Genetic control of compatibility in crosses between wheat and its wild or cultivated relatives.

In the recent years, the agricultural world has been progressing towards integrated crop protection, in the context of sustainable and reasoned agriculture to improve food security and quality, and to preserve the environment through reduced uses of water, pesticides, fungicides or fertilisers. For this purpose, one possible issue is to cross-elite varieties widely used in fields for crop productions with exotic or wild genetic resources in order to introduce new diversity for genes or alleles of agronomical interest to accelerate the development of new improved cultivars. However, crossing ability (or crossability) often depends on genetic background of the recipient varieties or of the donor, which hampers a larger use of wild resources in breeding programmes of many crops. In this review, we tried to provide a comprehensive summary of genetic factors controlling crossing ability between Triticeae species with a special focus on the crossability between wheat (Triticum aestivum L.) and rye (Secale cereale), which lead to the creation of Triticale (x Triticosecale Wittm.). We also discussed potential applications of newly identified genes or markers associated with crossability for accelerating wheat and Triticale improvement by application of modern genomics technologies in breeding programmes.

SPO11.2 is essential for programmed double-strand break formation during meiosis in bread wheat (Triticum aestivum L.).

Meiotic recombination is initiated by formation of DNA double-strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11-1 and SPO11-2. We analysed the sequences of SPO11-2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11-2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon-2 of the A copy lead to a premature stop codon and suggest that it encodes a non-functional protein. Remarkably, the mutation was absent from the diploid A-relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11-2 in meiotic recombination in wheat. In accordance with its 2-nucleotide deletion in exon-2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.

Bread wheat TaSPO11-1 exhibits evolutionarily conserved function in meiotic recombination across distant plant species.

The manipulation of meiotic recombination in crops is essential to develop new plant varieties rapidly, helping to produce more cultivars in a sustainable manner. One option is to control the formation and repair of the meiosis-specific DNA double-strand breaks (DSBs) that initiate recombination between the homologous chromosomes and ultimately lead to crossovers. These DSBs are introduced by the evolutionarily conserved topoisomerase-like protein SPO11 and associated proteins. Here, we characterized the homoeologous copies of the SPO11-1 protein in hexaploid bread wheat (Triticum aestivum). The genome contains three SPO11-1 gene copies that exhibit 93-95% identity at the nucleotide level, and clearly the A and D copies originated from the diploid ancestors Triticum urartu and Aegilops tauschii, respectively. Furthermore, phylogenetic analysis of 105 plant genomes revealed a clear partitioning between monocots and dicots, with the seven main motifs being almost fully conserved, even between clades. The functional similarity of the proteins among monocots was confirmed through complementation analysis of the Oryza sativa (rice) spo11-1 mutant by the wheat TaSPO11-1-5D coding sequence. Also, remarkably, although the wheat and Arabidopsis SPO11-1 proteins share only 55% identity and the partner proteins also differ, the TaSPO11-1-5D cDNA significantly restored the fertility of the Arabidopsis spo11-1 mutant, indicating a robust functional conservation of the SPO11-1 protein activity across distant plants. These successful heterologous complementation assays, using both Arabidopsis and rice hosts, are good surrogates to validate the functionality of candidate genes and cDNA, as well as variant constructs, when the transformation and mutant production in wheat is much longer and more tedious.

SNP markers for early identification of high molecular weight glutenin subunits (HMW-GSs) in bread wheat.

KEY MESSAGE: A set of eight SNP markers was developed to facilitate the early selection of HMW-GS alleles in breeding programmes. In bread wheat (Triticum aestivum), the high molecular weight glutenin subunits (HMW-GSs) are the most important determinants of technological quality. Known to be very diverse, HMW-GSs are encoded by the tightly linked genes Glu-1-1 and Glu-1-2. Alleles that improve the quality of dough have been identified. Up to now, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of grain proteins is the most widely used for their identification. To facilitate the early selection of HMW-GS alleles in breeding programmes, we developed DNA-based molecular markers. For each accession of a core collection (n = 364 lines) representative of worldwide bread wheat diversity, HMW-GSs were characterized by both genotyping and SDS-PAGE. Based on electrophoresis, we observed at least 8, 22 and 9 different alleles at the Glu-A1, Glu-B1 and Glu-D1 loci, respectively, including new variants. We designed a set of 17 single-nucleotide polymorphism (SNP) markers that were representative of the most frequent SDS-PAGE alleles at each locus. At Glu-A1 and Glu-D1, two and three marker-based haplotypes, respectively, captured the diversity of the SDS-PAGE alleles rather well. Discrepancies were found mainly for the Glu-B1 locus. However, statistical tests revealed that two markers at each Glu-B1 gene and their corresponding haplotypes were more significantly associated with the rheological properties of the dough than were the relevant SDS-PAGE alleles. To conclude, this study demonstrates that the SNP markers developed provide additional information on HMW-GS diversity. Two markers at Glu-A1, four at Glu-B1 and two at Glu-D1 constitute a useful toolbox for breeding wheat to improve end-use value.

