Separation of the effects of two reduced height (Rht) genes and genomic background to select for less Fusarium head blight of short-strawed winter wheat (Triticum aestivum L.) varieties.

KEY MESSAGE: FHB resistance shared pleiotropic loci with plant height and anther retention. Genomic prediction allows to select for genomic background reducing FHB susceptibility in the presence of the dwarfing allele Rht-D1b. With the high interest for semi-dwarf cultivars in wheat, finding locally adapted resistance sources against Fusarium head blight (FHB) and FHB-neutral reduced height (Rht) genes is of utmost relevance. In this study, 401 genotypes of European origin without/with dwarfing alleles of Rht-D1 and/or Rht24 were analysed across five environments on FHB severity and the morphological traits such as plant height (PH), anther retention (AR), number of spikelets per ear, ear length and ear density. Data were analysed by combined correlation and path analyses, association mapping and coupling single- and multi-trait genome-wide association studies (ST-GWAS and MT-GWAS, respectively) and genomic prediction (GP). All FHB data were corrected for flowering date or heading stage. High genotypic correlation (rg = 0.74) and direct path effect (0.57) were detected between FHB severity and anther retention (AR). Moderate correlation (rg = - 0.55) was found between FHB severity and plant height (PH) with a high indirect path via AR (- 0.31). Indirect selection for FHB resistance should concentrate on AR and PH. ST-GWAS identified 25 quantitative trait loci (QTL) for FHB severity, PH and AR, while MT-GWAS detected six QTL across chromosomes 2A, 4D, 5A, 6B and 7B conveying pleiotropic effects on the traits. Rht-D1b was associated with high AR and FHB susceptibility. Our study identified a promising positively acting pleiotropic QTL on chromosome 7B which can be utilized to improve FHB resistance while reducing PH and AR. Rht-D1b genotypes having a high resistance genomic background exhibited lower FHB severity and AR. The use of GP for estimating the genomic background was more effective than selection of GWAS-detected markers. We demonstrated that GP has a great potential and should be exploited by selecting for semi-dwarf winter wheat genotypes with higher FHB resistance due to their genomic background.

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.).

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997-2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01-3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype-phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.

Linkage mapping identifies a non-synonymous mutation in FLOWERING LOCUS T (FT-B1) increasing spikelet number per spike.

Total spikelet number per spike (TSN) is a major component of spike architecture in wheat (Triticum aestivum L.). A major and consistent quantitative trait locus (QTL) was discovered for TSN in a doubled haploid spring wheat population grown in the field over 4 years. The QTL on chromosome 7B explained up to 20.5% of phenotypic variance. In its physical interval (7B: 6.37-21.67 Mb), the gene FLOWERING LOCUS T (FT-B1) emerged as candidate for the observed effect. In one of the parental lines, FT-B1 carried a non-synonymous substitution on position 19 of the coding sequence. This mutation modifying an aspartic acid (D) into a histidine (H) occurred in a highly conserved position. The mutation was observed with a frequency of ca. 68% in a set of 135 hexaploid wheat varieties and landraces, while it was not found in other plant species. FT-B1 only showed a minor effect on heading and flowering time (FT) which were dominated by a major QTL on chromosome 5A caused by segregation of the vernalization gene VRN-A1. Individuals carrying the FT-B1 allele with amino acid histidine had, on average, a higher number of spikelets (15.1) than individuals with the aspartic acid allele (14.3) independent of their VRN-A1 allele. We show that the effect of TSN is not mainly related to flowering time; however, the duration of pre-anthesis phases may play a major role.

Transfer of the Dominant Virus Resistance Gene AV-1 pro From Asparagus prostratus to Chromosome 2 of Garden Asparagus A. officinalis L.

