Haploid Strategies for Functional Validation of Plant Genes

Abstract

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...

Novel technologies in doubled haploid line development

“…For single gene introgression, the expected probability of individuals with desired homozygous genotype is 1/4. The frequency of expected genotypes decreases exponentially following the formula 1/4 n , where n is the number of independently segregating genes (L€ ubberstedt and Frei, 2012;Ravi et al, 2014;Shen et al, 2015). In contrast, haploid target genotypes are generated with a frequency of 1/2 n .…”

State-of-the-art and novel developments of in vivo haploid technologies

“…The result is a substantial saving in both time and resources as compared to conventional practice. However, in many crop species, viable haploid technology is not yet available or only applicable to a limited number of genotypes and at exceedingly high costs, although the generation of haploid plants can be achieved by various approaches [reviewed by Dwivedi et al ( 2015 ), Ishii et al ( 2016 ), Kumlehn ( 2014 ) and Shen et al ( 2015 )]. Due to the application limits of current haploid technology, plant breeders are highly interested in any methodological improvements as well as in novel principles of haploidization.…”

Recent advances and perspectives in crop biofortification

“…Moreover, the homozygous make up of a target trait is often required in the breeding process. Notably, production of doubledhaploids could reduce the time needed for stabilization of the transgene in the genome, and then could be used in the functional validation of the gene and subsequent crossing (Shen et al 2015).…”