Epigenetic Variability Among Saffron Crocus (Crocus sativus L.) Accessions Characterized by Different Phenotypes

Abstract

This work represents the first epigenomic study carried out on saffron crocus. Five accessions of saffron, showing differences in tepal pigmentation, yield of saffron and flowering time, were analyzed at the epigenetic level by applying a methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach. Five accession-specific hypomethylomes plus a reference hypomethylome, generated by combining the sequence data from the single accessions, were obtained. Assembled sequences were annotated against existing online databases. In the absence of the Crocus genome, the rice genome was mainly used as the reference as it is the best annotated genome among monocot plants. Comparison of the hypomethylomes revealed many differentially methylated regions, confirming the high epigenetic variability present among saffron accessions, including sequences encoding for proteins that could be good candidates to explain the accessions’ alternative phenotypes. In particular, transcription factors involved in flowering process (MADS-box and TFL) and for the production of pigments (MYB) were detected. Finally, by comparing the generated sequences of the different accessions, a high number of SNPs, likely having arisen as a consequence of the prolonged vegetative propagation, were detected, demonstrating surprisingly high genetic variability. Gene ontology (GO) was performed to map and visualize sequence polymorphisms located within the GOs and to compare their distributions among different accessions. As well as suggesting the possible existence of alternative phenotypes with a genetic basis, a clear difference in polymorphic GO is present among accessions based on their geographic origin, supporting a possible signature of selection in the Indian accession with respect to the Spanish ones.

Environmental and genealogical effects on DNA methylation in a widespread apomictic dandelion lineage

“…This could be especially useful in species that are propagated clonally, and/or in which the phylogenetic relationships are difficult to infer with classical neutral genetic markers. For instance, a clonally propagated sterile crop such as saffron ( Crocus sativus ), in which the origin and relationships between close species have recently been discovered (Nemati et al, 2019; Schmidt et al, 2019), could benefit from the use of this molecular clock, as differences between accessions could be best explained by DNA methylation (Busconi et al, 2021).…”

Understanding saffron biology using omics- and bioinformatics tools: stepping towards a better Crocus phenome

“…AFLP analysis using methylation-sensitive restriction enzyme-sequencing (MRE-seq) gave more insight into saffron’s epigenome [ 52 ]. The study compared the epigenetic profile of 5 phenotypically different, but genetically similar accessions from WSCC germplasm.…”

“…A heatmap of the 50 highest polymorphic GOs shared between accessions highlighted the presence of two distinct clusters of Indian and Spanish accessions. Twelve GOs showed lower polymorphism in the Spanish accessions than Indian accessions [ 52 ].…”

“…has not been done There are contradictory results on the detection of polymorphisms using marker-based analysis Some studies conclude that saffron is a monomorphic species and whole genome sequencing is needed to discriminate between its isolates Some studies show that molecular markers are quite efficient in detecting polymorphism. Such studies conclude that saffron is not monomorphic and that there is diversity which could be useful for breeding purposes AFLP analysis using methylation-sensitive restriction enzyme-sequencing (MRE-seq) has shown that phenotypically different but genetically similar accessions vary in the methylation pattern of genomic regions encoding transcription factors and may result in alternative phenotypes Epigenetic structure in saffron is highly stable and may play a vital role in the constancy of saffron phenotype variability ISSR primers are reported to be capable of easily distinguishing genuine saffron from fake one [ 44 , 47 , 47 , 48 , 51 , 52 , 54 , 88 – 91 ] 2. Transcriptomics De novo transcriptome assemblies have been created from leaves, stamens, corm, tepals, and stigmas of Crocus sativus The most valued compounds of C. sativus are synthesised inside stigma in a developmental stage-specific manner During the transition from yellow stage to red stage stigmas there is an accumulation of zeaxanthin accompanied by sharp increase in the expression of phytoene synthase, phytoene desaturase, lycopene β cyclase, β carotene hydroxylase and zeaxanthin cleavage dioxygenase CsCCD2 (carotenoid cleavage dioxygenase) ESTs are prominent in the saffron stigma libraries obtained from early stages of stigma development UDP-glucosyltransferase is vital for conversion of crocetin to crocin, and therefore causes difference in metabolite accumulation between Crocus species 1 Deoxyxylulose 5 phosphate synthase (DXS) plays a vital role in apocarotenoid accumulation in stigma There is no direct concordance in the expression of Cs AP3 and Cs NAP gene expression in saffron Identification, isolation, and biochemical characterisation of uridine diphosphate glycosyltransferase (UGT709G1), which catalyses the HTCC glucosyltransferase reaction to yield picrocrocin, can provide a vital lead for the industrial production of picrocrocin/safranal Differentially expressed full-length transcripts of flowering and non-flowering saffron crocus have been identified and characterised Stigma development in field- and indoor-cultivated saffron is similar with respect to apocarotenoid content and gene expression profiles of 12 genes involved in apocarotenoid biosynthesis Carotenoid cleavage dioxygenase (CCD2) catalyzes the first step of crocin biosynthesis from carotenoid zeaxanthin and gets expressed at an extremely high level in the stigma as compared to corm, leaf, tepal, and stamen A C-class floral homeotic gene AGAMOUS (CsAG) gene is vital for stigma development of saffron.…”

Ancient Artworks and Crocus Genetics Both Support Saffron’s Origin in Early Greece

“…Hence, it is generally accepted that triploid saffron crocus emerged only once and was then distributed across the world (Rubio-Moraga et al, 2009;Fluch et al, 2010;Siracusa et al, 2013;Babaei et al, 2014;Alsayied et al, 2015;Nemati et al, 2019). However, a recent study by Busconi et al (2021) reported a surprising amount of genetic differences (singlenucleotide polymorphisms, SNPs). This was detected by a genomewide approach, in which primarily the global DNA methylation across five different saffron accessions was analyzed and a high epigenetic variability has been shown.…”