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https://doi.org//10.17113/ftb.58.01.20.6531

Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija

Mehmet Tekin ; Akdeniz University, Faculty of Agriculture, Department of Field Crops, 07059 Antalya, Turkey
Ahmet Cat ; Akdeniz University, Faculty of Agriculture, Department of Plant Protection, 07059 Antalya, Turkey
Sahriye Sönmez ; Akdeniz University, Faculty of Agriculture, Department of Soil Science, 07059 Antalya, Turkey
Taner Akar ; Akdeniz University, Faculty of Agriculture, Department of Field Crops, 07059 Antalya, Turkey

Puni tekst: hrvatski, pdf (1 MB) str. 49-56 preuzimanja: 7* citiraj
APA 6th Edition
Tekin, M., Cat, A., Sönmez, S. i Akar, T. (2020). Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija. Food Technology and Biotechnology, 58 (1), 49-56. Preuzeto s https://hrcak.srce.hr/237579
MLA 8th Edition
Tekin, Mehmet, et al. "Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija." Food Technology and Biotechnology, vol. 58, br. 1, 2020, str. 49-56. https://hrcak.srce.hr/237579. Citirano 04.07.2020.
Chicago 17th Edition
Tekin, Mehmet, Ahmet Cat, Sahriye Sönmez i Taner Akar. "Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija." Food Technology and Biotechnology 58, br. 1 (2020): 49-56. https://hrcak.srce.hr/237579
Harvard
Tekin, M., et al. (2020). 'Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija', Food Technology and Biotechnology, 58(1), str. 49-56. Preuzeto s: https://hrcak.srce.hr/237579 (Datum pristupa: 04.07.2020.)
Vancouver
Tekin M, Cat A, Sönmez S, Akar T. Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija. Food Technology and Biotechnology [Internet]. 2020 [pristupljeno 04.07.2020.];58(1):49-56. Dostupno na: https://hrcak.srce.hr/237579
IEEE
M. Tekin, A. Cat, S. Sönmez i T. Akar, "Identifikacija kultivara tvrde (durum) pšenice i njihovih tetraploida s malim udjelom kadmija", Food Technology and Biotechnology, vol.58, br. 1, str. 49-56, 2020. [Online]. Dostupno na: https://hrcak.srce.hr/237579. [Citirano: 04.07.2020.]
Puni tekst: engleski, pdf (1 MB) str. 49-56 preuzimanja: 27* citiraj
APA 6th Edition
Tekin, M., Cat, A., Sönmez, S. i Akar, T. (2020). Identification of Durum Wheat Cultivars and Their Tetraploid Relatives with Low Cadmium Content . Food Technology and Biotechnology, 58 (1), 49-56. Preuzeto s https://hrcak.srce.hr/237579
MLA 8th Edition
Tekin, Mehmet, et al. "Identification of Durum Wheat Cultivars and Their Tetraploid Relatives with Low Cadmium Content ." Food Technology and Biotechnology, vol. 58, br. 1, 2020, str. 49-56. https://hrcak.srce.hr/237579. Citirano 04.07.2020.
Chicago 17th Edition
Tekin, Mehmet, Ahmet Cat, Sahriye Sönmez i Taner Akar. "Identification of Durum Wheat Cultivars and Their Tetraploid Relatives with Low Cadmium Content ." Food Technology and Biotechnology 58, br. 1 (2020): 49-56. https://hrcak.srce.hr/237579
Harvard
Tekin, M., et al. (2020). 'Identification of Durum Wheat Cultivars and Their Tetraploid Relatives with Low Cadmium Content ', Food Technology and Biotechnology, 58(1), str. 49-56. Preuzeto s: https://hrcak.srce.hr/237579 (Datum pristupa: 04.07.2020.)
Vancouver
Tekin M, Cat A, Sönmez S, Akar T. Identification of Durum Wheat Cultivars and Their Tetraploid Relatives with Low Cadmium Content . Food Technology and Biotechnology [Internet]. 2020 [pristupljeno 04.07.2020.];58(1):49-56. Dostupno na: https://hrcak.srce.hr/237579
IEEE
M. Tekin, A. Cat, S. Sönmez i T. Akar, "Identification of Durum Wheat Cultivars and Their Tetraploid Relatives with Low Cadmium Content ", Food Technology and Biotechnology, vol.58, br. 1, str. 49-56, 2020. [Online]. Dostupno na: https://hrcak.srce.hr/237579. [Citirano: 04.07.2020.]