An introgression breeding programme was carried out to transfer the virus resistance gene AV-1 pro from the wild species Asparagus prostratus to the garden asparagus Asparagus officinalis. Serious crossing barriers caused by genetic distance and different ploidy levels of the crossing parents have been overcome using embryo rescue for the F1, BC1, and BC2 generations. The male and female fertility was widely restored in BC2 and was shown to be comparable to the cultivated asparagus. Five AV-1 resistant diploid (2n = 2x = 20) BC2 plants were selected and reciprocally backcrossed with asparagus cultivars. Segregation analyses of fourteen seedborne BC3 progenies suggested a monogenic dominant inheritance of the AV-1 resistance. Genotyping by sequencing analysis gave a strong hint for location of the resistance gene on asparagus Chromosome 2. Using an Axiom single nucleotide polymorphism (SNP) genotyping array for the analysis of three BC3 families with 10 AV-1 resistant and 10 AV-1 susceptible plants each, as well as 25 asparagus cultivars, the AV-1 pro locus on Chromosome 2 was further narrowed down. The SNP with the highest LOD score was converted to a kompetitive allele specific PCR (KASP) marker, shown to be useful for the further backcross programme and serving as the starting point for the development of a diagnostic marker.

Prospects of GWAS and predictive breeding for European winter wheat's grain protein content, grain starch content, and grain hardness.

Grain quality traits determine the classification of registered wheat (Triticum aestivum L.) varieties. Although environmental factors and crop management practices exert a considerable influence on wheat quality traits, a significant proportion of the variance is attributed to the genetic factors. To identify the underlying genetic factors of wheat quality parameters viz., grain protein content (GPC), grain starch content (GSC), and grain hardness (GH), we evaluated 372 diverse European wheat varieties in replicated field trials in up to eight environments. We observed that all of the investigated traits hold a wide and significant genetic variation, and a significant negative correlation exists between GPC and GSC plus grain yield. Our association analyses based on 26,694 high-quality single nucleotide polymorphic markers revealed a strong quantitative genetic nature of GPC and GSC with associations on groups 2, 3, and 6 chromosomes. The identification of known Puroindoline-b gene for GH provided a positive analytic proof for our studies. We report that a locus QGpc.ipk-6A controls both GPC and GSC with opposite allelic effects. Based on wheat's reference and pan-genome sequences, the physical characterization of two loci viz., QGpc.ipk-2B and QGpc.ipk-6A facilitated the identification of the candidate genes for GPC. Furthermore, by exploiting additive and epistatic interactions of loci, we evaluated the prospects of predictive breeding for the investigated traits that suggested its efficient use in the breeding programs.

Identification and validation of QTL for spike fertile floret and fruiting efficiencies in hexaploid wheat (Triticum aestivum L.).

KEY MESSAGE: This study identified and validated two QTL associated with spike fertile floret and fruiting efficiencies. They represent two new loci to use in MAS to improve wheat yield potential. The spike fruiting efficiency (FE-grains per unit spike dry weight at anthesis, GN/SDW) is a promising trait to improve wheat yield potential. It depends on fertile floret efficiency (fertile florets per unit SDW-FFE, FF/SDW) and grain set (grains per fertile floret-GST). Given its difficult measurement, it is often estimated as the grains per unit of nongrain spike dry weight at maturity (FEm). In this study, quantitative trait loci (QTL) were mapped using a double haploid population (Baguette 19/BIOINTA 2002, with high and low FE, respectively) genotyped with the iSelect 90 K SNP array and evaluated in five environments. We identified 37 QTL, but two were major with an R2 > 10% and stable for being at least present in three environments: the QFEm.perg-3A (on Chr. 3A, 51.6 cM, 685.12 Mb) for FEm and the QFFE.perg-5A (on Chr. 5A, 42.1 cM, 461.49 Mb) for FFE, FE and FEm. Both QTL were validated using two independent F2 populations and KASP markers. For the most promising QTL, QFFE.perg-5A, the presence of the allele for high FFE resulted in + 4% FF, + 9% GN, + 13% GST, + 16% yield gSDW-1 and + 5% yield spike-1. QFEm.perg-3A and QFFE.perg-5A represent two new loci to use in MAS to improve wheat yield potential.

