Genome analyses reveal population structure and a purple stigma color gene candidate in finger millet.

Finger millet is a key food security crop widely grown in eastern Africa, India and Nepal. Long considered a 'poor man's crop', finger millet has regained attention over the past decade for its climate resilience and the nutritional qualities of its grain. To bring finger millet breeding into the 21st century, here we present the assembly and annotation of a chromosome-scale reference genome. We show that this ~1.3 million years old allotetraploid has a high level of homoeologous gene retention and lacks subgenome dominance. Population structure is mainly driven by the differential presence of large wild segments in the pericentromeric regions of several chromosomes. Trait mapping, followed by variant analysis of gene candidates, reveals that loss of purple coloration of anthers and stigma is associated with loss-of-function mutations in the finger millet orthologs of the maize R1/B1 and Arabidopsis GL3/EGL3 anthocyanin regulatory genes. Proanthocyanidin production in seed is not affected by these gene knockouts.

Genome-wide identification and development of miniature inverted-repeat transposable elements and intron length polymorphic markers in tea plant (Camellia sinensis).

Marker-assisted breeding and tagging of important quantitative trait loci for beneficial traits are two important strategies for the genetic improvement of plants. However, the scarcity of diverse and informative genetic markers covering the entire tea genome limits our ability to achieve such goals. In the present study, we used a comparative genomic approach to mine the tea genomes of Camellia sinensis var. assamica (CSA) and C. sinensis var. sinensis (CSS) to identify the markers to differentiate tea genotypes. In our study, 43 and 60 Camellia sinensis miniature inverted-repeat transposable element (CsMITE) families were identified in these two sequenced tea genomes, with 23,170 and 37,958 putative CsMITE sequences, respectively. In addition, we identified 4912 non-redundant, Camellia sinensis intron length polymorphic (CsILP) markers, 85.8% of which were shared by both the CSS and CSA genomes. To validate, a subset of randomly chosen 10 CsMITE markers and 15 CsILP markers were tested and found to be polymorphic among the 36 highly diverse tea genotypes. These genome-wide markers, which were identified for the first time in tea plants, will be a valuable resource for genetic diversity analysis as well as marker-assisted breeding of tea genotypes for quality improvement.

Genomic, morphological, and biochemical analyses of a multi-metal resistant but multi-drug susceptible strain of Bordetella petrii from hospital soil.

Contamination of soil by antibiotics and heavy metals originating from hospital facilities has emerged as a major cause for the development of resistant microbes. We collected soil samples surrounding a hospital effluent and measured the resistance of bacterial isolates against multiple antibiotics and heavy metals. One strain BMCSI 3 was found to be sensitive to all tested antibiotics. However, it was resistant to many heavy metals and metalloids like cadmium, chromium, copper, mercury, arsenic, and others. This strain was motile and potentially spore-forming. Whole-genome shotgun assembly of BMCSI 3 produced 4.95 Mb genome with 4,638 protein-coding genes. The taxonomic and phylogenetic analysis revealed it, to be a Bordetella petrii strain. Multiple genomic islands carrying mobile genetic elements; coding for heavy metal resistant genes, response regulators or transcription factors, transporters, and multi-drug efflux pumps were identified from the genome. A comparative genomic analysis of BMCSI 3 with annotated genomes of other free-living B. petrii revealed the presence of multiple transposable elements and several genes involved in stress response and metabolism. This study provides insights into how genomic reorganization and plasticity results in evolution of heavy metals resistance by acquiring genes from its natural environment.

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.

Genome Comparison Identifies Different Bacillus Species in a Bast Fibre-Retting Bacterial Consortium and Provides Insights into Pectin Degrading Genes.

Retting of bast fibres requires removal of pectin, hemicellulose and other non-cellulosic materials from plant stem tissues by a complex microbial community. A microbial retting consortium with high-efficiency pectinolytic bacterial strains is effective in reducing retting-time and enhancing fibre quality. We report comprehensive genomic analyses of three bacterial strains (PJRB 1, 2 and 3) of the consortium and resolve their taxonomic status, genomic features, variations, and pan-genome dynamics. The genome sizes of the strains are ~3.8 Mb with 3729 to 4002 protein-coding genes. Detailed annotations of the protein-coding genes revealed different carbohydrate-degrading CAZy classes viz. PL1, PL9, GH28, CE8, and CE12. Phylogeny and structural features of pectate lyase proteins of PJRB strains divulge their functional uniqueness and evolutionary convergence with closely related Bacillus strains. Genome-wide prediction of genomic variations revealed 12461 to 67381 SNPs, and notably many unique SNPs were localized within the important pectin metabolism genes. The variations in the pectate lyase genes possibly contribute to their specialized pectinolytic function during the retting process. These findings encompass a strong foundation for fundamental and evolutionary studies on this unique microbial degradation of decaying plant material with immense industrial significance. These have preponderant implications in plant biomass research and food industry, and also posit application in the reclamation of water pollution from plant materials.

Genetic diversity and population structure detection in sponge gourd (Luffa cylindrica) using ISSR, SCoT and morphological markers.

Investigation of genetic diversity is essential for the selection of parents for crop breeding and conservation of genetic resources. To estimate the genetic variability and population structure in the midst of 45 accessions of sponge gourd brought together from different geographical areas of India, morphological traits and two molecular markers, ISSR and SCoT markers were compared. Principal components analysis of 20 morphological traits showed 72.70% variability and significant positive correlations between fruit traits. All three marker techniques clustered all accessions into two groups with few outgroups. High level of polymorphism was observed among ISSR (74.6%) and SCoT (71.5%) primers. The Bayesian model revealed the hidden grouping and showed admixture type of population. The diversity pattern is influenced by genetic marker used, as different molecular markers have different polymorphism evaluation efficiency. This study can be helpful in amplifying the genetic base and selection of specific traits for breeding. Thus, ISSR and SCoT markers are potential marker for identification in sponge gourd and provide valuable data on its genetic correlation and structure.

