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Journal of Integrative Agriculture 2016, 15(9): 1955–1967

RESEARCH ARTICLE

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Mapping QTLs for stomatal density and size under drought stress in wheat (Triticum aestivum L.) WANG Shu-guang1, JIA Shou-shan1, SUN Dai-zhen1, FAN Hua1, CHANG Xiao-ping2, JING Rui-lian2

1 College of Agronomy, Shanxi Agricultural University, Taigu 030801, P.R.China2 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

AbstractStomatal density and size affect plant water use efficiency, photosynthsis rate and yield. The objective of this study was to gain insights into the variation and genetic basis of stomatal density and size during grain filling under drought stress (DS) and well-watered (WW) conditions. The doubled haploid population derived from a cross of wheat cultivars Hanxuan 10 (H10), a female parent, and Lumai 14 (L14), a male parent, was used for phenotyping at the heading, flowering, and mid- and late grain filling stages along with established amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The stomatal density of doubled haploid (DH) lines was gradually increased, while the stomatal lengths and widths were gradually decreased during grain filling stage. Twenty additive QTLs and 19 pairs of epistatic QTLs for the 3 traits were identified under DS. The other 20 QTLs and 25 pairs epistatic QTLs were obtained under WW. Most QTLs made more than 10% contributions to the total phenotypic variations at one growth stage under DS or WW. Furthermore, QTLs for stomatal density near Xwmc74 and Xgwm291 located on chromosome 5A were tightly linked to previously reported QTLs regulating total number of spikelets per spike, number of sterile spikelets per spike and proportion of fertile spikelets per spike. Qsw-2D-1 was detected across stages, and was in the same marker region as a major QTL for plant height, QPH.cgb-2D.1. These indicate that these QTLs on chromosomes 5A and 2D are involved in regulating these agronomic traits and are valuable for molecular breeding.

Keywords: wheat (Triticum aestivum L.), stomatal density, stomatal length, stomatal width, quantitative trait loci

between the interior of the leaf and the atmosphere. The aperture of the stomatal pore and number of stomata that form on the epidermis regulate the amount of gas exchange (Hetherrington and Woodward 2003). Many studies showed the importance of stomatal density and size and their positive association with some physiological characters, such as stomatal conductance, photosynthetic characteristics in rice (Chen et al. 1990, 1995), barley (Miskin et al. 1972; Yoshida 1979), hybrid poplar (Reich 1984) and sorghum (Muchow and Sinclair 1989). However, Wang et al. (2013) found it was not significant relationship between stomatal density at the middle part of wheat flag leaf and stomatal conductance, photosynthesis rate and transpiration rate at 10 days after anthesis (DAA), while was strongly significant and negative at 20 DAA; instead

Received 31 August, 2015 Accepted 28 December, 2015Correspondence SUN Dai-zhen, Tel/Fax: +86-354-6288706, E-mail: [email protected]; JING Rui-lian, Tel/Fax: +86-10-82105829, E-mail: [email protected]

© 2016, CAAS. Published by Elsevier Ltd. This is an open access art ic le under the CC BY-NC-ND l icense (http:/ /creativecommons.org/licenses/by-nc-nd/4.0/)doi: 10.1016/S2095-3119(15)61264-3

1. Introduction

Stomata control the exchange of water vapour and CO2

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1956 WANG Shu-guang et al. Journal of Integrative Agriculture 2016, 15(9): 1955–1967

stomatal length and width was mostly positive with them. Yang et al. (1995) indicated that the change in leaf stomatal conductance is closely related to leaf aging. Thus it may be the change of stomatal density and size is associated with leaf senescence.

Earlier studies showed that rice leaves at later growth stages have higher stomatal densities (Rowland-Bamford et al. 1990; Kawamitsu et al. 1996). On the contrary, Chen et al. (2001) suggested that there were higher stomatal densities in younger leaves. Laza et al. (2010) also found that flag leaves in rice had higher stomatal densities at heading than at the mid-growth stage, and proposed that differences may be due to sampling time, the actual leaf sample and genotype.

Although stomatal density is affected by environmental factors and development stages, its genetic control is ev-ident (Nadeau and Sack 2002, 2003; Hetherrington and Woodward 2003). Miskin et al. (1972) showed that there were genetic differences in stomatal frequency (density) among genotypes in barley. More than a decade ago, it was found that the stomatal patterning in the erecta mutant of Arabidopsis were synergistically controlled by three genes ERECTA (Masle et al. 2005), ERL1 and ERL2 (Shpak et al. 2005). Another mutant SDD1 (STOMATAL DENSITY AND DISTRIBUTION1) was identified to do not interrupt the pattern formation but the density and distri-bution of stomata (Berger and Altmann 2000). Yang and Sack (1995) reported that the loss-of-FLP mutants showed an increased stomatal density and FLP may function in the late differentiation stage. Bergmann et al. (2004) showed loss-of-YODA mutants produce more stomata, while YODA overexpression lines produce no stomata in leaves. In rice, Ishimaru et al. (2001a) identified two QTLs for adaxial stomatal frequencies and two QTLs for abaxial stomatal frequencies in the middle part of the top fully expanded leaves, using Nipponbare/Kasalath backcross inbred lines (BILs). Ten QTLs for stomatal density and four QTLs for size were detected across growth stages and leaf surfaces (adaxial and abaxial) (Laza et al. 2010). In wheat, flag leaf photosynthesis contributes about 30–50% of the assimilates for grain filling (Sylvester-Bradley et al. 1990). However, the changing pattern and genetic basis of stomatal density and size in flag leaf during grain filling is not well understood. And to our knowledge, no analysis of the genetic control of these traits on the leaf surfaces and across growth stages has been reported in wheat.