TaAPO-A1, an ortholog of rice ABERRANT PANICLE ORGANIZATION 1, is associated with total spikelet number per spike in elite European hexaploid winter wheat (Triticum aestivum L.) varieties.

We dissected the genetic basis of total spikelet number (TSN) along with other traits, viz. spike length (SL) and flowering time (FT) in a panel of 518 elite European winter wheat varieties. Genome-wide association studies (GWAS) based on 39,908 SNP markers revealed highly significant quantitative trait loci (QTL) for TSN on chromosomes 2D, 7A, and 7B, for SL on 5A, and FT on 2D, with 2D-QTL being the functional marker for the gene Ppd-D1. The physical region of the 7A-QTL for TSN revealed the presence of a wheat ortholog (TaAPO-A1) to APO1-a rice gene that positively controls the spikelet number on the panicles. Interspecific analyses of the TaAPO-A1 orthologs showed that it is a highly conserved gene important for floral development and present in a wide range of terrestrial plants. Intraspecific studies of the TaAPO-A1 across wheat genotypes revealed a polymorphism in the conserved F-box domain, defining two haplotypes. A KASP marker developed on the polymorphic site showed a highly significant association of TaAPO-A1 with TSN, explaining 23.2% of the total genotypic variance. Also, the TaAPO-A1 alleles showed weak but significant differences for SL and grain yield. Our results demonstrate the importance of wheat sequence resources to identify candidate genes for important traits based on genetic analyses.

Gene Expression Profiling and Fine Mapping Identifies a Gibberellin 2-Oxidase Gene Co-segregating With the Dominant Dwarfing Gene Ddw1 in Rye (Secale cereale L.).

The gibberellin (GA)-sensitive dwarfing gene Ddw1 provides an opportunity to genetically reduce plant height in rye. Genetic analysis in a population of recombinant inbred lines confirmed a monogenetic dominant inheritance of Ddw1. Significant phenotypic differences in PH between homo- and heterozygotic genotypes indicate an incomplete dominance of Ddw1. De novo transcriptome sequencing of Ddw1 mutant as well as tall genotypes resulted in 113,547 contigs with an average length of 318 bp covering 36.18 Mbp rye DNA. A hierarchical cluster analysis based on individual groups of rye homologs of functionally characterized rice genes controlling morphological or physiological traits including plant height, flowering time, and source activity identified the gene expression profile of stems at the begin of heading to most comprehensively mirror effects of Ddw1. Genome-wide expression profiling identified 186 transcripts differentially expressed between semi-dwarf and tall genotypes in stems. In total, 29 novel markers have been established and mapped to a 27.2 cM segment in the distal part of the long arm of chromosome 5R. Ddw1 could be mapped within a 0.4 cM interval co-segregating with a marker representing the C20-GA2-oxidase gene ScGA2ox12, that is up-regulated in stems of Ddw1 genotypes. The increased expression of ScGA2ox12 observed in semi-dwarf rye as well as structural alterations in transcript sequences associated with the ScGA2ox12 gene implicate, that Ddw1 is a dominant gain-of-function mutant. Integration of the target interval in the wheat reference genome sequence indicated perfect micro-colinearity between the Ddw1 locus and a 831 kb segment on chromosome 5A, which resides inside of a 11.21 Mb interval carrying the GA-sensitive dwarfing gene Rht12 in wheat. The potential of Ddw1 as a breeder's option to improve lodging tolerance in rye is discussed.

Genome-wide Association Mapping and Prediction of Adult Stage Septoria tritici Blotch Infection in European Winter Wheat via High-Density Marker Arrays.