Rad u XML formatu

Sažetak
U ovom su radu okarakterizirani genotipovi 71 kultivara tvrde pšenice (Triticum durum Desf.), 22 kultivara dvozrnca (Triticum dicoccum L.) i 11 kultivara divlje dvozrne pšenice (Triticum dicoccoides L.) u svrhu pronalaska alela povezanih s većim udjelom kadmija. Nakon toga je uzgojem u posudi ispitan fenotip 14 odabranih kultivara s malim i velikim udjelom kadmija, radi potvrde genotipskih podataka. Identificirana su 32 genotipa tvrde (durum) pšenice, jedan kultivar dvozrnca i četiri kultivara divlje dvozrne pšenice koji sadržavaju alele povezane s velikim udjelom kadmija, te 68 genotipova koji sadržavaju alele povezane s malim udjelom kadmija, i to 39 kultivara durum pšenice, 21 dvozrnca i 7 divlje dvozrne pšenice. Povrh toga, fenotipske karakteristike uočene nakon uzgoja u posudama potvrdile su rezultate molekularne analize. Marker je uspješno upotrijebljen za klasifikaciju kultivara durum pšenice u one s velikim ili malim udjelom kadmija, pa je zaključeno da se može upotrijebiti u programu uzgoja novih kultivara durum pšenice koja sadržava alele povezane s malim udjelom kadmija. Zbog rutinske primjene fosfornih gnojiva na poljoprivrednim površinama i utjecaja ostalih antropogenih čimbenika što dovode do akumulacije toksičnog kadmija, treba čim prije razviti nove kultivare durum pšenice s malim udjelom kadmija za sigurnu proizvodnju makarona i ostalih tipova pšeničnih proizvoda za prehranu ljudi i ishranu stoke.

Ključne riječi
karakterizacija tvrde (durum) pšenice; mali udjel kadmija; tetraploidna pšenica; markerima potpomognuta selekcija

Hrčak ID: 237579

URI
https://hrcak.srce.hr/237579

▼ Article Information



INTRODUCTION

The accumulation of heavy metals in the soil has a major impact on the environment. Heavy metals accumulated in the soil are stored in plant tissues over time and, therefore, pose a threat to human and animal health (1, 2). Cadmium (Cd) is one of the toxic metals which causes very serious health problem for humans. So far, diseases caused by Cd such as Itai-itai have been diagnosed (2). Metal industry, fossil fuels, domestic waste, application of pesticide and phosphorus fertilizers are the greatest sources of Cd pollution (3). Moreover, 30 000 t of Cd are added to environment annually and 13 000 t of these are caused by human activities (4). Many countries have determined several restrictions for the mass fraction of Cd in phosphorus fertilizers. Germany for instance has limited the Cd mass fraction in phosphorus fertilizers to 200 mg/kg (5). In Sweden, phosphorus fertilizers have been subjected to taxes when the Cd mass fraction exceeds 5 mg/kg and imports of phosphorous fertilizers with a Cd mass fraction above 100 mg/kg are prohibited (6). This implementation encourages the production of low-Cd fertilizers and reduces Cd input into the soil.

Cd is mostly accumulated in the human body through food consumption, mainly thanks to a high intake of cereals. It is known that the contribution of cereal products to daily Cd intake ranges from 20 to 43% (7). Wheat (Triticum L.) is the first among the cultivated plants in terms of production and harvested area worldwide and wheat products provide 20% of the daily calories and also 20% of the protein, especially in 94 developing countries with more than 4.5 billion people (8). There is a large genetic variation especially in durum wheat (Triticum durum Desf.) regarding Cd accumulation in the grain and it is known that this species has higher Cd content than other cool season cereals: rye<barley<oat<bread wheat<durum wheat (6-9). Food and Agriculture Organization of the United Nations (FAO) and The International Codex Alimentarius Commission of World Health Organization (WHO) have standardized the maximum allowable Cd mass fraction in wheat grain to be 0.1 mg/kg (10). In order to reduce Cd uptake and toxicity, many alternative methods such as the use of plant growth regulators and plant nutrients are applied (2). However, the most environmentally friendly and efficient strategy are the development of new genetic materials with low cadmium content (2) and breeding programs have been carried out in many countries for this purpose. For instance, in Canada, where Cd is one of the most serious environmental problems, durum wheat cultivars have been developed since 2003 via marker-assisted selection (MAS) (11-13). A major gene Cdu1 (14), which is located in the long arm of chromosome 5B, controls the Cd accumulation in durum wheat (15, 16). Several tightly linked markers with gene Cdu1 have been developed to detect the allele associated with low content of Cd in durum wheat such as dominant SCAR marker ScOPC20 (15) and co-dominant CAPS marker usw47 (17). These markers can be used to characterize Cd accumulation in both tetraploid durum wheat and other tetraploid wheat relatives such as emmer (Triticum dicoccum L.) and wild emmer (Triticum dicoccoides L.).

The aim of the study was to molecularly characterize Turkish durum wheat gene pool and some emmer wheat and wild emmer genotypes via usw47 marker for allele associated with low Cd content. Additionally, some selected durum wheat cultivars with alleles associated with low or high Cd content were tested in pot experiment to verify the molecular data.