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.

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.

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.

UGbS-Flex, a novel bioinformatics pipeline for imputation-free SNP discovery in polyploids without a reference genome: finger millet as a case study.

BACKGROUND: Research on orphan crops is often hindered by a lack of genomic resources. With the advent of affordable sequencing technologies, genotyping an entire genome or, for large-genome species, a representative fraction of the genome has become feasible for any crop. Nevertheless, most genotyping-by-sequencing (GBS) methods are geared towards obtaining large numbers of markers at low sequence depth, which excludes their application in heterozygous individuals. Furthermore, bioinformatics pipelines often lack the flexibility to deal with paired-end reads or to be applied in polyploid species. RESULTS: UGbS-Flex combines publicly available software with in-house python and perl scripts to efficiently call SNPs from genotyping-by-sequencing reads irrespective of the species' ploidy level, breeding system and availability of a reference genome. Noteworthy features of the UGbS-Flex pipeline are an ability to use paired-end reads as input, an effective approach to cluster reads across samples with enhanced outputs, and maximization of SNP calling. We demonstrate use of the pipeline for the identification of several thousand high-confidence SNPs with high representation across samples in an F3-derived F2 population in the allotetraploid finger millet. Robust high-density genetic maps were constructed using the time-tested mapping program MAPMAKER which we upgraded to run efficiently and in a semi-automated manner in a Windows Command Prompt Environment. We exploited comparative GBS with one of the diploid ancestors of finger millet to assign linkage groups to subgenomes and demonstrate the presence of chromosomal rearrangements. CONCLUSIONS: The paper combines GBS protocol modifications, a novel flexible GBS analysis pipeline, UGbS-Flex, recommendations to maximize SNP identification, updated genetic mapping software, and the first high-density maps of finger millet. The modules used in the UGbS-Flex pipeline and for genetic mapping were applied to finger millet, an allotetraploid selfing species without a reference genome, as a case study. The UGbS-Flex modules, which can be run independently, are easily transferable to species with other breeding systems or ploidy levels.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants.

A promoter trap mutant line of Arabidopsis carrying a promoterless ß-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

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).

Genetic variation among species, races, forms and inbred lines of lac insects belonging to the genus Kerria (Homoptera, Tachardiidae).

THE LAC INSECTS (HOMOPTERA: Tachardiidae), belonging to the genus Kerria, are commercially exploited for the production of lac. Kerria lacca is the most commonly used species in India. RAPD markers were used for assessing genetic variation in forty-eight lines of Kerria, especially among geographic races, infrasubspecific forms, cultivated lines, inbred lines, etc., of K. lacca. In the 48 lines studied, the 26 RAPD primers generated 173 loci, showing 97.7% polymorphism. By using neighbor-joining, the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups. The major cluster, comprising 32 lines, included mainly cultivated lines of the rangeeni form, geographic races and inbred lines of K. lacca. The second cluster consisted of eight lines of K. lacca, seven of the kusmi form and one of the rangeeni from the southern state of Karnataka. The remaining eight lines formed a series of outgroups, this including a group of three yellow mutant lines of K. lacca and other species of the Kerria studied, among others. Color mutants always showed distinctive banding patterns compared to their wild-type counterparts from the same population. This study also adds support to the current status of kusmi and rangeeni, as infraspecific forms of K. lacca.

Genetic variation among species, races, forms and inbred lines of lac insects belonging to the genus Kerria (Homoptera, Tachardiidae)

The lac insects (Homoptera: Tachardiidae), belonging to the genus Kerria, are commercially exploited for the production of lac. Kerria lacca is the most commonly used species in India. RAPD markers were used for assessing genetic variation in forty-eight lines of Kerria, especially among geographic races, infrasubspecific forms, cultivated lines, inbred lines, etc., of K. lacca. In the 48 lines studied, the 26 RAPD primers generated 173 loci, showing 97.7 percent polymorphism. By using neighbor-joining, the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups. The major cluster, comprising 32 lines, included mainly cultivated lines of the rangeeni form, geographic races and inbred lines of K. lacca. The second cluster consisted of eight lines of K. lacca, seven of the kusmi form and one of the rangeeni from the southern state of Karnataka. The remaining eight lines formed a series of outgroups, this including a group of three yellow mutant lines of K. lacca and other species of the Kerria studied, among others. Color mutants always showed distinctive banding patterns compared to their wild-type counterparts from the same population. This study also adds support to the current status of kusmi and rangeeni, as infraspecific forms of K. lacca.

In Silico analysis of the lateral organ junction (LOJ) gene and promoter of Arabidopsis thaliana.

A T-DNA based promoter trapped mutant has led to the identification of a novel lateral organ junction specific promoter upstream of the pentatricopeptide repeat (PPR) protein coding gene LOJ in Arabidopsis thaliana by our laboratory. Various in silico based prediction tools are employed to characterize the upstream sequence of the LOJ gene. Out of numerous cis-elements detected in the LOJ promoter a few are considered important based on the expression pattern of the LOJ gene. These elements would provide a basis for designing experiments for more accurate promoter function annotation. A comparative search for conserved elements in the 5'-upstream region of a few genes involved in lateral organ development and meristem related expression reveals a few common relevant regulatory motifs. The coding region of the LOJ gene is intron-less and contains 19 PPR units. Based on in silico analysis, LOJ protein is predicted to be hydrophobic in nature and targeted to mitochondria. A partial 3D model of LOJ protein has been suggested using a homology-based modeling program.