Improving preharvest sprouting resistance in durum wheat with bread wheat genes.

Preharvest sprouting (PHS) of durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) is an important problem in Japan, where the rainy season overlaps with the harvest season. Since there are few PHS-resistant genetic resources in durum wheat, we introduced an R-gene for red seeds, the MFT gene, and the QPhs-5AL QTL, all of which are associated with PHS resistance, into durum wheat from a PHS-resistant bread wheat (T. aestivum L.) cultivar, 'Zenkoujikomugi' (Zen), by backcross breeding. Developed near isogenic lines (NILs) with red seeds had a lower percentage germination (PG) and germination index (GI) than the recurrent parent, and seed color had the greatest effect. A NIL combining all three sequences had the lowest GI and PG, with a similar GI to that of 'Shiroganekomugi' bread wheat. Among NILs with white seeds, a NIL combining MFT and QPhs-5AL had the lowest GI and PG. As the combination of all three sequences from Zen conferred PHS resistance on durum wheat, PHS-resistant genetic resources in bread wheat can be used in breeding durum wheat.

Introduction

Pasta consumption has increased in Japan since the late 1980s to about 270,000 t per year (Japan Customs Yokohama 2012). Half of the pasta is imported, mainly from Italy, and the other half is made in Japan from durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) imported from Canada (MAFF 2016).

The durum wheat harvest season in Japan overlaps with the rainy season. Preharvest sprouting (PHS) of wheat is induced by rainfall at or after the mature stage. PHS causes starch degradation through high alpha-amylase activity (Dick ), and dough made from PHS seeds has a low moisture content (Dexter ). Almost no durum wheat is grown commercially in Japan; Kai concluded that durum wheat culture in Japan would be difficult owing to its late maturity and very weak resistance to PHS and Fusarium head blight. Recently, the first Japanese durum wheat cultivar, ‘Setodure’, with early maturity, was released at the Western Region Agricultural Research Center (WARC) of the National Agriculture and Food Research Organization (NARO). Its early maturity makes ‘Setodure’ less likely to encounter rain, but durum wheats still have insufficient PHS resistance.

Wheat PHS resistance is determined by the seed dormancy level. There are several reports of PHS resistance genes and quantitative trait loci (QTLs) in durum wheat, such as ‘SC8021-V2’ (DePauw ). QTLs in durum wheat include QPhsd-spa-1A.1, -2A.1, and -7B.1 derived from ‘Kyle’ (Knox ), and QTLs on chromosomes (Chrs.) 3BL and 5BS from ‘Chahba 88’ and on 6BL from ‘IACT12’ (Gelin ). However, it is not known how effective these cultivars and QTLs would be under the severe climate of Japan.

More information is available on genes and QTLs in bread wheat (T. aestivum L.). Several QTLs of PHS resistance have been located on Chrs. 1A, 1B, 2B, 2D, 3A, 3B, 3D, 4A, 4D, 5A, 5B, 5D, 6D, 7B and 7D (Groos , Kottearachchi , Kulwal , Munkvold ).

Seed color is associated with PHS, and red wheats have stronger PHS resistance than white wheats (Nilsson-Ehle 1914). Red seed color is controlled by three genes—R-A1, R-B1, and R-D1—on Chrs. 3A, 3B and 3D, respectively (Groos , Sears 1944). R-gene encoding the R2R3-type MYB domain-containing transcription factor TaMYB10 on Chr. 3AL regulates flavonoid biosynthesis and has pleiotropic effects on PHS resistance (Himi ).

A QTL for PHS resistance on Chr. 3A, QPhs-3A.1, appears to have a large and stable effect (Mori ). Nakamura identified MOTHER OF FT AND TFL 1 (MFT), a key temperature-dependent regulator of PHS resistance, at QPhs-3A.1.

The Japanese bread wheat cultivar ‘Zenkoujikomugi’ (Zen) has strong PHS resistance (Miura , Osanai and Amano 1993) and has been used in Japanese wheat breeding and in research on PHS (Mori ). Nakamura showed that a single nucleotide polymorphism (SNP) in the Zen MFT promoter increased the transcription of MFT, resulting in strong grain dormancy. Chono reported a high frequency of the Zen-type SNP in Japanese commercial cultivars in southern Japan, and cultivars carrying that SNP had stronger grain dormancy. Zen also has red seeds and QPhs-5AL for PHS resistance (Himi , Kottearachchi ). Thus, the strong PHS resistance of Zen seems to derive from the red seed color, MFT, and QPhs-5AL, and these are promising PHS resistance sources under the severe climate of Japan.

