Genetic analysis of saturated fatty acid composition and other seed quality traits in rapeseed (Brassica napus L.)
Cumulative thesis
Date of Examination:2024-05-28
Date of issue:2024-06-20
Advisor:Dr. Christian Möllers
Referee:Prof. Dr. Wolfgang Link
Referee:Prof. Dr. Konstantin Krutovsky
Referee:Dr. Wolfgang Ecke
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Abstract
English
Genetic improvement of seed oil content and oil quality had been the main objective of rapeseed (Brassica napus L.) breeders until recently, when interest in improving rapeseed meal (RSM) as a secondary trait increased. Seed oil content is the main source of vegetable oil while RSM is a valuable source of animal feed. RSM is the protein-rich by-product after oil extraction from the seed. The quality of oil in rapeseed, as a source of diet is highly dependent on the relative amount of fatty acids in the seed oil. Total saturated fatty acid content (SFA) in the oil is the sum of palmitic, stearic, arachidic and behenic acid contents. The recommended dietary intake of SFA should be as low as possible (especially as far as palmitic acid is concerned) because in humans, higher SFA consumption is associated with cardiovascular diseases. Rapeseed breeders in Northwest Europe overlooked the need to reduce SFA content because it is in relatively low quantity in rapeseed compared to other oilseeds. As a result, breeding and release of low SFA rapeseed cultivar and vegetable oil (canola oil) is rare. However, recent studies revealed that SFA content in rapeseed oil in several rapeseed varieties is higher than the EU’s recommended 7.5% SFA dietary intake. For RSM protein, presence of high content of crude fibre and content of anti-nutritive glucosinolate limits its application in food industry. Seed fibre consists of hemicellulose, cellulose and indigestible lignin (ADL or LC). It contributes to the dark color of the seed hulls, affects digestions in animal thereby reduces RSM quality. The seeds of yellow-seeded types of rapeseed have lower fibre than the black-seeded types. However, yellow-seeded commercial varieties do presently not exist yet due to agronomic challenges. This offers the breeders the task and opportunity to breed for low fibre rapeseed lines. To further improve quality of seed oil and RSM protein, reduced contents of SFA and of fibre is desired; but there is negative correlation between seed oil and RSM protein. Marker assisted selection (MAS) offered a possibility – if the associated QTL are linked with DNA markers that could contribute positive alleles to both traits or if such markers allow to select for rare recombination between unfavorably linked seed oil and protein genes. QTL mapping studies for SFA are limited in winter types of rapeseed which are mostly cultivated in Europe. In addition, QTL for studies of seed fibre components and their relationship to seed oil and protein contents are also required. Generally, QTL studies as MAS tool in multiple populations for a broader application to breeding for low content SFA in oil and for low seed fibre content are rare. To this end, here, 4-6 composition of SFA (contents of arachidic acid, behenic acid, C16/C18, palmitic acid, SFA, and stearic acid) and of seed fibre (acid detergent fibre (ADF), cellulose (CC), hemicellulose (HC), lignin (ADL/LC), and neutral detergent fibre (NDF) contents) and of seed protein and glucosinolate content were analyzed in field experiments. This study was based on three populations (i.e. three sets of lines): two half sib doubled-haploid (DH) line sets and a diversity set of rapeseed. The objectives of the study were i) to analyze the genetic variation and inheritance of SFA contents and their genetic relationship with contents of other fatty acids and seed oil under field trials, ii) to analyze genetic variation and inheritance of seed fibre contents, their interrelationship and relationship to seed oil, protein and glucosinolate contents, iii) to identify QTL of SFA content, seed fibre, oil, protein and glucosinolate contents and to identify putative candidate genes associated to SNPs in the QTL confidence intervals. To address these aims, three field experiments were conducted under Northwest European field conditions. The field experiments were conducted as unreplicated randomized complete block design in each environment. At maturity, open-pollinated seeds were manually bulk harvested per plot from each rapeseed line from plants’ terminal raceme. Fatty acid contents in seed oil were measured using Gas Chromatography (GC) while seed fibre, oil, protein and glucosinolate contents were predicted using near infrared reflectance spectroscopy (NIRS). All the rapeseed lines in the three populations were genotyped with Brassica Illumina SNP chip. Linkage maps were constructed for the two DH populations using ASMap package in R, based on minimum spanning tree (MST) algorithms. QTL were analyzed with R/qtl using multiple interval mapping and Haley-Knott regression method. The candidate genes were searched in the reference genomes of rapeseed lines “ZS11” and “Express 617”. The first experiment consisted of a population of 170 F1 (DH) lines segregating for one erucic acid gene derived from a cross between the canola line cultivar Adriana and the DH line number 13 (DH13) derived from previous studies. Adriana has low contents of erucic acid, moderate palmitic acid