Cationic and anionic detergent buffers in sequence yield high-quality genomic DNA from diverse plant species.

Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75-1.85 against A260/280 and an A260/230 ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.

DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications.

BACKGROUND: High-purity RNA serves as the basic requirement for downstream molecular analysis of plant species, especially the differential expression of genes to various biotic and abiotic stimuli. But, the extraction of high-quality RNA is usually difficult from plants rich in polysaccharides and polyphenols, and their presence usually interferes with the downstream applications. The aim of the study is to optimize the extraction of high-quality RNA from diverse plant species/tissues useful for downstream molecular applications. RESULTS: Extraction of RNA using commercially available RNA extraction kits and routine hexadecyltrimethylammonium bromide (CTAB) methods did not yield good quality DNA-free RNA from Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera. A reliable protocol for the extraction of high-quality RNA from mature leaves of these difficult-to-extract trees was optimized after screening nine different methods. The DNase I-, and proteinase K treatment-free modified method, consisting of extraction with CTAB method followed by TRIzol, yielded high-quality DNA-free RNA with an A260/A280 and A260/A230 ratios > 2.0. Extraction of RNA from Conocarpus, the most difficult one, was successful by avoiding the heat incubation of ground tissue in a buffer at 65 oC. Pre-warming of the buffer for 5-10 min was sufficient to extract good-quality RNA. RNA integrity number of the extracted RNA samples ranged between 7 and 9.1, and the gel electrophoresis displayed intact bands of 28S and 18S RNA. A cDNA library constructed from the RNA of P. cineraria was used for the downstream applications. Real-time qPCR analysis using the cDNA from P. cineraria RNA confirmed the quality. The extraction of good quality RNA from samples of the desert-growing P. cineraria (> 20-years-old) collected in alternate months of the year 2021 (January to December covering winter, spring, autumn, and the very dry and hot summer) proved the efficacy of the protocol. The protocol's broad applicability was further validated by extracting good-quality RNA from 36 difficult-to-extract plant species, including tissues such as roots, flowers, floral organs, fruits, and seeds. CONCLUSIONS: The modified DNase I and Proteinase K treatment-free protocol enables to extract DNA-free, high-quality, intact RNA from a total of 39 difficult-to-extract plant species belonging to 32 angiosperm families is useful to extract good-quality RNA from dicots and monocots irrespective of tissue types and growing seasons.

Protocol for the High-quality Plasmid Isolation from Different Recalcitrant Bacterial Species: Agrobacterium spp., Rhizobium sp., and Bacillus thuringiensis.

High yield of good quality plasmid DNA from gram -ve bacteria (Agrobacterium tumefaciens, A. rhizogenes, and Rhizobium sp.) and gram +ve bacterium (Bacillus thuringiensis) is difficult. The widely used plasmid extraction kits for Escherichia coli yield a low quantity of poor-quality plasmid DNA from these species. We have optimized an in-house modification of the QIAprep Spin Miniprep kit protocol of Qiagen, consisting of two extraction steps. In the first, the centrifugation after adding neutralization buffer is followed by ethanol (absolute) precipitation of plasmid DNA. In the second extraction step, the precipitated DNA is dissolved in Tris-EDTA (TE) buffer, followed by an addition of 0.5 volumes of 5 M sodium chloride and 0.1 volumes of 20% (w/v) sodium dodecyl sulfate. After incubation at 65 °C for 15 min, the plasmid DNA is extracted with an equal volume of chloroform:isoamyl alcohol (CIA). RNase (20 mg/mL) is added to the upper phase retrieved after centrifugation and is incubated at 37 °C for 15 min. The extraction of the plasmid DNA with an equal volume of CIA is followed by centrifugation and is precipitated from the retrieved upper phase by adding an equal volume of absolute ethanol. The pellet obtained after centrifugation is washed twice with 70% (v/v) ethanol, air dried, dissolved in TE buffer, and quantified. This easy-to-perform protocol is free from phenol extraction, density gradient steps, and DNA binding columns, and yields high-quality plasmid DNA. The protocol opens an easy scale up to yield a large amount of high-quality plasmid DNA, useful for high-throughput downstream applications. Key features The protocol is free from density gradient steps and use of phenol. The protocol is an extension of the QIAprep Spin Miniprep kit (Qiagen) and is applicable for plasmid DNA isolation from difficult-to-extract bacterial species. The protocol facilitates the direct transformation of the ligation product into Agrobacterium by skipping the step of E. coli transformation. The plasmids isolated are of sequencing grade and the method is useful for extracting plasmids for metagenomic studies. Graphical overview Overview of the plasmid isolation protocol (modified QIAprep Spin Miniprep kit) of the present study.