The use of hexaploid bread wheat (AABBDD) in the breeding of tetraploid durum wheat (AABB) is limited by the requirement to use only the A and B genomes. However, Zen has red seed color, MFT and QPhs-5AL on the A or B genome. It is unknown whether the effect of PHS resistance genes and/or QTLs derived from bread wheat is compatible in durum wheat. Thus, in this study, we introduced the PHS resistance trait of Zen into durum wheat by interbreeding and backcross breeding and assessed the effects of the sources of resistance in durum wheat. We also evaluated the possibility to improve PHS resistance in durum wheat by using bread wheat as a donor, carrying PHS resistance genes and QTL on A and B genomes.

Materials and Methods

Plant materials

To develop near isogenic lines (NILs), we used the durum wheat cultivar ‘Setodure’, which is susceptible to PHS, as the recurrent parent, and the bread wheat cultivar Zen as the resistance donor (Table 1). Zen contains the R-gene encoding TaMYB10 on B genome (Himi ), MFT and QPhs-5AL on A genome (Kottearachchi , Nakamura ). Through backcrossing to ‘Setodure’, we developed BC4F1 plants heterozygous for MFT and QPhs-5AL by DNA marker-assisted selection (MAS) and selected plants with red seeds by eye confirming at each BCnF2 generations (Supplemental Fig. 1). BC4F2 plants homozygous for MFT and QPhs-5AL were selected for propagation to the BC4F4 generation. Red seed color was finally confirmed in the BC4F5 and BC4F6 generations. Selected NILs were named for their introduced sequences by seed color (red or white: R or W), MFT allele type (Seto or Zen: S or Z), and QPhs-5AL allele type (S or Z). We developed NILs with 7 of the 8 possible combinations (except R-MFTS-QPhs-5ALZ).

Table 1

Sources of PHS resistance introduced from bread wheat ‘Zenkoujikomugi’ into durum wheat ‘Setodure’

Introduced source of resistance (chromosome position)	Phenotype or selection marker
R-gene for seed color	red or white
MFT (3AS)	MFT
QPhs-5AL (5AL)	Xcfa2163

The 7 NILs, ‘Setodure’, and 3 Japanese bread wheat cultivars (‘Shiroganekomugi’, ‘Minaminokaori’ and Zen) were grown in a research field at WARC, Fukuyama, Hiroshima, Japan (34°30′4″ north latitude and 133°23′12″ east longitude). All materials were sown 12 cm apart in 2-m rows in November and harvested in June. There were 4 replicates of ‘Setodure’ and 6 of the NILs (3 of W-MFTS-QPhs-5ALS) in 2014, and 4 replicates of ‘Setodure’ and the NILs and 2 of the bread wheats in 2015.

Marker-assisted selection

DNA was extracted by the potassium acetate method (Dellaporta ). For MAS of MFT, a cleaved amplified polymorphic sequence (CAPS) marker was used (Nakamura ). DNA templates were amplified in a PCR Thermal Cycler Dice (TaKaRa Bio, Shiga, Japan) with ExTaq DNA polymerase (TaKaRa Bio) and an MFT primer set (forward 5′-GTAGCGGGTGAAATCTGCAT-3′; reverse 5′-GGGACGTACGAGGGTGTAGA-3′) under conditions of an initial 95°C for 30 s; 40 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 60 s; and a final 72°C for 5 min. The amplified PCR fragments were digested with ClaI at 37°C for 1 h, separated by 2% agarose gel electrophoresis, and visualized with Gel Red stain (Biotium, Fremont, CA, USA). For MAS of QPhs-5AL, we used its tightly linked codominant DNA marker Xcfp2163 (Kottearachchi ). The single primer set was applied for MAS of QPhs-5AL region, therefore we regarded QPhs-5AL region as QPhs-5AL in this paper. DNA templates were amplified in the same thermal cycler as above with HotStarTaq DNA polymerase (QIAGEN, Hilden, Germany) and the Xcfp2163 primer set (forward 5′-TTGATCCTTGATGGGAGGAG-3′, reverse 5′-CATCATTGTGTTTACGTTCTTTCA-3′) under conditions of an initial 95°C for 15 min; 35 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min; and a final 72°C for 10 min. Amplified PCR fragments were separated by 8% polyacrylamide gel electrophoresis and visualized as above.

