Nondestructive assessment of protein content in single seeds of rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy

The potential of near-infrared reflectance spectroscopy (NIRS) to detectwithin-plant differences for seed protein content was investigated. Fourhundred and fifty-one single seeds were scanned by NIRS using a specialadapter. After non-destructive NIRS scanning, the seeds were analysed forprotein content by the Dumas combustion method and a calibrationequation was developed. A validation set of 117 additional seeds fromthree individual plants from the cultivars Bristol, Lirajet and Maplus wasanalysed for protein content both by NIRS and combustion. The coefficientof determination between NIRS and combustion values in the validation setwas 0.94, with a standard error of performance (SEP) of 0.77% and aratio of the SEP to the standard deviation (SD) of the validation set of0.28. The coefficient of variation (CV) for seed protein content inindividual plants, as determined by the combustion method, was 11.7%for Bristol, 8.9% for Lirajet, and 9.5% for Maplus. The comparison ofsuch variation with the standard error (SE) of NIRS analysis, defined as thecombination of the SE of the combustion method and the SEP of NIRScalibration equation, revealed that the maximum explainable variance withinindividual plants that can be detected using NIRS analysis of proteincontent in single seeds was 0.86 for Bristol, 0.83 for Lirajet, and 0.85 forMaplus. These results demonstrated that NIRS is a powerful tool fornon-destructive assessment of within-plant variation for seed protein contentin rapeseed.     

Mapping of QTL controlling tocopherol content in winter oilseed rape

Tocopherols are natural antioxidants in vegetable oils and are important dietary nutrients. Enhanced tocopherol content has become an important objective in oilseed rape breeding. A segregating DH population was tested for 2 years at two locations in replicated field trials. Genotypic differences occurred for α‐, γ‐ and total tocopherol content as well as α/γ‐tocopherol ratio, but highly significant genotype x environment interactions resulted in low heritabilities. Using a mixed‐model composite interval mapping approach between one and five QTL with additive and/or additive x environment interaction effects could be mapped for α‐, γ‐ and total tocopherol content and α/β‐tocopherol ratio. In addition, one to six locus pairs with epistatic interaction effects were identified, indicating a strong contribution of epistasis to trait variation. In total, the additive and epistatic effects explained between 28% (α‐tocopherol content) and 73% (total tocopherol content) of the genotypic variance in the population, with individual QTL and locus pairs contributing between 7.5 and 29.2% of variance. Considering the low heritabilities of the tocopherol traits, the results of this study indicate that marker‐assisted selection may be an efficient strategy in a breeding program for enhanced tocopherol content in rapeseed.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

The measurements of acid detergent fibre in rapeseed by visible and near-infrared spectroscopy

Visible and near‐infrared spectroscopy (NIRS) calibrations for acid detergent fibre (ADF) in intact rapeseed (Brassica napus L.) were performed for two different sample volumes (10 ml, 500 seeds approximately; and 1 ml, 50 seeds approximately). The inclusion of brown and yellow‐seeded cultivars in this work has allowed the whole range of ADF currently described in the literature for this character to be covered. Chemometric techniques have been used for developing calibration equations for both procedures when measuring the two different seed sample volumes. On the basis of the coefficient of determination in the cross‐validation (R²cv) obtained for the 10 and 1 ml assays (0.80 and 0.73), and SECV/SEL ratios (2.30 and 2.57), respectively, both equations showed an accuracy sufficient for screening purposes in an ADF range from 6.80 to 13.46% dry wt, which is presented in this work.     

Genetic variation for sinapate ester content in winter rapeseed (Brassica napus L.) and development of NIRS calibration equations

