Michael Florian Mette

Bridging the gap between marker-assisted and genomic selection of heading time and plant height in hybrid wheat

Based on data from field trials with a large collection of 135 elite winter wheat inbred lines and 1604 F1 hybrids derived from them, we compared the accuracy of prediction of marker-assisted selection and current genomic selection approaches for the model traits heading time and plant height in a cross-validation approach. For heading time, the high accuracy seen with marker-assisted selection severely dropped with genomic selection approaches RR-BLUP (ridge regression best linear unbiased prediction) and BayesCπ, whereas for plant height, accuracy was low with marker-assisted selection as well as RR-BLUP and BayesCπ. Differences in the linkage disequilibrium structure of the functional and single-nucleotide polymorphism markers relevant for the two traits were identified in a simulation study as a likely explanation for the different trends in accuracies of prediction. A new genomic selection approach, weighted best linear unbiased prediction (W-BLUP), designed to treat the effects of known functional markers more appropriately, proved to increase the accuracy of prediction for both traits and thus closes the gap between marker-assisted and genomic selection.

The accuracy of prediction of genomic selection in elite hybrid rye populations surpasses the accuracy of marker-assisted selection and is equally augmented by multiple field evaluation locations and test years

BackgroundMarker-assisted selection (MAS) and genomic selection (GS) based on genome-wide marker data provide powerful tools to predict the genotypic value of selection material in plant breeding. However, case-to-case optimization of these approaches is required to achieve maximum accuracy of prediction with reasonable input.ResultsBased on extended field evaluation data for grain yield, plant height, starch content and total pentosan content of elite hybrid rye derived from testcrosses involving two bi-parental populations that were genotyped with 1048 molecular markers, we compared the accuracy of prediction of MAS and GS in a cross-validation approach. MAS delivered generally lower and in addition potentially over-estimated accuracies of prediction than GS by ridge regression best linear unbiased prediction (RR-BLUP). The grade of relatedness of the plant material included in the estimation and test sets clearly affected the accuracy of prediction of GS. Within each of the two bi-parental populations, accuracies differed depending on the relatedness of the respective parental lines. Across populations, accuracy increased when both populations contributed to estimation and test set. In contrast, accuracy of prediction based on an estimation set from one population to a test set from the other population was low despite that the two bi-parental segregating populations under scrutiny shared one parental line. Limiting the number of locations or years in field testing reduced the accuracy of prediction of GS equally, supporting the view that to establish robust GS calibration models a sufficient number of test locations is of similar importance as extended testing for more than one year.ConclusionsIn hybrid rye, genomic selection is superior to marker-assisted selection. However, it achieves high accuracies of prediction only for selection candidates closely related to the plant material evaluated in field trials, resulting in a rather pessimistic prognosis for distantly related material. Both, the numbers of evaluation locations and testing years in trials contribute equally to prediction accuracy.

Telomere-mediated truncation of barley chromosomes

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.

Intraspecific hybrids of Arabidopsis thaliana revealed no gross alterations in endopolyploidy, DNA methylation, histone modifications and transcript levels

Arabidopsis accessions Col-0 and C24 and their reciprocal hybrids were employed as a model system to investigate the potential relationship between changes in DNA methylation, chromatin structure, endopolyploidization and gene expression in heterotic genotypes. Nucleolus size, endopolyploidization level and distribution of DNA and histone H3 methylation at the microscopic level does not differ between parents and their hybrids. Methylation sensitive amplified polymorphism revealed a largely constant pattern of DNA methylation (97% of signals analyzed) after intraspecific crosses. The parental expression profile of selected genes was maintained in hybrid offspring. No correlation was found between expression pattern and DNA methylation levels at restriction sites within 5′ regulatory regions. Thus, the results revealed only minor changes of chromatin properties and other nuclear features in response to intraspecific hybridization in Arabidopsis thaliana.

Developmentally non-redundant SET domain proteins SUVH2 and SUVH9 are required for transcriptional gene silencing in Arabidopsis thaliana

In plants, RNA-directed DNA methylation (RdDM) and related transcriptional gene silencing (TGS) involve members of the suppressor of variegation 3-9-homologous (SUVH) group of putative histone methyltransferases. Utilizing a reverse genetic approach in Arabidopsis thaliana, we demonstrate that two closely related SUVH members, SUVH2 and SUVH9, act partially non-redundant in RdDM. DNA methylation, transcript accumulation and association with histone modifications were analyzed at the endogenous RdDM target AtSN1 (a SINE-like retroelement) in suvh2 and suvh9 single as well as suvh2suvh9 double mutants. SUVH2 was found to be required for full DNA methylation at AtSN1 in early seed development and was also higher expressed in seeds than at later developmental stages. SUVH9 had its impact on RdDM later during vegetative development of the plant and was also higher expressed during that stage than at earlier developmental stages. The strongest reduction of RdDM at AtSN1 was found in suvh2suvh9 double mutant plants. Histone 3-lysine 9-dimethylation (H3K9me2) associated with AtSN1 was reduced only in the simultaneous absence of functional SUVH2 and SUVH9. Thus, SUVH2 and SUVH9 functions in RdDM and TGS are overlapping in spite of some developmental specialization. Pol V specific transcripts were reduced in suvh2suvh9 plants. This might indicate a role of these SUVH proteins in Pol V complex recruitment.