Germination experiment

Ten mature spikes in 2015 and 16 in 2016 were harvested from all replicates. To generate various dormant conditions, half of the spikes were dried shortly after harvest as a deep dormancy condition and the other half were kept at room temperature for 1 week to fully ripen as a light dormancy condition and then dried. All samples were dried at 35°C for 1 day and stored at −20°C. Before the germination experiment, all spikes were returned to room temperature and the seeds were threshed by hand. We laid 100 seeds on 2 pieces of 85-mm grade 1 filter paper (Whatman, Maidstone, UK) in a 90-mm Petri dish, added 6 mL of water, and incubated them at 15°C as an easy germination condition or 20°C as a difficult germination condition. Germinated seeds were counted on 3rd, 5th and 7th day of imbibition (DI) in 2015 and daily until 7 DI. We calculated percentage germination (PG), except for the fully ripened seeds incubated at 15°C in 2015, of which we did not have enough.

The germination index (GI, Reddy ) of all conditions in 2016 was calculated as:

where n is the number of germinated seeds on day i. Values of ‘Setodure’, NILs, ‘Shiroganekomugi’, ‘Minaminokaori’, and Zen were calculated.

Statistical analysis

All PGs and GIs were arc-sin-transformed for analysis of variance (ANOVA). To assess the PHS resistance in each NILs, the PGs of ‘Setodure’ and NILs in 2015 and 2016 were analyzed by two-way ANOVA and Tukey’s HSD (at P < 0.05). Variance components were calculated from the mean squares except for W-MFTS-QPhs-5ALS, which was not significantly different from ‘Setodure’ and was carrying same genotype of ‘Setodure’ (see Table 3 later). And to assess the contribution of introduced sequences for the PHS resistance, the PGs in 2015 and 2016 and the GIs in 2016 of ‘Setodure’ and NILs (except for W-MFTS-QPhs-5ALS) were analyzed by multi-way ANOVA, and variance components were calculated from the mean squares except for Seed color × MFT × QPhs-5A, which is included in residuals, because we could not develop the NIL of R-MFTS-QPhs-5ALZ.

Table 3

Average percentage germination among ‘Setodure’ and NILs

Cultivar or NIL	Source of resistance			Mature stage on incubation at 20°C			Full-ripe stage on incubation at 20°C			Mature stage on incubation at 15°C
	Seed color	MFT	QPhs-5AL	3 DI	5 DI	7 DI	3 DI	5 DI	7 DI	3 DI	5 DI	7 DI
Setodure	White	Seto	Seto	76.3 a	91.7 a	95.0 a	69.3 a	90.0 a	95.2 a	85.5 a	95.9 a	98.0 a
W-MFTS-QPhs-5ALS	White	Seto	Seto	74.0 a	90.7 a	96.1 a	76.4 a	94.6 ab	97.7 a	80.0 ab	95.3 ab	99.3 ab
W-MFTS-QPhs-5ALZ	White	Seto	Zen	64.3 ab	81.5 ab	86.5 a	63.8 a	87.9 ab	92.7 a	80.4 ab	94.1 abc	98.1 abc
W-MFTZ-QPhs-5ALS	White	Zen	Seto	58.1 bc	86.1 abc	92.1 b	68.1 ab	92.5 ab	96.6 ab	73.9 abc	93.5 abc	97.8 ac
W-MFTZ-QPhs-5ALZ	White	Zen	Zen	50.5 c	74.4 bcd	80.5 b	50.3 ac	77.9 ac	86.5 bc	67.5 bc	87.0 bcd	93.6 abc
R-MFTS-QPhs-5ALS	Red	Seto	Seto	32.1 d	69.4 b	84.0 b	50.2 ac	81.4 abc	94.6 abc	57.9 bd	81.2 bd	94.6 abc
R-MFTZ-QPhs-5ALS	Red	Zen	Seto	20.4 d	44.3 d	56.7 c	30.7 c	65.7 c	79.7 c	41.1 de	76.1 d	92.0 c
R-MFTZ-QPhs-5ALZ	Red	Zen	Zen	7.1 e	30.5 e	46.4 c	11.7 d	34.0 d	57.1 d	22.9 e	53.3 e	78.6 d

“Seto” allele derived from ‘Setodure’ (non-dormant); “Zen” allele derived from Zen (dormant).