and normal oil while, DH13 is characterized by high erucic acid, moderate palmitic acid and higher oil contents. The parental and DH lines (termed ASG) were tested in three growing seasons in five field environments located in Germany and Poland. Since a parent of this population (DH13) contained high erucic acid which is controlled by major loci, SFA palmitic acid and seed oil contents were corrected for erucic acid using regression method. A linkage map with 3763 polymorphic SNP markers distributed across 19 linkage groups was used to develop a frame work map of 870 markers which spanned a map distance of 1683.5 centi-Morgan (cM). The second experiment consisted of a population of 95 F1 DH lines derived from a cross between the (already above-mentioned) German cultivar Adriana and a Chinese cultivar Zheyou 50. Zheyou 50 is a semi-winter cultivar characterized by high oil and oleic acid contents. The parental and DH lines (termed AZH) were tested in four growing seasons in one environment located in Germany. A linkage map comprising 5742 polymorphic SNP markers distributed in 19 linkage groups was used to develop a frame work map of 766 markers covered a map distance of 2126.7 centi-Morgan (cM). The third experiment consisted of a diversity set of 306 rapeseed accessions tested in five field environments over three seasons. The population was genotyped with polymorphic 28,554 SNP markers from Brassica 60K Illumina chips. Genome-wide association (GWAS) mapping was performed for 12 fatty acid compositions and for seed oil content using the adjusted phenotypic mean across environments and SNP marker data. First three principal components (PC) from principal component analysis (PCA) and kinship matrix were used as covariates to control false positives using a Random-SNP Multi-locus Model (MrMLM) for the GWAS analysis. Candidate genes were searched within 800 kbp region flanking the associated SNPs. Genomic prediction (GP) was implemented using Bayesian ridge regression with 5000 iterations and burning-in and a five-fold cross validation and 10 independent sample runs for contents of six SFAs, oleic acid and seed oil. Predictive ability for each trait was compared using all the polymorphic SNP markers and using only SNPs that were significant in GWAS analysis. For SFA contents in the two DH experiments, there was a significant effect of genotypes and environments with heritability ranging from 33 to 89% for behenic and palmitic acid contents, respectively. There was a normal distribution of all SFA contents in both populations confirming they were all quantitatively inherited. There were low phenotypic correlations among the SFA contents. There were low negative phenotypic correlations between all SFA contents with seed oil content, except palmitic acid content which had non-significant correlations with seed oil content. A total of 32 QTL was detected for the contents of five SFA, 31 QTL for five other fatty acid contents and nine QTL for seed oil content altogether, in both DH populations. Most of the QTL detected, except for seed oil content, did not overlap between the two DH populations. A major QTL was detected for palmitic acid content on chromosome A09 (q16:0-A9) in ASG DH population and on another chromosome C08, in AZH DH population, resulting in the identification of the Fatty acyl thioesterase –B (FATB) as the candidate gene for both QTL. The result also raised speculation that the region on both A09 and C08 may be homoeologous in rapeseed genome. The QTL for palmitic acid content also co-located with QTL for C16/C18 in both populations. In ASG DH population, there was overlapping of QTL confidence interval between QTL for contents of palmitic acid and stearic with oil content on chromosome A01 and C05, respectively. Other candidate genes identified in the confidence interval of minor QTL for content of stearic acid include stearoyl-acyl carrier protein desaturase (SAD) and ß-ketoacyl-synthase II (KASII); and for seed oil content include lysophosphatidic acid acyltransferase (LPAT) and glycerol-3-phosphate acyltransferase (GPAT) in both populations. A major QTL was also mapped for oil content on chromosome C02 in AZH DH population which was not previously reported. In conclusion, the work elaborated interaction between contents of SFA and of seed oil, which can be applied to breeding winter rapeseed simultaneously for (1) reduced SFA content and for (2) for improved seed oil quality. QTL detected on A09 and C08 for palmitic acid content will be interesting for MAS towards low SFA. Also, it confirmed that reducing palmitic acid content can further reduce SFA content in rapeseed oil. For seed fibre composition, the heritability ranged from 67 to 95% for hemicellulose and lignin content, respectively. The heritability was higher in the ASG than in the AZH DH population. There were high positive phenotypic correlations between both cellulose and lignin with other seed fibre composition in the ASG DH population, while there were positive correlations between hemicellulose and other fibre composition in AZH DH population. As expected, there were positive phenotypic correlations between glucosinolate and protein contents and negative correlations between lignin and oil contents in both populations. A total of 37 QTL were detected for five seed fibre compositions, 13 QTL for protein and two QTL for glucosinolate contents in both