identify three QTL strongly associated with AX content on chromosome 5A of tetraploid wheat. The possible reason for this discrepancy could be that the chromosome survey sequences do not cover the whole of the Ae. umbellulata genome (data not shown).

The ratio of AXOS released by endoxylanase digestion was affected by Ae. biuncialis chromosomes 2M^b, 3M^b, and 7M^b in wheat, as their addition increased the ratio of monosubstituted to disubstituted AXOS. However, this increase in the M/D ratio was due to an increase in the amount of monosubstituted AXOS in addition line 2M^b, but due to a decrease in the amount of disubstituted AXOS in the addition lines 3M^b and 7M^b. These results indicate that AX biosynthesis could be modified by these Aegilops chromosomes. However, other genes were also observed to have smaller effects (Figure 5G). Interestingly, several enzymes involved in the AX biosynthesis pathway were identified on the group 2, 3, and 7 chromosomes of wheat (Group 2: glucuronosyltransferase, cis-zeatin O-glucosyltransferase 1, glycosyltransferase; group 3: glycosyl hydrolase; group 7: 1,3-β-D-glucan synthase, Glycosyl hydrolase, β-1,4-endoglucanase) (Marcotuli et al., 2015). The fact that the Ae. geniculata and Ae. biuncialis accessions exhibited significant phenotypic variations for the grain β-glucan and AX content supports the notion that these species have considerable genetic variability for improving the edible fiber content of bread wheat.