Values followed by the same letter are not significantly different by Tukey’s HSD at P = 0.05.

The GIs in 2016 were analyzed by one-way ANOVA and Tukey’s HSD (at P < 0.05). All statistics were analyzed in R version 3.3.1 software (R Core Team 2016).

Results

Percentage germination

PG was significantly affected by genotype in all conditions; by year at mature stage on incubation at 20°C at 3 and 7 DI, and at full-ripe stage at 20°C at 7 DI, and by their interaction at mature stage at 20°C at 3 and 5 DI and at full-ripe stage at 20°C at 3 and 7 DI (Table 2). Thus, one or more sources of resistance affected PG.

Table 2

Two-way ANOVA of percentage germination among ‘Setodure’ and NILs

Mature stage on incubation at 20°C
Factor	3 DI				5 DI				7 DI
	Df	Mean Sq	F value	P value	Df	Mean Sq	F value	P value	Df	Mean Sq	F value	P value
Year	1	0.2336	21.593	2.75E-05	1	0.0326	3.892	0.054	1	0.4259	46.688	1.47E-08
Genotype	7	0.6806	62.905	<2.00E-16	7	0.4201	50.111	<2.00E-16	7	0.4663	51.113	<2.00E-16
Year × Genotype	7	0.5430	5.023	2.64E-05	7	0.0664	7.919	2.53E-06	7	0.0136	1.496	0.192
Residuals	47	0.0108			47	0.0084			47	0.0091

DI = days of imbibition.

By Tukey’s HSD, the PGs of R-MFTZ-QPhs-5ALS (20.4–92.0%) and R-MFTZ-QPhs-5ALZ (7.1–78.6%) were significantly lower than those of ‘Setodure’ (76.3–98.0%); R-MFTZ-QPhs-5ALZ had the lowest PGs in all conditions (Table 3). In contrast, the PGs of W-MFTS-QPhs-5ALS (74.0–99.3%) and W-MFTS-QPhs-5ALZ (63.8–98.1%) were not significantly different from those of ‘Setodure’ in all conditions. The PGs of W-MFTZ-QPhs-5ALS (58.1–97.8%) were significantly lower than those of ‘Setodure’ only at the mature stage on incubation at 20°C at 3 and 7 DI. The PGs of W-MFTZ-QPhs-5ALZ (50.5–93.6%) were the lowest among ‘Setodure’ and NILs with white seeds. The PGs of R-MFTS-QPhs-5ALS (32.1–94.6%) were the highest among NILs with red seeds and were significantly lower than those of ‘Setodure’ at the mature stage at 20°C and at the mature stage at 15°C at 3 and 5 DI. Thus, red seed color plus MFT, with or without QPhs-5AL, from Zen clearly promoted PHS resistance.

Multi-way ANOVA of genotype effect on percentage germination

The variance components of genotype in two-way ANOVA ranged from 18.3% to 72.2% (data not shown). We tested the condition that gave the greatest value—full-ripe stage at 20°C at 3 DI (Table 4)—by multi-way ANOVA for additive or epistatic effects among sources of resistance in ‘Setodure’ and the NILs. The variance components of seed color (49.0%), MFT (31.6%), QPhs-5AL (3.0%), and seed color × QPhs-5AL (9.3%) were significant. (Tables 4, 5). Thus, seed color and MFT (additive effects) had the largest effect on PG.

Table 4

Multi-way ANOVA of percentage germination among sources of resistance at full-ripe stage on incubation at 20°C at 3 days of imbibition

Source of variance	df	Mean Sq	F value	P value
Seed color	1	1.1732	84.230	3.21E-12
MFT	1	0.7217	35.100	3.06E-07
QPhs-5AL	1	0.1963	9.547	0.003
Seed color × MFT	1	0.0400	1.947	0.169
Seed color × QPhs-5AL	1	0.1186	5.769	0.020
MFT × QPhs-5AL	1	0.0350	1.701	0.198
Residuals	49	0.0206

Seed color × MFT × QPhs-5AL is included in residuals.

