Novel PHOTOPERIOD-1 gene variants associate with yield-related and root-angle traits in European bread wheat.

KEY MESSAGE: PHOTOPERIOD-1 homoeologous gene copies play a pivotal role in regulation of flowering time in wheat. Here, we show that their influence also extends to spike and shoot architecture and even impacts root development. The sequence diversity of three homoeologous copies of the PHOTOPERIOD-1 gene in European winter wheat was analyzed by Oxford Nanopore amplicon-based multiplex sequencing and molecular markers in a panel of 194 cultivars representing breeding progress over the past 5 decades. A strong, consistent association with an average 8% increase in grain yield was observed for the PpdA1-Hap1 haplotype across multiple environments. This haplotype was found to be linked in 51% of cultivars to the 2NS/2AS translocation, originally introduced from Aegilops ventricosa, which leads to an overestimation of its effect. However, even in cultivars without the 2NS/2AS translocation, PpdA1-Hap1 was significantly associated with increased grain yield, kernel per spike and kernel per m2 under optimal growth conditions, conferring a 4% yield advantage compared to haplotype PpdA1-Hap4. In contrast to Ppd-B1 and Ppd-D1, the Ppd-A1 gene exhibits novel structural variations and a high number of SNPs, highlighting the evolutionary changes that have occurred in this region over the course of wheat breeding history. Additionally, cultivars carrying the photoperiod-insensitive Ppd-D1a allele not only exhibit earlier heading, but also deeper roots compared to those with photoperiod-sensitive alleles under German conditions. PCR and KASP assays have been developed that can be effectively employed in marker-assisted breeding programs to introduce these favorable haplotypes.

Frequent spontaneous structural rearrangements promote rapid genome diversification in a Brassica napus F1 generation.

In a cross between two homozygous Brassica napus plants of synthetic and natural origin, we demonstrate that novel structural genome variants from the synthetic parent cause immediate genome diversification among F1 offspring. Long read sequencing in twelve F1 sister plants revealed five large-scale structural rearrangements where both parents carried different homozygous alleles but the heterozygous F1 genomes were not identical heterozygotes as expected. Such spontaneous rearrangements were part of homoeologous exchanges or segmental deletions and were identified in different, individual F1 plants. The variants caused deletions, gene copy-number variations, diverging methylation patterns and other structural changes in large numbers of genes and may have been causal for unexpected phenotypic variation between individual F1 sister plants, for example strong divergence of plant height and leaf area. This example supports the hypothesis that spontaneous de novo structural rearrangements after de novo polyploidization can rapidly overcome intense allopolyploidization bottlenecks to re-expand crops genetic diversity for ecogeographical expansion and human selection. The findings imply that natural genome restructuring in allopolyploid plants from interspecific hybridization, a common approach in plant breeding, can have a considerably more drastic impact on genetic diversity in agricultural ecosystems than extremely precise, biotechnological genome modifications.

Long-Amplicon Single-Molecule Sequencing Reveals Novel, Trait-Associated Variants of VERNALIZATION1 Homoeologs in Hexaploid Wheat.

