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3.3 Materials and Methods
3.3.1 Plant material
The seed material consisted of 19 European breeding lines and open pollination (OP) cultivars of winter oilseed rape with different quality background, ranging from double zero (0,0) to double high (+,+) quality (Table 3.1). All cultivars are inbred lines except Visby that is a hybrid cultivar. The line SGDH14 is a doubled haploid line that was developed based on F1-microspore culture technic from a cross between the old German cultivar Sollux and the Chinese cultivar Gaoyou (Zhao et al. 2005). Furthermore, SGEDH13 is a doubled haploid line derived of a cross between line SGDH14 and the cultivar Express. The seed material was obtained from breeding companies and the Department of Crop Sciences at Göttingen University.
3.3 Materials and Methods 19 breeding lines and cultivars
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Table 3.1: Name and features of the 19 breeding lines and cultivars of winter oilseed rape
Genotype name Seed quality Genetic background
Adriana
00 Line cultivar
Akela 00 Line cultivar
Apex
00 Line cultivar
Express 617 00 Line cultivar, inbred line
Gaoyou
00 less than 2% erucic acid in the oil and less than 25 μmolg-1 glucosinolates in the seed 0+ less than 2% erucic acid in the oil and more than 25 μmolg-1 glucosinolates in the seed ++ more than 2% erucic acid in the oil and more than 25 μmolg-1 glucosinolates in the seed
3.3 Materials and Methods 19 breeding lines and cultivars
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3.3.2 Field sown experiments
All 19 breeding lines and cultivars, named 19 genotypes in this study, were phenotypically tested in field sown experiments in the two different environments: autumn sown and spring sown. Since the two environments diffed, they were named the two mega environments.
3.3.2.1 Autumn sown environment
The seed material of 19 genotypes were sown in North-Western Germany at four locations with two replicates during growing seasons 2014/15 and no replicate in 2015/16. In 2014/15 the locations were Peine (Limagrain GmbH), sown on 21 August, and Einbeck (KWS Saat SE), sown on 4 September. In 2015/16 the locations were Peine (Limagrain GmbH), sown on 20 August, and Göttingen-Reinshof, sown on 28 August. Hundred seeds from each line were sown in small field-plots with double rows in Peine and Göttingen with 2 m length, 0.5 m space between plots and plant-to-plant distance in the row was 10 cm. In Einbeck, seeds were sown as one row with 3 m length and 0.8 m space between the rows and plant-to-plant distance in the row was 6 cm. All agronomic practices, such as fertilizer, herbicide and insecticides were applied at each location according to common practices. Three to four months after sowing date, five representative plants were harvested by cutting the stem below the root neck (crown). Harvesting time in 2014/15 for Peine and Einbeck were 8 and 24 December 2014 and in 2015/16 for Peine and Göttingen were 30 November 2015 and 11 January 2016, respectively. Shoot length from root neck (crown) to shoot apex and shoot diameter at the root neck were measured using a slide gauge and a metering rule and they were called in this study shoot length before winter and shoot diameter before winter.
3.3.2.2 Spring sown environment
The seed material of 19 genotypes were sown with two replicates at four location in 2013 and 2014 and with no replicate at two locations in 2015. Four locations were Göttingen-Reinshof, sown on 19 and 30 April 2013, 20 March 2014 and 24 March 2015 (no replicate). Two locations were in Einbeck (KWS Saat SE), sown on 4 April 2014 and 10 April 2015 (no replicate). All
3.3 Materials and Methods 19 breeding lines and cultivars
22 plants were grown by sowing 100 seeds in 2 m long double rows and 0.5 m space between plots with 10 cm distance between plants in the row in Göttingen and 3 m single row and 8 cm space in Einbeck. Three to four months after sowing date (Göttingen 18.07.2013, 7.08.2013, 02.07.2014, 26.06.2016; Einbeck 06.07.2014, 13.07.2015), seven representative plants were harvested by cutting the stem below the root neck (crown). Shoot length from root neck to shoot apex and shoot diameter, at the root neck, were measured using metering rule and slide gauge.
Percentage of tendency to form inflorescence was scored as 100% for visible buds or flower and 0% for lack of buds in each of the seven plants.
3.3.3 Statistical analysis
Analysis of variance and descriptive statistics were done by PLABSTAT (Utz 2011) and R (i386 3.0.3). Analysis of variance was done for the autumn sown and spring sown environment, separately. In each mega environment, location and year were defined as experiment, meaning four experiments in the autumn sown environment and six experiments in the spring sown environment. Due to unbalance replicates at different locations, genotype’s mean over replicates were used for ANOVA. Linear mixed-effect model (R package {nlme} version 3.1-125) was applied to test significant difference for the studied traits. Therefore, experiment and sample plants were random factor versus, genotypes were fixed factor. The statistic model used for ANOVA is shown as follow:
Xijk = µ + gi + ej + giej + pk:giej
where Xijk is phenotypic observation of genotype i in experiment j and plant k, µ is overall mean, gi and ej are effects of genotype i and experiment j, respectively, giej is residual error to test gi and ej andpk:giej is sampling residual error to test giej effect. Broad sense heritability (h2) of genotypes mean over experiments was calculated by following equations suggested by Hill et al.
(1998)
h2 = 2G / (2G + 2GE/E+2p:ge/EP)
3.3 Materials and Methods 19 breeding lines and cultivars
23 where 2G and 2GE are genetic and genetic xexperiment variance components, respectively. E is number of experiments and P is number of sample plants. Least significant difference (LSD) was utilized for mean comparisons at P<0.05 for shoot length before winter and shoot length in the spring sown environment. Mean values of the genotypes across the experiments were used to calculate Spearman’s rank correlation coefficients between the traits that was done by R software (i386 3.0.3).
Results 19 breeding lines and cultivars
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