Heiko C. Becker

Conditional QTL mapping of oil content in rapeseed with respect to protein content and traits related to plant development and grain yield

Oil content in rapeseed (Brassica napus L.) is generally regarded as a character with high heritability that is negatively correlated with protein content and influenced by plant developmental and yield related traits. To evaluate possible genetic interrelationships between these traits and oil content, QTL for oil content were mapped using data on oil content and on oil content conditioned on the putatively interrelated traits. Phenotypic data were evaluated in a segregating doubled haploid population of 282 lines derived from the F1 of a cross between the old German cultivar Sollux and the Chinese cultivar Gaoyou. The material was tested at four locations, two each in Germany and in China. QTLMapper version 1.0 was used for mapping unconditional and conditional QTL with additive (a) and locus pairs with additive × additive epistatic (aa) effects. Clear evidence was found for a strong genetic relationship between oil and protein content. Six QTL and nine epistatic locus pairs were found, which had pleiotropic effects on both traits. Nevertheless, two QTL were also identified, which control oil content independent from protein content and which could be used in practical breeding programs to increase oil content without affecting seed protein content. In addition, six additional QTL with small effects were only identified in the conditional mapping. Some evidence was apparent for a genetic interrelationship between oil content and the number of seeds per silique but no evidence was found for a genetic relationship between oil content and flowering time, grain filling period or single seed weight. The results indicate that for closely correlated traits conditional QTL mapping can be used to dissect the genetic interrelationship between two traits at the level of individual QTL. Furthermore, conditional QTL mapping can reveal additional QTL with small effects that are undetectable in unconditional mapping.

Genetic Analysis of Heterosis for Yield and Yield Components in Rapeseed (Brassica napus L.) by Quantitative Trait Locus Mapping

The main objective in this research was the genetic analysis of heterosis in rapeseed at the QTL level. A linkage map comprising 235 SSR and 144 AFLP markers covering 2045 cM was constructed in a doubled-haploid population from a cross between the cultivar “Express” and the resynthesized line “R53.” In field experiments at four locations in Germany 250 doubled-haploid (DH) lines and their corresponding testcrosses with Express were evaluated for grain yield and three yield components. The heterosis ranged from 30% for grain yield to 0.7% for kernel weight. QTL were mapped using three different data sets, allowing the estimation of additive and dominance effects as well as digenic epistatic interactions. In total, 33 QTL were detected, of which 10 showed significant dominance effects. For grain yield, mainly complete dominance or overdominance was observed, whereas the other traits showed mainly partial dominance. A large number of epistatic interactions were detected. It was concluded that epistasis together with all levels of dominance from partial to overdominance is responsible for the expression of heterosis in rapeseed.

Comparative mapping of quantitative trait loci involved in heterosis for seedling and yield traits in oilseed rape (Brassica napus L.)

Little is known about the genetic control of heterosis in the complex polyploid crop species oilseed rape (Brassica napus L.). In this study, two large doubled-haploid (DH) mapping populations and two corresponding sets of backcrossed test hybrids (THs) were analysed in controlled greenhouse experiments and extensive field trials for seedling biomass and yield performance traits, respectively. Genetic maps from the two populations, aligned with the help of common simple sequence repeat markers, were used to localise and compare quantitative trait loci (QTL) related to the expression of heterosis for seedling developmental traits, plant height at flowering, thousand seed mass, seeds per silique, siliques per unit area and seed yield. QTL were mapped using data from the respective DH populations, their corresponding TH populations and from mid-parent heterosis (MPH) data, allowing additive and dominance effects along with digenic epistatic interactions to be estimated. A number of genome regions containing numerous heterosis-related QTL involved in different traits and at different developmental stages were identified at corresponding map positions in the two populations. The co-localisation of per se QTL from the DH population datasets with heterosis-related QTL from the MPH data could indicate regulatory loci that may also contribute to fixed heterosis in the highly duplicated B. napus genome. Given the key role of epistatic interactions in the expression of heterosis in oilseed rape, these QTL hotspots might harbour genes involved in regulation of heterosis (including fixed heterosis) for different traits throughout the plant life cycle, including a significant overall influence on heterosis for seed yield.

