The Aegilops ventricosa 2NvS segment in bread wheat: cytology, genomics and breeding.

KEY MESSAGE: The first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield. The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, 'Jagger' and 'CDC Stanley,' and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

Production of synthetic wheat lines to exploit the genetic diversity of emmer wheat and D genome containing Aegilops species in wheat breeding.

Due to the accumulation of various useful traits over evolutionary time, emmer wheat (Triticum turgidum subsp. dicoccum and dicoccoides, 2n = 4x = 28; AABB), durum wheat (T. turgidum subsp. durum, 2n = 4x = 28; AABB), T. timopheevii (2n = 4x = 28; AAGG) and D genome containing Aegilops species offer excellent sources of novel variation for the improvement of bread wheat (T. aestivum L., AABBDD). Here, we made 192 different cross combinations between diverse genotypes of wheat and Aegilops species including emmer wheat × Ae. tauschii (2n = DD or DDDD), durum wheat × Ae. tauschii, T. timopheevii × Ae. tauschii, Ae. crassa × durum wheat, Ae. cylindrica × durum wheat and Ae. ventricosa × durum wheat in the field over three successive years. We successfully recovered 56 different synthetic hexaploid and octaploid F2 lines with AABBDD, AABBDDDD, AAGGDD, D1D1XcrXcrAABB, DcDcCcCcAABB and DvDvNvNvAABB genomes via in vitro rescue of F1 embryos and spontaneous production of F2 seeds on the Fl plants. Cytogenetic analysis of F2 lines showed that the produced synthetic wheat lines were generally promising stable amphiploids. Contribution of D genome bearing Aegilops and the less-investigated emmer wheat genotypes as parents in the crosses resulted in synthetic amphiploids which are a valuable resource for bread wheat breeding.

Tissue-Specific Transcriptome Analysis Reveals Candidate Transcripts Associated with the Process of Programmed B Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from programmed elimination of chromosomes. Here, we present the first transcriptome analysis of programmed chromosome elimination using laser capture microdissection (LCM)-based isolation of the central meristematic region of Aegilops speltoides embryos where B chromosome (B) elimination occurs. The comparative RNA-seq analysis of meristematic cells of embryos with (Bplus) and without Bs (B0) allowed the identification of 14,578 transcript isoforms (35% out of 41,615 analyzed transcript isoforms) that are differentially expressed during the elimination of Bs. A total of 2908 annotated unigenes were found to be up-regulated in Bplus condition. These genes are either associated with the process of B chromosome elimination or with the presence of B chromosomes themselves. GO enrichment analysis categorized up-regulated transcript isoforms into 27 overrepresented terms related to the biological process, nine terms of the molecular function aspect and three terms of the cellular component category. A total of 2726 annotated unigenes were down-regulated in Bplus condition. Based on strict filtering criteria, 341 B-unique transcript isoforms could be identified in central meristematic cells, of which 70 were functionally annotated. Beside others, genes associated with chromosome segregation, kinetochore function and spindle checkpoint activity were retrieved as promising candidates involved in the process of B chromosome elimination.

Dissection and physical mapping of wheat chromosome 7B by inducing meiotic recombination with its homoeologues in Aegilops speltoides and Thinopyrum elongatum.

KEY MESSAGE: We constructed a homoeologous recombination-based bin map of wheat chromosome 7B, providing a unique physical framework for further study of chromosome 7B and its homoeologues in wheat and its relatives. Homoeologous recombination leads to the dissection and diversification of the wheat genome. Advances in genome sequencing and genotyping have dramatically improved the efficacy and throughput of homoeologous recombination-based genome studies and alien introgression in wheat and its relatives. In this study, we aimed to physically dissect and map wheat chromosome 7B by inducing meiotic recombination of chromosome 7B with its homoeologues 7E in Thinopyrum elongatum and 7S in Aegilops speltoides. The special genotypes, which were double monosomic for chromosomes 7B' + 7E' or 7B' + 7S' and homozygous for the ph1b mutant, were produced to enhance 7B - 7E and 7B - 7S recombination. Chromosome-specific DNA markers were developed and used to pre-screen the large recombination populations for 7B - 7E and 7B - 7S recombinants. The DNA marker-mediated preselections were verified by fluorescent genomic in situ hybridization (GISH). In total, 29 7B - 7E and 61 7B - 7S recombinants and multiple chromosome aberrations were recovered and delineated by GISH and the wheat 90 K SNP assay. Integrated GISH and SNP analysis of the recombinants physically mapped the recombination breakpoints and partitioned wheat chromosome 7B into 44 bins with 523 SNPs assigned within. A composite bin map was constructed for chromosome 7B, showing the bin size and physical distribution of SNPs. This provides a unique physical framework for further study of chromosome 7B and its homoeologues. In addition, the 7B - 7E and 7B - 7S recombinants extend the genetic variability of wheat chromosome 7B and represent useful germplasm for wheat breeding. Thereby, this genomics-enabled chromosome engineering approach facilitates wheat genome study and enriches the gene pool of wheat improvement.

