DNA hypomethylation is the plausible driver of heat stress adaptation in Linum usitatissimum.

Heat stress has a significant impact on the climatic adaptation of flax, a cool-season economic crop. Genome-wide DNA methylation patterns are crucial for understanding how flax cultivars respond to heat adversities. It is worth noting that the DNA methylome in flax has yet to be investigated at the nucleotide level. Although heat stress above 40°C caused oxidative damage in flax leaves, 5-azacytidine, a hypomethylating agent, reduced this effect by 15%-24%. Differences in the expression of the LuMET1 (DNA methyltransferase) gene suggested that DNA methylation/demethylation may play a major role in the flax heat stress response. Thus, whole-genome bisulfite sequencing-derived DNA methylation profiles in flax, with or without heat stress and 5-azaC, were developed and analyzed here. In response to heat stress, a high percentage of significant differentially methylated regions (DMRs), particularly hypomethylated DMRs, were identified in the CHH nucleotide sequence context (H = A/T/C). Some of these DMRs overlapped with transposable element insertions. The majority of DMRs were discovered in intergenic regions, but several DMR loci were also found near genes relevant to heat stress response and epigenetic processes. These DMRs, in particular, are linked to CpG islands, implying a possible role in promoter methylation and gene silencing. The DMRs discovered in this study are crucial for understanding and identifying the key players in heat stress response in flax, which will help in developing climate-smart flax varieties.

Resolving population structure and genetic differentiation associated with RAD-SNP loci under selection in tossa jute (Corchorus olitorius L.).

The genetic basis of selection for geographic adaptation and how it has contributed to population structure are unknown in tossa jute (Corchorus olitorius), an important bast fibre crop. We performed restriction site-associated DNA (RAD) sequencing-based (1115 RAD-SNPs) population genomic analyses to investigate genetic differentiation and population structure within a collection of 221 fibre-type lines from across nine geographic regions of the world. Indian populations, with relatively higher overall diversity, were significantly differentiated (based on FST and PCA) from the African and the other Asian populations. There is strong evidence that African C. olitorius was first introduced in peninsular India that could perhaps be its secondary centre of origin. However, multiple later introductions have occurred in central, eastern and northern India. Based on four assignment tests with different statistical bases, we infer that two ancestral subpopulations (African and Indian) structure the C. olitorius populations, but not in accordance with their geographic origins and patterns of diversity. Our results advocate recent migration of C. olitorius through introduction and germplasm exchange across geographical boundaries. We argue that high intraspecific genetic admixture could be associated with increased genetic variance within Indian populations. Employing both subpopulation (FST/GST-outlier) and individual-based (PCAdapt) tests, we detected putative RAD-SNP loci under selection and demonstrated that bast fibre production was an artificial, while abiotic and biotic stresses were natural selection pressures in C. olitorius adaptation. By reinferring the population structure without outlier loci, we propose ad interim that C. olitorius was possibly domesticated as a fibre crop in the Indian subcontinent.

Genome-wide regulatory gene-derived SSRs reveal genetic differentiation and population structure in fiber flax genotypes.

We designed a set of 580 simple sequence repeat markers; 506 from transcription factor-coding genes, and 74 from long non-coding RNAs and designated them as regulatory gene-derived simple sequence repeat (ReG-SSR) markers. From this set, we could anchor 559 ReG-SSR markers on 15 flax chromosomes with an average marker distance of 0.56 Mb. Thirty-one polymorphic ReG-SSR primers, amplifying SSR loci length of at least 20 bp were chosen from 134 screened primers. This primer set was used to characterize a diversity panel of 93 flax accessions. The panel included 33 accessions from India, including released varieties, dual-purpose lines and landraces, and 60 fiber flax accessions from the global core collection. Thirty-one ReG-SSR markers generated 76 alleles, with an average of 2.5 alleles per primer and a mean allele frequency of 0.77. These markers recorded 0.32 average gene diversity, 0.26 polymorphism information content and 1.35% null alleles. All the 31 ReG-SSR loci were found selectively neutral and showed no evidence of population reduction. A model-based clustering analysis separated the flax accessions into two sub-populations-Indian and global, with some accessions showing admixtures. The distinct clustering pattern of the Indian accessions compared to the global accessions, conforms to the principal coordinate analysis, genetic dissimilarity-based unweighted neighbor-joining tree and analysis of molecular variance. Fourteen flax accessions with 99.3% allelic richness were found optimum to adopt in breeding programs. In summary, the genome-wide ReG-SSR markers will serve as a functional marker resource for genetic and phenotypic relationship studies, marker-assisted selections, and provide a basis for selection of accessions from the Indian and global gene pool in fiber flax breeding programs.