blotch (STB) caused by the fungus is a devastating foliar disease of wheat ( L.) that can lead to substantial yield losses. Quantitative genetic resistance has been proposed as a durable strategy for STB control. In this study, we dissected the genetic basis of STB infection in 371 European wheat varieties based on 35k and 90k single nucleotide polymorphism marker arrays. The phenotypic data analyses suggested that large genetic variance exists for STB infection with a broad-sense heritability of 0.78. Genome-wide association studies (GWAS) propose the highly quantitative nature of STB infection with potential associations on chromosomes 1A, 1B, 2D, 4A, 5A, 6A, 6D, 7A, and 7B. Increased marker density in GWAS by combining markers from both arrays helped to detect additional markers explaining increased genotypic variance. Linkage disequilibrium analyses revealed genes with a possible role in disease resistance. The potential of genomic prediction (GP) assessed via two models accounting for additive effects and additive plus epistatic interactions among the loci suggested the possibility of genomic selection for improved STB resistance. Genomic prediction results also indicated that the higher-order epistatic interactions are not abundant and that both marker platforms are equally suitable for GP of STB infection. Our results provide further understanding of the quantitative genetic nature of STB infection, serve as a resource for marker-assisted breeding, and highlight the potential of genomic selection for improved STB resistance.

Whole-Genome Association Mapping and Genomic Prediction for Iron Concentration in Wheat Grains.

Malnutrition of iron (Fe) affects two billion people worldwide. Therefore, enhancing grain Fe concentration (GFeC) in wheat (Triticum aestivum L.) is an important goal for breeding. Here we study the genetic factors underlying GFeC trait by genome-wide association studies (GWAS) and the prediction abilities using genomic prediction (GP) in a panel of 369 European elite wheat varieties which was genotyped with 15,523 mapped single-nucleotide polymorphism markers (SNP) and a subpanel of 183 genotypes with 44,233 SNP markers. The resulting means of GFeC from three field experiments ranged from 24.42 to 52.42 µg·g-1 with a broad-sense heritability (H²) equaling 0.59 over the years. GWAS revealed 41 and 137 significant SNPs in the whole and subpanel, respectively, including significant marker-trait associations (MTAs) for best linear unbiased estimates (BLUEs) of GFeC on chromosomes 2A, 3B and 5A. Putative candidate genes such as NAC transcription factors and transmembrane proteins were present on chromosome 2A (763,689,738⁻765,710,113 bp). The GP for a GFeC trait ranged from low to moderate values. The current study reported GWAS of GFeC for the first time in hexaploid wheat varieties. These findings confirm the utility of GWAS and GP to explore the genetic architecture of GFeC for breeding programs aiming at the improvement of wheat grain quality.

Identifying Candidate Genes for Enhancing Grain Zn Concentration in Wheat.

Wheat (Triticum aestivum L.) is one of the major staple food crops worldwide. Despite efforts in improving wheat quality, micronutrient levels are still below the optimal range for human nutrition. In particular, zinc (Zn) deficiency is a widespread problem in human nutrition in countries relying mainly on a cereal diet; hence improving Zn accumulation in grains is an imperative need. This study was designed to understand the genetic architecture of Zn grain concentrations in wheat grains. We performed a genome-wide association study (GWAS) for grain Zn concentrations in 369 European wheat genotypes, using field data from 3 years. The complete wheat panel was genotyped by high-density arrays of single nucleotide polymorphic (SNP) markers (90k iSELECT Infinium and 35k Affymetrix arrays) resulting in 15,523 polymorphic markers. Additionally, a subpanel of 183 genotypes was analyzed with a novel 135k Affymetrix marker array resulting in 28,710 polymorphic SNPs for high-resolution mapping of the potential genomic regions. The mean grain Zn concentration of the genotypes ranged from 25.05-52.67 µg g-1 dry weight across years with a moderate heritability value. Notably, 40 marker-trait associations (MTAs) were detected in the complete panel of varieties on chromosomes 2A, 3A, 3B, 4A, 4D, 5A, 5B, 5D, 6D, 7A, 7B, and 7D. The number of MTAs in the subpanel was increased to 161 MTAs whereas the most significant and consistent associations were located on chromosomes 3B (723,504,241-723,611,488 bp) and 5A (462,763,758-466,582,184 bp) having major effects. These genomic regions include newly identified putative candidate genes, which are related to Zn uptake and transport or represent bZIP and mitogen-activated protein kinase genes. These findings provide the basis for understanding the genetic background of Zn concentration in wheat grains that in turn may help breeders to select high Zn-containing genotypes to improve human health and grain quality.

