Genetic Analysis And Development Of Allele-specific Markers For High Oleic And Low Linolenic Acid Contents In Brassica Napus L.

Oilseed rape(Brassica napus L.) is one of the most important oil crops worldwide. The edible and processing quality of rapeseed oil is mainly determined by the fatty acid composition of the triglycerol lipid in its seeds. High C18:1and low C18:3content in rapeseed oil is not sensitive to the oxidation when processing, storage and frying, therefore desirable for its good thermal stability and long shelf life. In addition, high oleic acid oil is also a renewable raw material for biodiesel and environmentally friendly lubricants production. Therefore, one of the major goals for quality breeding after removal of erucic acid in oilseed rape is to increase the C18:1content and reduce the C18:3content in the seeds of oilseed rape cultivars. The fatty acid desaturase genes FAD2and FAD3have been shown as the major genes for the control of C18:1and C18:3contents. However, the copy number, locus distribution, genes structure, gene expression and gene function of the two gene families in amphidiploid Brassica napus are still not completely understood to date. Based on QTL mapping, molecular cloning and bioinforamtic analysis of the transcriptome data from Brassica napus, we studied the copy number, locus distribution, genes structure, gene expression and gene function of the FAD2gene and FAD3gene families of amphidiploid Brassica napus in this study. Furthermore, based on the results of genetic mapping and identified sequences, allele-specific markers of FAD2and FAD3genes were developed for breeding of high oleic, low linolenic acid rapeseed. The main results are as follows:1. In the present study, all copies of FAD2and FAD3genes in the A-and C-genome of B. napus and its two diploid progenitor species, B.rapa and B.oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2genes exist in B.rapa and B.oleracea, and four copies of FAD2genes exist in B.napus. Three and six copies of FAD3genes were identified in diploid species and amphidiploid species, respectively. Through data mining and molecular cloning of FAD2, FAD3genes in Brassica species, we showed that:1) there is a naturally mutated ORF for one of the FAD2locus in A-genome (Al) of both B.rapa and B.napus. The locus contained five insertions or deletions, which resulted in a frame shift starting and premature termination codon (PTC), likely representing a pseudogene of FAD2in A-genome.2) Based on the protein sequences, the FAD2and FAD3genes from B.napus and their corresponding counterparts in two diploid species could be grouped into two, three clusters, respectively. And members belonging to the same category were more closely related to each other regardless of their genome and species origins.3) The sequences of the FAD2gene in Arabidopsis and three examined Brassica species were highly similar among the loci. On the other hand, the genomic sequences of the FAD3gene are rather divergent, mainly reflected in the length and similarity of the introns sequences. Interestingly, at protein and cDNA levels, both FAD2and FAD3members among the loci share high similarities.2. The genetic control of high C18:1and low C18:3contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5chromosome was responsible for the high C18:1content, which accounting for89%of the trait variation. A4bp insertion mutation in the BnaA.FAD2.a locus was uncovered, which resulted in a frame shift starting and PTC. And that represented a previously unidentified allele for the high oleic variation in Brassica napus species. Two major QTLs on A4and C4chromosomes were found to be responsible for the low C18:3content in the DH population as well as in SW Hickory, which explained about31%,60%of the variation of C18:3content, respectively. Two single nucleotide mutations in the parents SW Hickory were identified in the locus of BnaA.FAD3.b (LG A4). The first one was a T to C substitution in exon2leading to a synonymous mutation, and the second a C to T transition in exon7, which resulted in an amino acid substitution from arginine to cysteine. BnaC.FAD3.b (LG C4) was a G to A substitution in the5'-end of intron6in SW Hickory, which resulted in an abnormal splicing with the entire sixth intron being retained in the mature BnaC.FAD3.b transcript, eventually resulted in frameshift and the PTC. Meanwhile, LNAA5and LNAA6were detected in three and one years, which explained about7%,2%of the variation of C18:3content, respectively. BnaA.FAD3.a (LG A5) was a T deletion mutation in269th base of the genome and cDNA sequences in SW Hickory, which resulted in frameshift and eventually led to the PTC.3. With the RNA sequencing data of Brassica napus, gene expression in FAD2and FAD3genes has been compared in different tissues and developmental stages. The results showed that, in the mid-late stages of seed development (14-25DAF), which is the most vigorous in fatty acid synthesis in rapeseed, the expressions of function locus(BnaA.FAD2.a) of oleic acid content, and three functional loci (BnaA.FAD3.a, BnaA.FAD3.b and BnaC.FAD3.b) of linolenic acid content exhibited a sharp rise, as much as7-20time higher than in other developmental stages. The gene expression pattern of each locus is highly consistent with their contributions to the variation of oleic acid or linolenic acid content.4. Based on the polymorphism information of BnaA.FAD2.a, BnaA.FAD3.a, BnaA.FAD3.b and BnaC.FAD3.b genes sequence, allele-specific codominant SCAR marker were developed for oleic acid. And single-nucleotide amplified polymorphisms (SNAP) codominant markers for FADS genes by utilizing the SNAPER program. The allele-specific codominant markers aided selection can greatly enhance the efficiency and effectiveness of plant breeding relative to conventional methods, thereby overcoming the shortcomings of phenotypic selection in the process of the HOLL breeding of Brassica napus. Genotyping can be reliably performed at an early stage of plant growth, avoiding the need to conduct large scale of field trials. The allele-specific marker is demonstrated to be time-and cost-effective in developing HOLL varieties.