MATERIALS AND METHODS

Genetic materials

Seventy-one durum wheat (Triticum durum Desf.) cultivars and advanced breeding lines with 2 universal controls, 24 emmer wheat (Triticum dicoccum L.) genotypes and 11 wild emmer (Triticum dicoccoides L.) genotypes (Table 1) were used as genetic materials in this study. Canadian durum wheat cultivar “Commander” and advanced line “Dt 812” kindly provided by Dr Y. Ruan from Agriculture and Agri-Food Canada were used as negative and positive control, respectively. Ten emmer wheat and all wild emmer genotypes were obtained from Turkish Seed Gene Bank (Ankara, Turkey). Other emmer wheat lines used in the study were developed via selection breeding in a project funded by TUBITAK (The Scientific and Technological Research Council of Turkey, Project No: 214O401).

Table 1 Different tetraploid germplasm materials used in the study
No.SpeciesGenotypeNo.SpeciesGenotypeNo.SpeciesGenotype
1Triticum durum Desf.Zenit37Triticum durum Desf.Tunca 7973Triticum durum Desf.Dt 812
2Triticum durum Desf.Svevo38Triticum durum Desf.Ankara 9874Triticum dicoccum L.TR79489
3Triticum durum Desf.Saragolla39Triticum durum Desf.Sis gd 14 v75Triticum dicoccum L.TR61225
4Triticum durum Desf.Claudio40Triticum durum Desf.Sis gd 14 e76Triticum dicoccum L.TR69596
5Triticum durum Desf.Aydın 9341Triticum durum Desf.Sis gd 14 c77Triticum dicoccum L.TR69623
6Triticum durum Desf.Ege 8842Triticum durum Desf.578Triticum dicoccum L.TR68784
7Triticum durum Desf.Fırat 9343Triticum durum Desf.679Triticum dicoccum L.TR68789
8Triticum durum Desf.Fuatbey 200044Triticum durum Desf.1280Triticum dicoccum L.TR68817
9Triticum durum Desf.Gap45Triticum durum Desf.1481Triticum dicoccum L.TR68857
10Triticum durum Desf.Gediz 7546Triticum durum Desf.2282Triticum dicoccum L.TR69632
11Triticum durum Desf.Harran 9547Triticum durum Desf.2983Triticum dicoccum L.TR72183
12Triticum durum Desf.Sarıçanak 9848Triticum durum Desf.4784Triticum dicoccum L.Advanced line 5
13Triticum durum Desf.Şölen 200249Triticum durum Desf.5085Triticum dicoccum L.Advanced line 6
14Triticum durum Desf.Turabi50Triticum durum Desf.5786Triticum dicoccum L.Advanced line 11
15Triticum durum Desf.Tüten 200251Triticum durum Desf.6987Triticum dicoccum L.Advanced line 13
16Triticum durum Desf.Diyarbakır 8152Triticum durum Desf.7188Triticum dicoccum L.Advanced line 14
17Triticum durum Desf.Altıntaş 9553Triticum durum Desf.7289Triticum dicoccum L.Advanced line 18
18Triticum durum Desf.Amanos 9754Triticum durum Desf.7590Triticum dicoccum L.Advanced line 24
19Triticum durum Desf.Maestrale55Triticum durum Desf.10191Triticum dicoccum L.Advanced line 35
20Triticum durum Desf.Aurea56Triticum durum Desf.10292Triticum dicoccum L.Advanced line 42
21Triticum durum Desf.Normanno57Triticum durum Desf.10393Triticum dicoccum L.Advanced line 50
22Triticum durum Desf.Gracale58Triticum durum Desf.10694Triticum dicoccum L.Advanced line 52
23Triticum durum Desf.Levante59Triticum durum Desf.10795Triticum dicoccum L.Advanced line 53
24Triticum durum Desf.Kunduru 114960Triticum durum Desf.11096Triticum dicoccum L.Advanced line 57
25Triticum durum Desf.Eminbey61Triticum durum Desf.11197Triticum dicoccum L.Advanced line 60
26Triticum durum Desf.Kamut62Triticum durum Desf.Dww tk 498Triticum dicoccoides LTGB 00777
27Triticum durum Desf.Çeşit 125263Triticum durum Desf.Dww tk 1199Triticum dicoccoides LTGB 00791
28Triticum durum Desf.Dumlupınar64Triticum durum Desf.Dww tk 12100Triticum dicoccoides LTGB 000792
29Triticum durum Desf.Kızıltan 9165Triticum durum Desf.Dww tk 14101Triticum dicoccoides LTGB 045861
30Triticum durum Desf.Meram 200266Triticum durum Desf.Dww tk 15102Triticum dicoccoides LTGB 045871
31Triticum durum Desf.Mirzabey 200067Triticum durum Desf.Kocasarı 2-3103Triticum dicoccoides L.TGB 045910
31Triticum durum Desf.Yelken68Triticum durum Desf.Kocasarı 4-1104Triticum dicoccoides LTGB 045911
33Triticum durum Desf.Selçuklu 9769Triticum durum Desf.Kocasarı 8-2105Triticum dicoccoides LTGB 045912
34Triticum durum Desf.Altın 40/9870Triticum durum Desf.Kocasarı 9-1106Triticum dicoccoides LTGB 045913
35Triticum durum Desf.Kunduru414/4471Triticum durum Desf.Kocasarı 16-1107Triticum dicoccoides LTGB 045920
36Triticum durum Desf.Yılmaz 9872Triticum durum Desf.Commander108Triticum dicoccoides LTGB 038501