In this study we used 150 doubled haploid (DH) lines derived from a cross between cultivars Hanxuan 10 (H10) and Lumai 14 (L14) to study the change of stomatal density and size on wheat flag leaf surfaces, and QTLs controlling these traits were identified at different growth stages. The

purpose was to gain insights into the changing pattern and molecular basis of stomatal density and size in flag leaf during grain filling.

2. Materials and methods

2.1. Plant materials and field designs

The wheat DH population comprised of 150 lines derived from a H10×L14 cross was constructed at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing (Jing et al. 1999; Hao et al. 2003; Zhou et al. 2005). All 150 lines and parents were grown at the experimental farm (37°25´N, 112°35´E, 799.6 m a.s.l.) of Shanxi Agricul-tural University, China in 2011. The experimental field was divided into two parts for different water environments. The field design of each part consisted of randomized complete blocks with three replications. Each block was a length of 2 m and a width of 40 m. 150 lines and two parents with 0.25 m between rows and 40 seeds per row were sown. One water regime was rainfed and was used as the drought stress (DS) environment with a total of 198.2 mm rainfall during the whole growing season; the other was well-wa-tered (WW) with 65 mm applied at the pre-overwintering, seedling establishment, jointing and mid-grain filling stages.

2.2. Measurement and calculation of stomatal density and size

Three heading plants in each line and parents were tagged. 2 cm2 samples from the central portion of three flag leaves of each line at four phenological stages of heading, flowering, mid- (10 DAA) and late-grain filling (20 DAA) were placed in 2 mL centrifuge tube with 2.5% glutaraldehyde. Every tube was quickly shaken to prevent leaf adhesion, then placed in a refrigerator at 4°C.

After a sample was taken out, its surface was cleaned using a degreased cotton ball dipped in alcohol, and then carefully smeared with a thin film of nail varnish on the un-der epidermis. When dry, the film was peeled from the leaf surface, mounted on a glass slide, and immediately covered with a cover slip. The numbers of stomata per view were scored, and stomatal lengths and widths were measured under a 40X objective lens of a photomicroscope fitted with objective and eyepiece micrometers (Olympus Corporation, Tokyo, Japan). Stomatal averages of 12 view areas (S=πr2, r=View radius) were calculated, and stomatal density was defined as N/S (number of stomata mm–2). Six random stomata per view were randomly selected for measuring lengths and widths which were then meaned as the value for each plant.

1957WANG Shu-guang et al. Journal of Integrative Agriculture 2016, 15(9): 1955–1967

2.3. Data analysis

Analyses of variance (ANOVA) of the data were conducted by the SPSS ver. 17.0 statistical package to assess the variances among doubled haploid lines (DHLs) and to cal-culate the mean, variance, standard deviation, coefficient of variation, kurtosis and skewness of DHLs for stomatal density and size. Broad sense heritabilities (hB

2) were computed from the estimates of genetic (s2

g) and residual (s2

e) variances derived from the expected mean squares as hB

2=sg2/(sg

2+se2/k), where k was the number of replications

(Kong 2005).A genetic linkage map, consisting of 395 marker loci,

including 132 amplified fragment length polymorphisms (AFLP) and 263 simple sequence repeats (SSRs), was con-structed from data on the 150 DHLs using MAPMAKER/Exp ver. 3.0 software (Lander et al. 1987; Lincoln et al. 1993). The map covered 3 904 centiMorgans (cM) with an average distance of 9.9 cM between adjacent markers.

QTLs for stomatal density and size in two water regimes were detected using QTL Mapper ver. 2.0 (Wang et al. 1999) set for composite interval mapping (CIM) of a mixed linear model. The threshold LOD score to declare the presence of a QTL was 2.50, and significance levels of P<0.001 and P<0.005 were adopted for identifying additive and epistatic effects of QTLs. QTLs were named according to the rule of “QTL+trait+lab+chromosome+gene number” (McIntosh et al. 1999). For simplicity the lab code ‘cgb’ is not used in this paper.

3. Results

3.1. Phenotypic variation of stomatal density and size in the DHLs and parents

The leaf stomatal density and width of H10 were greater than those of L14, but stomatal length showed only small differences (Fig. 1) and the averages of DHLs were in-

H10 under DS L14 under DS H10 under WW L14 under WW

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Fig. 1 Stomata for the parents of doubled haploid (DH) lines at different growth stages under drought stress (DS) and well-watered (WW). HS, heading stage; FS, flowering stage; MS, middle grain-filling stage; LS, late grain-filling stage. H10, Hanxuan 10; L14, Lumai 14. The same as below.


termediate between the parents for all four stages under two water conditions. The coefficients of variation of the stomatal density were obviously greater than those of the stomatal length and width, indicating that stomatal density had greater plasticity across stages (Table 1). Stomatal density and size of DHLs showed continuous transgressive segregation with skewness and kurtosis values close to 0, suggesting normal distribution. All target traits were thus quantitatively controlled by multiple genes and were suitable for QTL mapping (Fig. 2).

