Detection of induced mutations in CaFAD2 genes by next-generation sequencing leading to the production of improved oil composition in Crambe abyssinica.

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.

Genetic Architecture of Flowering Time and Sex Determination in Hemp (Cannabis sativa L.): A Genome-Wide Association Study.

Flowering time and sex determination in hemp (Cannabis sativa L.) strongly influence fiber quality and seed production of this crop. The control of these traits is paramount for the breeding of new cultivars. Yet, little is known about the genetics underlying such complex traits and a better understanding requires in depth knowledge of the molecular mechanisms responsible for these traits. In this report, the genetic architecture of flowering time and sex determination in hemp was studied using a Genome-Wide Association Studies (GWAS) approach. Association studies were performed on a panel of 123 hemp accessions, tested in three contrasting environments, using a set of 600 K SNP markers. Altogether, eight QTLs were identified across environments; six for flowering time traits and two for sex determination. These QTLs covered genomic regions with 33 transcripts predicted to be involved in flowering and sex determination as well as a microRNA, miR156. Genes related to perception and transduction of light and transcription factors well-known to regulate flowering were identified in QTLs for flowering time traits. Transcription factors and genes involved in regulating the balance of phytohormones, specially auxins and gibberellic acid, were identified in QTLs for sex determination. Sex determination QTLs were associated with the development of male flowers in female plants and thus with the stability of sex determination in monecious plants. The present study elucidates relevant knowledge on the genetic mechanisms of flowering and sex determination traits in hemp, and provides new tools for hemp breeding.

Elucidating the Genetic Architecture of Fiber Quality in Hemp (Cannabis sativa L.) Using a Genome-Wide Association Study.

Hemp (Cannabis sativa L.) is a bast-fiber crop with a great potential in the emerging bio-based economy. Yet, hemp breeding for fiber quality is restricted and that is mainly due to the limited knowledge of the genetic architecture of its fiber quality. A panel of 123 hemp accessions, with large phenotypic variability, was used to study the genetic basis of seven cell wall and bast fiber traits relevant to fiber quality. These traits showed large genetic variance components and high values of broad sense heritability in this hemp panel, as concluded from the phenotypic evaluation across three test locations with contrasting environments. The hemp panel was genotyped using restriction site associated DNA sequencing (RAD-seq). Subsequently, a large set (> 600,000) of selected genome-wide single nucleotide polymorphism (SNP) markers was used for a genome-wide association study (GWAS) approach to get insights into quantitative trait loci (QTLs) controlling fiber quality traits. In absence of a complete hemp genome sequence, identification of QTLs was based on the following characteristics: (i) association level to traits, (ii) fraction of explained trait variance, (iii) collinearity between QTLs, and (iv) detection across different environments. Using this approach, 16 QTLs were identified across locations for different fiber quality traits, including contents of glucose, glucuronic acid, mannose, xylose, lignin, and bast fiber content. Among them, six were found across the three environments. The genetic markers composing the QTLs that are common across locations are valuable tools to develop novel genotypes of hemp with improved fiber quality. Underneath the QTLs, 12 candidate genes were identified which are likely to be involved in the biosynthesis and modification of monosaccharides, polysaccharides, and lignin. These candidate genes were suggested to play an important role in determining fiber quality in hemp. This study provides new insights into the genetic architecture of fiber traits, identifies QTLs and candidate genes that form the basis for molecular breeding for high fiber quality hemp cultivars.

Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.