Transcriptome Analysis Reveals Important Candidate Genes Related to Nutrient Reservoir, Carbohydrate Metabolism, and Defence Proteins during Grain Development of Hexaploid Bread Wheat and Its Diploid Progenitors.

Wheat grain development after anthesis is an important biological process, in which major components of seeds are synthesised, and these components are further required for germination and seed vigour. We have made a comparative RNA-Seq analysis between hexaploid wheat and its individual diploid progenitors to know the major differentially expressed genes (DEGs) involved during grain development. Two libraries from each species were generated with an average of 55.63, 55.23, 68.13, and 103.81 million reads, resulting in 79.3K, 113.7K, 90.6K, and 121.3K numbers of transcripts in AA, BB, DD, and AABBDD genome species respectively. Number of expressed genes in hexaploid wheat was not proportional to its genome size, but marginally higher than that of its diploid progenitors. However, to capture all the transcripts in hexaploid wheat, sufficiently higher number of reads was required. Functional analysis of DEGs, in all the three comparisons, showed their predominance in three major classes of genes during grain development, i.e., nutrient reservoirs, carbohydrate metabolism, and defence proteins; some of them were subsequently validated through real time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Further, developmental stage-specific gene expression showed most of the defence protein genes expressed during initial developmental stages in hexaploid contrary to the diploids at later stages. Genes related to carbohydrates anabolism expressed during early stages, whereas catabolism genes expressed at later stages in all the species. However, no trend was observed in case of different nutrient reservoirs gene expression. This data could be used to study the comparative gene expression among the three diploid species and homeologue-specific expression in hexaploid.

Genetic analysis of Aegilops tauschii-derived seedling resistance to leaf rust in synthetic hexaploid wheat.

Seedling resistance to leaf rust available in the synthetic hexaploid wheat line Syn137 was characterised by means of cytogenetic and linkage mapping. Monosomic analysis located a single dominant gene for leaf rust resistance on chromosome 5D. Molecular mapping not only confirmed this location but also positioned the gene to the distal part of the long arm of chromosome 5D. A test of allelism showed that the gene, tentatively named LrSyn137, is independent but closely linked to Lr1. It appears that Syn137 is occasionally heterogeneous for Lr1 since the analysis of the Lr1-specific marker RGA567-5 in the genetic mapping population indicated the presence of Lr1. Syn137 represents another source of genetic variation that can be useful for the diversification of leaf rust resistance in wheat cultivars.

Early Sibling Conflict May Ultimately Benefit the Family.

Relatives often interact differently with each other than with nonrelatives, and whether kin cooperate or compete has important consequences for the evolution of mating systems, seed size, dispersal, and competition. Previous research found that the larger of the size dimorphic seeds produced by the annual plant Aegilops triuncialis suppressed germination of their smaller sibs by 25%-60%. Here, we found evidence for kin recognition and sibling rivalry later in life among Aegilops seedlings that places seed-seed interactions in a broader context. In experiments with size dimorphic seeds, seedlings reduced the growth of sibling seedlings by ∼40% but that of nonsibling seedlings by ∼25%. These sequential antagonistic interactions between seeds and then seedlings provide insight into conflict and cooperation among kin. Kin-based conflict among seeds may maintain dormancy for some seeds until the coast is clear of more competitive siblings. If so, biotically induced seed dormancy may be a unique form of cooperation, which increases the inclusive fitness of maternal plants and offspring by minimizing competition among kin.

Genetic Contribution of Synthetic Hexaploid Wheat to CIMMYT's Spring Bread Wheat Breeding Germplasm.