The hB2 of stomatal density and length and width under

the two water conditions across growth stages varied from 58.16 to 81.26%, 44.81 to 93.28% and 66.21 to 87.37%, respectively, indicating that environmental factors also had a larger influence on inheritance of the stomatal traits.

3.2. Variation of stomatal density and size at different growth stages

The stomatal density of DHLs under DS were higher than those under WW conditions but the stomatal length and width were lower across growth stages. Although the stomatal density of DHLs was gradually increased, the stomatal length and width were gradually decreased under two water environments from the heading to the late filling stages (Fig. 3).

3.3. Correlation analysis for stomatal density and size

Stomatal density showed a highly significant negative cor-relation with stomatal length across stages under the two

Table 1 Phenotypic variation in stomatal density and size in doubled haploid (DH) lines and their parents at different growth stages under two water regimes

Trait1) Environment2) Stage3)Parents4) DH lines

H10 L14 Mean SD Skewness Kurtosis Variation CV (%) hB2 (%)5)

SD DS HS 75.43 61.02 64.81 6.86 0.315 0.427 44.14–83.68 10.6 69.90

FS 75.57 59.55 65.33 6.71 1.268 2.622 52.03–94.09 10.3 62.44

MS 68.41 57.72 63.71 6.36 0.872 0.750 53.39–87.09 10.0 65.28

LS 60.82 58.06 63.34 7.20 0.805 0.553 49.13–84.55 11.4 81.91

WW HS 75.42 64.34 64.64 7.40 0.191 0.041 46.08–87.87 11.4 58.16

FS 70.82 63.40 63.80 5.81 –0.034 –0.430 49.40–77.02 9.1 81.26

MS 71.88 62.31 62.84 5.42 –0.036 0.012 47.55–76.01 8.6 67.15

LS 71.06 65.42 63.33 5.56 0.319 0.135 51.18–80.05 8.8 62.81

SL DS HS 46.21 47.99 46.80 2.23 0.031 –0.054 41.46–53.54 4.8 44.81

FS 47.97 49.28 48.52 2.13 –0.546 2.071 39.10–54.72 4.4 64.46

MS 50.37 49.95 49.17 1.99 –0.392 0.751 42.22–54.58 4.0 50.25

LS 50.56 49.49 49.01 2.03 –0.736 0.611 41.94–53.19 4.1 84.97

WW HS 47.52 46.76 47.96 1.72 –0.233 –0.018 42.43–51.81 3.6 78.19

FS 48.33 47.43 48.58 1.91 –0.083 –0.230 44.24–53.33 3.9 88.15

MS 49.24 49.68 49.49 1.58 –0.120 –0.184 45.00–52.64 3.2 61.87

LS 50.56 50.42 49.99 1.75 –0.240 0.410 45.56–54.72 3.3 93.28

SW DS HS 27.20 25.14 26.13 1.81 1.064 2.812 21.88–34.58 6.9 79.96

FS 26.86 24.62 25.87 1.21 0.014 0.280 22.92–29.44 4.7 77.18

MS 27.87 25.81 26.59 1.15 0.126 0.395 23.61–29.86 4.3 71.11

LS 27.64 25.60 26.17 1.36 –0.094 0.425 22.08–29.79 5.3 87.37

WW HS 27.09 25.74 26.67 1.32 0.428 –0.132 23.85–31.15 4.9 66.66

FS 27.08 25.19 26.81 1.30 0.345 –0.413 24.38–30.52 4.8 70.20

MS 27.64 25.97 26.91 1.13 0.355 0.231 24.65–30.69 4.2 66.21

LS 27.92 26.44 26.84 1.22 0.211 0.224 23.13–30.63 5.1 83.441) SD, stomatal density; SL, stomatal length; SW, stomatal width.2) DS, drought stress; WW, well-watered.3) HS, heading stage; FS, flowering stage; MS, middle grain-filling stage; LS, late grain-filling stage. 4) H10, Hanxuan 10; L14, Lumai 14.5) hB

2, broad sense heritabilities.


water environments. Stomatal density was negatively associated with stomatal width across stages under the two water regimes, with the exceptions of being significant at the heading stage under WW and the flowering and

late grain filling stages under DS. Stomatal length was significantly and positively correlated with stomatal width except at heading under DS and at flowering stage under WW (Table 2).

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Fig. 2 Frequency distributions for stomatal density, length and width for DH lines at heading stage (A), flowering stage (B), mid-grain filling stage (C) and late grain filling stage (D) under two water regimes. Dotted and solid arrowheads represent the H10 and L14 parents, respectively.


3.4. Additive QTL for stomatal density and size in wheat flag leaves at different growth stages

A total of 40 additive QTLs were detected for the three traits. The QTLs were located on 18 of 21 chromosomes, except for 4B, 5B and 6D (Table 3 and Fig. 4). Among them, 13 additive QTLs involved interaction.