The gene VERNALIZATION1 (VRN1) is a key controller of vernalization requirement in wheat. The genome of hexaploid wheat (Triticum aestivum) harbors three homoeologous VRN1 loci on chromosomes 5A, 5B, and 5D. Structural sequence variants including small and large deletions and insertions and single nucleotide polymorphisms (SNPs) in the three homoeologous VRN1 genes not only play an important role in the control of vernalization requirement, but also have been reported to be associated with other yield related traits of wheat. Here we used single-molecule sequencing of barcoded long-amplicons to assay the full-length sequences (∼13 kbp plus 700 bp from the promoter sequence) of the three homoeologous VRN1 genes in a panel of 192 predominantly European winter wheat cultivars. Long read sequences revealed previously undetected duplications, insertions and single-nucleotide polymorphisms in the three homoeologous VRN1 genes. All the polymorphisms were confirmed by Sanger sequencing. Sequence analysis showed the predominance of the winter alleles vrn-A1, vrn-B1, and vrn-D1 across the investigated cultivars. Associations of SNPs and structural variations within the three VRN1 genes with 20 economically relevant traits including yield, nodal root-angle index and quality related traits were evaluated at the levels of alleles, haplotypes, and copy number variants. Cultivars carrying structural variants within VRN1 genes showed lower grain yield, protein yield and biomass compared to those with intact genes. Cultivars carrying a single vrn-A1 copy and a unique haplotype with a high number of SNPs were found to have elevated grain yield, kernels per spike and kernels per m2 along with lower grain sedimentation values. In addition, we detected a novel SNP polymorphism within the G-quadruplex region of the promoter of vrn-A1 that was associated with deeper roots in winter wheat. Our findings show that multiplex, single-molecule long-amplicon sequencing is a useful tool for detecting variants in target genes within large plant populations, and can be used to simultaneously assay sequence variants among target multiple gene homoeologs in polyploid crops. Numerous novel VRN1 haplotypes and alleles were identified that showed significantly associations to economically important traits. These polymorphisms were converted into PCR or KASP assays for use in marker-assisted breeding.

Development of Breeder-Friendly KASP Markers from Genome-Wide Association Studies Results.

Array-based SNP markers are commonly used in genome-wide association studies (GWAS) to identify genomic regions involved in important agronomical traits. However, conversion of these SNP markers into breeder-friendly kompetitive allele-specific PCR (KASP) markers for use in marker-assisted selection is often challenging. In this chapter we describe general considerations and successfully applied protocols for the conversion of Illumina array SNP markers into locus-specific KASP markers with a special emphasis and examples on how to overcome difficulties in polyploid wheat.

A toolkit to rapidly modify root systems through single plant selection.

BACKGROUND: The incorporation of root traits into elite germplasm is typically a slow process. Thus, innovative approaches are required to accelerate research and pre-breeding programs targeting root traits to improve yield stability in different environments and soil types. Marker-assisted selection (MAS) can help to speed up the process by selecting key genes or quantitative trait loci (QTL) associated with root traits. However, this approach is limited due to the complex genetic control of root traits and the limited number of well-characterised large effect QTL. Coupling MAS with phenotyping could increase the reliability of selection. Here we present a useful framework to rapidly modify root traits in elite germplasm. In this wheat exemplar, a single plant selection (SPS) approach combined three main elements: phenotypic selection (in this case for seminal root angle); MAS using KASP markers (targeting a root biomass QTL); and speed breeding to accelerate each cycle. RESULTS: To develop a SPS approach that integrates non-destructive screening for seminal root angle and root biomass, two initial experiments were conducted. Firstly, we demonstrated that transplanting wheat seedlings from clear pots (for seminal root angle assessment) into sand pots (for root biomass assessment) did not impact the ability to differentiate genotypes with high and low root biomass. Secondly, we demonstrated that visual scores for root biomass were correlated with root dry weight (r = 0.72), indicating that single plants could be evaluated for root biomass in a non-destructive manner. To highlight the potential of the approach, we applied SPS in a backcrossing program which integrated MAS and speed breeding for the purpose of rapidly modifying the root system of elite bread wheat line Borlaug100. Bi-directional selection for root angle in segregating generations successfully shifted the mean root angle by 30° in the subsequent generation (P ≤ 0.05). Within 18 months, BC2F4:F5 introgression lines were developed that displayed a full range of root configurations, while retaining similar above-ground traits to the recurrent parent. Notably, the seminal root angle displayed by introgression lines varied more than 30° compared to the recurrent parent, resulting in lines with both narrow and wide root angles, and high and low root biomass phenotypes. CONCLUSION: The SPS approach enables researchers and plant breeders to rapidly manipulate root traits of future crop varieties, which could help improve productivity in the face of increasing environmental fluctuations. The newly developed elite wheat lines with modified root traits provide valuable materials to study the value of different root systems to support yield in different environments and soil types.