Abstract
Key Message: The loss of mini-exon 2 in the 1-FEH IIb glycosyl-hydrolase results in a putative non-functional allele. This loss of function has a strong impact on the susceptibility to post-harvest inulin depolymerization. Significant variation of copy number was identified in its close paralog 1-FEH IIa, but no quantitative effect of copy number on carbohydrates-related phenotypes was detected.Inulin polyfructan is the second most abundant storage carbohydrate in flowering plants. After harvest, it is depolymerized by fructan exohydrolases (FEHs) as an adaptive response to end-season cold temperatures. In chicory, the intensity of this depolymerization differs between cultivars but also between individuals within a cultivar. Regarding this phenotypic variability, we recently identified statistically significant associations between inulin degradation and genetic polymorphisms located in three FEHs. We present here new results of a systematic analysis of copy number variation (CNV) in five key members of the chicory (Cichorium intybus) GH32 multigenic family, including three FEH genes and the two inulin biosynthesis genes: 1-SST and 1-FFT. qPCR analysis identified a significant variability of relative copy number only in the 1-FEH IIa gene. However, this CNV had no quantitative effect. Instead, cloning of the full length gDNA of a close paralogous sequence (1-FEH IIb) identified a 1028 bp deletion in lines less susceptible to post-harvest inulin depolymerization. This region comprises a 9 bp mini-exon containing one of the three conserved residues of the active site. This results in a putative non-functional 1-FEH IIb allele and an observed lower inulin depolymerization. Extensive genotyping confirmed that the loss of mini-exon 2 in 1-FEH IIb and the previously identified 47 bp duplication located in the 3′UTR of 1-FEH IIa belong to a single haplotype, both being statistically associated with reduced susceptibility to post-harvest inulin depolymerization. Emergence of these haplotypes is discussed.
Highlights
Industrial chicory is the main commercial source of inulin, a linear fructose polymer used by the agro-industry as texturizer, fat substitute (Mendoza et al, 2001; Keenan et al, 2014; Karimi et al, 2015) or low calories sweetener
A paper reporting the validation of reference genes for real-time gene expression analysis (RT-PCR) in chicory evaluated the expression stability of nicotinamide adenine dinucleotide dehydrogenase (NADHD), ACT, TUB, glyceraldehyde 3-phosphatedehydrogenase (GAPDH), histone H3 (H3), elongation factor 1-alpha (EF), and 18S rRNA (rRNA) and concluded that ACT, EF, rRNA, H3, and TUB could be used for normalization (Maroufi et al, 2010)
Our results indicate that, based on a preliminary analysis performed on 20 individuals (Pool 1, P1), for our chicory sampling population, estimates of relative copy number of 1-FFT gene using ACT as normalization gene resulted in a higher variability and standard error deviation than when normalized against β-tubulin
Summary
Industrial chicory is the main commercial source of inulin, a linear fructose polymer used by the agro-industry as texturizer, fat substitute (Mendoza et al, 2001; Keenan et al, 2014; Karimi et al, 2015) or low calories sweetener. Like in most Asteraceae (Hendry, 1993), inulin is the main reserve carbohydrate. It is accumulated in the taproot during growing season and it is hydrolyzed by FEHs as an adaptive response to the exposure to end-season cold temperatures (Dauchot et al, 2014). The 1-SST enzyme initiates inulin synthesis by transferring a fructose moiety from a sucrose donor molecule (GF) to a second sucrose molecule, which acts as fructose acceptor, to produce 1-kestotriose (GFF) and free glucose (G). This 1-kestotriose molecule is used as fructose donor by the enzyme fructan:fructan 1-fructosyltransferase (1-FFT) to elongate inulin molecules (GFFn+1), releasing free sucrose (GF). The coding sequences of chicory 1-FEH I, 1-FEH IIa, and 1-FEH IIb inulin hydrolyzing genes were isolated short after (Van den Ende et al, 2000, 2001)
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