For stomatal density, 18 additive QTLs comprised 11 under DS and 7 under WW located on different regions of chromosomes 1A, 3A, 4A, 5A, 6A, 7A, 2B, 1D and 3D. Phe-notypic variation of these QTLs ranged from 7.56 to 17.49%, with LOD scores from 3.74 to 8.76 (Table 3 and Fig. 4). Among these QTLs, six mapped at adjacent marker intervals on chromosome 5A, and with additive effects from favorable alleles of H10. They are Qsd-5A.1 in the marker interval Xwmc410–Xwmc74 detected at heading and mid-grain filling stages, and Qsd-5A.2 in marker interval Xwmc74–Xgwm291

identified at flowering and mid-grain filling stages under DS conditions, as well as Qsd-5A.3 and Qsd-5A.4 in the marker interval Xgwm291–Xgwm410–Xwmc340 detected at late grain filling stage under WW conditions. In addition, Qsd-6A.1 was mapped in the marker interval P1832–Xgwm617 at flowering and late grain filling stages under DS. Qsd-6A.2 was mapped in the marker interval Xgwm617–Xcwm487 at the late grain filling stage under DS conditions. All had the same additive effect directions from H10. The remaining nine QTLs were detected only at one growth stage and in one water regime.

Nine additive QTLs for stomatal length were detected with LOD values from 3.90 to 7.74 and explaining from 7.65 to 30.93% of the phenotypic variation under the two water conditions (Table 3 and Fig. 4). Seven loci, mapping on chromosomes 4A, 5A, 6A, 1B, and 2B, derived their additive effects from favorable alleles of L14, whereas the additive effects of the other two, located on 4A and 1B, came from favorable alleles in H10. Among nine additive QTLs, Qsl-5A.1 was detected at the heading and flower-ing stages under DS, explaining 30.93 and 24.29% of the phenotypic variation, respectively. The remaining seven QTLs were detected at one stage and in one water regime. In additon, Qsl-5A.1 and Qsl-6A.1 were also involved in gene interaction.

Thirteen additive QTLs controlling stomatal width were located on chromosomes 3A, 4A, 6A, 6B, 2D, and 3D, ac-counting for 7.69 to 22.83% of the phenotypic variation, with LOD values from 2.55 to 7.74 under both water regimes. Four of them were detected under DS, including Qsw-2D.1 mapped at heading and flowering, and Qsw-3A.1 identified at the mid- and late grain filling stages with favorable alleles from L14. Another nine QTLs were all detected under WW. Qsw-2D.1 was mapped again at the flowering, mid- and late grain filling stages, and was also involved in gene interaction at flowering and middle grain filling under WW.

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Fig. 3 Changes in stomatal traits of DH lines at different growth stages under two water regimes.

Table 2 Correlations of stomatal density and size in DH lines at different growth stages under two water regimes

Environment Stage Trait SW SLWW HS SD –0.33** –0.46**

SL 0.45** MS SD –0.11 –0.38**

SL 0.38** FS SD –0.07 –0.35**

SL 0.02LS SD –0.09 –0.42**

SL 0.20* DS HS SD –0.04 –0.57**

SL 0.04MS SD –0.14 –0.67**

SL 0.20* FS SD –0.36** –0.69**

SL 0.26** LS SD –0.40** –0.64**

SL 0.53** * and ** represent significance at P=0.05 and 0.01, respectively. The same as below.


3.5. Epistatic QTL for stomatal density and size in wheat flag leaves at different growth stages

All three traits were influenced by epistatic effects of the additive×additive type. A total of 44 pairs of epistatic QTLs were identified (Table 4). These epistatic QTLs, explaining phenotypic variations ranging from 0.01 to 29.11%, showed more interaction of non-allelic genes between the A and B genomes. Among them, 22 pairs had positive effects, i.e., the parent-type effects were higher than the recom-

binant-type effects, whereas the other 22 pairs showed negative effects where recombinant-type effects were higher than parent-type effects.

Among 17 pairs of epistatic QTLs for stomatal density, 9 and 8 were detected under DS and WW with LOD values from 5.23 to 9.11, and 11 pairs explained more than 10% of the phenotypic variation (Table 4). Sixteen pairs of epi-static QTLs involved stomatal length; among them, 6 and 8 were detected under DS and WW, explaining phenotypic variations ranging from 0.39 to 28.66%, with LOD values

Table 3 Additive effect QTLs for stomatal density, length and width in wheat leaves

Trait Stage Treatment QTL Flanking markers Site (cM)1) LOD A2) PVE (%)3)

SD HS DS Qsd-5A.1 Xwmc410–Xwmc74 0.4 4.20 2.86** 17.49WW Qsd-3D Xgdm72–Xgwm341 0 4.86 1.87** 12.50

FS DS Qsd-1A.1 Xwmc59–Xwmc254 0.8 4.55 2.28** 12.64Qsd-5A.2 Xwmc74–Xgwm291 0 5.43 2.32** 13.09Qsd-6A.1 P1832–Xgwm617 1.8 5.80 2.55** 15.82

WW Qsd-7A.1 Xwmc488–P2071 0 6.17 –1.76** 17.29MS DS Qsd-3A.1 Xwmc21–Xwmc505.2 2.4 4.47 1.87** 7.70