Increasing the meal and protein quality of winter rapeseed (Brassica napus L.) for food and feed purposes is gaining importance in rapeseed breeding programmes. Rapeseed meal has a high content of phenolic acid esters, mainly sinapate esters, which have been shown to cause a dark colour and a bitter taste in rapeseed meal and derived protein products. The aim of the present study was to analyse the genetic variation for individual and total sinapate ester content, to develop Near Infrared Reflectance Spectroscopic (NIRS) calibrations, and to identify genotypes with a low sinapate ester content after testing in the field. The following sinapate esters were analysed by HPLC: sinapoylcholine (sinapine), sinapoylglucose, and a minor group of ‘other sinapate esters’ which includes free sinapate. A genotypically diverse set of seed samples of winter oilseed rape (old and new cultivars, breeding lines, resynthesized rapeseed) from different years and locations was collected, their NIRS spectra recorded and the samples were further analysed by HPLC. The complete NIRS calibration seed sample set (n = 575) showed a large variation in total sinapate ester content, ranging from 3.2 to 12.7 mg sinapate equivalents per g seeds. The NIRS calibration equations showed high fractions of explained variances in cross validation () ranging from 0.75 (other sinapate esters) to 0.85 (sinapoylglucose). The standard errors of cross validation (SECV) ranged from 0.38 (other sinapate esters) to 0.70 mg/g seed (total sinapate esters). In validation and in independent validations the predicted results were not always acceptable, indicating that the NIRS calibrations need to be extended by analysing samples from new populations. Following replicated field experiments, a doubled haploid line obtained from the old Dutch cultivar Mansholts’ Hamburger Raps, and related DH lines from the cross DH Mansholts’ × Express were confirmed to have a 30–40% lower sinapate ester content compared to check cultivars.     

Feeding quality assessment of fresh maize stover by means of near-infrared spectroscopy with a new sample presentation unit

Stover quality traits are important in breeding programmes of maize cultivars. However, conventional procedures for evaluation of stover quality are expensive and thus limit the full exploitation of the available genetic variability. Our objective was to assess the potential of near‐infrared spectroscopy (NIRS) with a new sample presentation unit to determine feeding quality of maize stover. The plant material comprised maize inbreds and hybrids. The sample presentation unit was equipped with a near‐infrared diode‐array spectrometer. Coefficient of determination in cross‐validation ranged between 0.77 and 0.94 for contents of dry matter, nitrogen, neutral detergent fibre and in vitro fermentability expressed as gas volume after 24h incubation time, but it was lower for ash content. It was concluded that NIRS with the new sample presentation unit was less accurate than laboratory NIRS to predict the standard reference methods. However, the new sample presentation unit might be used as a fast and efficient technique to perform large screenings of maize stover accessions in breeding programmes.     

Genetic analysis of tocopherol content and composition in winter rapeseed

The improvement of the nutritional value and the stability properties of rapeseed oil is partly hindered by the lack of information on the genetic control of tocopherols. The objectives of this investigation were to characterize the inheritance of tocopherols by using two factorial mating designs (North Carolina Design II, NC_II). The first (NC_II‐A) was produced with two sets of nine parents each, one selected for high and the other for low total tocopherol contents. In the second (NC_II‐B), each set consisted of six parents, which were either high or low for the α/γ‐tocopherol ratio. Parents and F₁ hybrids from both experiments were tested under field conditions in 1998 and 1999 using a completely randomized design with two replications. Only small differences were detected between mean values of parents and F₁ hybrids. General combining ability (GCA) variation in both experiments was highly significant (P < 0.01) for all tocopherol traits. Significant specific combining ability (SCA) effects were only detected for a‐tocopherol in NC_IIA and therefore for the α‐/γ‐tocopherol ratio. These results indicate that tocopherol contents and composition are controlled mainly by genes with additive effects. Interactions of F₁ hybrids and GCA effects with the environment were significant for only tocopherol contents and not for tocopherol composition.     

Genetic and functional analysis of tocopherol biosynthesis pathway genes from rapeseed (Brassica napus L.)

Brassica napus L. is one of the most important oilseed crops in the world. Improving oil quality by increasing vitamin E content is a major target of rapeseed breeding. Vitamin E compounds, or tocopherols, are lipid‐soluble antioxidants that are essential nutrients for mammals. In this study, we report the characterization of rapeseed orthologs of the Arabidopsis tocopherol genes VTE1, VTE2 and PDS1. For each gene, at least two homologous sequences were found, and their expression was analysed in different tissues from three rapeseed genotypes. Genetic complementation experiments demonstrated that BnaX.VTE1.b and BnaA.VTE2.b homologs are capable of recovering seed γ‐tocopherol in Arabidopsis mutants. Overexpression of the genes in Arabidopsis Col‐0 shifted the seed tocopherol composition towards higher α‐tocopherol without increasing the total tocopherol content. To address the functionality of BnPDS1 sequences, we performed overexpression tests in Escherichia coli and enzymatic activity analyses. Overall, our results show that the identified sequences from rapeseed are functional orthologs of the Arabidopsis VTE genes and thus have considerable potential as molecular markers for selecting rapeseed with improved seed oil quality.     

