Differential evolution of Brassica flowering time genes

Many gene families seem to have a common ancestor, so obviously this ancestor has duplicated and diversified afterwards. Those processes of sub- and neofunctionalisation are central to gene evolution, but generally hard to observe due to the long periods involved. Brassica species are particularly well suited to study those processes, as they contain both mesohexaploid species and recent allotetraploids. It is therefore possible to study both long and short-term refunctionalisation.

This does not only have theoretical value, but is also important for understanding selective responses in breeding: Breeders need to know which copies they need to check for allelic variance and which copies have lower or no importance. The papers presented in this chapter show studies of increasing evidence for subfunctionalisation across the flowering time genes Bna.FT, Bna.FLC and Bna.SRR1. The invited Spotlight paper in Chapter 2.1 highlights that allelic variance in only one copy of each Bna.FT and Bna.FLC is responsible for ecotype differences, while the other copies show no relevant allelic variance. The experimental paper in Chapter 2.2 shows that all nine annotated Bna.FLC copies have different expression patterns. Three of them do not react to cold anymore, so did loose part of their original function. Chapter 2.3 is an experimental paper showing that different copies of Bna.SRR1 have different abilities to restore srr1 mutants in A. thaliana and also identifies possible protein domains responsible for this change.

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Chapter 2.1: Illuminating Crop Adaptation Using Population Genomics

Chapter II:

Snowdon RJ, Schiessl S (2019) Illuminating Crop Adaptation Using Population Genomics. Molecular Plant 12:27–29. doi: 10.1016/j.molp.2018.12.014

Illuminating Crop Adaptation Using Population Genomics

In this issue,Wu et al. (2019)describe the largest whole-genome resequencing dataset published to date for rapeseed (Brassica napus), an allopolyploid crop species that originated just a few thousand years ago under anthropogenic influence and rapidly evolved into one of the world’s most important oilseed crops.

In almost 1000 accessions spanning species-wide germplasm for oilseed rape, a comprehensive analysis of sequence diver-sity related to flowering-related traits uncovered selective sweeps associated with eco-geographic adaptation and human selection, attributable particularly to divergence among homoe-ologs of key flowering-time regulation and ethylene synthesis/

signaling genes. The authors further extend their analysis to retrospective, genome-based documentation of diversity foot-prints that trace the global expansion of the species across a century of breeding. The study contributes not only a rich catalog of genome-wide diversity for genetic analysis and future breeding of important agro-economic traits but also a unique conceptional framework for ongoing selective adaptation of oilseed rape crops to emerging challenges presented by chang-ing climatic conditions in key production areas. Selective sweeps were found to contain genes involved in stress adapta-tion and development, especially flowering time, indicating that climatic adaptation both in terms of local stress factors and of life-cycle adaptation was the major factor underlying agronomic selection for improved seed yield.

Modulation of flowering behavior and morphotype evolution via post-polyploidization genome restructuring and homoeologous expression changes has previously been identified as a decisive factor in the success ofB.napusas a diverse, globally adapted crop (Samans et al., 2017; Schiessl et al., 2017). This new study expands that knowledge by combining information on genome-wide SNP diversity and linkage disequilibrium (LD) with genome-wide association studies (GWAS) and gene expression data. The resulting picture underlines the pivotal role of mutations creating novel flowering and stress response phenotypes as a driver of geographic expansion. Moreover, the data show that this selection inB.napuswas very specific to distinct gene copies and did not act in parallel on copies of the same gene.

Interestingly, functional variation apparently not only arose by mutations within coding sequences but rather via mutations in promoter regions. The two most prominent flowering-time genes, FLOWERING LOCUS T (FT)andFLOWERING LOCUS C(FLC), were strong candidates for selection. Indeed, the expression pat-terns ofB.napus FTand FLChomoeologs vary in expression between different ecotypes (Figure 1), indicating that promoter variation in floral regulatory genes is an essential factor in the creation of adaptive variation and needs to be considered in knowledge-based breeding approaches. The phenomenon of promoter-driven adaptation may also hold true for other closely related crops: For example, selection against premature bolting in spring-typeBrassica rapaecotypes was shown to be highly

associated with promoter variation in a copy ofVERNALIZATION INSENSITIVE 3 (VIN3) (Su et al., 2018). Interestingly, similar effects are observed in cereals, where mutations in promoter sequences of the cereal geneVERNALIZATION 1(VRN1) also associate with eco-geographic adaptation (Deng et al., 2015).

Moreover, potential variation in promoter sequences was recently postulated as a possible driver of pleiotropic effects that appear to simultaneously modulate flowering responses and root architecture in wheat and barley (Voss-Fels et al., 2018). Such findings underline the importance of detailed knowledge about the genetic intricacies of flowering-time regulation and abiotic stress responses at the DNA sequence and regulatory expression levels. To provide the knowledge needed for targeted breeding of climate-adapted crops to cope with future challenges, we urgently need more and better re-sources for illuminating genome-wide and genome-deep data.