74-83 emmer and 98-108 wild emmer genotypes were obtained from Turkish Seed Gene Bank (Ankara, Turkey), 84-97 emmer genotypes were developed in a project funded by TUBITAK (The Scientific and Technological Research Council of Turkey, project no. 214O401)

Genetic characterization

Seeds of all genotypes were sown on trays and then leaf samples were collected from plants for DNA extraction at 2-3 leaf stage. DNA was extracted according to cetyl trimethylammonium bromide (CTAB) method (18). The extracted DNA samples were loaded on agarose gel (Biomax, Thomas Scientific, Swedesboro, NJ, USA) with a DNA standard in order to determine the quality and concentration of the DNA and then were stored in sterile distilled water at -20 °C until use. To amplify Cdu1 gene alleles by PCR, the co-dominant CAPS marker usw47, which was derived from an expressed sequence tag (EST) XBF474090 co-segregating with Cdu1 (16), was used.

PCR was carried out as follows: the total volume of the reaction mixture was 15 μL containing 100 ng genomic DNA, 1× PCR buffer (Sigma Aldrich, Merck, St. Louis, MO, USA), 1.5 mM MgCI2 (Sigma Aldrich, Merck), 0.2 mM of dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA) 0.4 μM of usw47 forward primer (5’-GCTAGGACTTGATTCATTGAT-3’), 0.4 μM of usw47 reverse primer (5’-AGTGATCTAAACGTTCTTATA-3’), 1.25 U Taq DNA polymerase (Sigma Aldrich, Merck). Amplification was performed in a thermocycler (MyGenieTM 96; Bioneer, Daejon, Korea) under the following conditions: 94 °C initial denaturation for 5 min, 30 cycles at 94 °C for 30 s, annealing temperature 55 °C for 30 s, 72 °C for 1 min, and then a final extension of 10 min at 72 °C.

The PCR products were digested by Hpy188I (New England Biolabs, Ipswich, MA, USA) restriction enzyme after amplification. The total volume of the reaction mixture for enzymatic digestion was 15 μL containing 4 μL PCR product, 0.25 μL Hpy188I gene from Helicobacter pylori, 1× NEBuffer 4 (New England Biolabs) and 9.25 μL distilled water. Enzymatic digestion was performed in a thermo-shaker (Biosan, Riga, Latvia) under the following conditions: 37 °C for 1 h, 65 °C for 20 min and holding at 10 °C for 5 min and then the products were loaded in 2% agarose gel and visualized under UV light after staining with ethidium bromide (Sigma Aldrich, Merck).

Pot experiment and elemental analysis

After molecular analysis, a small set of commonly cultivated 14 genotypes (Ege-88, Amanos-97, Sarıçanak 98, Şölen 2002, Turabi, Svevo, Zenit, Fırat-93, Fuatbey 2000, GAP, Gediz 75, Tüten 2002, Diyarbakır and Levante) was grown in pots in three replicates. The soil was mixed with acidic peat, in 1:1 ratio to increase the Cd uptake by plants, and then each pot was filled in with 2 kg of the mixture. A volume of 10 mL of CdCI2·H2O (Merck, Darmstadt, Germany) was added to each pot with automatic pipette to achieve final Cd mass fraction of 8 mg/kg. At physiologically ripening stage based on Zadoks growth scale (Z 98), grain and stem parts were sampled for each genotype and the samples were dried at 70 °C to constant mass. Dried plant samples of 0.5 g each were digested with 10 mL HNO3/HClO4 acid (4:1; Merck) mixture on a hotplate. The samples were then heated until a clear solution was obtained. The same procedure was repeated several times. The samples were filtered and diluted to 100 mL using distilled water, and then Cd mass fraction of the combusted samples with other elements such as P, Mg, Ca, K, Zn, Cu, Fe and Mn was measured by inductively coupled plasma-optical emission spectrometer (ICP-OES) (Optima, PerkinElmer Inc., Waltham, MA, USA). Additionally, soil in each pot was analyzed to determine total Cd accumulation from the soil in the biomass of each genotype at the end of the pot experiment.

