Large-Scale Rice Mutant Establishment and High-Throughput Mutant Manipulation Help Advance Rice Functional Genomics

Abstract

Rice (Oryza sativa L.) is a stable food for over half of the world population, contributing 50-80% of the daily calorie intake. The completion of rice genome sequencing marks a significant milestone in understanding functional genomics, yet the systematic identification of gene functions remains a bottleneck for rice improvement. Large-scale mutant libraries in which the functions of genes are lost or gained (e.g., through chemical/physical treatments, T-DNA, transposons, RNAi, CRISPR/Cas9) have proven to be powerful tools for the systematic linking of genotypes to phenotypes. So far, using different mutagenesis approaches, a million mutant lines have been established and about 5-10% of the predicted rice gene functions have been identified due to the high demands of labor and low-throughput utilization. DNA-barcoding-based large-scale mutagenesis offers unprecedented precision and scalability in functional genomics. This review summarizes large-scale loss-of-function and gain-of-function mutant library development approaches and emphasizes the integration of DNA barcoding for pooled analysis. Unique DNA barcodes can be tagged to transposons/retrotransposons, DNA constructs, miRNA/siRNA, gRNA, and cDNA, allowing for pooling analysis and the assignment of functions to genes that cause phenotype alterations. In addition, the integration of high-throughput phenotyping and OMICS technologies can accelerate the identification of gene functions.

Keywords: gain-of-function; loss-of-function; mutagenesis; mutant library and DNA barcoding; rice (Oryza sativa L.).

Figure 1

Insertional mutagenesis approaches to develop loss-of-function mutants. This figure illustrates three distinct approaches for developing loss-of-function mutants. (a) The T-DNA insertion method utilizes Agrobacterium tumefaciens harboring the binary vector to genetically transform rice, leading to the integration of T-DNA into the genome and the disruption of gene functions. (b) The Ac/Ds mutant lines are generated by crossing independently transformed Ac and Ds lines, which facilitates the disruption of gene functions in the subsequent generations. (c) The Tos17 insertion method involves tissue or callus culture activation to integrate Tos17 into the genome, resulting in the disruption of gene functions.

Figure 2

miRNA/siRNA-mediated RNA interference and CRISPR/Cas9-mediated mutagenesis approaches. This figure describes two advanced approaches for knockdown and loss-of-function mutagenesis. (a) The miRNA/siRNA mediated RNA silencing method involves genetic transformation using Agrobacterium that harbors pools of miRNA/siRNA constructs, enabling the generation of large-scale knockdown-mutant lines. (b) The CRISPR/Cas9-mediated mutagenesis approach utilizes genetic transformation with Agrobacterium containing pools of sgRNA constructs, facilitating the creation of large-scale knockout-mutant lines.

Figure 3

Gain-of-function mutagenesis approaches. This figure illustrates two key gain-of-function mutagenesis approaches. (a) Activation tagging: this approach involves the insertion of a strong enhancer construct, using Agrobacterium tumefaciens transformation, near a gene of interest, thereby enhancing its expression and resulting in the gain of function. BAR, glufosinate resistance; LB, left border; RB, right border. (b) FOX hunting. In this approach, full-length cDNA is cloned between a strong promoter and terminator and then transformed using Agrobacterium tumefaciens to drive the overexpression of specific genes. PUbi-1, ubiquitin promoter; Ter, terminator.

Figure 4

This figure illustrates a simplified dual-index barcoding approach, facilitating the simultaneous pooling of various sample types, including cDNA libraries for FOX hunting mutagenesis, gRNA for CRISPR/Cas9-mediated mutagenesis, and genomic DNA for identifying transposon or T-DNA insertion sites. This innovative design enhances the efficiency of next-generation sequencing (NGS) by allowing for the multiplexing of samples, thereby reducing costs and improving the throughput in genotyping. The color is to indicate the unique combinatorial barcoding method.