Genome Wide Association Study of Frost Tolerance in Wheat

Winter wheat growing areas in the Northern hemisphere are regularly exposed to heavy frost. Due to the negative impact on yield, the identification of genetic factors controlling frost tolerance (FroT) and development of tools for breeding is of prime importance. Here, we detected QTL associated with FroT by genome wide association studies (GWAS) using a diverse panel of 276 winter wheat genotypes that was phenotyped at five locations in Germany and Russia in three years. The panel was genotyped using the 90K iSelect array and SNPs in FroT candidate genes. In total, 17,566 SNPs were used for GWAS resulting in the identification of 53 markers significantly associated (LOD ≥4) to FroT, corresponding to 23 QTL regions located on 11 chromosomes (1A, 1B, 2A, 2B, 2D, 3A, 3D, 4A, 5A, 5B and 7D). The strongest QTL effect confirmed the importance of chromosome 5A for FroT. In addition, to our best knowledge, seven FroT QTLs were discovered for the first time in this study comprising QTLs on chromosomes 3A, 4A, 1B, and two on chromosomes 2D, 3D, and 7D. Identification of novel FroT candidate genes will help to better understand the FroT mechanism in wheat and to develop more effective combating strategies.

A Digital PCR Assay to Quantify the Percentages of Hulled vs. Hulless Wheat in Flours and Flour-Based Products

Several food products, made from hulled wheats, are now offered by the market, ranging from grains and pasta to flour and bakery products. The possibility of verifying the authenticity of wheat species used at any point in the production chain is relevant, in defense of both producers and consumers. A chip digital PCR assay has been developed to detect and quantify percentages of hulless (i.e., common and durum wheat) and hulled (i.e., einkorn, emmer and spelt) wheats in grains, flours and food products. The assay has been designed on a polymorphism in the miRNA172 target site of the AP2-5 transcription factor localized on chromosome 5A and involved in wheat spike morphogenesis and grain threshability. The assay has been evaluated even in a real-time PCR system to assess its applicability and to compare the analytical costs between dPCR and real-time PCR approaches.

Comparison of Single-Trait and Multi-Trait Genome-Wide Association Models and Inclusion of Correlated Traits in the Dissection of the Genetic Architecture of a Complex Trait in a Breeding Program

Traits with an unknown genetic architecture make it difficult to create a useful bi-parental mapping population to characterize the genetic basis of the trait due to a combination of complex and pleiotropic effects. Seedling emergence of wheat (Triticum aestivum L.) from deep planting is a vital factor affecting stand establishment and grain yield, has a poorly understood genetic architecture, and is historically correlated with coleoptile length. The creation of bi-parental mapping populations can be overcome by using genome-wide association studies (GWAS). This study aimed to dissect the genetic architecture of seedling emergence while accounting for correlated traits using one multi-trait GWAS model (MT-GWAS) and three single-trait GWAS models (ST-GWAS) with the inclusion of covariates for correlated traits. The ST-GWAS models included one single locus model (MLM), and two multiple loci models (FarmCPU and BLINK). We conducted the GWAS using two populations, the first consisting of 473 varieties from a diverse association mapping panel (DP) phenotyped from 2015-2019, and the other population used as a validation population consisting of 279 breeding lines (BL) phenotyped in 2015 in Lind, WA, with 40,368 markers. We also compared the inclusion of coleoptile length and markers associated with reduced height as covariates in our ST-GWAS models for the DP. ST-GWAS found 107 significant markers across 19 chromosomes, while MT-GWAS found 82 significant markers across 14 chromosomes. MT-GWAS models were able to identify large-effect markers on chromosome 5A. FarmCPU and BLINK models were able to identify many small effect markers, and the inclusion of covariates helped to identify the large effect markers on chromosome 5A. Therefore, by using multi-locus models combined with pleiotropic covariates, breeding programs can uncover the complex nature of traits to help identify candidate genes and the underlying architecture of a trait, such as seedling emergence of deep-sown winter wheat.

Identification of a Major and Stable QTL on Chromosome 5A Confers Spike Length in Wheat (Triticum aestivum L.)

Spike length (SL) is the key determinant of plant architecture and yield potential. In this study, 193 recombinant inbred lines (RILs) derived from a cross between 13F10 and Chuanmai 42 (CM42) were evaluated for spike length in six environments. Sixty RILs consisting of 30 high and 30 low SLs were genotyped using the bulked segregant analysis exome sequencing (BSE-Seq) analysis for preliminary quantitative trait locus (QTL) mapping. A 6.69 Mb (518.43-525.12 Mb) region on chromosome 5AL was found to have a significant effect on the SL trait. Fifteen kompetitive allele specific PCR (KASP) markers were successfully converted from the single nucleotide polymorphisms (SNPs) in the SL target region. Combined with four novel simple sequence repeat (SSR) markers, a genetic linkage map spanning 21.159 cM was constructed. The mapping result confirmed the identity of a major and stable QTL named QSl.cib-5A in the targeted region that explained 7.88–26.60% of the phenotypic variation in SL. QSl.cib-5A was narrowed to a region of 4.84 cM interval corresponding to a 4.67 Mb (516.60-521.27 Mb) physical region in the Chinese Spring RefSeq v2.0 containing 17 high-confidence genes with 25 transcripts. In addition, this QTL exhibited pleiotropic effects on spikelet density (SD), with the phenotypic variances proportion ranging from 11.34–19.92%. This study provides a foundational step for cloning the QSl.cib-5A, which is involved in the regulation of spike morphology in common wheat.