Statistical analysis

Basic statistical parameters such as mean and standard error of mean were determined. Analysis of variance (ANOVA) was performed with least significant difference (LSD) test at the 95% confidence level using SAS statistical software (19). Additionally, correlation and principal component analyses (PCA) were performed to determine relationships among the elements by XLSTAT statistical software (20).

RESULTS AND DISCUSSION

Genetic characterization

Fig. 1 shows the results of PCR analysis of alleles associated with the accumulation of Cd obtained from usw47 marker. According to banding patterns of usw47, there are three possible alleles: for low Cd content, high Cd content and heterogeneous. Fig. S1 and Fig. S2 show all gel visualizations obtained from molecular analysis. Genotyping results of 108 tested tetraploid wheats are shown in Table 2.

Fig. 1 Results of PCR analysis of durum wheat alleles associated with the accumulation of Cd obtained from usw47 marker. Red and blue symbols illustrate alleles associated with high and low content of Cd, respectively
FTB-58-49-f1
Table 2 Determination of alleles of durum wheat cultivars associated with low and high Cd content based on molecular analysis
No.Molecular evaluationNo.Molecular evaluationNo.Molecular evaluation
1High37High73Low
2High38Low74Low
3High39Low75Low
4Low40Low76Low
5High41High77Low
6Low42Low78High
7High43High79Low
8High44Low80Low
9High45High81Low
10High46Low82Low
11High47Low83Low
12Low48Low84Low
13Low49Low85Low
14Low50Low86Low
15High51Low87Low
16High52High88Low
17High53High89Low
18Low54High90Low
19High55Low91Low
20High56Low92Low
21High57Low93Low
22Low58High94Low
23High59Low95Low
24Low60High96Low
25Low61Low97Low
26High62Low98Low
27Low63High99Low
28Low64High100High
29Low65High101Low
30Low66High102High
31Low67Low103High
31Low68Low104Low
33High69Low105Low
34Low70Low106Low
35Low71Low107Low
36High72High108High

Based on the molecular analysis, 21 (52.5%) out of 40 durum wheat cultivars had alleles associated with high and 12 (47.5%) with low Cd content, and 19 (36.4%) out of 33 advanced breeding lines had alleles associated with high and 21 (63.6%) with low Cd content (Table 2 and Fig. S1). Additionally, only 1 (4%) of the 24 emmer wheat genotypes had alleles associated with high Cd content, and 7 (63.6%) of 11 wild emmer genotypes with low and 4 (36.4%) with high Cd content (Table 2 and Fig. S2). Similar results were obtained by Zimmerl et al. (17), who reported 166 (53%) of 314 tetraploid wheat genotypes associated with low Cd content and usw47 marker can be successfully used to determine low Cd accumulators in tetraploid wheat accessions. Moreover, Vergine et al. (21) also genetically characterized tetraploid genotypes by a sequence-characterized amplified region (SCAR) marker, ScOPC20 in terms of Cd accumulation. However, this marker allows to display two different alleles: one associated with low Cd content (band absent) and another with high Cd content (band present), therefore, heterogeneous state cannot be detected.

Elemental analysis based on pot experiment

Fig. 2 shows Cd mass fractions in the grain, stem and underground parts of fourteen durum wheat cultivars. The results in Fig. 2a demonstrate that Cd addition (8 mg/kg) to the soil mixture clearly increases Cd accumulation in the grain. The cultivar Diyarbakır was the highest accumulator of Cd in the grain in both control and samples with added Cd (0.38 and 0.91 mg/kg respectively, Fig. 2a), while the control sample of Turabi cultivar and the sample of Amanos-97 cultivar grown in the soil with added Cd had the lowest Cd content in grain (0.1 and 0.12 mg/kg respectively, Table S1). All cultivars used in pot experiment except Amanos-97 and Sarıçanak 98 accumulated more Cd in grains after the addition of Cd to the soil (Table S1). In addition to cadmium, phosphorus, potassium, calcium, magnesium, iron, zinc, copper and manganese mass fractions were also determined (Table S1). A two-way ANOVA showed a significant difference (p<0.01) in the mass fractions of these elements among cultivars. Moreover, taking into account cultivar × treatment interaction, a significant difference was found in the mass fractions of all elements at the p=0.01 except for phosphorus (p<0.05). As expected, phenotypic data obtained from pot experiment for Cd accumulation in the grain were similar to molecular data. Low mass fraction of Cd was found in cultivars Ege-88, Amanos-97, Sarıçanak 98, Sölen 2002 and Turabi, which have the allele associated with low Cd content (Fig. 2a). Zimmerl et al. (17) and Perrier et al. (22) reported that varieties with the allele associated with high Cd content had 2.4-fold more Cd in the grain than the varieties with the allele associated with low Cd content.