Qsd-4A Xgwm601–Xgwm610 0 4.17 2.02** 8.99Qsd-5A.1 Xwmc410–Xwmc74 2 3.74 2.11** 9.81Qsd-5A.2 Xwmc74–Xgwm291 0.6 3.79 2.05** 9.26

WW Qsd-1D Xcwm1–Xwmc432 1 6.12 –2.13** 15.38Qsd-7A.2 P8150–P4114.1 0 5.57 –2.03** 13.97

LS DS Qsd-2B Xgwm374–Xwmc179.2 0 8.76 –2.83** 15.67Qsd-6A.1 P1832–Xgwm617 1.6 8.61 2.76** 14.90Qsd-6A.2 Xgwm617–Xcwm487 0.2 5.44 2.08** 8.46

WW Qsd-3A.2 P3614–EST47 2.2 6.46 –2.92** 16.43Qsd-5A.3 Xgwm291–Xgwm410 1.8 3.95 2.05** 8.10Qsd-5A.4 Xgwm410–Xwmc340 0.2 3.99 1.98** 7.56

SL HS DS Qsl-5A.1 Xgwm410–Xwmc340 4.2 7.74 –1.12** 30.93WW Qsl-2B.1 Xgwm319–Xwmc441 0 4.66 –0.64** 11.98

FS DS Qsl-5A.1 Xgwm410–Xwmc340 3.8 5.29 –1.17** 24.29WW Qsl-4A.1 P8922–P8222.2 0 6.25 0.91** 25.14

Qsl-4A.2 P2454.3–P3465.1 0.2 5.65 –0.94** 26.83MS DS Qsl-5A.2 Xwmc524–Xgwm595 0.6 4.95 –0.74** 10.21

WW Qsl-6A.1 Xgwm570–Xwmc179.3 0 4.23 –0.63** 19.32LS DS Qsl-1B.1 Xgwm273–Xgwm131 0.2 4.02 0.68** 7.65

Qsl-6A.2 P1832–Xgwm617 1.6 3.90 –0.83** 17.05SW HS DS Qsw-2D.1 Xwmc453.1–Xwmc18 4.2 7.74 –1.12** 10.30

WW Qsw-4A.1 P8922–P8222.2 0.6 4.50 0.43** 8.89Qsw-6A.1 Xcwm487–P3465.4 0 4.05 –0.40** 7.69Qsw-6B.1 P6901.3–P1142.1 0.8 5.76 –0.49** 11.54Qsw-6B.2 P1142.1–P8166.1 0.6 6.18 –0.49** 11.54

FS DS Qsw-2D.1 Xwmc453.1–Xwmc18 0 2.45 –0.4185** 11.53WW Qsw-2D.1 Xwmc453.1–Xwmc18 0.4 4.29 –0.54** 22.83

MS DS Qsw-3A.1 Xwmc532–Xgwm369 1 5.35 –0.41** 14.38WW Qsw-2D.1 Xwmc453.1–Xwmc18 0 5.00 –0.46** 17.00

LS DS Qsw-3A.1 Xwmc532–Xgwm369 1 4.09 –0.43** 11.33WW Qsw-2D.1 Xwmc453.1–Xwmc18 0.2 3.83 –0.40** 8.65

Qsw-3D.1 Xwmc437–Xwmc529.1 3.4 5.04 0.45** 10.95　　　 Qsw-3D.2 Xwmc529.1–Xgdm72 0.8 3.92 0.40** 8.651) Site (cM) means genetic distance of the putative QTL from the left flanking marker.2) A, additive effect, a positive value indicates that the allele is from H10, while a negative value means that the allele is from L14.3) PVE (%) indicates the phenotypic variance explained by the additive QTL.The same as below.


QTLs for SD at HS, FS, MS and LS under WW QTLs for SD at HS, FS, MS and LS under DS

QTLs for SL at HS, FS, MS and LS under DS

QTLs for SW at HS, FS, MS and LS under DS

QTLs for SL at HS, FS, MS and LS under WW

QTLs for SW at HS, FS, MS and LS under WW

HS : heading stage, FS : flowering stage, MS : mid-grain filling stage, LS : late grain filling stage. WW DS

Chr. 2D

Chr. 3D

Chr. 1D

Chr. 6BChr. 2B

Chr. 1B

Chr. 1A Chr. 3A Chr. 4A Chr. 5A Chr. 6A Chr. 7A

Fig. 4 Chromosome locations of additive effect QTLs for stomatal traits in the DH population.


Table 4 Epistatic effect QTLs for stomatal density, length and width in wheat

Trait Stage Treatment QTL Flanking markersSite (cM)

QTL Flanking markersSite (cM)

LOD score

AA1) PVE (%)

SD HS DS Qsd-3D Xgdm8–Xgwm645 2.6 Qsd-4A.1 P8222.2–P5611.1 0.2 5.36 –1.38* 16.35FS DS Qsd-6A.3 P4232.4–Xcwm306 2.8 Qsd-7B.1 P1123.2–Xgwm611 0 5.25 0.77** 20.30

Qsd-6A.1 P1832–Xgwm617 1.6 Qsd-7A-3 P5611.2–Xwmc 422 0.2 5.45 2.50 0.73WW Qsd-3D Xgdm8–Xgwm645 0 Qsd-6B-1 P4232.5–P6901.3 0 6.25 0.28** 18.99

