Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Wheat in vivo RNA structure landscape reveals a prevalent role of RNA structure in modulating translational subgenome expression asymmetry

Background Polyploidy, especially allopolyploidy, which entails merging divergent genomes via hybridization and whole-genome duplication (WGD), is a major route to speciation in plants. The duplication among the parental genomes (subgenomes) often leads to one subgenome becoming dominant over the other(s), resulting in subgenome asymmetry in gene content and expression. Polyploid wheats are allopolyploids with most genes present in two (tetraploid) or three (hexaploid) functional copies, which commonly show subgenome expression asymmetry. It is unknown whether a similar subgenome asymmetry exists during translation. We aim to address this key biological question and explore the major contributing factors to subgenome translation asymmetry. Results Here, we obtain the first tetraploid wheat translatome and reveal that subgenome expression asymmetry exists at the translational level. We further perform in vivo RNA structure profiling to obtain the wheat RNA structure landscape and find that mRNA structure has a strong impact on translation, independent of GC content. We discover a previously uncharacterized contribution of RNA structure in subgenome translation asymmetry. We identify 3564 single-nucleotide variations (SNVs) across the transcriptomes between the two tetraploid wheat subgenomes, which induce large RNA structure disparities. These SNVs are highly conserved within durum wheat cultivars but are divergent in both domesticated and wild emmer wheat. Conclusions We successfully determine both the translatome and in vivo RNA structurome in tetraploid wheat. We reveal that RNA structure serves as an important modulator of translational subgenome expression asymmetry in polyploids. Our work provides a new perspective for molecular breeding of major polyploid crops.

Detecting quantitative trait loci and exploring chromosomal pairing in autopolyploids using polyqtlR

Motivation: The investigation of quantitative trait loci (QTL) is an essential component in our understanding of how organisms vary phenotypically. However, many important crop species are polyploid (carrying more than two copies of each chromosome), requiring specialised tools for such analyses. Moreover, deciphering meiotic processes at higher ploidy levels is not straightforward, but is necessary to understand the reproductive dynamics of these species, or uncover potential barriers to their genetic improvement. Results: Here we present polyqtlR, a novel software tool to facilitate such analyses in (auto)polyploid crops. It performs QTL interval mapping in F1 populations of outcrossing polyploids of any ploidy level using identity-by-descent (IBD) probabilities. The allelic composition of discovered QTL can be explored, enabling favourable alleles to be identified and tracked in the population. Visualisation tools within the package facilitate this process, and options to include genetic co-factors and experimental factors are included. Detailed information on polyploid meiosis including prediction of multivalent pairing structures, detection of preferential chromosomal pairing and location of double reduction events can be performed. Availability and implementation: polyqtlR is freely available from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polyqtlR.

Genome editing of polyploid crops: prospects, achievements and bottlenecks

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

Regulation and Subfunctionalization of Flowering Time Genes in the Allotetraploid Oil Crop Brassica napus

Flowering is a vulnerable, but crucial phase in building crop yield. Proper timing of this period is therefore decisive in obtaining optimal yields. However, genetic regulation of flowering integrates many different environmental signals and is therefore extremely complex. This complexity increases in polyploid crops which carry two or more chromosome sets, like wheat, potato or rapeseed. Here, I summarize the current state of knowledge about flowering time gene copies in rapeseed (Brassica napus), an important oil crop with a complex polyploid history and a close relationship to Arabidopsis thaliana. The current data show a high demand for more targeted studies on flowering time genes in crops rather than in models, allowing better breeding designs and a deeper understanding of evolutionary principles. Over evolutionary time, some copies of rapeseed flowering time genes changed or lost their original role, resulting in subfunctionalization of the respective homologs. For useful applications in breeding, such patterns of subfunctionalization need to be identified and better understood.

Edition of complex gene families in tobacco with GoldenBraid 4.0, a multipurpose web-based platform for plant genome engineering

ABSTRACTCRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL mutants harboring up to 9 biallelic mutations in a single generation. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and part’s collection, GB4.0 turns an unprecedentedly comprehensive open platform for plant genetic engineering.

Validation of Bread Wheat KASP Markers in Durum Wheat Lines in Kazakhstan

Development of efficient DNA markers plays an important role in modern breeding projects of many crops, including cultivated hexaploid bread wheat (BW) and tetraploid durum wheat (DW). Findings of genome-wide association studies on major polyploid crops, such as BW, may also help in molecular breeding studies in relative cultivated species with a similar genetic background, including DW. Therefore, the validation of identified quantitative trait loci or marker-trait associations is an important preliminary step in marker-assisted selection (MAS) oriented projects. In this study, thirty-two SNP (single nucleotide polymorphism) markers of six agronomic traits identified in BW, harvested in Kazakhstan, were converted to KASP (Kompetitive Allele-Specific PCR) as-says. Generated 32 KASP assays were used in the analysis of 29 DW accessions from Kazakhstan. Firstly, the group of DW accessions was tested using replicated and randomised one-metre blocks in field conditions of southeast Kazakhstan and evaluated for main agronomic traits. The analysis showed that 14 KASP assays were polymorphic in the scoring of 29 DW accessions. The t-test suggested that the segregation in eight KASP assays was significantly associated with five agronomic traits. The study confirms robustness of KASP assays in MAS of DW breeding projects for the improvement of yield potential.