Development and Evaluation of a Barley 50k iSelect SNP Array.

High-throughput genotyping arrays continue to be an attractive, cost-effective alternative to sequencing based approaches. We have developed a new 50k Illumina Infinium iSelect genotyping array for barley, a cereal crop species of major international importance. The majority of SNPs on the array have been extracted from variants called in exome capture data of a wide range of European barley germplasm. We used the recently published barley pseudomolecule assembly to map the exome capture data, which allowed us to generate markers with accurate physical positions and detailed gene annotation. Markers from an existing and widely used barley 9k Infinium iSelect array were carried over onto the 50k chip for backward compatibility. The array design featured 49,267 SNP markers that converted into 44,040 working assays, of which 43,461 were scorable in GenomeStudio. Of the working assays, 6,251 are from the 9k iSelect platform. We validated the SNPs by comparing the genotype calls from the new array to legacy datasets. Rates of agreement averaged 98.1 and 93.9% respectively for the legacy 9k iSelect SNP set (Comadran et al., 2012) and the exome capture SNPs. To test the utility of the 50k chip for genetic mapping, we genotyped a segregating population derived from a Golden Promise × Morex cross (Liu et al., 2014) and mapped over 14,000 SNPs to genetic positions which showed a near exact correspondence to their known physical positions. Manual adjustment of the cluster files used by the interpreting software for genotype scoring improved results substantially, but migration of cluster files between sites led to a deterioration of results, suggesting that local adjustment of cluster files is required on a site-per-site basis. Information relating to the markers on the chip is available online at https://ics.hutton.ac.uk/50k.

Genetic and physical mapping of anther extrusion in elite European winter wheat.

The production and cultivation of hybrid wheat is a possible strategy to close the yield gap in wheat. Efficient hybrid wheat seed production largely depends on high rates of cross-pollination which can be ensured through high anther extrusion (AE) by male parental lines. Here, we report the AE capacity and elucidate its genetics in 514 elite European winter wheat varieties via genome-wide association studies (GWAS). We observed a wide range of variation among genotypes and a high heritability (0.80) for AE. The whole panel was genotyped with the 35k Affymetrix and 90k iSELECT single nucleotide polymorphism (SNP) arrays plus Ppd-D1, Rht-B1 and Rht-D1 candidate markers. GWAS revealed 51 marker-trait associations (MTAs) on chromosomes 1A, 1B, 2A, 4D and 5B, with Rht-D1 (4D) being the most significant marker. Division of whole panel according to the Rht-D1 genotype resulted in 212 and 294 varieties harboring Rht-D1a and Rht-D1b allele, respectively. The presence of Rht-D1a compared to Rht-D1b (mutant) allele had a large phenotypic influence on AE resulting in its ~17% increase. GWAS performed on the sub-panels detected novel MTAs on chromosomes 2D, 3B and 6A with increased phenotypic variance imparted by individual markers. Our study shows that AE is a highly quantitative trait and wild type Rht-D1a allele greatly improves AE. Moreover, demarcating the quantitative trait loci regions based on intra-chromosomal linkage disequilibrium revealed AE's candidate genes/genomic regions. Understanding the genetics of AE in elite European wheat and utilizing the linked markers in breeding programs can help to enhance cross-pollination for better exploitation of heterosis.

The roles of pleiotropy and close linkage as revealed by association mapping of yield and correlated traits of wheat (Triticum aestivum L.).