Near-infrared spectroscopy on combine harvesters to measure maize grain dry matter content and quality parameters

Dry matter (DM) content and quality parameters of maize grain are important traits in breeding of maize hybrids. New developments in near‐infrared spectroscopy (NIRS) allow to determine DM content and product quality parameters directly on plot combine harvesters. The main objective of our study was to examine the potential of NIRS on combine harvester (NOCH) for determination of DM, crude protein (CP) and starch (ST) contents in maize grain. Plot combine harvesters equipped with spectrometers were used. Eleven locations were harvested in Europe during 2003 and 2004. The NOCH calibration models were developed with standard error of prediction (SEP) and coefficient of determination for validation (R) of 1.2% and 0.95 for DM, 0.3% and 0.88 for CP, and 1.0% and 0.79 for ST, respectively. NOCH is a promising technology for determination of DM, CP and ST contents of maize grain for breeding purposes, because it automates the assessment of each harvested plot, and the information for making selection decision is rapidly available to plan the next generation.     

Genetic variation of tocopherol content in a germplasm collection of Brassica napus L.

The genetic variation for tocopherol contents was investigated in a very divergent collection of 87 winter rapeseed genotypes grown in the greenhouse and in two years in the field. Genotypic and environmental effects were highly significant for alpha-, gamma- and total tocopherol contents and the alpha-/gamma-tocopherol ratio. Field and greenhouse environments differed significantly (p < 0.001) for tocopherol traits, with greenhouse means up to 19% higher than field means. Alpha-tocopherol content ranged from 63 to157 mg kg^-1 seed, gamma-tocopherol content from 114 to 211 mg kg^-1 seed, total tocopherol content from 182 to367 mg kg^-1 seed, and the alpha-/gamma-tocopherol contents ratio from0.36 to 1.23. The resynthesized lines often have a remarkably low alpha-tocopherol content and consequently a low alpha-/gamma-tocopherol contents ratio. The lines with altered fatty acid composition displayed the highest variation for both tocopherol content and composition. No significant differences were observed among groups with different seed quality types [00], [0+] and [++]. Total tocopherol content was not correlated with the alpha-/gamma-tocopherol ratio, indicating that total tocopherol content is independent from tocopherol composition. Alpha- and gamma-tocopherol contents were also not correlated. Gamma-tocopherol contents showed a significant positive correlation with oil content (r = 0.34^**). This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and genetic characterization of a safflower mutant with a modified tocopherol profile

Safflower (Carthamus tinctorius L.) seeds have a tocopherol fraction dominated by α‐tocopherol, which accounts for more than 95% of the tocopherols. α‐Tocopherol exerts a high vitamin E activity, but a low in vitro antioxidant action. For non‐food applications, replacement of high α‐tocopherol by γ‐tocopherol is preferred. Because of the limited variability found in germplasm of cultivated safflower, the objective of the present research was to search for variability for tocopherol profile in germplasm of wild safflower species. Bulk seed samples of 77 accessions from six species were analysed for total tocopherol content and profile. One accession of C. oxyacanthus showed increased γ‐tocopherol content (36%). Single‐seed analysis showed the existence of a bimodal distribution, which included high α‐tocopherol (>90%) and high γ‐tocopherol (HGT) seeds (>85%). Plants from the HGT seeds bred true for the trait and showed introgression of cultivated safflower, which allowed a rapid selection for cultivated safflower traits. Genetic analysis revealed that the HGT trait was controlled by partially recessive alleles at a single locus Tph1, which will facilitate the transference of the novel trait to diverse safflower germplasms.     

