Morten Lillemo

Characterization of polyploid wheat genomic diversity using a high-density 90 000 single nucleotide polymorphism array

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker–trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence–absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

Association analysis of historical bread wheat germplasm using additive genetic covariance of relatives and population structure

Linkage disequilibrium can be used for identifying associations between traits of interest and genetic markers. This study used mapped diversity array technology (DArT) markers to find associations with resistance to stem rust, leaf rust, yellow rust, and powdery mildew, plus grain yield in five historical wheat international multienvironment trials from the International Maize and Wheat Improvement Center (CIMMYT). Two linear mixed models were used to assess marker–trait associations incorporating information on population structure and covariance between relatives. An integrated map containing 813 DArT markers and 831 other markers was constructed. Several linkage disequilibrium clusters bearing multiple host plant resistance genes were found. Most of the associated markers were found in genomic regions where previous reports had found genes or quantitative trait loci (QTL) influencing the same traits, providing an independent validation of this approach. In addition, many new chromosome regions for disease resistance and grain yield were identified in the wheat genome. Phenotyping across up to 60 environments and years allowed modeling of genotype × environment interaction, thereby making possible the identification of markers contributing to both additive and additive × additive interaction effects of traits.

A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat

As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat (Triticum aestivum), single genes that provide resistance to multiple pathogens are valuable in crop improvement. The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars

Kernel hardness that is conditioned by puroindoline genes has a profound effect on milling, baking and end-use quality of bread wheat. In this study, 219 landraces and 166 historical cultivars from China and 12 introduced wheats were investigated for their kernel hardness and puroindoline alleles, using molecular and biochemical markers. The results indicated that frequencies of soft, mixed and hard genotypes were 42.7, 24.3, and 33.0%, respectively, in Chinese landraces and 45.2, 13.9, and 40.9% in historical cultivars. The frequencies of PINA null, Pinb-D1b and Pinb-D1p genotypes were 43.8, 12.3, and 39.7%, respectively, in hard wheat of landraces, while 48.5, 36.8, and 14.7%, respectively, in historical hard wheats. A new Pinb-D1 allele, designated Pinb-D1t, was identified in two landraces, Guangtouxianmai and Hongmai from the Guizhou province, with the characterization of a glycine to arginine substitution at position 47 in the coding region of Pinb gene. Surprisingly, a new Pina-D1 allele, designated Pina-D1m, was detected in the landrace Hongheshang, from the Jiangsu province, with the characterization of a proline to serine substitution at position 35 in the coding region of Pina gene; it was the first novel mutation found in bread wheat, resulting in a hard endosperm with PINA expression. Among the PINA null genotypes, an allele designed as Pina-D1l, was detected in five landraces with a cytosine deletion at position 265 in Pina locus; while another novel Pina-D1 allele, designed as Pina-D1n, was identified in six landraces, with the characterization of an amino acid change from tryptophan-43 to a ‘stop’ codon in the coding region of Pina gene. The study of puroindoline polymorphism in Chinese wheat germplasm could provide useful information for the further understanding of the molecular basis of kernel hardness in bread wheat.

Anther extrusion and plant height are associated with Type I resistance to Fusarium head blight in bread wheat line ‘Shanghai-3/Catbird’

Fusarium head blight (FHB) is a destructive wheat disease of global importance. Resistance breeding depends heavily on the Fhb1 gene. The CIMMYT line Shanghai-3/Catbird (SHA3/CBRD) is a promising source without this gene. A recombinant inbred line (RIL) population from the cross of SHA3/CBRD with the German spring wheat cv. Naxos was evaluated for FHB resistance and related traits in field trials using spray and spawn inoculation in Norway and point inoculation in China. After spray and spawn inoculation, FHB severities were negatively correlated with both anther extrusion (AE) and plant height (PH). The QTL analysis showed that the Rht-B1b dwarfing allele co-localized with a QTL for low AE and increased susceptibility after spawn and spray inoculation. In general, SHA3/CBRD contributed most of the favorable alleles for resistance to severity after spray and spawn inoculation, while Naxos contributed more favorable alleles for reduction in FDK and DON content and resistance to severity after point inoculation. SHA3/CBRD contributed a major resistance QTL close to the centromere on 2DLc affecting FHB severity and DON after all inoculation methods. This QTL was also associated with AE and PH, with high AE and tall alleles contributed by SHA3/CBRD. Several QTL for AE and PH were detected, and low AE or reduced PH was always associated with increased susceptibility after spawn and spray inoculation. Most of the other minor FHB resistance QTL from SHA3/CBRD were associated with AE or PH, while the QTL from Naxos were mostly not. After point inoculation, no other QTL for FHB traits was associated with AE or PH, except the 2DLc QTL which was common across all inoculation methods. Marker-assisted selection based on the 2DLc QTL from SHA3/CBRD combined with phenotypic selection for AE is recommended for resistance breeding based on this valuable source of resistance.

Genetic analysis of partial resistance to powdery mildew in bread wheat line Saar

Powdery mildew, caused by Blumeria graminis (syn. Erysiphe graminis) f. sp. tritici, is an important disease of bread wheat (Triticum aestivum) in many countries. The CIMMYT bread wheat line Saar has exhibited a high level of partial resistance to powdery mildew in field trials conducted in Europe, Asia, and South America, and represents a valuable source of resistance in wheat breeding. A set of 114 random F5 inbred lines from the cross Saar × Avocet-YrA (susceptible) were evaluated in replicated field trials at two locations in southeastern Norway to determine the number of genes involved in partial resistance to powdery mildew. Narrow-sense heritability estimates were high (0.83 to 0.92). Based on both quantitative and qualitative genetic analyses, the minimum number of genes with additive effects segregating for powdery mildew resistance in the population was four. Transgressive segregation indicated that Avocet-YrA might have contributed one minor gene for resistance. It is concluded that partial resistance to powdery mildew in Saar is controlled by at least three genes. Such resistance conferred by multiple genes having additive effects is expected to be durable.