Grain yield (GY) of bread wheat (Triticum aestivum L.) is quantitatively inherited. Correlated GY-syndrome traits such as plant height (PH), heading date (HD), thousand grain weight (TGW), test weight (TW), grains per ear (GPE), and ear weight (EW) influence GY. Most quantitative genetics studies assessed the multiple-trait (MT) complex of GY-syndrome using single-trait approaches, and little is known about its underlying pleiotropic architecture. We investigated the pleiotropic architecture of wheat GY-syndrome through MT association mapping (MT-GWAS) using 372 varieties phenotyped in up to eight environments and genotyped with 18 832 single nucleotide polymorphisms plus 24 polymorphic functional markers. MT-GWAS revealed a total of 345 significant markers spread genome wide, representing 8, 40, 11, 40, 34, and 35 effective GY-PH, GY-HD, GY-TGW, GY-TW, GY-GPE, and GY-EW associations, respectively. Among them, pleiotropic roles of Rht-B1 and TaGW2-6B loci were corroborated. Only one marker presented simultaneous associations for three traits (i.e. GY-TGW-TW). Close linkage was difficult to differentiate from pleiotropy; thus, the pleiotropic architecture of GY-syndrome was dissected more as a cause of pleiotropy rather than close linkage. Simulations showed that minor allele frequencies, along with sizes and distances between quantitative trait loci for two traits, influenced the ability to distinguish close linkage from pleiotropy.

Genome-wide association mapping of resistance to eyespot disease (Pseudocercosporella herpotrichoides) in European winter wheat (Triticum aestivum L.) and fine-mapping of Pch1.

KEY MESSAGE: Genotypes with recombination events in the Triticum ventricosum introgression on chromosome 7D allowed to fine-map resistance gene Pch1, the main source of eyespot resistance in European winter wheat cultivars. Eyespot (also called Strawbreaker) is a common and serious fungal disease of winter wheat caused by the necrotrophic fungi Oculimacula yallundae and Oculimacula acuformis (former name Pseudocercosporella herpotrichoides). A genome-wide association study (GWAS) for eyespot was performed with 732 microsatellite markers (SSR) and 7761 mapped SNP markers derived from the 90 K iSELECT wheat array using a panel of 168 European winter wheat varieties as well as three spring wheat varieties and phenotypic evaluation of eyespot in field tests in three environments. Best linear unbiased estimations (BLUEs) were calculated across all trials and ranged from 1.20 (most resistant) to 5.73 (most susceptible) with an average value of 4.24 and a heritability of H 2 = 0.91. A total of 108 SSR and 235 SNP marker-trait associations (MTAs) were identified by considering associations with a -log10 (P value) ≥3.0. Significant MTAs for eyespot-score BLUEs were found on chromosomes 1D, 2A, 2D, 3D, 5A, 5D, 6A, 7A and 7D for the SSR markers and chromosomes 1B, 2A, 2B, 2D, 3B and 7D for the SNP markers. For 18 varieties (10.5%), a highly resistant phenotype was detected that was linked to the presence of the resistance gene Pch1 on chromosome 7D. The identification of genotypes with recombination events in the introgressed genomic segment from Triticum ventricosum harboring the Pch1 resistance gene on chromosome 7DL allowed the fine-mapping of this gene using additional SNP markers and a potential candidate gene Traes_7DL_973A33763 coding for a CC-NBS-LRR class protein was identified.

Validating the prediction accuracies of marker-assisted and genomic selection of Fusarium head blight resistance in wheat using an independent sample.

KEY MESSAGE: Compared with independent validation, cross-validation simultaneously sampling genotypes and environments provided similar estimates of accuracy for genomic selection, but inflated estimates for marker-assisted selection. Estimates of prediction accuracy of marker-assisted (MAS) and genomic selection (GS) require validations. The main goal of our study was to compare the prediction accuracies of MAS and GS validated in an independent sample with results obtained from fivefold cross-validation using genomic and phenotypic data for Fusarium head blight resistance in wheat. In addition, the applicability of the reliability criterion, a concept originally developed in the context of classic animal breeding and GS, was explored for MAS. We observed that prediction accuracies of MAS were overestimated by 127% using cross-validation sampling genotype and environments in contrast to independent validation. In contrast, prediction accuracies of GS determined in independent samples are similar to those estimated with cross-validation sampling genotype and environments. This can be explained by small population differentiation between the training and validation sets in our study. For European wheat breeding, which is so far characterized by a slow temporal dynamic in allele frequencies, this assumption seems to be realistic. Thus, GS models used to improve European wheat populations are expected to possess a long-lasting validity. Since quantitative trait loci information can be exploited more precisely if the predicted genotype is more related to the training population, the reliability criterion is also a valuable tool to judge the level of prediction accuracy of individual genotypes in MAS.