The genetic architecture of genome-wide recombination rate variation in allopolyploid wheat revealed by nested association mapping.

Recombination affects the fate of alleles in populations by imposing constraints on the reshuffling of genetic information. Understanding the genetic basis of these constraints is critical for manipulating the recombination process to improve the resolution of genetic mapping, and reducing the negative effects of linkage drag and deleterious genetic load in breeding. Using sequence-based genotyping of a wheat nested association mapping (NAM) population of 2,100 recombinant inbred lines created by crossing 29 diverse lines, we mapped QTL affecting the distribution and frequency of 102 000 crossovers (CO). Genome-wide recombination rate variation was mostly defined by rare alleles with small effects together explaining up to 48.6% of variation. Most QTL were additive and showed predominantly trans-acting effects. The QTL affecting the proximal COs also acted additively without increasing the frequency of distal COs. We showed that the regions with decreased recombination carry more single nucleotide polymorphisms (SNPs) with possible deleterious effects than the regions with a high recombination rate. Therefore, our study offers insights into the genetic basis of recombination rate variation in wheat and its effect on the distribution of deleterious SNPs across the genome. The identified trans-acting additive QTL can be utilized to manipulate CO frequency and distribution in the large polyploid wheat genome opening the possibility to improve the efficiency of gene pyramiding and reducing the deleterious genetic load in the low-recombining pericentromeric regions of chromosomes.

Meiotic gene evolution: can you teach a new dog new tricks?

Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit," we investigated the fate of meiotic gene duplicates following whole genome duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counterselected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.

ASYNAPSIS 1 ensures crossover fidelity in polyploid wheat by promoting homologous recombination and suppressing non-homologous recombination.

During meiosis, the chromosome axes and synaptonemal complex mediate chromosome pairing and homologous recombination to maintain genomic stability and accurate chromosome segregation. In plants, ASYNAPSIS 1 (ASY1) is a key component of the chromosome axis that promotes inter-homolog recombination, synapsis and crossover formation. Here, the function of ASY1 has been cytologically characterized in a series of hypomorphic wheat mutants. In tetraploid wheat, asy1 hypomorphic mutants experience a reduction in chiasmata (crossovers) in a dosage-specific manner, resulting in failure to maintain crossover (CO) assurance. In mutants with only one functional copy of ASY1, distal chiasmata are maintained at the expense of proximal and interstitial chiasmata, indicating that ASY1 is required to promote chiasma formation away from the chromosome ends. Meiotic prophase I progression is delayed in asy1 hypomorphic mutants and is arrested in asy1 null mutants. In both tetraploid and hexaploid wheat, single asy1 mutants exhibit a high degree of ectopic recombination between multiple chromosomes at metaphase I. To explore the nature of the ectopic recombination, Triticum turgidum asy1b-2 was crossed with wheat-wild relative Aegilops variabilis. Homoeologous chiasmata increased 3.75-fold in Ttasy1b-2/Ae. variabilis compared to wild type/Ae. variabilis, indicating that ASY1 suppresses chiasma formation between divergent, but related chromosomes. These data suggest that ASY1 promotes recombination along the chromosome arms of homologous chromosomes whilst suppressing recombination between non-homologous chromosomes. Therefore, asy1 mutants could be utilized to increase recombination between wheat wild relatives and elite varieties for expediting introgression of important agronomic traits.

TaRECQ4 contributes to maintain both homologous and homoeologous recombination during wheat meiosis.