For a long time, polyploidy was a major obstacle to providing such resources. The first genome assembly for B. napus (Chalhoub et al., 2014) represented one of the most highly duplicated and structurally rearranged polyploid plant genomes to be completed at the time. It was also one of the last crop genomes to be assembled on a backbone of DNA reads generated from tiled BAC clones using Titanium Roche 454 and Sanger sequencing technologies. Unimaginable just a decade ago for a crop species with such a complex genome, the dimensions of this new resequencing study underline the enormous power of large-scale genome interrogation. However, as sequencing power and cost no longer present significant bar-riers to mining for useful genetic diversity, even in complex crop genomes, new questions arise with regard to the maximum exploitation of large-scale sequence datasets. One key concern for breeders and researchers is the need for effective, standard-ized, and integrated (e.g., across species) platforms to manage, mine, and utilize genomic data from extensive crop plant collections, coupled with methods and standards to unambigu-ously link specific genotypes and seed lots to corresponding genomic and phenotypic data. Given that data handling, storage, and downstream analysis represent potentially greater future challenges than the actual generation of large-scale genomic sequence datasets, public bioinformatics infrastructure for effective sharing and exploitation of published genome datasets are essential prerequisites to maximize the added value of genome sequences for crop genetics and breeding.

Enhancing the opportunities to mine genome sequence data for previously invisible diversity (Gabur et al., 2018a) is particularly relevant for species likeB.napus with dynamic, allopolyploid genomes where homeologous chromosome exchanges are Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 12, 27–29, January 2019ªThe Author 2018. 27

prolific (Hurgobin et al., 2017; Gabur et al., 2018b). For example, there is growing evidence that novel genome structural variants in B.napusare created even during self-pollination of homozygous cultivars or during generation of doubled haploid lines (He et al., 2017). In this context, genome-wide sequences can potentially reveal a scale of structural genome diversity within ‘‘fixed’’ geno-types that was hitherto undiscoverable. The closer we look, the clearer it becomes that small-scale deletions affecting duplicates ofB.napusgenes or their promoter regions, many of which may be remnants of structural variants caused by illicit pairing of ho-moeologous chromosomes, are an important source of new adaptive and/or agronomic trait variation (Harper et al., 2012;

Qian et al., 2016; Schiessl et al., 2017) with enormous potential value for breeding.

With this in mind, a further key issue of particular relevance for crops with highly duplicated and strongly restructured genomes is the quality and choice of reference genome assemblies for read mapping from short-read sequence data. The potential for ascertainment bias caused by an inability to interrogate chromo-some regions not represented in specific accessions due to genome structural variation is rarely considered in resequencing studies. This is because the huge advantages of comprehensive genome sequence data outweigh any potential shortfalls in com-parison with less comprehensive genotyping platforms based on fixed SNP panels that were identified in specific germplasm col-lections. Nevertheless, the value of enormous genome datasets such as the one described by Wu et al. (2019) will increase even further when gold-standard pan-genomes representing the full extent of available morphotype diversity become avail-able. Here, short reads were mapped to assemblies of the winter oilseed rape cultivars Darmor-bzh (Chalhoub et al., 2014) and Tapidor (Bayer et al., 2017). The differences in SNP and InDel calling were small and led to the same conclusions. However, structural variants within selective sweeps were not evaluated, so the full extent of genetic diversification for crop evolution and adaptation resulting from genome restructuring has yet to be discovered. Further analysis of such expansive genome

sequencing datasets in the context of pan-genomic references thus has considerable potential to deliver key information to future breeders. Powerful new methods to generate platinum-quality assemblies of complex Brassica genomes using long-read sequence data (Belser et al., 2018), in combination with population-scale resequencing using inexpensive short-read technologies, can help provide an ordered catalog of pan-genomic sequence diversity with huge relevance to breeders.

ACKNOWLEDGMENTS

The authors confirm that they have no conflict of interest.

Received: December 17, 2018 Revised: December 17, 2018 Accepted: December 18, 2018 Published: December 21, 2018

Rod J. Snowdon* and Sarah Schiessl

Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany

*Correspondence: Rod J. Snowdon (rod.snowdon@agrar.uni-giessen.de) https://doi.org/10.1016/j.molp.2018.12.014

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Eco-geographic selection acting on the flowering-time gene copies BnaA02g12130D (FT copy on chromosome A02, orange box) and BnaA10g22080D (FLC copy on chromosome A10, blue box) inBrassica napus. The thin orange arrows indicate the positions of the remaining FT copies in theB. napus genome, while the thin blue arrows indicate the positions of the remaining FLC copies. Ecotypes are represented by colored symbols (see legend). The genetic fixation index (Fst) between ecotypes is indicated by the tone of gray in the star symbols, with darker tones indicating higher values and therefore stronger selection. The average expression of each gene in young leaves of non-vernalized plants is indicated with a triangle, with darker triangles indicating higher expression. The table in the black box shows the degree of conservation (in %,±SE) of decisive sequence polymorphisms within the promotor regions of the two gene copies for each ecotype.

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Chapter 2.2: The vernalisation regulator FLOWERING LOCUS C is differentially expressed in biennial and annual Brassica napus

Schiessl SV, Quezada-Martinez D, Tebartz E, Snowdon RJ, Qian L (2019): The vernalisation regulator FLOWERING LOCUS C is differentially expressed in biennial and annual Brassica napus. Sci Rep 9:

14911. doi: 10.1038/s41598-019-51212-x.

the vernalisation regulator