Identification and Fine-Mapping of Quantitative Trait Loci Controlling Plant Height in Central European Winter Triticale (×Triticosecale Wittmack)

The quantitatively inherited trait plant height is routinely evaluated in triticale breeding programs as it substantially influences lodging and disease susceptibility, is a main contributor to biomass yield, and is required to improve hybrid seed production by fine-tuning plant height in the female and male parental pools in hybrid breeding programs. In this study, we evaluated a panel of 846 diverse Central European triticale genotypes to dissect the genetic architecture underlying plant height by genome-wide association mapping. This revealed three medium- to large-effect QTL on chromosomes 5A, 4B, and 5R. Genetic and physical fine-mapping of the putative QTL revealed that the QTL on chromosome 5R most likely corresponds to Ddw1 and that the QTL on chromosome 5A is likely to be Rht12. Furthermore, we observed a temporal trend in registered cultivars with a decreasing plant height during the past decades, accompanied by an increasing use of the height-reducing alleles at the identified QTL. In summary, our results shed new light on the genetic control of plant height in triticale and open new avenues for future improvement by breeding.

Overgrowth mutants determine the causal role of gibberellin GA2oxidaseA13 in Rht12 dwarfism of wheat

The induced dwarf mutant Rht12 was previously shown to have agronomic potential to replace the conventional DELLA mutants Rht-B1b/Rht-D1b in wheat. The Rht12 dwarfing gene is not associated with reduced coleoptile length (unlike the DELLA mutants) and it is dominant, characteristics which are shared with the previously characterized dwarfing genes Rht18 and Rht14. Using the Rht18/Rht14 model, a gibberellin (GA) 2-oxidase gene was identified in the Rht12 region on chromosome 5A. A screen for suppressor mutants in the Rht12 background identified tall overgrowth individuals that were shown to contain loss-of-function mutations in GA2oxidaseA13, demonstrating the role of this gene in the Rht12 dwarf phenotype. It was concluded that Rht12, Rht18, and Rht14 share the same height-reducing mechanism through the increased expression of GA 2-oxidase genes. Some of the overgrowth mutants generated in this study were semi-dwarf and taller than the original Rht12 dwarf, providing breeders with new sources of agronomically useful dwarfism.

Identification and genetic analysis of EMS-mutagenized wheat mutants conferring lesion-mimic premature aging

Background: Lesion-mimic and premature aging ( lmpa ) mutant lm pa 1 was identified from the ethyl methane sulfonate (EMS) mutant library in the bread wheat variety Keda 527 (KD527) background. To reveal the genetic basis of l mpa 1 mutant, phenotypic observations and analyses of chlorophyll content and photosynthesis were carried out in l mpa 1 , KD527 and their F 1 and F 2 derivatives. Further, bulked segregation analysis (BSA) in combination with a 660K SNP Chip were conducted on the F 2 segregation population of l mpa 1 /Chinese spring(CS) to locate the l mpa 1 gene. Results: Most agronomic traits of l mpa 1 were similar to those of KD527 before lesion-like spots appeared. Genetic analysis indicated that the F 1 plants from the crossing of l mpa 1 and KD527 exhibited the l mpa phenotype and the F 2 progenies showed a segregation of normal (wild type, WT) and l mpa , with the ratios of l mpa :WT=124:36（χ 2 =1.008<=3.841), indicating that l mpa is a dominant mutation. The combination of BSA and the SNP Chip analysis of CS, l mpa 1 and l mpa 1 /CS F 2 WT pool (50 plants) and l mpa pool (50 plants) showed that polymorphic SNPs were enriched on chromosome 5A, within a region of 30-40 Mb, indicating that the wheat premature aging gene Lmpa1 was probably located on the short arm of chromosome 5A. Conclusions: EMS-mutagenized mutant lmpa 1 deriving from elite wheat line KD527 conferred lmpa . lmpa phenotype of l mpa 1 mutant is controlled by a single dominant allele designated as Lmpa1 , which affected wheat growth and development and reduced the thousand grain weight ( tgw ) of single plant in wheat. The gene Lmpa1 was tentatively located within the region of 30-40 Mb near to the short arm of chromosome 5A.

Misexpression of a transcriptional repressor candidate provides a molecular mechanism for the suppression of awns by Tipped 1 in wheat

Awns are bristle-like structures formed at the tip of the lemma on the florets of some cereal grasses. Wild-type wheat is awned, but awnletted and awnless variants have been selected and nowadays all forms are cultivated. In this study, we dissected the genetic control underlying variation of this characteristic feature by association mapping in a large panel of 1110 winter wheat cultivars of worldwide origin. We identified the B1 (Tipped 1) locus on chromosome 5A as the major determinant of awnlessness globally. Using a combination of fine-mapping and expression analysis, we identified a putative C2H2 zinc finger protein with an EAR domain, characteristic of transcriptional repressors, as a likely candidate for Tipped 1. This gene was found to be up-regulated in awnless B1 compared with awned b1 plants, indicating that misexpression of this transcriptional regulator may contribute to the reduction of awn length in B1 plants. Taken together, our study provides an entry point towards a better molecular understanding of the evolution of morphological features in cereals through selection and breeding.