**2. Materials and methods**

### **2.1 Plant material**

For the evaluation there were used 46 amaranth genotypes from Crop Research Institute in Prague, Czech Republic (CRI) and from USDA, ARS, NCRPIS Iowa State University. In these samples, there were 6 accessions of wild weed and 40 of the cultivated species. The acronyms used for the wild species were as follows: De - *A. deflexus*, Au - *A. australis*, Wr - *A. wrightii*, Tu - *A. tuberculatus*, Cn - *A. cannabinus*, Re - *A. retroflexus*. The cultivated samples were evaluated in the field conditions in 2008 and 2009 according to the list of descriptors for amaranths created for purposes of the Czech Gene Bank. The morphological and phenological characters are evaluated according to List of Descriptors for amaranth created in the Czech Gene Bank. Following traits were evaluated in the field conditions:


The first two traits were assessed when 50% of plants were in this stage. The numbers of day from emergence to inflorescence observation and the numbers of days from emergence to flowering are important characters due to fact, that certain amaranth genotypes are sensitive to day-length. Maturity was estimated when 75% of the grains were mature. Plant height was measured from the soil surface to the top of the main stem in cm. Length of inflorescence was measured from the downmost branch to the top of inflorescence of the main stem in cm. Weight of thousand seeds (WTS) was weight of thousand seeds in g.

#### **2.2 Total seed protein content and protein fractions content determination**

The measurements of total seed protein content and protein fraction content were performed by the Kjeldahl method (Czech state norm 56 0512-12) in Kjeltec automatic analyzer (Kjeltec 2300, Foss Tecator, Sweden) with the protein-nitrogen coefficient set to 6.025. Protein fractions (albumins, globulins, prolamins and total glutelins) were extracted according to the protocol developed for the wheat protein fraction separation by Dvoracek (2006) with some modifications. For the determination of protein fractions content was used 0.5 g of milled amaranth seeds. The protein fractions were extracted by adding 5 ml of solvent (distilled water for albumins, 0.5 M NaCl for globulins, cold 60% ethanol for prolamins), vortexing and centrifuging by 10 000 × g for 15 minutes (Universal 32R HettichCentrifugen, Germany). This procedure was repeated twice and the supernatants from each extraction were saved and poured together. In the case of prolamins, after first addition of solvent, tubes were vortexed and chilled to 4°C for 4 hours; after that the procedure was performed exactly as for albumins and globulins. The protein content of whole seed was also measured by milling 1g of amaranth seeds. For the boiling in the automatic digestion system (2015lift, 2020 digestor, Foss Tecator, Denmark) were used 10 ml from the obtained 15 ml of each fraction extract. Into the each 250 ml tube one catalyser tablet, 3.5 g of K2SO4 and CuSO4 mixture and 10 ml of H2SO4 were added. In one tube was a blank sample. Tubes were let to boil to the temperature of 420°C for about 1 h 40 min. After cooling for about 10 min, 75 ml of distilled water was added. The content of glutelin and the residual nitrogen fraction was calculated as the difference between the content of the total seed protein and three measured fractions.

### **2.3 Electrophoresis of the proteins**

462 Genetic Diversity in Plants

For the evaluation there were used 46 amaranth genotypes from Crop Research Institute in Prague, Czech Republic (CRI) and from USDA, ARS, NCRPIS Iowa State University. In these samples, there were 6 accessions of wild weed and 40 of the cultivated species. The acronyms used for the wild species were as follows: De - *A. deflexus*, Au - *A. australis*, Wr - *A. wrightii*, Tu - *A. tuberculatus*, Cn - *A. cannabinus*, Re - *A. retroflexus*. The cultivated samples were evaluated in the field conditions in 2008 and 2009 according to the list of descriptors for amaranths created for purposes of the Czech Gene Bank. The morphological and phenological characters are evaluated according to List of Descriptors for amaranth created

The first two traits were assessed when 50% of plants were in this stage. The numbers of day from emergence to inflorescence observation and the numbers of days from emergence to flowering are important characters due to fact, that certain amaranth genotypes are sensitive to day-length. Maturity was estimated when 75% of the grains were mature. Plant height was measured from the soil surface to the top of the main stem in cm. Length of inflorescence was measured from the downmost branch to the top of inflorescence of the main stem in cm. Weight of thousand seeds (WTS) was weight of thousand seeds in g.

The measurements of total seed protein content and protein fraction content were performed by the Kjeldahl method (Czech state norm 56 0512-12) in Kjeltec automatic analyzer (Kjeltec 2300, Foss Tecator, Sweden) with the protein-nitrogen coefficient set to 6.025. Protein fractions (albumins, globulins, prolamins and total glutelins) were extracted according to the protocol developed for the wheat protein fraction separation by Dvoracek (2006) with some modifications. For the determination of protein fractions content was used 0.5 g of milled amaranth seeds. The protein fractions were extracted by adding 5 ml of solvent (distilled water for albumins, 0.5 M NaCl for globulins, cold 60% ethanol for prolamins), vortexing and centrifuging by 10 000 × g for 15 minutes (Universal 32R HettichCentrifugen, Germany). This procedure was repeated twice and the supernatants from each extraction were saved and poured together. In the case of prolamins, after first addition of solvent, tubes were vortexed and chilled to 4°C for 4 hours; after that the procedure was performed exactly as for albumins and globulins. The protein content of whole seed was also measured by milling 1g of amaranth seeds. For the boiling in the automatic digestion system (2015lift, 2020 digestor, Foss Tecator, Denmark) were used 10 ml from the obtained 15 ml of each fraction extract. Into the each 250 ml tube one catalyser tablet, 3.5 g of K2SO4 and CuSO4 mixture and 10 ml of H2SO4 were added. In one tube was a blank sample. Tubes were let to boil to the temperature of 420°C for about 1 h 40 min. After cooling for about 10 min, 75 ml of distilled water was added. The content of glutelin and the residual nitrogen fraction was calculated as the difference between the content of the total

in the Czech Gene Bank. Following traits were evaluated in the field conditions:

**2.2 Total seed protein content and protein fractions content determination** 

number of days from emergence to inflorescence observation,

 number of days from emergence to flowering, number of days from emergence to maturity.

seed protein and three measured fractions.