Introduction: Meiotic recombination (or crossover, CO) is essential for gamete fertility as well as for alleles and genes reshuffling that is at the heart of plant breeding. However, CO remains a limited event, which strongly hampers the rapid production of original and improved cultivars. RecQ4 is a gene encoding a helicase protein that, when mutated, contributes to improve recombination rate in all species where it has been evaluated so far. Methods: In this study, we developed wheat (Triticum aestivum L.) triple mutant (TM) for the three homoeologous copies of TaRecQ4 as well as mutants for two copies and heterozygous for the last one (Htz-A, Htz-B, Htz-D). Results: Phenotypic observation revealed a significant reduction of fertility and pollen viability in TM and Htz-B plants compared to wild type plants suggesting major defects during meiosis. Cytogenetic analyses of these plants showed that complete absence of TaRecQ4 as observed in TM plants, leads to chromosome fragmentation during the pachytene stage, resulting in problems in the segregation of chromosomes during meiosis. Htz-A and Htz-D mutants had an almost normal meiotic progression indicating that both TaRecQ4-A and TaRecQ4-D copies are functional and that there is no dosage effect for TaRecQ4 in bread wheat. On the contrary, the TaRecQ4-B copy seems knocked-out, probably because of a SNP leading to a Threonine>Alanine change at position 539 (T539A) of the protein, that occurs in the crucial helicase ATP bind/DEAD/ResIII domain which unwinds nucleic acids. Occurrence of numerous multivalents in TM plants suggests that TaRecQ4 could also play a role in the control of homoeologous recombination. Discussion: These findings provide a foundation for further molecular investigations into wheat meiosis regulation to fully understand the underlying mechanisms of how TaRecQ4 affects chiasma formation, as well as to identify ways to mitigate these defects and enhance both homologous and homoeologous recombination efficiency in wheat.

Variation in crossover rates across a 3-Mb contig of bread wheat (Triticum aestivum) reveals the presence of a meiotic recombination hotspot.

In bread wheat (Triticum aestivum L.), initial studies using deletion lines indicated that crossover (CO) events occur mainly in the telomeric regions of the chromosomes with a possible correlation with the presence of genes. However, little is known about the distribution of COs at the sequence level. To investigate this, we studied in detail the pattern of COs along a contig of 3.110 Mb using two F2 segregating populations (Chinese Spring × Renan (F2-CsRe) and Chinese Spring × Courtot (F2-CsCt)) each containing ~2,000 individuals. The availability of the sequence of the contig from Cs enabled the development of 318 markers among which 23 co-dominant polymorphic markers (11 SSRs and 12 SNPs) were selected for CO distribution analyses. The distribution of CO events was not homogeneous throughout the contig, ranging from 0.05 to 2.77 cM/Mb, but was conserved between the two populations despite very different contig recombination rate averages (0.82 cM/Mb in F2-CsRe vs 0.35 cM/Mb in F2-CsCt). The CO frequency was correlated with the percentage of coding sequence in Cs and with the polymorphism rate between Cs and Re or Ct in both populations, indicating an impact of these two factors on CO distribution. At a finer scale, COs were found in a region covering 2.38 kb, spanning a gene coding for a glycosyl transferase (Hga3), suggesting the presence of a CO hotspot. A non-crossover event covering at least 453 bp was also identified in the same interval. From these results, we can conclude that gene content could be one of the factors driving recombination in bread wheat.

Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat.

BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.

Crop Improvement: Where Are We Now?

Improving the production of all crops is crucial to meeting the challenge of the growing needs related to the simultaneous increase in the world population and demands from farmers and end-users [...].

Generation of Deletion Lines in Allohexaploid Bread Wheat.

Positional cloning in bread wheat (Triticum aestivum L.) remains a daunting task because of its large genome, high density of repeats, low recombination rate especially in pericentromeric regions and its allopolyploidy. One way to face this challenge is to decrease the size of the interval bearing the gene of interest both genetically and physically, in order to reduce significantly the number of potential candidate genes. In this chapter, we describe a technical approach to produce chromosome-specific deletion lines to locate precisely genes of interest onto wheat chromosomes, a step forward to their cloning.