Relatedness severely impacts accuracy of marker-assisted selection for disease resistance in hybrid wheat.

The accuracy of genomic selection depends on the relatedness between the members of the set in which marker effects are estimated based on evaluation data and the types for which performance is predicted. Here, we investigate the impact of relatedness on the performance of marker-assisted selection for fungal disease resistance in hybrid wheat. A large and diverse mapping population of 1739 elite European winter wheat inbred lines and hybrids was evaluated for powdery mildew, leaf rust and stripe rust resistance in multi-location field trials and fingerprinted with 9 k and 90 k SNP arrays. Comparison of the accuracies of prediction achieved with data sets from the two marker arrays revealed a crucial role for a sufficiently high marker density in genome-wide association mapping. Cross-validation studies using test sets with varying degrees of relationship to the corresponding estimation sets revealed that close relatedness leads to a substantial increase in the proportion of total genotypic variance explained by the identified QTL and consequently to an overoptimistic judgment of the precision of marker-assisted selection.

Identification of Quantitative Trait Loci Underlying Plant Height and Seed Weight in Soybean

For clarifying the genetic base of the variation of yield‐related traits in soybean [Glycine max (L.) Merr.], we mapped quantitative trait loci (QTLs) for plant height and seed weight using a recombinant inbred line population derived from a cross between Chinese elite line Zhongpin03‐5373 and cultivar Zhonghuang13. We detected 11 QTLs for plant height and 18 QTLs for seed weight across six diverse environments. These included three pairs of plant height‐ vs. seed weight‐related QTLs located in close proximity to each other, with two pairs, qPH‐7 vs. qSW‐7‐2 and qPH‐19‐2 vs. qSW‐19, sharing the same direction of additive effects. Individual QTLs explained 2.02 to 47.60% of the variation in plant height and 2.13 to 14.35% in seed weight. Two and five of the major QTLs discovered for plant height and seed weight, respectively, that were stable across environments in our study have been reported previously. Among them, four QTLs, qPH‐13, qSW‐11, qSW‐12‐2, and qSW‐18, were not involved in digenic epistatic interaction in our biparental population, indicating that these QTLs will be useful for marker‐assisted selection and should be targeted for the future identification of candidate genes. Moreover, eight QTLs for both plant height and seed weight were newly identified in our population.

IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation

In plants, a particular class of short interfering (si)RNAs can serve as a signal to induce cytosine methylation at homologous genomic regions. If the targeted DNA has promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing (RdTGS) of transgenes provides a versatile system for the study of epigenetic gene regulation. We used transcription of a nopaline synthase promoter (ProNOS)-inverted repeat (IR) to provide a RNA signal that triggers de novo cytosine methylation and TGS of a homologous ProNOS copy in trans. Utilizing a ProNOS-NPTII reporter gene showing high sensitivity to silencing in this two component system, a forward genetic screen for EMS-induced no rna-directed transcriptional silencing (nrd) mutations was performed in Arabidopsis thaliana. Three nrd mutant lines were found to contain one novel loss-of-function allele of idn2/rdm12 and two of nrpd2a/nrpe2a. IDN2/RDM12 encodes a XH/XS domain protein that is able to bind double-stranded RNA with 5′ overhangs, while NRPD2a/NRPE2a encodes the common second-largest subunit of the plant specific DNA-dependent RNA polymerases IV and V involved in silencing processes. Both idn2/rdm12 and nrpd2a/nrpe2a release target transgene expression and reduce CHH context methylation at transgenic as well as endogenous RdDM target regions to similar extents. Nevertheless, accumulation of IR-derived siRNA is not affected, allowing us to present a refined model for the pathway of RdDM and RdTGS that positions function of IDN2 downstream of siRNA formation and points to an important role for its XH domain.

Potential and limits to unravel the genetic architecture and predict the variation of Fusarium head blight resistance in European winter wheat ( Triticum aestivum L.)

Genome-wide mapping approaches in diverse populations are powerful tools to unravel the genetic architecture of complex traits. The main goals of our study were to investigate the potential and limits to unravel the genetic architecture and to identify the factors determining the accuracy of prediction of the genotypic variation of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum L.) based on data collected with a diverse panel of 372 European varieties. The wheat lines were phenotyped in multi-location field trials for FHB resistance and genotyped with 782 simple sequence repeat (SSR) markers, and 9k and 90k single-nucleotide polymorphism (SNP) arrays. We applied genome-wide association mapping in combination with fivefold cross-validations and observed surprisingly high accuracies of prediction for marker-assisted selection based on the detected quantitative trait loci (QTLs). Using a random sample of markers not selected for marker–trait associations revealed only a slight decrease in prediction accuracy compared with marker-based selection exploiting the QTL information. The same picture was confirmed in a simulation study, suggesting that relatedness is a main driver of the accuracy of prediction in marker-assisted selection of FHB resistance. When the accuracy of prediction of three genomic selection models was contrasted for the three marker data sets, no significant differences in accuracies among marker platforms and genomic selection models were observed. Marker density impacted the accuracy of prediction only marginally. Consequently, genomic selection of FHB resistance can be implemented most cost-efficiently based on low- to medium-density SNP arrays.

Live-cell CRISPR imaging in plants reveals dynamic telomere movements

Summary Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.