Selection for contrasting tocopherol content and profile in Ethiopian mustard

Tocopherols are compounds present in oilseeds with major effect on nutritional and technological properties of seed oils. Brassica spp. mainly contain varying concentrations of α‐tocopherol, with high vitamin E value, and γ‐tocopherol, which contributes to oil stability. The objective of this research was to select for contrasting tocopherol content and profile in Ethiopian mustard (Brassica carinata A. Braun). A collection of 211 F_4:5 lines was evaluated for tocopherol content and profile, and divergent selection was conducted till the S_3:4 generation. Selection resulted in two lines with increased tocopherol content (244 and 243 mg/kg seed as average of four environments), one line with reduced tocopherol content (117 mg/kg), two lines with reduced α/γ‐tocopherol ratio (0.22 and 0.28) and two lines with increased α/γ‐tocopherol ratio (3.74 and 3.34). Control line C‐101 averaged 196 mg/kg with α/γ‐tocopherol ratio of 1.10. Reduced α/γ‐tocopherol ratio was controlled by recessive alleles at a single locus. Seed to plant correlation was high (r = 0.92), which suggests the feasibility of selecting at the single‐seed level.     

Identification of quantitative trait loci and candidate genes controlling the tocopherol synthesis pathway in soybean (Glycine max)

A recombinant inbred line (RIL) population was used to identify quantitative trait loci (QTLs) and their candidate genes controlling the tocopherol (Toc) synthesis pathway. The RIL population was cultivated in field conditions in 3 years. A genetic map constructed using 1624 DNA markers was used for QTL analysis. We identified 22 QTLs for seed tocopherol contents and their ratios, of which two QTL clusters on chromosomes (Chr) 9 and 14 exerted consistent large effects on tocopherol composition across the 3 years. The QTL cluster localized on Chr 9 might correspond to γ-TMT3, which controls the conversion of γ-Toc into α-Toc. The QTL cluster localized on Chr 14 was novel, which might regulate the conversion of MPBQ (a precursor of δ-Toc) into DMPBQ (the precursor of γ-Toc). The effect of the QTL cluster on Chr 14 was validated in a pair of near isogenic lines, and its candidate gene was mined. The identified QTLs and their candidate genes might be used in breeding programmes to improve α-Toc content in soybean seeds.     

Genetic variability of tocopherol composition in sunflower seeds as a basis of breeding for improved oil quality

The variability of seed tocopherol content in wild sunflower species, the expressivity of tph1 and tph2 mutations in different lines and the oxidative stability of sunflower oil with altered tocopherol and fatty acid composition were objectives of this research. Near-isogenic lines for three genes, i.e. Tph1, Tph2, and Ol, were developed and investigated. Tocopherol content was determined with TLC and HPLC, as well as fatty acid composition with GC of methyl esters. Rancimat tests were used to estimate the oxidative stability of the oil. The seed tocopherol composition of wild sunflower species was shown to be uniform with a prevailing content of the α-homologue (90-99%). The genetic background of different near-isogenic lines was found to influence expressivity of mutations for tocopherol composition. High content of strong antioxidants, such as β-, γ-, and δ-tocopherols increased oil oxidative stability of linoleic and oleic types of oil by 1.2–3.0 times. The breeding model of sunflower hybrids should include antioxidant and vitamin parameters balanced for oils of different applications.     

Near infrared reflectance spectroscopy analysis of digestibility traits of maize stover

Summary Near infrared reflectance spectroscopy (NIRS) is emerging as a potentially useful tool in breeding plants for quality traits. Information is lacking, however, on its use in forage maize (Zea mays L.). The objectives of the present investigation were to evaluate the prediction of digestibility traits of maize stover using NIRS technique and to study the effect of laboratory (Lab) and NIRS assays on the estimates of variation and covariation. Twelve inbred lines, 66 diallel crosses among them and eight hybrid checks were evaluated at silage and grain harvests for 2 years at two agro-climatically diverse locations in the Federal Republic of Germany. Standard methods were used for Lab analysis of in vitro digestible organic matter (IVDOM), neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL). In NIRS evaluation, calibration equations were developed by modified stepwise regression. The standard error of calibration was 2.5, 1.7, 1.4 and 0.4 for IVDOM, NDF, ADF and ADL, respectively. The coefficient of multiple determination was high (0.9) except for ADL. The validation statistics (standard error and correlation coefficient) were similar. In the diallel crosses, the estimates of variation (heritabilities in broad and narrow sense, genotypic and error coefficients of variation), generally, did not vary appreciably and consistently in the comparisons between Lab and NIRS methods particulary at silage harvest. Simple and rank correlations between Lab and NIRS analyses were positive and significant. These correlation coefficients based on the mean performance of the diallel crosses at silage harvest were >0.9 and at least 16 hybrids were common between the two analyses, among the upper one-third or lower one-third (22) hybrids. The study showed that NIRS analysis should be useful in maize breeding programmes wherein a large number of genotypes need to be evaluated.Abbreviations NIRS near infrared reflectance spectroscopy - Lab laboratory - IVDOM in vitro digestible organic matter - NDF neutral detergent fibre - ADF acid detergent fibre - ADL acid detergent lignin - SEC standard error of calibration - SEV standard error of validation - SD standard deviation - r simple correlation coefficient - r_s rank correlation coefficient - R² coefficient of multiple determination - h_b ² heritability in broad sense - h_n ² heritability in narrow sense - CV_g genotypic coefficient of variation - CV_e error coefficient of variation - GCA general combining ability effect     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular mapping of soybean seed tocopherols in the cross ‘OAC Bayfield’ × ‘OAC Shire’

