A haploid genetics toolbox for Arabidopsis thaliana

Ravi, Maruthachalam; Marimuthu, Mohan Prem Anand; Tan, Ek Han; Maheshwari, Shamoni; Henry, Isabelle; Marin-Rodriguez, Brenda; Urtecho, Guillaume; Tan, James S.; Thornhill, Kristina; Zhu, Fan; Panoli, Aneesh; Sundaresan, Venkatesan; Britt, Anne B.; Comai, Luca; Chan, Simon W. L.

doi:10.1038/ncomms6334

Cited by 107 publications

(118 citation statements)

References 49 publications

Supporting

Mentioning

107

Contrasting

Order By: Relevance

“…Even the most recently developed CRISPR/Cas method requires production of the T1 generation for functional evaluation of modified plant genes [36,37]. When multiple genes in a gene family are knocked out to understand gene function redundancies, additional generations and a larger population will be needed for obtaining homozygous genotypes [34]. The required resources are an important constraint and bottleneck of functional validation in plants.…”

Section: Time and Resources Required For Current Approachesmentioning

confidence: 99%

“…Independent of the approach, diploid plants are usually heterozygous for the modified region in the first generation (T0 or M1) [25,34]. Altered genes and sequence motifs are often recessive and, therefore, without phenotypic effect in mutagenized T0 or M1 plants [34]. One or more additional generations are thus required to obtain homozygous genotypes for altered genes or sequence motifs that can actually be used for validation of their phenotypic effect [34,35].…”

Section: Time and Resources Required For Current Approachesmentioning

confidence: 99%

Section: Time and Resources Required For Current Approachesmentioning

confidence: 99%

“…Some plant species such as maize, Arabidopsis and barley can produce haploids by uniparental genome elimination via a male inducer [34,43,44].…”

Section: Strategies: Using Haploids For Functional Validation Of Planmentioning

confidence: 99%

“…Altered genes and sequence motifs are often recessive and, therefore, without phenotypic effect in mutagenized T0 or M1 plants [34]. One or more additional generations are thus required to obtain homozygous genotypes for altered genes or sequence motifs that can actually be used for validation of their phenotypic effect [34,35]. Even the most recently developed CRISPR/Cas method requires production of the T1 generation for functional evaluation of modified plant genes [36,37].…”

mentioning

confidence: 99%

See 4 more Smart Citations

Haploid Strategies for Functional Validation of Plant Genes

Shen

Pan

Lübberstedt

2015

Trends in Biotechnology

View full text Add to dashboard Cite

Increasing knowledge of plant genome sequences requires the development of more reliable and efficient genetic approaches for genotype-phenotype validation. Functional identification of plant genes is generally achieved by a combination of creating genetic modifications and observing the according phenotype, which begins with forward-genetic methods represented by random physical and chemical mutagenesis and move towards reverse-genetic tools as targeted genome editing. A major bottleneck is time need to produce modified homozygous genotypes that can actually be used for phenotypic validation. Herein, we comprehensively address and compare available experimental approaches for functional validation of plant genes, and propose haploid strategies to reduce the time needed and cost consumed for establishing gene function. Classic and current approaches to establish plant gene functionThe classic approach for plant gene function establishment began with loss-of-function mutagenesis after treatment with mutagens such as radiation with X-rays or neutrons, or chemicals that introduce random small deletions or point mutations in plant genomes [5,8]. Chemical mutagens such as ethyl methanesulfonate (EMS) were more popular as they are less destructive, easier available, and have a higher efficiency than physical mutagens [9]. Theoretically, we can find a EMS mutation in any given gene by screening no more than 5000 plants from the mutagenized M1 generation for the model plant Arabidopsis [3,8]. Conventional mutagenesis has been widely used in forward genetic strategies that start with a phenotype of interest and address identification of genes affecting this phenotype [9][10][11].As an alternative forward-genetic tool, insertional mutagenesis, including T-DNA (Transferred DNA) and transposon tagging, facilitate the identification of genes disrupted by these elements [5]. Currently, T-DNA-tagged lines have been generated in large numbers, becoming a popular resource for plant gene function [12,13].Superior to T-DNA, mobilizable transponsons can provide a verification about mutational effects of insertions when they are remobilized from the insertion site to recover a potential phenotype [14]. Insertional mutagenesis is less practicable for species, for which a systematic transformation platform has not been established [15].As the number of characterized plant genes increases, reverse-genetic methodologies play an increasingly important role in gene function validation [16]. Targeting Induced Local Lesions in Genomes (TILLING) is the first reverse-genetic tool, in which chemical or physical mutagenesis is followed by a high-throughput screening for point mutations [3,4,16]. TILLING is practicable for plant species with large-sized genomes and without transformation system because it is not different from traditional chemical or physical mutagenesis in creating mutations [12].Changes of gene expression levels may result in modified phenotypes, which can be another powerful approach for elucidating gene function [5...

show abstract

Section: Time and Resources Required For Current Approachesmentioning

confidence: 99%