Qsd-5A.5 Xwmc524–Xgwm595 1.8 Qsd-5D-1 Xgwm205.2–Xgdm68 0 7.94 0.50** 24.75MS DS Qsd-1B.1 Xgwm582–Xgwm273 0 Qsd-7B-2 Xwmc276–Xgwm577 0 5.91 –0.21** 21.10

WW Qsd-1A-2 Xpsp3027–Xgwm164 0 Qsd-6B-2 Xwmc341–Xgwm193 0 5.23 –2.65 17.90Qsd-1B-2 Xcwm547–P5140.3 0 Qsd-7A-2 P8150–P4114.1 0.4 5.48 0.96** 2.35Qsd-1B-2 Xcwm547–P5140.3 0.2 Qsd-7B-3 Xwmc526–Xwmc273 0.4 5.37 2.34 13.96

LS DS Qsd-2B Xgwm374–Xwmc179.2 0 Qsd-3A-3 Xwmc388–Xwmc21 0 8.81 0.08 0.01Qsd-3B Xwmc231–Xgwm284 0 Qsd-5A-6 Xgwm304–P2470 0.2 6.20 1.98** 7.80Qsd-4A-2 P6431.1–Xgwm160 0.6 Qsd-5D-2 Xgdm3–Xgdm43 0 5.55 –2.39** 11.36Qsd-4D Xgwm192–Xwmc331 0 Qsd-7B-4 Xpsp3033–Xgwm297 0 5.55 2.25 10.07Qsd-6A-4 Xwmc179.1–Xwmc256 0 Qsd-6A-1 P1832–Xgwm617 1.6 9.11 –0.38 0.29

WW Qsd-1A-3 Xwmc120–Xgwm135 0 Qsd-5A-4 Xgwm410–Xwmc340 2.2 7.67 2.90** 13.90Qsd-1A-1 Xwmc59–Xwmc254 0.2 Qsd-2D Xgwm261–Xwmc112 0 7.72 –2.68** 11.87Qsd-3A-4 EST47–P3716.2 0 Qsd-3A-3 Xwmc388–Xwmc21 0.4 7.27 –0.89 1.31

SL HS DS Qsl-2A-1 Xwmc522–Xwmc453.2 0 Qsl-4A-3 P3613.2–P8922 0 5.89 –0.02** 9.11Qsl-3A-1 Xwmc388–Xwmc21 0.4 Qsl-5A-1 Xgwm410–Xwmc340 4.2 9.28 0.11 2.28Qsl-6A-1 Xgwm570–Xwmc179.3 0 Qsl-7B-1 Xgwm297–Xgwm68.1 0.2 5.79 –0.02** 10.59Qsl-7A-1 P3465.2–Xcwm48.2 0.4 Qsl-7A-2 Xwmc116–P8443.2 0 7.09 0.52** 22.68

WW Qsl-1B-2 Xwmc367–P8444.5 0 Qsl-2A-2 Xwmc27.2–P5166.2 0 8.08 0.23** 13.19Qsl-2A-3 Xpsp3088–Xwmc296 0 Qsl-7A-3 Xgwm635.2–Xgwm635.1 0.8 5.69 –0.03** 14.81Qsl-2A-4 P2478.2–Xwmc27.2 0.4 Qsl-2D Xgwm261–Xwmc112 0 5.31 0.01** 10.24Qsl-2B-1 Xgwm319–Xwmc441 0.2 Qsl-3A-1 Xwmc388–Xwmc21 0.4 5.89 –0.76 0.61

FS DS Qsl-1B-3 Xcwm547–P5140.3 0 Qsl-5A-1 Xgwm410–Xwmc340 4.2 5.68 0.19 0.39WW Qsl-1A Xwmc93–Xwmc304 0.4 Qsl-3A-2 P6934.5–Xcwm48.1 0 8.00 –0.23** 17.20

Qsl-1B-4 P3470.4–P4133 1.6 Qsl-5A-3 Xgwm291–Xgwm410 0.2 6.66 0.05** 12.86Qsl-1B-3 Xcwm547–P5140.3 0 Qsl-4A-2 P2454.3–P3465.1 0.2 5.91 0.12 0.48

MS DS Qsl-3A-3 Xgwm391–P8422 0 Qsl-5A-2 Xwmc524–Xgwm595 1.2 6.67 0.63** 9.19WW Qsl-4A-4 Xgwm265–Xwmc161 0 Qsl-7A-4 P3156.3–Xwmc83 0 6.84 –0.59** 11.42

Qsl-7B-2 Xwmc273–Xwmc276 0 Qsl-7D Xwmc436–Xgwm44 0.4 8.62 –0.73** 17.49LS WW Qsl-2B-2 Xpsp3034–Xgwm630 0 Qsl-3B P2478.1–Xwmc505.1 0 5.39 –0.20** 28.66

SW　

HS DS Qsw-2B-1 Xgwm319–Xwmc441 0 Qsw-4A-2 Xcwm145–P3613.2 0 6.87 –0.01** 15.83WW Qsw-2B-2 P4233.1–Xcwm529 0 Qsw-4A-3 P8222.2–P5611.1 0.2 8.51 0.24** 19.92