Fig. 2 Mass fractions of Cd in: a) grain, b) stem and c) underground parts of durum wheat cultivars in both control and samples grown in the soil with the addition of w(Cd)=8 mg/kg
FTB-58-49-f2

In addition to grain, stem Cd mass fractions were determined (Fig. 2b). The addition of Cd (8 mg/kg) to the soil mixture increased the stem Cd mass fraction in almost all cultivars used in pot experiment. Moreover, among control samples, Gap cultivar had the highest stem Cd mass fraction, while Tüten 2002 cultivar had the lowest (Fig. 2b and Table S2). Durum wheat cultivars with low Cd mass fraction in their stems can be beneficial feed sources especially for small ruminants that graze wheat stems and leaves after grain harvest under rainfed conditions in Turkey. Svevo cultivar also had the highest stem Cd mass fraction in addition to its high grain Cd accumulation (Fig. 2b). The results of a two-way ANOVA show that there were significant differences (p<0.01) in mass fractions of all elements determined in stem for cultivar and cultivar × treatment interaction (Table S2). On the other hand, Cd mass fractions in underground parts (roots and stubble) of cultivars grown in the soil with the addition of 8 mg/kg Cd were also determined and the results showed that most of the added Cd was accumulated by the plants (Fig. 2c). While Amanos-97 cultivar had the lowest Cd mass fraction in each organ in general, Ege-88 cultivar had the highest Cd mass fraction in the underground parts in particular (Fig 2c). Considering Cd distribution in plant organs, most of the Cd was found in the underground parts, in which cultivars with the alleles associated with low Cd content had 4.13 mg/kg i.e. 67% total Cd (Fig. 3a), whereas those containing alleles associated with high Cd content had 3.75 mg/kg, i.e. 60% of the total Cd (Fig. 3b).

Fig. 3 Distribution of Cd content in durum wheat organs of: a) cultivars with alleles associated with low and b) high Cd content
FTB-58-49-f3

Correlation and multivariate analyses of Cd mass fraction in the grain

In order to understand the relationships among elements, correlation analysis was performed (Table 3). There was a negative correlation between the grain Cd and Cu (r=-0.76, p<0.01) and Mn (r=-0.56, p<0.01) in the control samples. Grain Cd was also negatively correlated with Mg (r=-0.55, p<0.01) in the grain samples grown in soil with added Cd (Table 3). An opposite finding was reported by Perrier et al. (22) that grain Cd was positively correlated with Mn (r=0.61, p<0.01) and Mg (r= 0.38, p<0.05). In addition to these, there was a non-significant correlation between the grain Cd and Cu (22). Liu et al. (23) also studied correlations between Cd and mineral nutrients in parts of roots and leaves in rice and they reported that Cd2+ was generally correlated with Fe3+, Mn2+, Cu2+ and Mg2+. Jalil et al. (24) conducted a similar study of durum wheat with different Cd mass fraction added to nutrient solution and they reported that for all of them, the mass fractions of Mn, Zn, Cu and Fe were not affected significantly but Cd additions to the solution depressed the uptake of Zn and Mn. A similar negative interaction between Cd and Mn was also found in this study.

Table 3 Correlations between Cd and other elements in the grain of control samples and samples grown in soil with the addition of w(Cd)=8 mg/kg
TreatmentPKCaMgFeZnCuMn
Control-0.32-0.08-0.280.15-0.26-0.21-0.76*-0.56*
w(Cd)=8 mg/kg-0.270.360.27-0.55*-0.35-0.19-0.48-0.31