Synthetic hexaploid (SH) wheat (AABBD'D') is developed by artificially generating a fertile hybrid between tetraploid durum wheat (Triticum turgidum, AABB) and diploid wild goat grass (Aegilops tauschii, D'D'). Over three decades, the International Maize and Wheat Improvement Center (CIMMYT) has developed and utilized SH wheat to bridge gene transfer from Ae. tauschii and durum wheat to hexaploid bread wheat. This is a unique example of success utilizing wild relatives in mainstream breeding at large scale worldwide. Our study aimed to determine the genetic contribution of SH wheat to CIMMYT's global spring bread wheat breeding program. We estimated the theoretical and empirical contribution of D' to synthetic derivative lines using the ancestral pedigree and marker information using over 1,600 advanced lines and their parents. The average marker-estimated D' contribution was 17.5% with difference in genome segments suggesting application of differential selection pressure. The pedigree-based contribution was correlated with marker-based estimates without providing chromosome segment specific variation. Results from international yield trials showed that 20% of the lines were synthetic derived with an average D' contribution of 15.6%. Our results underline the importance of SH wheat in maintaining and enhancing genetic diversity and genetic gain over years and is important for development of a more targeted introgression strategy. The study provides retrospective view into development and utilization of SH in the CIMMYT Global Wheat Program.

Chromosome engineering-mediated introgression and molecular mapping of novel Aegilops speltoides-derived resistance genes for tan spot and Septoria nodorum blotch diseases in wheat.

KEY MESSAGE: We identified, mapped and introduced novel Aegilops speltoides-derived resistance genes for tan spot and SNB diseases into wheat, enhancing understanding and utilization of host resistance to both diseases in wheat. Tan spot and Septoria nodorum blotch (SNB) are two important fungal diseases of wheat. Resistance to these diseases is often observed as the lack of sensitivity to the necrotrophic effectors (NE) produced by the fungal pathogens and thus exhibits a recessive inheritance pattern. In this study, we identified novel genes for resistance to tan spot and SNB on Aegilops speltoides (2n = 2x = 14, genome SS) chromosome 2S. These genes confer dominant resistance in the wheat background, indicating a distinct NE-independent mechanism of resistance. Ae. speltoides chromosome 2S was engineered for resistance gene introgression and molecular mapping by inducing meiotic homoeologous recombination with wheat chromosome 2B. Twenty representative 2B-2S recombinants were evaluated for reaction to tan spot and SNB and were delineated by genomic in situ hybridization and high-throughput wheat 90 K SNP assay. The resistance genes physically mapped to the sub-telomeric region (~ 8 Mb) on the short arm of chromosome 2S and designated TsrAes1 for tan spot resistance and SnbAes1 for SNB resistance. In addition, we developed SNP-derived PCR markers closely linked to TsrAes1/SnbAes1 for marker-assisted selection in wheat breeding. TsrAes1 and SnbAes1 are the first set of NE-independent tan spot, and SNB resistance genes are identified from Ae. speltoides. The 2SS-2BS·2BL recombinants with minimal amounts of Ae. speltoides chromatin containing TsrAes1/SnbAes1 were produced for germplasm development, making the wild species-derived resistance genes usable in wheat breeding. This will strengthen and diversify resistance of wheat to tan spot and SNB and facilitate understanding of resistance to these two diseases.

Drought stress affects the protein and dietary fiber content of wholemeal wheat flour in wheat/Aegilops addition lines.

Wild relatives of wheat, such as Aegilops spp. are potential sources of genes conferring tolerance to drought stress. As drought stress affects seed composition, the main goal of the present study was to determine the effects of drought stress on the content and composition of the grain storage protein (gliadin (Gli), glutenin (Glu), unextractable polymeric proteins (UPP%) and dietary fiber (arabinoxylan, ß-glucan) components of hexaploid bread wheat (T. aestivum) lines containing added chromosomes from Ae. biuncialis or Ae. geniculata. Both Aegilops parents have higher contents of protein and ß-glucan and higher proportions of water-soluble arabinoxylans (determined as pentosans) than wheat when grown under both well-watered and drought stress conditions. In general, drought stress resulted in increased contents of protein and total pentosans in the addition lines, while the ß-glucan content decreased in many of the addition lines. The differences found between the wheat/Aegilops addition lines and wheat parents under well-watered conditions were also manifested under drought stress conditions: Namely, elevated ß-glucan content was found in addition lines containing chromosomes 5Ug, 7Ug and 7Mb, while chromosomes 1Ub and 1Mg affected the proportion of polymeric proteins (determined as Glu/Gli and UPP%, respectively) under both well-watered and drought stress conditions. Furthermore, the addition of chromosome 6Mg decreased the WE-pentosan content under both conditions. The grain composition of the Aegilops accessions was more stable under drought stress than that of wheat, and wheat lines with the added Aegilops chromosomes 2Mg and 5Mg also had more stable grain protein and pentosan contents. The negative effects of drought stress on both the physical and compositional properties of wheat were also reduced by the addition of these. These results suggest that the stability of the grain composition could be improved under drought stress conditions by the intraspecific hybridization of wheat with its wild relatives.