An optimized isolation protocol yields high‐quality <scp>RNA</scp> from cassava tissues (<i>Manihot esculenta</i> Crantz)

Behnam, Babak; Bohórquez-Chaux, Adriana; Castañeda-Méndez, Oscar; Tsuji, Hiroyuki; Ishitani, Manabu; López-Lavalle, Luis Augusto Becerra

doi:10.1002/2211-5463.12561

Cited by 19 publications

(22 citation statements)

References 32 publications

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“…S19, S20). RNA extraction protocols and commercial kits are limited to specific species and/or tissue 3,17,36 , whereas the Fig. 3.…”

Section: Resultsmentioning

confidence: 99%

“…S19 , S20 ). RNA extraction protocols and commercial kits are limited to specific species and/or tissue 3 , 17 , 36 , whereas the modified SDS-LiCl method was suitable for extracting high quality RNA from various crop tissues differing in developmental stages and exposed to different abiotic stresses under controlled environment and field conditions.…”

Section: Resultsmentioning

confidence: 99%

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A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber

Vennapusa

Somayanda

Doherty

et al. 2020

Sci Rep

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Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7–9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.

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“…S19, S20). RNA extraction protocols and commercial kits are limited to specific species and/or tissue 3,17,36 , whereas the Fig. 3.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber

Vennapusa

Somayanda

Doherty

et al. 2020

Sci Rep

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“…Plants from accessions G25230 (Mesoamerican wild) and G27455 (Mesoamerican landrace) were grown under greenhouse conditions at the Centro Internacional de Agricultura Tropical (CIAT; Palmira, Valle del Cauca, Colombia). Total RNA was extracted from leaves, pods, and flowers with a specific protocol for each tissue 76 , 77 . For the analysis of differential gene expression related to pod dehiscence, RNA was extracted for the wild and domesticated accession at two pod developmental stages (with three replicates each), at the initiation of pod elongation (T1) and before seed filling (T2).…”

Section: Methodsmentioning

confidence: 99%

Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean

et al. 2021

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Lima bean (Phaseolus lunatus L.), one of the five domesticated Phaseolus bean crops, shows a wide range of ecological adaptations along its distribution range from Mexico to Argentina. These adaptations make it a promising crop for improving food security under predicted scenarios of climate change in Latin America and elsewhere. In this work, we combine long and short read sequencing technologies with a dense genetic map from a biparental population to obtain the chromosome-level genome assembly for Lima bean. Annotation of 28,326 gene models show high diversity among 1917 genes with conserved domains related to disease resistance. Structural comparison across 22,180 orthologs with common bean reveals high genome synteny and five large intrachromosomal rearrangements. Population genomic analyses show that wild Lima bean is organized into six clusters with mostly non-overlapping distributions and that Mesomerican landraces can be further subdivided into three subclusters. RNA-seq data reveal 4275 differentially expressed genes, which can be related to pod dehiscence and seed development. We expect the resources presented here to serve as a solid basis to achieve a comprehensive view of the degree of convergent evolution of Phaseolus species under domestication and provide tools and information for breeding for climate change resiliency.

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“…Total RNA was extracted using the methodology described by Behnam et al [80]. RNA was quantified using a Nanodrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA).…”

Section: Rna Extraction and Quality Assessmentmentioning

confidence: 99%

Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid

et al. 2020

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Background: Whiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/ subtropical regions. Understanding the molecular mechanisms regulating cassava's responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors. Results: The cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families. Conclusions: This study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA.

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An optimized isolation protocol yields high‐quality RNA from cassava tissues (Manihot esculenta Crantz)

Cited by 19 publications

References 32 publications

A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber

A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber

Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean

Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid

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