Estimation of seed weight, oil content and fatty acid composition in intact single seeds of rapeseed ( Brassica napus} L.) by near-infrared reflectance spectroscopy

The potential of near-infrared reflectance spectroscopy (NIRS) for the simultaneous analysis of seed weight, total oil content and its fatty acid composition in intact single seeds of rapeseed was studied. A calibration set of 530 single seeds was analysed by both NIRS and gas-liquid chromatography (GLC) and calibration equations for the major fatty acids were developed. External validation with a set of 75 seeds demonstrated a close relationship between NIRS and GLC data for oleic (r = 0.92) and erucic acid (r = 0.94), but not for linoleic (r = 0.75) and linolenic acid (r = 0.73). Calibration equations for seed weight and oil content were developed from a calibration set of 125 seeds. A gravimetric determination was used as reference method for oil content. External validation revealed a coefficient of correlation between NIRS and reference methods of 0.92 for both traits. The performance of the calibration equations for oleic and erucic acid was further studied by analysing two segregating F2 seed populations not represented in the calibration set. The results demonstrated that a reliable selection for both fatty acids in segregating populations can be made by using NIRS. We concluded that a reliable estimation of seed weight, oil content, oleic acid and erucic acid content in intact, single seeds of rapeseed is possible by using NIRS technique.

Genotypic diversity and patterns of variation in a germplasm material of Ethiopian mustard (Brassica carinata A. Braun)

The amphidiploid species Brassica carinata A. Braun is believed to have originated in the plateaus of Ethiopia and has been cultivated there as an oilseed crop since antiquity. The species possesses agronomically important genes of rare occurrence. Although there is a large number of collections in Ethiopia, information on the extent of their genetic diversity is very limited. Thirty-six accessions of ecologically diverse regions were tested at three locations and multivariate analyses on 13 morphological and seed characters were performed. There was generally a large amount of divergence in all characters. Geographic isolation of genes, however, was not observed. Both principal component and cluster analyses disclosed complex relationships among the accessions and characters. Accessions with potential genes of interest to improve earliness, yield components and oil and protein contents have been identified. Length of growing period and yield components contributed most for divergence and clustering pattern. Oil, glucosinolate and protein contents also varied among the accessions but not so much between clusters. Increasing protein in high-glucosinolate genotypes reduced oil content and should be more efficient in genotypes of low-glucosinolate genetic background.

Variability for the fatty acid composition of the seed oil in a germplasm collection of the genus Brassica

A germplasm collection consisting of 1475 entries from 21 species of Brassica, including 36 lower taxa, was evaluated for the fatty acid composition of the seed oil. A total of 358 entries representing the taxonomic variability in the collection were selected and analysed by gas-liquid chromatography (GLC). The remaining 1117 entries were analysed by near-infrared reflectance spectroscopy (NIRS), after developing multi-species calibration equations. The results demonstrated that NIRS is an effective technique to assess variability for oleic, linoleic, linolenic and erucic acid in intact-seed samples of multiple Brassica species, provided that calibration equations be developed from sets containing large taxonomic and chemical variability. Some fatty acid ratios were used to estimate the efficiency of the different biosynthetic pathways. Two well-defined patterns were observed. The first one was characterised by high elongation efficiency and accumulation of high levels of erucic acid. The highest erucic acid content (>55% of the total fatty acids) was found in the cultivated species B. napus L., B. oleracea L., and B. rapa L., and in the wild species B. incana Tenore, B. rupestris Raf., and B. villosa Bivona-Bernardi, the three latter belonging to the B. oleracea group (n=9). The second pattern was characterised by high desaturation efficiency, resulting in the accumulation of high levels of the polyunsaturated linoleic and linolenic acid (up to more than 55%). The highest levels of these fatty acids were found in samples of B. elongata Ehrh., especially of the var. integrifolia Boiss. The utility of the reported variability for plant breeding is discussed.

Estimating the fatty acid composition of the oil in intact-seed rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy

The objective of this work was to evaluate the potential of near-infrared reflectance spectroscopy (NIRS) as a rapid method to estimate the fatty acid composition of the oil in intact-seed samples of rapeseed. A total of 549 samples (3 g intact seed) from selected mutant and breeding lines were scanned by NIRS, and 220 of them were selected and scanned again by using two different adapters, which reduced the sample size to 300 and 60 mg, respectively. Selected samples were analysed by gas liquid chromatography and calibration equations for individual fatty acids were developed. Calibrations for oleic, linoleic, linolenic, and erucic acid were highly accurate, with values of r2 in cross validation from 0.95 to 0.98 (samples of 3 g), from 0.93 to 0.97 (300 mg), and from 0.84 to 0.96 (60 mg). Calibrations for palmitic and stearic acid were less accurate, with values of r2 in cross validation always lower than 0.8, probably because of the narrow range available for these fatty acids. The accuracy of the calibration equations for eicosenoic acid was very low (r2 = 0.69 in 3 g samples), although improved equations were developed (r2 from 0.78 to 0.91) when the relationship between erucic and eicosenoic acid was taken into account. We conclude that NIRS is a powerful technique to estimate the fatty acid composition of the oil in rapeseed, provided that samples covering a wide range of fatty acid levels are available, with the advantage that such estimation is possible with few additional costs when NIRS is used for the determination of other seed quality traits.