The Genome of the Mimosoid Legume Prosopis cineraria, a Desert Tree.

The mimosoid legumes are a clade of ~40 genera in the Caesalpinioideae subfamily of the Fabaceae that grow in tropical and subtropical regions. Unlike the better studied Papilionoideae, there are few genomic resources within this legume group. The tree Prosopis cineraria is native to the Near East and Indian subcontinent, where it thrives in very hot desert environments. To develop a tool to better understand desert plant adaptation mechanisms, we sequenced the P. cineraria genome to near-chromosomal assembly, with a total sequence length of ~691 Mb. We predicted 77,579 gene models (76,554 CDS, 361 rRNAs and 664 tRNAs) from the assembled genome, among them 55,325 (~72%) protein-coding genes that were functionally annotated. This genome was found to consist of over 58% repeat sequences, primarily long terminal repeats (LTR-)-retrotransposons. We find an expansion of terpenoid metabolism genes in P. cineraria and its relative Prosopis alba, but not in other legumes. We also observed an amplification of NBS-LRR disease-resistance genes correlated with LTR-associated retrotransposition, and identified 410 retrogenes with an active burst of chimeric retrogene creation that approximately occurred at the same time of divergence of P. cineraria from a common lineage with P. alba~23 Mya. These retrogenes include many biotic defense responses and abiotic stress stimulus responses, as well as the early Nodulin 93 gene. Nodulin 93 gene amplification is consistent with an adaptive response of the species to the low nitrogen in arid desert soil. Consistent with these results, our differentially expressed genes show a tissue specific expression of isoprenoid pathways in shoots, but not in roots, as well as important genes involved in abiotic salt stress in both tissues. Overall, the genome sequence of P. cineraria enriches our understanding of the genomic mechanisms of its disease resistance and abiotic stress tolerance. Thus, it is a very important step in crop and legume improvement.

Transcriptome Profiling and Functional Validation of RING-Type E3 Ligases in Halophyte Sesuvium verrucosum under Salinity Stress.

Owing to their sessile nature, plants have developed a tapestry of molecular and physiological mechanisms to overcome diverse environmental challenges, including abiotic stresses. Adaptive radiation in certain lineages, such as Aizoaceae, enable their success in colonizing arid regions and is driven by evolutionary selection. Sesuvium verrucosum (commonly known as Western sea-purslane) is a highly salt-tolerant succulent halophyte belonging to the Aizoaceae family; thus, it provides us with the model-platform for studying plant adaptation to salt stress. Various transcriptional and translational mechanisms are employed by plants to cope with salt stress. One of the systems, namely, ubiquitin-mediated post-translational modification, plays a vital role in plant tolerance to abiotic stress and other biological process. E3 ligase plays a central role in target recognition and protein specificity in ubiquitin-mediated protein degradation. Here, we characterize E3 ligases in Sesuvium verrucosum from transcriptome analysis of roots in response to salinity stress. Our de novo transcriptome assembly results in 131,454 transcripts, and the completeness of transcriptome was confirmed by BUSCO analysis (99.3% of predicted plant-specific ortholog genes). Positive selection analysis shows 101 gene families under selection; these families are enriched for abiotic stress (e.g., osmotic and salt) responses and proteasomal ubiquitin-dependent protein catabolic processes. In total, 433 E3 ligase transcripts were identified in S. verrucosum; among these transcripts, single RING-type classes were more abundant compared to multi-subunit RING-type E3 ligases. Additionally, we compared the number of single RING-finger E3 ligases with ten different plant species, which confirmed the abundance of single RING-type E3 ligases in different plant species. In addition, differential expression analysis showed significant changes in 13 single RING-type E3 ligases (p-value < 0.05) under salinity stress. Furthermore, the functions of the selected E3 ligases genes (12 genes) were confirmed by yeast assay. Among them, nine genes conferred salt tolerance in transgenic yeast. This functional assay supports the possible involvement of these E3 ligase in salinity stress. Our results lay a foundation for translational research in glycophytes to develop stress tolerant crops.