*p<0.01

Additionally, principal component analysis (PCA) was performed to determine the relationships between genotypes and plant organs (Fig. 4). PCA showed that the first two components (PC1 and PC2) accounted for 96.31% of the total variance. PC1 explained 70.18% variance, while PC2 elucidated 26.13% of the total variance (Fig. 4). Moreover, contribution of each plant organ to the PC1 and PC2 shows that Cd in the underground parts of plant (43.51) was major contributor to PC1, whereas Cd in the grain (79.72) mainly contributed to PC2 (Table 4). Vergine et al. (21) similarly performed PCA for determination of Cd mass fraction in durum wheat and they reported that roots and kernels contributed to PC1 and grains mostly contributed to PC2. As a result of biplot visualization, different groups were revealed for each plant organ such as underground part (shoots and roots), stem and grain (Fig. 4). Each circle represents different group of Cd mass fractions in each plant organ in the bi-plot graph. The Diyarbakır and Levante cultivars, marked with yellow color, accumulate the highest mass fractions of Cd in the grain. The second group marked with green consists of Svevo and Tüten 2002 cultivars, which had the highest mass fractions of Cd in the stem. The third group marked with blue color comprises Amanos-97 and Sarıçanak 98 cultivars, which accumulate high mass fractions of Cd in the root. All other cultivars, Fırat-93, GAP, Fuatbey-2000, Zenit, Gediz 75, Turabi and Ege-88, had lower Cd mass fractions in all plant organs (Fig. 4). Svevo cultivars accumulated the highest mass fraction of Cd in both grain and stem, while Amanos-97 cultivar had the lowest mass fraction of Cd in the stem and grain. At this point, difference in the root to grain translocation of Cd among durum wheat genotypes is very important to develop new cultivars that can be grown in Cd-contaminated soils. If this translocation is weak or root sequestrates the Cd efficiently, grain Cd content will be low (25, 26). In addition to this, partitioning of Cd among plant organs is the second important strategy for low Cd mass fraction in the grain. Perrier et al. (22) highlighted that growing long-stemmed cultivars may have advantages since lower mass fractions of Cd are moved to plant organs such as stem, leaves, bracts, rachis and grains. Arduini et al. (27) found that partitioning to shoots and grains with increasing Cd supply was markedly higher in Svevo cultivar. They also reported that high Cd content in grains of Svevo cultivar may be related to the high allocation of biomass in roots during vegetative growth stage coupled with high post-heading dry matter accumulation and root to grain re-mobilization. Higher accumulation of elements from the soil in the plant is a desired trait to obtain higher yield and quality; therefore, breeding studies have focused on improving yield components to increase crop yield (28). Due to these concerns, modern wheat varieties tend to accumulate elements such as Cd in the grain (21, 22, 28). However, since high Cd content in the grain is not a desirable trait, cultivars with alleles associated with low Cd content and high yield should be given first priority in durum wheat breeding.

Fig. 4 Bi-plot obtained with principal component analysis (PCA) of Cd mass fractions in plant organs of different durum wheat cultivars
FTB-58-49-f4
Table 4 The results of principal component analysis (PCA) of plant organ (underground parts, stems and grains) contribution to Cd accumulation in durum wheat cultivars
Plant organPC1PC2
Underground parts (root and stubble)43.513.19
Stem38.8217.10
Grain17.6879.72

CONCLUSIONS

In a nutshell, Cd is released into the environment in many ways, including the use of intensive phosphate fertilizers, sewage sludge and fossil fuel combustion in addition to natural Cd sources, and therefore Cd contamination of the soils has increased worldwide. In recent years, lower accumulation of Cd has been a breeding priority in addition to other quality traits especially in Cd-contaminated soils, and many wheat varieties have been developed with marker-assisted breeding. In this study, molecular analysis showed that 24 durum wheat cultivars, one emmer wheat and four wild emmer genotypes accumulated high mass fractions of Cd, while 68 genotypes had the allele associated with low Cd accumulation. Moreover, these molecular findings were supported by elemental analyses performed after pot experiment using a small set of 14 cultivars. In conclusion, since chemical or elemental analyses are expensive and time consuming for selection of genotypes with low levels of Cd, marker-assisted studies can be effectively used for both selection and introgression of Cdu1 alleles to adapted common durum wheat cultivars with low grain Cd content.

Notes

[1] Financial disclosure This study was financially supported by The Scientific Research Projects Unit of Akdeniz University (grant number: FBA-2017-2402).

[2] Conflicts of interest The authors declare that they have no any conflicts of interest.

SUPPLEMENTARY MATERIAL

All supplementary material is available at www.ftb.com.hr.