Soybean (Glycine max [L.] Merr.) is cultivated primarily for its protein and oil in the seed. In addition, soybean seeds contain nutraceutical compounds such as tocopherols (vitamin E), which are powerful antioxidants with health benefits. The objective of this study was to identify molecular markers linked to quantitative trait loci (QTL) that affect accumulation of soybean seed tocopherols. A recombinant inbred line (RIL) population derived from the cross ‘OAC Bayfield’ × ‘OAC Shire’ was grown in three locations over 2 years. A total of 151 SSR markers were polymorphic of which a one‐way analysis of variance identified 42 markers whereas composite interval mapping identified 26 markers linked to tocopherol QTL across 17 chromosomes. Individual QTL explained from 7% to 42% of the total phenotypic variation. Significant two‐locus epistatic interactions were identified for a total of 122 combinations in 2009 and 152 in 2010. The multiple‐locus models explained 18.4–72.2% of the total phenotypic variation. The reported QTL may be used in marker‐assisted selection (MAS) to develop high tocopherol soybean cultivars.     

Novel variation for the tocopherol profile in a sunflower created by mutagenesis and recombination

α‐Tocopherol is the main tocopherol in sunflower seeds (>90%). Because it exerts a weak antioxidant action in vitro, its partial replacement by other tocopherols is an important breeding objective in this crop. The objective of this research was to develop novel tocopherol profiles in sunflower through mutagenesis and genetic recombination. Seeds of four ‘Peredovik’ accessions were used for chemical mutagenesis with ethyl methane sulfonate (EMS). Single‐seed screening in the M₂ generation resulted in two M₂ seeds, derived from different M₁ plants, with increased γ‐tocopherol contents of 19.2% and 96.7%, respectively. M₃ progeny from the M₂ seed with the 96.7% content bred true for high c‐tocopherol content, containing more than 90%γ‐tocopherol. M₃ progeny from the M₂ seed with only 19.2%γ‐tocopherol segregated in a range from 0 to 84.6%. Selection for high c‐tocopherol content produced an M_4:5 line, designated IAST‐1, with a stable high concentration of γ‐tocopherol. Crosses between IAST‐1 and T589, with an increased b‐tocopherol content, produced F₂ segregants with trans‐gressive levels of up to 77%β‐tocopherol or up to 68% d‐tocopherol. Both novel tocopherol profiles were confirmed in the F₃ generation.     

应用近红外反射光谱法整粒测定小样品油菜籽含油量的研究

以完整油菜籽为样品,通过光谱预处理和回归统计方法来优化油菜籽含油量的近红外反射光谱法所建立回归方程,同时采用三种不同用量的样品杯进行近红外扫描分析.结果表明,光谱预处理对较正结果影响较大,不同光谱数学处理以3级导数处理较好,综合考虑定标组和检验组的效果,采用"2,3,3,1"、 "2,6,6,1"和"3,3,3,1"的三种处理组合较好     

The multilocular trait of rapeseed is ideal for high‐yield breeding

The multilocular trait is generally considered as a desirable agricultural trait for the high‐yield breeding of Brassica crops and has attracted considerable attention. The siliques of multilocular plants contain more chambers and seeds than those of bilocular rapeseed varieties and, thus, have a higher yield. Furthermore, multilocular plants are precious germplasm resources for the breeding of Brassica crops. In this article, we provide a summary of researches regarding the multilocular trait in rapeseed, including its natural germplasms, silique morphology, genetic models, and regulatory mechanisms. The study will highlight the potential of multilocular rapeseed for high‐yield breeding.