Table 5

Analysis of estimated variance components of percentage germination among sources of resistance at full-ripe stage on incubation at 20°C at 3 days of imbibition

Variance component	Variance value	Percentage of the total (%)
σSeed color2	0.259	49.0
σMFT2	0.167	31.6
σQPhs-5AL2	0.016	3.0
σ(Seed color × MFT)2	0.010	1.8
σ(Seed color × QPhs-5AL)2	0.049	9.3
σ(MFT × QPhs-5AL)2	0.007	1.4
σResiduals2	0.021	3.9
σTotal2	0.528	100.0

Estimated variance values were calculated from mean squares of multi-way ANOVA in Table 4.

Germination index

The GIs of ‘Setodure’ (65.1–79.7) tended to be highest and those of Zen (1.4–25.1) were lowest in all conditions (Table 6). The GIs of R-MFTZ-QPhs-5ALZ (19.9–55.8) were significantly lower than those of ‘Setodure’ in all conditions, and tended to be lowest among NILs with red seeds. The GIs of R-MFTS-QPhs-5ALS (48.1–77.3) and R-MFTZ-QPhs-5ALS (24.9–71.5) were significantly lower than those of ‘Setodure’ except at full-ripe stage at 15°C. The GIs of W-MFTZ-QPhs-5ALZ (46.8–70.8) tended to be lowest among ‘Setodure’ and NILs with white seeds, and were significantly lower than those of ‘Setodure’ except at the mature stage at 20°C. The GIs of W-MFTS-QPhs-5ALS (66.1–79.3) and W-MFTS-QPhs-5ALZ (49.0–73.3) were not significantly different from those of ‘Setodure’ in all conditions. The GIs of W-MFTZ-QPhs-5ALS (49.0–72.0) were not significantly different from those of ‘Setodure’ except at full-ripe stage at 20°C. The GIs of W-MFTZ-QPhs-5ALZ and R-MFTZ-QPhs-5ALZ were respectively similar to those of ‘Minaminokaori’ (54.5–79.5) and ‘Shiroganekomugi’ (30.9–57.6).

Table 6

Average germination index among ‘Setodure’, NILs, and current Japanese standard bread wheat cultivars in 2016

Cultivar or NIL	N	Source of resistance			20°C		15°C
		Seed color	MFT	QPhs-5AL	Mature stage	Full-ripe stage	Mature stage	Full-ripe stage
Setodure	4	White	Seto	Seto	65.1 a	71.8 a	77.2 a	79.7 a
W-MFTS-QPhs-5ALS	4	White	Seto	Seto	66.1 a	72.7 a	72.3 ab	79.3 a
W-MFTS-QPhs-5ALZ	4	White	Seto	Zen	58.8 ab	67.1 ab	65.9 abc	73.3 ab
W-MFTZ-QPhs-5ALS	4	White	Zen	Seto	49.0 ab	57.3 bc	69.2 abc	72.0 ab
W-MFTZ-QPhs-5ALZ	4	White	Zen	Zen	46.8 ab	49.2 c	61.8 bcd	70.8 b
R-MFTS-QPhs-5ALS	4	Red	Seto	Seto	48.1 bc	56.7 bc	56.9 cd	77.3 ab
R-MFTZ-QPhs-5ALS	4	Red	Zen	Seto	24.9 bc	42.2 cd	51.5 d	71.5 ab
R-MFTZ-QPhs-5ALZ	4	Red	Zen	Zen	19.9 c	21.6 d	34.0 e	55.8 c
Minaminokaori	2	Red	non-Zen	*2	54.5	73.7	64.1	79.5
Shiroganekomugi	2	Red	Zen*1	*2	30.9	22.9	39.0	57.6
Zenkoujikomugi	2	Red	Zen	Zen	1.4	3.4	15.9	25.1

“Seto” allele derived from ‘Setodure’ (non-dormant); “Zen” allele derived from Zen (dormant); “non-Zen” allele derived from non-dormant parent (Chono ).

Although ‘Shiroganekomugi’ had Zen-type MFT, its pedigree did not include Zen (Chono ).

Non-dormant: neither ‘Setodure’ nor Zen.