DH populations. Clusters of QTL were detected for the contents of four seed fibre compositions (ADF, hemicellulose, lignin, and NDF contents) and for seed oil and protein contents. The QTL were mapped at a similar position with a previously reported major QTL on chromosome C05 and it explained between 8 to 66% of the phenotypic variation. The physical position of the QTL clusters on C05 was also similar in both DH populations. There was increasing allele for both oil and protein contents and decreasing allele for all the four seed fibre contents for this major QTL speculating the loci could increase seed oil and protein content while reducing the contents of fibre. The positive alleles for QTL for seed oil and protein contents were derived from cultivar Adriana in both populations. The negative alleles for QTL for lignin content were derived from parents DH13 line and Zheyou 50 in ASG and AZH populations respectively. It was also speculated that these two parents shared a common origin. The most likely candidate gene for the major QTL for seed fibre contents was phenylalanine ammonia-lyase 4 (PAL4). A second cluster of QTL for three seed fibre contents (ADF, NDF and lignin), seed oil and protein contents were identified on the same region on chromosome A04 in AZH population explaining between 4 to 27% of phenotypic variation, and likely candidate gene was Acetyl-CoA synthase (ACS). A major QTL for glucosinolate content not previously reported was identified on chromosome A02 in AZH population explaining 26% of phenotypic variation. Two well-known candidate genes and transcription factors thioglucoside glucohydrolase 1 and 2 (TGG1 and TGG2) and MYB28 and MYB34 were found within this QTL confidence interval. QTL detected on chromosome C05 in both populations and on A04 in AZH DH population are interesting because of their possible pleiotropic effect on seed fibre, protein and seed oil contents. This suggests that individual QTL alleles for the fibre components can be used to further reduce overall fibre components and increase seed oil and protein contents. The simultaneous investigation of two half-sib DH populations also gave clues into the direction that QTL effect is depending on the individual parents could complicate breeding for seed quality traits in rapeseed. In the third experiment involving the diversity set of 306 rapeseed accessions, heritability ranged from 46 to 84% for contents of six SFA; 86 to 99% for five unsaturated fatty acid contents; and 85% for seed oil contents. The population structure analysis revealed 2 sub-populations among the rapeseed accessions roughly separating the winter and the spring types. GWAS analysis identified a total of 26 SNP loci significantly associated with the contents of five SFA, 47 SNP loci for the contents of four unsaturated fatty acids and 10 for seed oil content. There was QTL collocation on chromosome A07 with significant SNP markers associated to palmitic and stearic acid contents between regions between 22.6 Mbp to 22.8 Mbp explaining between 5 to 6% of variation. Identified significant SNP loci for SFA content on chromosome A09 in this experiment was in the same physical regions with QTL for palmitic acid content in the two DH experiments and at 758 kbp to Brassica napus candidate gene FATB. A total of 34 candidate genes related to fatty acid synthesis and oil metabolism were identified within the confidence interval regions of all the significantly associated SNP markers. Most prominent candidate genes identified for contents of five SFA are fatty acyl-ACP thioesterase B (FATB), beta-ketoacyl reductase 1 (KCR1) and steoryl ACP desaturase (SAD), while phosphatidate phosphatase (PAP) involved in oil synthesis Kennedy Pathway, phosphatidate phosphohydrolase 1 (PAH1) and glycerol-3-phosphate acyltransferase 5 (GPAT 5) was identified for seed oil content. The result of the genomic prediction showed higher predictive ability for six SFA contents and seed oil content when used only the GWAS significant SNPs. It also demonstrated that using GWAS significant SNPs could improve predictive ability for traits with low heritability and that marker-assisted selection can improve genomic selection. In conclusion, the results from the three experiments revealed insights into the inheritance and relationship between contents of SFA, seed fibre and seed oil in rapeseed. The results from QTL mapping can be exploited for marker-assisted selection in rapeseed. Contents of SFA and fibre are quantitative traits but QTL analysis in biparental populations could detect major loci that explained larger phenotypic variation and revealed pleiotropic connections with other traits. A region on chromosome A09 was a QTL hotspot for SFA contents and a region on C05 with marked impact on seed fibre, oil and protein contents was confirmed in all three populations. A combination of QTL identified, both minor and major, are recommended for MAS to understand better, the genetic variation for seed oil and further seed quality traits in rapeseed. The results also underlined the challenges of rapeseed breeding for improved seed oil and meal quality traits. The identified loci will help in marker assisted selection towards improved seed oil quality with particular consideration of low saturated fatty acid and fibre contents.
Keywords: Rapeseed oil; Saturated fatty acid; QTL; GWAS; Seed oil quality; Seed fibres