Progress and innovations of gene cloning in wheat and its close relatives

Abstract

Wheat and its close relatives have large and complex genomes, making gene cloning difficult. Nevertheless, developments in genomics over the past decade have made it more feasible. The large and complex genomes of cereals, especially bread wheat, have always been a challenge for gene mapping and cloning. Nevertheless, recent advances in genomics have led to significant progress in this field. Currently, high-quality reference sequences are available for major wheat species and their relatives. New high-throughput genotyping platforms and next-generation sequencing technologies combined with genome complexity reduction techniques and mutagenesis have opened new avenues for gene cloning. In this review, we provide a comprehensive overview of the genes cloned in wheat so far and discuss the strategies used for cloning these genes. We highlight the advantages and drawbacks of individual approaches and show how particular genomic progress contributed to wheat gene cloning. A wide range of new resources and approaches have led to a significant increase in the number of successful cloning projects over the past decade, demonstrating that it is now feasible to perform rapid gene cloning of agronomically important genes, even in a genome as large and complex as that of wheat.

Fig. 1

Workflow of map-based cloning. The first step of map-based cloning involves the development of a mapping population by crossing parents with contrasting phenotypes. The mapping population is genotyped with markers equally distributed across the genome and phenotyped for the trait of interest. Obtained markers are used for the construction of genetic map, which is used together with the phenotypes for QTL analysis. QTL analysis results in the identification of locus/loci determining the trait of interest. In the next step, recombinant lines, obtained by genotyping individuals descendent from plants heterozygous in the mapped region with flanking markers (Marker A and B), are phenotyped and genotyped with new markers (C, D, E, F, G, H) to saturate the mapped locus. This results in markers flanking the target gene within a more narrowly defined interval. The final step is to obtain the sequence between the markers flanking the gene of interest. Created with BioRender

Fig. 2

Workflow of mutagenesis-based cloning approaches. The first step of mutagenesis-based approaches involves development of mutant population and identification of loss-of-function mutants. Depending on the method used, the next step is genome complexity reduction. In MutRenSeq, wild-type parent and several independent EMS-derived mutants are subjected to disease resistance gene enrichment targeting the NLR sequences. The NLR sequences are NGS sequenced and wild-type reads are de novo assembled into NLR contigs. In MutChromSeq, the chromosome with the locus of interest from the wild-type parent and several independent EMS-derived mutants (black and red, respectively) are flow sorted. The chromosomes are NGS sequenced and wild-type reads are de novo assembled into contigs. In MutRNAseq and MutIsoSeq/STAM the complexity reduction is achieved by short-read transcriptome sequencing. The difference between these methods lies in sequencing of the wild type. In the case of MutRNAseq, the wild-type parent is subjected to long-read whole-genome sequencing, while in MutIsoSeq/STAM the wild-type parent is subjected to Iso-seq. The final step in all these approaches is the identification of candidate genes by mapping the reads from mutants to obtained contigs/transcripts of the wild type. The contig/transcript, for which a mutation is found in all examined mutants (Contig/Transcript 3), is subjected to candidate gene identification. Created with BioRender

Fig. 3

Dynamics of wheat gene cloning from 1997 to 2024. Note the significant increase in the number of successfully cloned wheat genes since 2014, driven by the publication of the draft bread wheat genome sequence and advances in the NGS sequencing and gene function validation approaches of reverse genomics