Diversity analysis and genomic prediction of Sclerotinia resistance in sunflower using a new 25 K SNP genotyping array.

KEY MESSAGE: We have developed a SNP array for sunflower containing more than 25 K markers, representing single loci mostly in or near transcribed regions of the genome. The array was successfully applied to genotype a diversity panel of lines, hybrids, and mapping populations and represented well the genetic diversity of cultivated sunflower. Results of PCoA and population substructure analysis underlined the complexity of the genetic composition of current elite breeding material. The performance of this genotyping platform for genome-based prediction of phenotypes and detection of QTL with improved resolution could be demonstrated based on the re-evaluation of a population segregating for resistance to Sclerotinia midstalk rot. Given our results, the newly developed 25 K SNP array is expected to be of great utility for the most important applications in genome-based sunflower breeding and research. ABSTRACT: Genotyping with a large number of molecular markers is a prerequisite to conduct genome-based genetic analyses with high precision. Here, we report the design and performance of a 25 K SNP genotyping array for sunflower (Helianthus annuus L.). SNPs were discovered based on variant calling in de novo assembled, UniGene-based contigs of sunflower derived from whole genome sequencing and amplicon sequences originating from four and 48 inbred lines, respectively. After inclusion of publically available transcriptome-derived SNPs, in silico design of the Illumina(®) Infinium iSelect HD BeadChip yielded successful assays for 22,299 predominantly haplotype-specific SNPs. The array was validated in a sunflower diversity panel including inbred lines, open-pollinated varieties, introgression lines, landraces, recombinant inbred lines, and F2 populations. Validation provided 20,502 high-quality bi-allelic SNPs with stable cluster performance whereby each SNP marker represents a single locus mostly in or near transcribed regions of the sunflower genome. Analyses of population structure and quantitative resistance to Sclerotinia midstalk rot demonstrate that this array represents a significant improvement over currently available genomic tools for genetic diversity analyses, genome-wide marker-trait association studies, and genetic mapping in sunflower.

Analysis of main effect QTL for thousand grain weight in European winter wheat (Triticum aestivum L.) by genome-wide association mapping.

Grain weight, an essential yield component, is under strong genetic control and at the same time markedly influenced by the environment. Genetic analysis of the thousand grain weight (TGW) by genome-wide association study (GWAS) was performed with a panel of 358 European winter wheat (Triticum aestivum L.) varieties and 14 spring wheat varieties using phenotypic data of field tests in eight environments. Wide phenotypic variations were indicated for the TGW with BLUEs (best linear unbiased estimations) values ranging from 35.9 to 58.2 g with a mean value of 45.4 g and a heritability of H(2) = 0.89. A total of 12 candidate genes for plant height, photoperiodism and grain weight were genotyped on all varieties. Only three candidates, the photoperiodism gene Ppd-D1, dwarfing gene Rht-B1and the TaGW-6A gene were significant explaining up to 14.4, 2.3, and 3.4% of phenotypic variation, respectively. For a comprehensive genome-wide analysis of TGW-QTL genotyping data from 732 microsatellite markers and a set of 7769 mapped SNP-markers genotyped with the 90k iSELECT array were analyzed. In total, 342 significant (-log10 (P-value) ≥ 3.0) marker trait associations (MTAs) were detected for SSR-markers and 1195 MTAs (-log10(P-value) ≥ 3.0) for SNP-markers in all single environments plus the BLUEs. After Bonferroni correction, 28 MTAs remained significant for SSR-markers (-log10 (P-value) ≥ 4.82) and 58 MTAs for SNP-markers (-log10 (P-value) ≥ 5.89). Apart from chromosomes 4B and 6B for SSR-markers and chromosomes 4D and 5D for SNP-markers, MTAs were detected on all chromosomes. The highest number of significant SNP-markers was found on chromosomes 3B and 1B, while for the SSRs most markers were significant on chromosomes 6D and 3D. Overall, TGW was determined by many markers with small effects. Only three SNP-markers had R(2) values above 6%.