Values followed by the same letter are not significantly different by Tukey’s HSD at P = 0.05.

We also conducted multi-ANOVA using GI to access the effect of resistance source. Similar tendencies were found as shown in Tables 3 and 4 by ANOVA and variance component using PG. Single effects of seed color (44.6%), MFT (15.0%) and QPhs-5AL (35.7%) were significant on GI on the full-ripe stage on incubation at 20°C (Supplemental Tables 1, 2).

Discussion

Among the introduced sources of resistance, the R-gene encoding the TaMYB10 transcription factor, which is associated with red seed color, was the most effective for promoting PHS resistance (Tables 3, 6). The R-gene increased seed dormancy and decreased germination in a NIL of the white-seeded bread wheat ‘Spica’ (Kottearachchi ). In general, bread wheat cultivars with red seeds have a higher PHS resistance than those with white seeds. Our results agree, and show that red seed color controlled by an R-gene can promote PHS resistance with strong seed dormancy in durum wheat. Thus, the R-gene for TaMYB10 may confer PHS resistance in Triticum species regardless of ploidy.

Mori reported a QTL on Chr. 3A for strong PHS resistance. Nakamura revealed that MFT was a causal gene of the QTL; seeds of the red-seeded bread wheat cultivar ‘Chinese Spring’ overexpressing MFT had clearly increased PHS resistance. We found that the introduction of MFT alone into white-seeded ‘Setodure’ was not enough to improve PHS resistance, but its introduction (with or without QPhs-5AL) with the R-gene for red seed color increased PHS resistance (Table 3). In addition, MFT alone could not inhibit germination at 15°C or at the full-ripe stage. We conclude that the effect of the introduction of MFT alone on PHS resistance is different from the case in bread wheat. Single introduction MFT may be effective but not sufficient for promoting PHS resistance in durum wheat, thus it may need to pyramid with other PHS resistance genes and/or QTLs.

The introduction of QPhs-5AL alone did not seem to promote PHS resistance in durum wheat, but its introduction with R-gene or MFT was effective (Tables 3, 6), albeit with a weaker effect (Tables 4, 5). Kottearachchi reported QPhs-5AL as a minor QTL for PHS resistance in a white-seeded recombinant inbred line population of a Zen/‘Spica’ bread wheat cross. Our results suggest that single introduction QPhs-5AL may almost not be effective for promoting PHS resistance in durum wheat, however, QPhs-5AL pyramiding with red seed color and MFT can be improving PHS resistance.

From their GI scores, W-MFTZ-QPhs-5ALZ and R-MFTS-QPhs-5ALS have similar PHS resistance to ‘Minaminokaori’, a major red-seeded bread wheat in Japan with non-Zen-type MFT and QPhs-5AL (Table 6). In addition, R-MFTZ-QPhs-5ALZ has similar PHS resistance to ‘Shiroganekomugi’, a leading red-seeded bread wheat in Japan with Zen-type MFT. These results suggest that breeding of durum wheat for PHS resistance using genetic resources derived from bread wheat can increase resistance to levels similar to those of current Japanese bread wheat cultivars, and thus these durum wheats may grow successfully where ‘Minaminokaori’ or ‘Shiroganekomugi’ are currently grown.

Red seed color is one of the most important traits for PHS resistance in wheat. Chao assessed PHS resistance on many durum wheat genotypes including red-seeded, and concluded red-seeded wheats carrying R-gene had the strongest resistance among them. However, Kai reported that red-seeded durum wheats did not show sufficient PHS resistance for the Japanese severe climate. Red-seeded bread wheats can experience PHS due to rain at harvest in the Japanese climate. In addition, most durum wheats grown for pasta production are white-seeded, and there is no precedent for the use of red-seeded durum wheats for pasta production, so it will be necessary to assess the acceptability of red-seeded durum wheat for pasta production. If red-seeded durum wheats are not acceptable, we need to conduct durum wheat breeding based on white-seeded.

In summary, the introduction of an R-gene for red seed color, MFT, and QPhs-5AL from bread wheat into durum wheat by interbreeding was very effective at introducing PHS resistance into durum wheat. This strategy can compensate for the insufficiency of genetic resources for PHS resistance in durum wheat. The pyramiding of several genes and/or QTLs for PHS resistance may further promote PHS resistance in durum wheat.