Qsw-2D-2 P4233.2–P6411.4 2.6 Qsw-5D Xgwm205.2–Xgdm68 0.6 6.69 0.12** 17.81FS DS Qsw-1D P3470.7–Xcwm170 0 Qsw-3A-2 Xwmc21–Xwmc505.2 1.8 6.60 –0.01** 20.45

WW Qsw-2D-1 Xwmc453.1–Xwmc18 0.4 Qsw-6A-2 Xwmc179.1–Xwmc256 2 5.32 –0.63** 5.42MS DS Qsw-1A Xwmc120–Xgwm135 0 Qsw-6B-3 Xgwm193–P3470.2 0 7.96 0.72** 29.11

WW Qsw-2D-1 Xwmc453.1–Xwmc18 0 Qsw-6A-3 Xwmc179.1–Xwmc256 0 5.92 –0.10 0.08LS DS Qsw-2A Xwmc522–Xwmc453.2 0 Qsw-4A-4 Xwmc89–Xwmc420 0.2 5.88 0.56** 22.44

WW Qsw-1B-1 Xcwm547–P5140.3 0 Qsw-3D-1 Xwmc437–Xwmc529.1 3.2 5.27 –0.11 0.01Qsw-1B-2 Xwmc44–Xgwm259 1 Qsw-7A P8443.2–Xgwm282 1 5.61 –0.12** 27.45

　　 Qsw-2D-3 Xgwm261–Xwmc112 0 Qsw-3A-3 Xcwm48.1–Xwmc532 0 6.66 –0.01** 13.931) AA, epistatic effect, positive values indicate parent type effect>recombinant type effect; negative values indicate the opposite.

from 5.31 to 9.28. Finally, 11 pairs of epistatic QTLs were detected for stomatal width, of which 4 and 7 were detected under DS and WW, with LOD values from 5.27 to 8.51, ex-plaining phenotypic variations ranging from 0.01 to 29.11%.

4. Discussion

4.1. Variation of stomatal density and size at different growth stages under the two water environments

In the present study, the middle part of wheat flag leaves had

higher stomatal densities and shorter stomatal lengths and widths under drought stress than those under well-watered conditions across growth stages. This may be mainly due to the decrease of flag leaf area under drought stress, but it is not ruled out the possibility that drought stress influenced the development and differentiation of stomata (Yang and Wang 2001). Aasamaa et al. (2001) reported that stoma-tal pore length and sensitivity to increasing drought were negatively related in six forest tree species. Laza et al. (2010) suggested that small stomata, which generally occur at higher densities, can open and close more rapidly and


thus provide a capacity for rapid increases in leaf stomatal conductance. Spence et al. (1986) reported that small guard cells may cause stomata to remain open under drought, demonstrating a balance between carbon gain through pho-tosynthesis and prevention of excessive water loss through transpiration in an adaptive response to dry conditions. Thus it seems that more and smaller stomata is a mechanism of plant adaptation to drought stress. On the other hand, the stomatal density was gradually decreased, while the sto-matal lengths and widths were gradually increased under two water environments from the heading to the late grain filling stages. We referred that this change may be related to the development and differentiation of stomata, because the flag leaf area in wheat was largest at the flowering stage, then gradually decreased (Li 2011).

Several reports have indicated that stomatal density and size in rice leaves are strongly negatively correlated (Ishi-maru et al. 2001b; Ohsumi et al. 2007). Stomatal density was also negatively correlated with stomatal length under different water conditions in jujube (Liu et al. 2006) and Platanus acerifolia leaves (Zhang et al. 2004). The present study found that stomatal density was always significantly and negatively correlated with stomatal length at all four stages, but a negative relationship between stomatal den-sity and width was significant only at heading under WW conditions and at flowering and late grain filling under DS. Stomatal length and width showed positive and significant correlations, except at heading under DS and flowering under WW. These results indicated that stomatal density and size may have higher plasticity in response to different water situations. Therefore, under different water conditions and at different growth stages, wheat plants can improve their adaptability to different water situations through co-or-dinated or compensatory variation in stomatal density and length, stomatal density and width, or stomatal length and width. Response patterns of stomatal number and size to water stress depend on the actual stage of leaf development (Lecoeur et al. 1995; Yang et al. 1995; Laza et al. 2010) and actual water status.

4.2. Genetic dissection and gene expression

In the present study, among the 18 additive and 17 pairs of epistatic QTLs for stomatal density, 15 and 10 pairs, respectively, that increased stomatal density were alleles from H10. Except for Qsl-4A.1 and Qsl-1B.1, all additive QTLs increasing stomatal length were L14 alleles. And of 13 additive QTLs for stomatal width, 10 increasing stomatal width were L14 alleles. The findings confirmed large genetic differences exsited between the two parents.