**2. Materials and methods** 

**2.1 Plant material** 

#### **2.3.1 Extraction of the total seed storage proteins**

Five different approaches to the extraction were tested for the development of the best extraction approach:


Seed samples were crushed separately and mixed with extraction solution (consisted of 0.0625 M Tris-HCl pH 8.8, 5% (w/v) 2- mercaptoethanol, 2% (w/v) SDS, 10% (w/v) glycerol, 0.01% (w/v) bromphenol blue) by vortexing (MS2 Minishaker, IKA, Germany) several times in 1.5 ml tubes. Tubes were allowed to stand at 4 °C for three hours. After this extraction time, the tubes were centrifuged at 12 000 x g for 15 min (Universal 32R HettichCentrifugen, Germany). After the replacement of the samples to the new tubes, the samples were heated in a boiling water bath for 2 min.

Ten seeds from each variety were selected randomly, crushed and put into 2 ml micro tube. The protein fractions were extracted by adding 100 µl of solvent (distilled water for albumins, 0.5 M NaCl for globulins, cold 60% ethanol for prolamins), vortexing and centrifuging by 10 000 × g for 15 minutes (Universal 32R HettichCentrifugen, Germany). This procedure was repeated twice but the supernatants of the second and third wash were always discarded. In the case of prolamins, after first addition of solvent, tubes were vortexed and chilled to 4°C for 4 hours; after that the procedure was performed as in the case of albumins and globulins. Tubes containing protein fractions extract and the seed pellets (glutelins) were freezed to -25°C. After the supernatant in the tubes became solid, the top of the tubes was perforated by a needle to form small holes what serve to prevent the loss of the sample by lyofilisation. The lyofilisation was performed by freeze dryer (Christ, Germany) during 24 h at -58°C and 0.018 mBar. The lyophilized solid samples were mixed with 100 µl extraction solution (consisted of 0.0625 M Tris-HCl pH 8.8, 5% (w/v) 2 mercaptoethanol, 2% (w/v) SDS, 10% (w/v) glycerol, 0.01% (w/v) bromphenol blue) by vortexing several times in 1.5 ml tubes. Tubes were allowed to stand at 4 °C for three hours. After this extraction time, the tubes were centrifuged at 12000 x g for 15 min. The supernatants were put into new tubes and heated in boiling water for 2 min.

#### **2.3.2 Protein separation by SDS PAGE**

The amaranth protein extracts were separated in conditions of discontinuous electrophoresis (SDS-PAGE) according to Laemmli (1970) 4% stacking gel of pH 6.8, 10% separation gel of pH 8.8 on the polyacrylamide gels of the size 180 x 160 x 0.75 mm.

On the gel was loaded:


The electrophoresis was performed on 90 mA (45 mA / gel) and let to run for about 4 hours. The gels were stained with a solution of 0.1% (w/v) Coomasie Brilliant Blue (CBB) R250, 50% (w/v) methanol, 10% acetic acid, 0.02% (w/v) bromphenol blue salt for 1 day and destained with a solution of 25% (w/v) denatured alcohol and 3.5% (w/v) acetic acid, what lasted also 1 day. Gels were preserved in solution: 45% (w/v) denatured alcohol, 3% (w/v) glycerol for 2 hours, then dried and stored into cellophane sheets. The whole procedure including the test of the different extraction concentrations, the protein fraction separation procedure and the electrophoresis was repeated for the control of the correct experiment performance.

#### **2.3.3 Chip electrophoresis**

All the extracted protein fraction samples were analyzed by chip capillary electrophoresis using commercial Experion Pro260 Analysis Kit for 10 Chips and the Experion automated electrophoresis system (Bio-Rad Laboratories, USA) for protein quantification according to the manufacturer's instructions. Experion automated electrophoresis station performs automatically all the steps of the gel-based electrophoresis (samples separation, staining, destaining, imaging, band detections, and data analysis).

#### **2.4 Statistical analysis**

For the statistical evaluation of morphological traits, analysis of variance (ANOVA) and the Tukey HSD test were used (software -Statistica 7.0 CZ). In the case of protein fraction proportion in accessions with different seed colour, the basic statistics of R statistics 2.10.0 software were used for calculation of mean x, standard deviation sx and p-values (adjusted by Holm correction, two sided Welch Two Sample t-test used).

The SDS-PAGE spectra of total seed storage proteins and protein fractions were compared and confronted with the spectra of the chip capillary electrophoresis. The bands in the spectra were analyzed regarding the positions of the bands and also the relative intensity of the bands. The intensity of the bands was analyzed individually for each sample considering the intensity of the internal markers of the chip electrophoresis and the general intensity of all the bands in the sample. The intensity of the bands was expressed as the relative protein concentration measured by chip capillary electrophoresis what was the multiplication of numbers 0, 1, 2, 3 used in our statistics (0– no band, 1- light band, 2 – medium intensity band, 3 – dark band). The spectra expressed as the numerical values were analyzed by R statistics 2.10.0 software. The relationships between accessions were expressed by Pearson correlation using single linkage. The hierarchical clustering dendrogram was cut at the level of correlation 0.99 to show the well defined clusters.