Location and Identification on Chromosome 3B of Bread Wheat of Genes Affecting Chiasma Number.

Understanding meiotic crossover (CO) variation in crops like bread wheat (Triticum aestivum L.) is necessary as COs are essential to create new, original and powerful combinations of genes for traits of agronomical interest. We cytogenetically characterized a set of wheat aneuploid lines missing part or all of chromosome 3B to identify the most influential regions for chiasma formation located on this chromosome. We showed that deletion of the short arm did not change the total number of chiasmata genome-wide, whereas this latter was reduced by ~35% while deleting the long arm. Contrary to what was hypothesized in a previous study, deletion of the long arm does not disturb the initiation of the synaptonemal complex (SC) in early meiotic stages. However, progression of the SC is abnormal, and we never observed its completion when the long arm is deleted. By studying six different deletion lines (missing different parts of the long arm), we revealed that at least two genes located in both the proximal (C-3BL2-0.22) and distal (3BL7-0.63-1.00) deletion bins are involved in the control of chiasmata, each deletion reducing the number of chiasmata by ~15%. We combined sequence analyses of deletion bins with RNA-Seq data derived from meiotic tissues and identified a set of genes for which at least the homoeologous copy on chromosome 3B is expressed and which are involved in DNA processing. Among these genes, eight (CAP-E1/E2, DUO1, MLH1, MPK4, MUS81, RTEL1, SYN4, ZIP4) are known to be involved in the recombination pathway.

Breeding for Economically and Environmentally Sustainable Wheat Varieties: An Integrated Approach from Genomics to Selection.

There is currently a strong societal demand for sustainability, quality, and safety in bread wheat production. To address these challenges, new and innovative knowledge, resources, tools, and methods to facilitate breeding are needed. This starts with the development of high throughput genomic tools including single nucleotide polymorphism (SNP) arrays, high density molecular marker maps, and full genome sequences. Such powerful tools are essential to perform genome-wide association studies (GWAS), to implement genomic and phenomic selection, and to characterize the worldwide diversity. This is also useful to breeders to broaden the genetic basis of elite varieties through the introduction of novel sources of genetic diversity. Improvement in varieties particularly relies on the detection of genomic regions involved in agronomical traits including tolerance to biotic (diseases and pests) and abiotic (drought, nutrient deficiency, high temperature) stresses. When enough resolution is achieved, this can result in the identification of candidate genes that could further be characterized to identify relevant alleles. Breeding must also now be approached through in silico modeling to simulate plant development, investigate genotype × environment interactions, and introduce marker-trait linkage information in the models to better implement genomic selection. Breeders must be aware of new developments and the information must be made available to the world wheat community to develop new high-yielding varieties that can meet the challenge of higher wheat production in a sustainable and fluctuating agricultural context. In this review, we compiled all knowledge and tools produced during the BREEDWHEAT project to show how they may contribute to face this challenge in the coming years.

Investigation of genetic diversity and population structure of common wheat cultivars in northern China using DArT markers.

BACKGROUND: In order to help establish heterotic groups of Chinese northern wheat cultivars (lines), Diversity arrays technology (DArT) markers were used to investigate the genetic diversity and population structure of Chinese common wheat (Triticum aestivum L.). RESULTS: In total, 1637 of 7000 DArT markers were polymorphic and scored with high confidence among a collection of 111 lines composed mostly of cultivars and breeding lines from northern China. The polymorphism information content (PIC) of DArT markers ranged from 0.03 to 0.50, with an average of 0.40, with P > 80 (reliable markers). With principal-coordinates analysis (PCoA) of DArT data either from the whole genome or from the B-genome alone, all lines fell into one of two major groups reflecting 1RS/1BL type (1RS/1BL and non-1RS/1BL). Evidence of geographic clustering of genotypes was also observed using DArT markers from the A genome. Cluster analysis based on the unweighted pair-group method with algorithmic mean suggested the existence of two subgroups within the non-1RS/1BL group and four subgroups within the 1RS/1BL group. Furthermore, analysis of molecular variance (AMOVA) revealed highly significant (P < 0.001) genetic variance within and among subgroups and among groups. CONCLUSION: These results provide valuable information for selecting crossing parents and establishing heterotic groups in the Chinese wheat-breeding program.