Mapping a high oleic acid mutation in winter oilseed rape (Brassica napus L.)

Abstract Two winter oilseed rape mutant lines, 7488 and 19661, with a high oleic (HO) acid content in the seed oil were characterized phenotypically. In both mutant lines the HO trait was monogenically inherited. Segregation analysis in an F2 population derived from a cross between 7488 and 19661 showed the two mutations to be allelic. From a comparison of seed, leaf and root fatty acid composition it was concluded that fad2, the endoplasmic oleic acid desaturase, is affected by the mutation. In a bulked segregant analysis three AFLP markers linked to this mutation were detected and localized on the genetic map of Brassica napus. The markers mapped near the locus of one copy of the fad2 gene in the rapeseed genome.

Comparison of phenotypic and molecular distances to predict heterosis and F1 performance in Ethiopian mustard (Brassica carinata A. Braun)

Predicting heterosis and F1 performance from the parental generation could largely enhance the efficiency of breeding hybrid or synthetic cultivars. This study was undertaken to determine the relationship between parental distances estimated from phenotypic traits or molecular markers with heterosis, F1 performance and general combining ability (GCA) in Ethiopian mustard (Brassica carinata). Nine inbred lines representing seven different geographic regions of Ethiopia were crossed in half-diallel. The nine parents along with their 36 F1s were evaluated in a replicated field trail at three locations in Ethiopia. Distances among the parents were calculated from 14 phenotypic traits (Euclidean distance, ED) and 182 random amplified polymorphic DNA (RAPD) markers (Jaccard’s distances, JD), and correlated with heterosis, F1 performance and GCA sum of parents (GCAsum). The correlation between phenotypic and molecular distances was low (r=0.34, P≤0.05). Parents with low molecular distance also had low phenotypic distance, but parents with high molecular distance had either high, intermediate or low phenotypic distance. Phenotypic distance was highly significantly correlated with mid-parent heterosis (r=0.53), F1 performance (r=0.61) and GCA (r=0.79) for seed yield. Phenotypic distance was also positively correlated with (1) heterosis, F1 performance and GCA for plant height and seeds plant−1, (2) heterosis for number of pods plant−1, and (3) F1 performance for 1,000 seed weight. Molecular distance was correlated with GCAsum (r=0.36, P≤0.05) but not significantly with heterosis and F1 performance for seed yield. For each parent a mean distance was calculated by averaging the distances to the eight other parents. Likewise, mean heterosis was estimated by averaging the heterosis obtained when each parent is crossed with the other eight. For seed yield, both mean ED and JD were significantly correlated with GCA (r=0.90, P≤0.01 for ED and r=0.68, P≤0.05 for JD) and mean heterosis (r=0.79, P≤0.05 for ED and r=0.77, P≤0.05 for JD). In conclusion, parental distances estimated from phenotypic traits better predicted heterosis, F1 performance and GCA than distances estimated from RAPD markers.

Reduction of sinapate ester content in transgenic oilseed rape (Brassica napus) by dsRNAi-based suppression of BnSGT1 gene expression

Seeds of oilseed rape (Brassica napus) accumulate high amounts of antinutritive sinapate esters (SE) with sinapoylcholine (sinapine) as major component, accompanied by sinapoylglucose. These phenolic compounds compromise the use of the protein-rich valuable seed meal. Hence, a substantial reduction of the SE content is considered essential for establishing rape as a protein crop. The present work focuses on the suppression of sinapine synthesis in rape. Therefore, rape (spring cultivar Drakkar) was transformed with a dsRNAi construct designed to silence seed-specifically the BnSGT1 gene encoding UDP-glucose:sinapate glucosyltransferase (SGT1). This resulted in a substantial decrease of SE content in T2 seeds with a reduction reaching 61%. In T2 seeds a high and significant correlation between the contents of sinapoylglucose and all other sinapate esters has been observed. Among transgenic plants, no significant difference in other important agronomic traits, such as oil, protein, fatty acid and glucosinolate content in comparison to the control plants was observed. Maximal reduction of total SE content by 76% was observed in seeds of one homozygous T2 plant (T3 seeds) carrying the BnSGT1 suppression cassette as a single copy insert. In conclusion, this study is an initial proof of principle that suppression of sinapoylglucose formation leads to a strong reduction of SE in rape seeds and is thus a promising approach in establishing rape, currently an important oil crop, as a protein crop as well.