Genomic insights into HSFs as candidate genes for high-temperature stress adaptation and gene editing with minimal off-target effects in flax.

Flax (Linum usitatissimum) is a cool season crop commercially cultivated for seed oil and stem fibre production. A comprehensive characterization of the heat shock factor (HSF) candidate genes in flax can accelerate genetic improvement and adaptive breeding for high temperature stress tolerance. We report the genome-wide identification of 34 putative HSF genes from the flax genome, which we mapped on 14 of the 15 chromosomes. Through comparative homology analysis, we classified these genes into three broad groups, and sub-groups. The arrangement of HSF-specific protein motifs, DNA-binding domain (DBD) and hydrophobic heptad repeat (HR-A/B), and exon-intron boundaries substantiated the phylogenetic separation of these genes. Orthologous relationships and evolutionary analysis revealed that the co-evolution of the LusHSF genes was due to recent genome duplication events. Digital and RT-qPCR analyses provided significant evidence of the differential expression of the LusHSF genes in various tissues, at various developmental stages, and in response to high-temperature stress. The co-localization of diverse cis-acting elements in the promoters of the LusHSF genes further emphasized their regulatory roles in the abiotic stress response. We further confirmed DNA-binding sites on the LusHSF proteins and designed guide RNA sequences for gene editing with minimal off-target effects. These results will hasten functional investigations of LusHSFs or assist in devising genome engineering strategies to develop high-temperature stress tolerant flax cultivars.

Transcriptome profiling uncovers ß-galactosidases of diverse domain classes influencing hypocotyl development in jute (Corchorus capsularis L.).

Enzyme ß-galactosidase (EC 3.2.1.23) is known to influence vascular differentiation during early vegetative growth of plants, but its role in hypocotyl development is not yet fully understood. We generated the hypocotyl transcriptome data of a hypocotyl-defect jute (Corchorus capsularis L.) mutant (52,393 unigenes) and its wild-type (WT) cv. JRC-212 (44,720 unigenes) by paired-end RNA-seq and identified 11 isoforms of ß-galactosidase, using a combination of sequence annotation, domain identification and structural-homology modeling. Phylogenetic analysis classified the jute ß-galactosidases into six subfamilies of glycoside hydrolase-35 family, which are closely related to homologs from Malvaceous species. We also report here the expression of a ß-galactosidase of glycoside hydrolase-2 family that was earlier considered to be absent in higher plants. Comparative analysis of domain structure allowed us to propose a domain-centric evolution of the five classes of plant ß-galactosidases. Further, we observed 1.8-12.2-fold higher expression of nine ß-galactosidase isoforms in the mutant hypocotyl, which was characterized by slower growth, undulated shape and deformed cell wall. In vitro and in vivo ß-galactosidase activities were also higher in the mutant hypocotyl. Phenotypic analysis supported a significant (P ≤ 0.01) positive correlation between enzyme activity and undulated hypocotyl. Taken together, our study identifies the complete set of ß-galactosidases expressed in the jute hypocotyl, and provides compelling evidence that they may be involved in cell wall degradation during hypocotyl development.

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions.

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST-SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI=0.46; He=0.31; P=85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P=0.01. The maximum Nei's unbiased genetic distance (D=0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.

The draft genome of Corchorus olitorius cv. JRO-524 (Navin).

Here, we present the draft genome (377.3 Mbp) of Corchorus olitorious cv. JRO-524 (Navin), which is a leading dark jute variety developed from a cross between African (cv. Sudan Green) and indigenous (cv. JRO-632) types. We predicted from the draft genome a total of 57,087 protein-coding genes with annotated functions. We identified a large number of 1765 disease resistance-like and defense response genes in the jute genome. The annotated genes showed the highest sequence similarities with that of Theobroma cacao followed by Gossypium raimondii. Seven chromosome-scale genetically anchored pseudomolecules were constructed with a total size of 8.53 Mbp and used for synteny analyses with the cocoa and cotton genomes. Like other plant species, gypsy and copia retrotransposons were the most abundant classes of repeat elements in jute. The raw data of our study are available in SRA database of NCBI with accession number SRX1506532. The genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession LLWS00000000, and the version described in this paper will be the first version (LLWS01000000).