Crop Improvement: Now and Beyond.

There is an urgent need to increase and improve the production of most agronomic species to meet the current food security challenge [...].

Evolution of Recombination Landscapes in Diverging Populations of Bread Wheat.

Reciprocal exchanges of DNA (crossovers) that occur during meiosis are mandatory to ensure the production of fertile gametes in sexually reproducing species. They also contribute to shuffle parental alleles into new combinations thereby fueling genetic variation and evolution. However, due to biological constraints, the recombination landscape is highly heterogeneous along the genome which limits the range of allelic combinations and the adaptability of populations. An approach to better understand the constraints on the recombination process is to study how it evolved in the past. In this work, we tackled this question by constructing recombination profiles in four diverging bread wheat (Triticum aestivum L.) populations established from 371 landraces genotyped at 200,062 SNPs. We used linkage disequilibrium (LD) patterns to estimate in each population the past distribution of recombination along the genome and characterize its fine-scale heterogeneity. At the megabase scale, recombination rates derived from LD patterns were consistent with family-based estimates obtained from a population of 406 recombinant inbred lines. Among the four populations, recombination landscapes were positively correlated between each other and shared a statistically significant proportion of highly recombinant intervals. However, this comparison also highlighted that the similarity in recombination landscapes between populations was significantly decreasing with their genetic differentiation in most regions of the genome. This observation was found to be robust to SNPs ascertainment and demography and suggests a relatively rapid evolution of factors determining the fine-scale localization of recombination in bread wheat.

Ph2 encodes the mismatch repair protein MSH7-3D that inhibits wheat homoeologous recombination.

Meiotic recombination is a critical process for plant breeding, as it creates novel allele combinations that can be exploited for crop improvement. In wheat, a complex allohexaploid that has a diploid-like behaviour, meiotic recombination between homoeologous or alien chromosomes is suppressed through the action of several loci. Here, we report positional cloning of Pairing homoeologous 2 (Ph2) and functional validation of the wheat DNA mismatch repair protein MSH7-3D as a key inhibitor of homoeologous recombination, thus solving a half-century-old question. Similar to ph2 mutant phenotype, we show that mutating MSH7-3D induces a substantial increase in homoeologous recombination (up to 5.5 fold) in wheat-wild relative hybrids, which is also associated with a reduction in homologous recombination. These data reveal a role for MSH7-3D in meiotic stabilisation of allopolyploidy and provides an opportunity to improve wheat's genetic diversity through alien gene introgression, a major bottleneck facing crop improvement.

Resistance of the Wheat Cultivar 'Renan' to Septoria Leaf Blotch Explained by a Combination of Strain Specific and Strain Non-Specific QTL Mapped on an Ultra-Dense Genetic Map.

Quantitative resistance is considered more durable than qualitative resistance as it does not involve major resistance genes that can be easily overcome by pathogen populations, but rather a combination of genes with a lower individual effect. This durability means that quantitative resistance could be an interesting tool for breeding crops that would not systematically require phytosanitary products. Quantitative resistance has yet to reveal all of its intricacies. Here, we delve into the case of the wheat/Septoria tritici blotch (STB) pathosystem. Using a population resulting from a cross between French cultivar Renan, generally resistant to STB, and Chinese Spring, a cultivar susceptible to the disease, we built an ultra-dense genetic map that carries 148,820 single nucleotide polymorphism (SNP) markers. Phenotyping the interaction was done with two different Zymoseptoria tritici strains with contrasted pathogenicities on Renan. A linkage analysis led to the detection of three quantitative trait loci (QTL) related to resistance in Renan. These QTL, on chromosomes 7B, 1D, and 5D, present with an interesting diversity as that on 7B was detected with both fungal strains, while those on 1D and 5D were strain-specific. The resistance on 7B was located in the region of Stb8 and the resistance on 1D colocalized with Stb19. However, the resistance on 5D was new, so further designated Stb20q. Several wall-associated kinases (WAK), nucleotide-binding and leucine-rich repeats (NB-LRR) type, and kinase domain carrying genes were present in the QTL regions, and some of them were expressed during the infection. These results advocate for a role of Stb genes in quantitative resistance and for resistance in the wheat/STB pathosystem being as a whole quantitative and polygenic.