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Floating objects

Table S1 Accumulation of cadmium and other elements in durum wheat grain (control samples and samples grown in soil with the addition of w(Cd)=8 mg/kg (dose))
Cultivarw/(mg/kg)
CdPKCaMgFeZnCuMn
ControlDoseControlDoseControlDoseControlDoseControlDoseControlDoseControlDoseControlDoseControlDose
Ege-880.180.302175.102394.678136.335296.00606.80358.10812.30854.3332.8920.4435.8927.813.942.3818.3319.58
Amanos-970.190.122684.672429.675246.005020.67429.60408.93869.00984.6321.5139.8437.5541.473.2310.7528.4530.57
Sarıçanak 980.150.132262.002408.675123.674813.67341.77346.87804.40984.6314.6224.5422.3027.092.273.2117.4923.40
Şölen 20020.130.242275.672240.006685.675907.33294.07379.00869.83946.8013.7532.7530.9129.683.842.9919.2224.68
Turabi0.100.402097.001936.005926.336598.67481.53525.33865.10841.1731.5427.0332.2725.754.924.4323.1929.04
Svevo0.270.652673.672420.006119.335406.33416.33492.03912.30904.0019.7613.1528.3026.112.922.4021.3723.25
Zenit0.210.462728.672448.007261.679801.67428.37551.10989.83728.2015.0128.3233.2726.473.232.6014.9420.51
Fırat-930.350.532298.002602.007208.677093.00429.17316.97965.43949.3318.7327.6034.5131.192.483.6516.4619.87
Fuatbey 20000.320.592371.672507.006500.677341.33336.83338.97877.50877.4716.4620.6128.5729.343.293.1216.5921.09
GAP0.280.472081.332535.334668.335177.33307.67371.13812.50870.0720.0524.1424.3825.452.082.4917.4621.70
Gediz 750.360.422113.672161.676691.005862.67348.40502.87811.43771.1717.4630.6032.7121.572.382.8415.3918.07
Tüten 20020.370.551997.671771.335074.005643.33246.97382.43844.33862.7017.0925.7525.9828.811.941.9818.3725.37
Diyarbakır0.380.911915.672074.334742.675819.33429.30457.17974.90768.1720.4123.7429.6232.442.002.3614.6125.16
Levante0.360.72169.672033.337278.008852.33418.27476.07803.03845.9322.3935.9526.0526.562.213.6213.3016.63
F (C)97.96**7.85**68.21**12.12**5.44**21.76**20.36**62.28**32.36**
F (C × T)28.12**2.13*21.65**7.89**9.20**26.92**8.45**44.85**3.41**
LSD (0.05)0.11250.91868.8980.9086.336.233.741.402.27

C=cultivar, T=treatment; *p<0.05, **p<0.01

Table S2 Accumulation of cadmium and other elements in durum wheat stem (control samples and samples grown in soil with the addition of w(Cd)=8 mg/kg (dose))
Cultivarw/(mg/kg)
CdPKCaMgFeZnCuMn
ControlDoseControlDoseControlDoseControlDoseControlDoseControlDoseControlDoseControlDoseControlDose
Ege-880.662.341124.331117.1015320.0013540.004072.003974.331197.67913.93124.47154.5499.22111.371.230.9420.9724.31
Amanos-970.701.201046.001533.3312613.3312830.004600.333922.33896.10977.90100.15148.7053.9657.300.703.6937.2532.62
Sarıçanak 980.571.20749.43727.9712550.0012370.003866.674234.001290.001272.33134.77142.9799.2470.623.733.5224.5527.96
Şölen 20020.502.23302.97251.7013156.6713443.332846.673722.00865.10987.1784.83108.7265.5972.122.813.1129.0446.75
Turabi0.571.78273.90302.1314536.6712846.333484.003454.00806.80875.9388.04199.2061.3547.812.131.2823.7340.12
Svevo0.563.50529.67437.9714973.3314560.003800.004665.67999.731041.77129.85120.9381.0297.141.063.1334.3553.43
Zenit0.631.67498.70494.7011513.3314066.673227.003572.00774.67633.70103.84109.83166.0942.963.974.8117.5526.66
Fırat-930.701.64247.00308.3014190.0013906.673367.004838.33624.17834.80140.30150.0753.9171.393.684.0616.5833.41
Fuatbey 20000.612.07330.30500.7317786.6718696.673716.003473.33760.63705.87104.54138.3649.4068.491.211.8616.9030.52
GAP0.821.33193.17243.7315181.0016236.673789.334537.67915.90911.6098.73181.4381.4579.893.895.6312.5135.60
Gediz 750.621.55342.00601.2713330.0015946.673187.003927.00875.001048.67135.00189.7348.7970.971.382.0714.7333.68
Tüten 20020.482.61360.43643.9712563.3318936.673728.336710.67804.731198.67146.10159.8461.3986.370.233.1527.4238.33
Diyarbakır0.601.71288.07472.2313506.6715360.003796.674892.33875.071121.30127.23151.2471.7792.440.622.8917.0435.24
Levante0.651.36277.40366.5315548.0017310.003466.674367.33640.53878.73150.07147.1051.8071.782.914.3016.1825.70
F (C)36.06**254.46**48.18**275.06**141.40**19.51**106.87**69.26**663.40**
F (C × T)48.83**15.22**19.72**16.56**10.36**17.25**126.73**17.81**76.58**
LSD (0.05)0.43146.581382.00609.37124.1822.9723.460.8122.97

C=cultivar, T=treatment; *p<0.05, **p<0.01

Fig. S1 Results of PCR analysis of alleles of: a-c) durum wheat cultivars, and d-f) lines associated with the accumulation of Cd obtained from usw47 marker
FTB-58-49-fS.1
Fig. S2 Results of PCR analysis of alleles of: a-c) emmer and d) wild emmer genotypes associated with the accumulation of Cd obtained from usw47 marker
FTB-58-49-fS.2

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