Material and methods

To study natural variation for selected seed constituent traits, a collection of 237 chickpea genotypes (180 desi, 49 kabuli and 8 pea shaped) was procured from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT, Patancheru, India). These genotypes were collected from 26 countries covering four continents throughout the world (Appendix 4).

Chickpea genotypes were grown in three trials with two replications each: two field trials in 2011 (F 2011) at ICRISAT (17 ° 53 ′ N latitude, 78 ° 27 ′ E longitude and 545 m altitude, Patancheru, India), in 2013 (F 2013) at Aberdeen (52 ° 34 ′ N latitude, 106 ° 29 ′ W longitude and 517 m altitude, SK, Canada) whereas one greenhouse (GH) trial in 2012 (G 2012) at University of Saskatchewan (52 ° 07 ′ N latitude, 106 ° 38 ′ W longitude and 481.5 m altitude, Saskatoon, SK, Canada). Field experiments were performed in a randomized complete bock design during October to mid-March and June to mid-September at ICRISAT and Aberdeen, respectively. Seeds of each lines were sown with 10 and 30 cm spacing between plants and rows, respectively. Fertilizers (22 kg/ha P2O5 and 40 kg/ha N) were applied before seeding. For

F 2011, mean minimum and maximum temperatures were 15.5 and 31.7 °C, respectively, with 7.2 mm of average precipitation during the growing season. The mean minimum and maximum temperatures were 11.1 and 23.0 °C, respectively, for F 2013 with a mean precipitation of 2.3 mm. In the GH, average daily minimum and maximum temperatures were 18 and 23 °C with an 18 h photoperiod. Plants were harvested and seeds were stored at room temperature. All genotypes were grown in two blocks. In each block, 50 seeds of one genotype were sown in a single row. When harvested, all seeds from the 50 plants of the same genotype were bulked. For each genotype, the selected seed constituent traits were analyzed two times for each block, giving two biological replicates for each block. For each biological replicate in each block, two technical replicates were analyzed, giving eight technical replicates in total. Field trial of 2011

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was carried out by researchers and employees at ICRISAT and seeds were sent to the University of Saskatchewan for analysis.

3.3.2 Seed weight analysis

One hundred seeds of each genotype were counted using electronic seed counter (Seedburo Equipment Co., Chicago, IL, USA) and weighed. The weight was multiplied by ten to obtain one thousand seed weight (TSW).

3.3.3 Grinding of seed material

Chickpea seeds (about 10 g) were ground into a fine meal using a UDY cyclone mill (Udy Corporation, Fort Collins, CO, USA) to pass through a 0.5 mm sieve. The seed meal was collected, stored at room temperature and used to determine total starch, amylose and protein concentration.

3.3.4 Determination of total starch concentration

Total starch concentration in chickpea seed meal (100 ± 0.5 mg) was determined by an enzymatic hydrolysis method using a commercial kit (Megazyme International Ireland Ltd., Wicklow, Ireland) (McCleary et al., 1997). Starch was sequentially hydrolyzed into dextrins and finally to D-glucose using α-amylase and amyloglucosidase, respectively. The D-glucose was treated with glucose oxidase/peroxidase reagent (GOPOD) producing red quinoneimine, the concentration of which was determined at A510 nm.

3.3.5 Amylose determination

Starch was purified, prior to amylose determination, from chickpea seed meal following a modified method (Peng et al., 1999) using cesium chloride (CsCl) density gradient centrifugation. Chickpea seed meal (200 ± 1 mg) was suspended in distilled water (5 mL), vortexed, filtered into a 15 mL disposable tube. The disposable tube was centrifuged at 3,000 × g for 10 min and supernatant was discarded. The sediment was placed on the top of cesium chloride (1 mL of 80 % v/v solution) in a 2 mL centrifuge tube. The tube was centrifuged at 16,000 ×g for 30 min. Supernatant was discarded; and the pellet was washed sequentially with distilled water, wash buffer [contained 55 mL of Tris-HCl (1 M; pH 6.8), sodium dodecyl sulfate (23 g), glycerol (100 mL) and distilled water (final volume up to 1 L)] and distilled water (twice) at 16,000 × g for 10 min. The pellet was finally washed with acetone (1 mL), centrifuged at 16,000 × g for 10 min and air-dried at room temperature.

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The purified starch was used to determine amylose concentration using an iodine based method with some modifications (Mahmooda et al., 2007); therefore, it is expressed as percentage of total starch. In brief, purified starch (5 mg) was weighed in a 2 mL centrifuge tube. The starch was sequentially suspended in 95% (v/v) ethanol (75 µL), 1 M NaOH (450 µL) and distilled water with proper shaking before adding the next solution. The mixture was mixed well and incubated at room temperature for 1 h. Thereafter, an aliquot (200 µL) was taken out in a 15 mL disposable tube and neutralized with 0.05 M citric acid (1 mL), followed by addition of 800 µL of I2/KI solution [0.8 g iodine (I2) and 8 g potassium iodide (KI) in 1 L

of distilled water]. The mixture was mixed well and the volume was made up to 12 mL with distilled water. The absorbance was observed at 535 (reddish brown) and 620 (blue) nm for amylopectin and amylose, respectively.

3.3.6 Estimation of protein concentration

Total nitrogen was determined by combustion method (FP-528 Protein/Nitrogen Analyser, Leco Corporation, St Joseph, MI, USA) (Frimpong et al., 2009). Protein concentration was calculated by using the following formula (Karaca et al., 2011):

Protein (%) = % N × nitrogen to protein conversion factor (6.25 for chickpea seeds)……...(3.1)

3.3.7 Statistical analysis

Shannon−Weaver diversity index (SDI) was calculated as

SDI = (− ∑𝑛_𝑖=1𝑃_𝑖× log_𝑒𝑃_𝑖)/ log_𝑒𝑛 ………..……(3.2) where, n represents the total number of phenotypic classes, and Pi is the proportion of total

number of entries in the ith _{class (Bhattacharjee et al., 2007). Statistical analysis including box-}

plots, analysis of variance (using general linear model) and Pearson’s correlation coefficients, was executed using MINITAB 14 statistical software (Minitab Inc., State College, PA, USA). Phenotypic classes were prepared by using MINITAB 14 statistical software (Minitab Inc., State College, PA, USA). Mixed model was used to calculate analysis of variance (ANOVA) in MINITAB 14 and resulted F values from ANOVA was utilized to determine the significance of the influences from genotype (G), environment (E) and their interaction (G × E). Covariance estimates of variance components were used to calculate heritability (H2_{) as described by Singh}

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