Partial resistance to powdery mildew in German spring wheat ‘Naxos’ is based on multiple genes with stable effects in diverse environments

Powdery mildew is one of the most important wheat diseases in temperate regions of the world. Resistance breeding is considered to be an economical and environmentally benign way to control this disease. The German spring wheat cv. ‘Naxos’ exhibits high levels of partial and race non-specific resistance to powdery mildew in the field and is a valuable source in resistance breeding. The main objective of the present study was to map the genetic factors behind the resistance in Naxos, based on a population of recombinant inbred lines (RIL) from a cross with the susceptible CIMMYT breeding line SHA3/CBRD. Powdery mildew severity was evaluated in six field trials in Norway and four field trials in China. The major quantitative trait locus (QTL) with resistance from Naxos was detected close to the Pm3 locus on 1AS in all environments, and explained up to 35% of the phenotypic variation. Naxos was shown to carry another major QTL on 2DL and minor ones on 2BL and 7DS. QTL with resistance from SHA3/CBRD were detected on 1RS, 2DLc, 6BL and 7AL. The QTL on the 1B/1R translocation showed highly variable effects across environments corresponding to known virulence differences against Pm8. SHA3/CBRD was shown to possess the Pm3 haplotype on 1AS, but none of the known Pm3a-g alleles. The RIL population did not provide any evidence to suggest that the Pm3 allele of SHA3/CBRD acted as a suppressor of Pm8.

Leaf Rust Resistance Gene LR34 is Involved in Powdery Mildew Resistance of Cimmyt Bread Wheat Line Saar

The CIMMYT bread wheat line Saar has a high level of adult plant resistance to leaf rust (LR, caused by Puccinia triticina) and stripe rust (YR, caused by P. striiformis f. sp. tritici) based on Lr34/Yr18 in combination with additional minor resistance genes. Because Saar also has good partial resistance to powdery mildew (PM, caused by Blumeria graminis f. sp. tritici), experiments were set up to test whether Lr34 could be involved in its resistance to PM. A population of 113 recombinant inbred F6 lines from a cross between Saar and Avocet-YrA was tested for all three diseases. Correlations among the disease data for LR, YR and PM were strong and highly significant, suggesting that the cross segregated for at least one common genetic factor that affected the resistance to all three diseases. Strong correlations with leaf tip necrosis (LTN), a phenotypic marker for Lr34, indicated that this gene was indeed involved in the PM resistance of Saar. Disease testing of near-isogenic lines for Lr34 and Lr46 in the genetic background of Avocet-YrA and YrLrPrl1 in the background of Lalbahadur showed that all three genes were associated with significantly reduced levels of LR, YR and PM compared to their susceptible genetic backgrounds. It is concluded that resistance to both rust and PM is not only confined to Lr34, but could be a general phenomenon of LTN-associated resistance genes, including Lr46 and YrLrPrl1

Molecular mapping of partial resistance to powdery mildew in winter wheat cultivar Folke

The Swedish winter wheat (Triticum aestivum L.) cultivar Folke has a long record of partial and race non-specific resistance to powdery mildew (caused by Blumeria graminis f. sp. tritici) in the field. The aim of the present study was to map the main genetic factors behind the partial resistance in Folke and identify molecular markers for use in marker-assisted selection. A population of 130 recombinant inbred lines was developed from a cross between Folke and the moderately susceptible spring wheat line T2038. The population was tested for powdery mildew resistance over two years at two locations in Norway and genotyped with DArT and SSR markers. Composite interval mapping detected a total of eight quantitative trait loci (QTL) for powdery mildew resistance; six with resistance from Folke on 2BS, 2DL, 5AL, 5BS and 6BS and two with resistance from T2038 on 5BS and 7AL. None of the loci with resistance from Folke mapped to chromosomal regions with known race-specific resistance genes, which confirmed the race non-specific nature of the resistance in this cultivar. The molecular markers linked to the reported QTL will be useful as a tool for selecting partial and potentially durable resistance to powdery mildew based on the resistance in Folke.

QTL Characterization of Fusarium Head Blight Resistance in CIMMYT Bread Wheat Line Soru#1

Fusarium head blight (FHB) resistant line Soru#1 was hybridized with the German cultivar Naxos to generate 131 recombinant inbred lines for QTL mapping. The population was phenotyped for FHB and associated traits in spray inoculated experiments in El Batán (Mexico), spawn inoculated experiments in Ås (Norway) and point inoculated experiments in Nanjing (China), with two field trials at each location. Genotyping was performed with the Illumina iSelect 90K SNP wheat chip, along with a few SSR and STS markers. A major QTL for FHB after spray and spawn inoculation was detected on 2DLc, explaining 15–22% of the phenotypic variation in different experiments. This QTL remained significant after correction for days to heading (DH) and plant height (PH), while another QTL for FHB detected at the Vrn-A1 locus on 5AL almost disappeared after correction for DH and PH. Minor QTL were detected on chromosomes 2AS, 2DL, 4AL, 4DS and 5DL. In point inoculated experiments, QTL on 2DS, 3AS, 4AL and 5AL were identified in single environments. The mechanism of resistance of Soru#1 to FHB was mainly of Type I for resistance to initial infection, conditioned by the major QTL on 2DLc and minor ones that often coincided with QTL for DH, PH and anther extrusion (AE). This indicates that phenological and morphological traits and flowering biology play important roles in resistance/escape of FHB. SNPs tightly linked to resistance QTL, particularly 2DLc, could be utilized in breeding programs to facilitate the transfer and selection of those QTL.