Although a multitude of studies on variation of stomatal

density and size under drought stress have been under-taken, the genetic basis of variation in stomatal density and size under drought stress at the molecular level was rarely examined. In rice, Laza et al. (2010) reported 6 and 2, and 4 and 2 additive QTLs controlling stomatal density and size, respectively, at the heading and mid-grain filling stages. These loci were located on different chromosome regions with different genetic effects. In the present study, a total of 40 additive and 44 pairs of epistatic QTLs with significant contributory percentages and covering 3 traits were identified under the 2 water regimes at 4 growth stages. Among the 40 additive QTLs, Qsd-5A.1 and Qsd-5A-2 mapped to the marker intervals Xwmc410–Xwmc74 and Xwmc74–Xgwm291, respectively. If these are at the same locus, then the same gene was responsible stable expression at the heading, flowering and mid-grain filling stages. In addition, Qsd-6A.1 was detected at flowering and late grain filling, Qsl-5A.1 was located at the heading and flowering, and Qsw-3A.1 was detected at mid- and late-grain filling. All of these QTLs were induced by DS. In contrast, Qsw-2D.1 was detected at all 4 stages under DS and/or WW, and was thus expressed independently of water status. The remaining 25 additive QTLs were each detected under DS or WW at one growth stage. In addition, among 44 pairs of epistatic QTLs covering 3 traits, 19 and 25 pairs were identified under DS and WW, respectively. These finding implied that stomatal density, length and width are each governed by a set of additive and epistatic QTLs, and are capable of conferring different expression patterns under different water conditions and different growth stages. Thus to alleviate damage caused by water stress plants can apparently induce the expression of genes that are otherwise silent under well watered conditions .

4.3. Pleiotropy of QTLs

Various studies have found that QTLs for closely correlated traits may be located at, or near, the same chromosomal positions (Hervé et al. 2001; Fracheboud et al. 2002; Tuberosa et al. 2002). Phenotypic correlations between stomatal density and length were found in our work and in previous studies (Zhang et al. 2004). And in this study, Qsd-5A.4 detected at late filling under WW and Qsl-5A.1 detected at heading and flowering under DS were the same marker region. The finding provided the molecular basis for close correlation between stomatal density and length. On the other hand, using the same population, Yang et al. (2007) mapped a QTL for stem water soluble carbohydrates at early flowering in marker interval Xwmc453.1–Xwmc18 on chromosome 2D. Liu et al. (2011) detected a QTL for root diameter near Xwmc18. The two QTLs were located


in the same marker interval as Qsw-2D.1 detected in our study. Co-location of these QTLs, and expression at different growth stages or in different environments mean that these QTLs may be pleiotropic (with different functions at different growth stages or in different environments) or are tightly linked genes. One way to further understand them could be to improve the genetic map by increasing the number mark-ers or number of progenies (Tuberosa et al. 2002). Another could be to generate inbred lines with the separate regions of interest and to analyse their phenotypes. In addition to chromosome 5A, additive QTLs for stomatal density and size appear also to be clustered in chromosomes 6A and 2D. These hot-spot regions could provide genetic information on stomatal characteristics of wheat leaves. Finally, the negative relationship between stomatal density and length and co-location of Qsd-5A.4 and Qsl-5A.1 may be useful in designing strategies for crop improvement.

4.4. Stomatal density and size in relation to yield-re-lated traits

Stomatal density and size are dominant factors determining leaf conductance, which is crucial for both photosynthesis and transpiration and consequently affect plant water use efficiency and yield (Wang et al. 2007; Mei et al. 2013). Ishimaru et al. (2001a) found that qabs3 for abax-ial stomatal frequencies and qads3 for adaxial stomatal frequencies overlapped with QTL controlling yield, and there were significant correlations between yield and ADS (adaxial stomatal frequencies) and ABS (abaxial stomatal frequencies). The major QTNSS.cgb-5A (total number of spikelets per spike), QNSSS.cgb-5A (Number of sterile spikelets per spike), QRFSS.cgb-5A.2 (Proportion of fertile spikelets per spike) identified by Wu et al. (2012) located the same marker region on Chromosome 5A as Qsd-5A-1, Qsd-5A-2 and Qsd-5A-3 detected in the study. Qsw-2D-1 detected across stages in the study were the same marker region as a major QPH.cgb-2D.1 (Wu et al. 2012). Le-dent and Jouret (1978) reported that the correlations of stomatal densities (adaxial and abaxial epidermies) with grain yield per culm or yield ha–1 are mostly negative and no-siginicant. Obviously, the different genetic backgrounds of the plant species used for such studies would certainly complicate the interpretation of the results. Furthermore, yield is a complex trait, and is regulated by a number of elementary factors, but it is likely that the factors are not equally effective.

5. Conclusion

Qsd-5A.1, Qsd-5A.2, Qsd-5A.3 and Qsd-5A.4 for stomatal density near marker Xwmc74 and Xgwm291 were tightly

linked to previously reported QTLs regulating total num-ber of spikelets per spike, number of sterile spikelets per spike and proportion of fertile spikelets per spike. Qsw-2D-1 for stomatal width detected across stages was in the same marker region as a major QTL for plant height, QPH.cgb-2D.1. These QTLs are involved in regulating these agronomic traits and are valuable for molecular breeding.

Acknowledgements

This work was supported by the National Science and Technology Major Projects for Cultivation of New Transgenic Varieties, Ministry of Agriculture of China (2014ZX0800203B-003), the Natural Science Foundation of Shanxi Province, China (2014011004-3), the Specialized Research Fund for the Doctoral Program of Higher Educa-tion, China (20121403110005), a Program of Consultative Group for International Agricultural Research (CGIAR) Project, Generation Challenge Programme (G7010.02.01).

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