Whole genome association mapping of plant height in winter wheat (Triticum aestivum L.).

The genetic architecture of plant height was investigated in a set of 358 recent European winter wheat varieties plus 14 spring wheat varieties based on field data in eight environments. Genotyping of diagnostic markers revealed the Rht-D1b mutant allele in 58% of the investigated varieties, while the Rht-B1b mutant was only present in 7% of the varieties. Rht-D1 was significantly associated with plant height by using a mixed linear model and employing a kinship matrix to correct for population stratification. Further genotyping data included 732 microsatellite markers, resulting in 770 loci, of which 635 markers were placed on the ITMI map plus a set of 7769 mapped SNP markers genotyped with the 90 k iSELECT chip. When Bonferroni correction was applied, a total of 153 significant marker-trait associations (MTAs) were observed for plant height and the SSR markers (-log10 (P-value) ≥ 4.82) and 280 (-log10 (P-value) ≥ 5.89) for the SNPs. Linear regression between the most effective markers and the BLUEs for plant height indicated additive effects for the MTAs of different chromosomal regions. Analysis of syntenic regions in the rice genome revealed closely linked rice genes related to gibberellin acid (GA) metabolism and perception, i.e. GA20 and GA2 oxidases orthologous to wheat chromosomes 1A, 2A, 3A, 3B, 5B, 5D and 7B, ent-kaurenoic acid oxidase orthologous to wheat chromosome 7A, ent-kaurene synthase on wheat chromosome 2B, as well as GA-receptors like DELLA genes orthologous to wheat chromosomes 4B, 4D and 7A and genes of the GID family orthologous to chromosomes 2B and 5B. The data indicated that besides the widely used GA-insensitive dwarfing genes Rht-B1 and Rht-D1 there is a wide spectrum of loci available that could be used for modulating plant height in variety development.

Genetic architecture of main effect QTL for heading date in European winter wheat.

A genome-wide association study (GWAS) for heading date (HD) was performed with a panel of 358 European winter wheat (Triticum aestivum L.) varieties and 14 spring wheat varieties through the phenotypic evaluation of HD in field tests in eight environments. Genotyping data consisted of 770 mapped microsatellite loci and 7934 mapped SNP markers derived from the 90K iSelect wheat chip. Best linear unbiased estimations (BLUEs) were calculated across all trials and ranged from 142.5 to 159.6 days after the 1st of January with an average value of 151.4 days. Considering only associations with a -log10 (P-value) ≥ 3.0, a total of 340 SSR and 2983 SNP marker-trait associations (MTAs) were detected. After Bonferroni correction for multiple testing, a total of 72 SSR and 438 SNP marker-trait associations remained significant. Highly significant MTAs were detected for the photoperiodism gene Ppd-D1, which was genotyped in all varieties. Consistent associations were found on all chromosomes with the highest number of MTAs on chromosome 5B. Linear regression showed a clear dependence of the HD score BLUEs on the number of favorable alleles (decreasing HD) and unfavorable alleles (increasing HD) per variety meaning that genotypes with a higher number of favorable or a low number of unfavorable alleles showed lower HD and therefore flowered earlier. For the vernalization gene Vrn-A2 co-locating MTAs on chromosome 5A, as well as for the photoperiodism genes Ppd-A1 and Ppd-B1 on chromosomes 2A and 2B were detected. After the construction of an integrated map of the SSR and SNP markers and by exploiting the synteny to sequenced species, such as rice and Brachypodium distachyon, we were able to demonstrate that a marker locus on wheat chromosome 5BL with homology to the rice photoperiodism gene Hd6 played a significant role in the determination of the heading date in wheat.