Identification of Ug99 stem rust resistance loci in winter wheat germplasm using genome-wide association analysis

The evolution of a new race of stem rust, generally referred to as Ug99, threatens global wheat production because it can overcome widely deployed resistance genes that had been effective for many years. To identify loci conferring resistance to Ug99 in wheat, a genome-wide association study was conducted using 232 winter wheat breeding lines from the International Winter Wheat Improvement Program. Breeding lines were genotyped with diversity array technology, simple sequence repeat and sequence-tagged site markers, and phenotyped at the adult plant stage for resistance to stem rust in the stem rust resistance screening nursery at Njoro, Kenya during 2009-2011. A mixed linear model was used for detecting marker-trait associations. Twelve loci associated with Ug99 resistance were identified including markers linked to known genes Sr2 and Lr34. Other markers were located in the chromosome regions where no Sr genes have been previously reported, including one each on chromosomes 1A, 2B, 4A and 7B, two on chromosome 5B and four on chromosome 6B. The same data were used for investigating epistatic interactions between markers with or without main effects. The marker csSr2 linked to Sr2 interacted with wPt4930 on 6BS and wPt729773 in an unknown location. Another marker, csLV34 linked to Lr34, also interacted with wPt4930 on 6BS and wPt4916 on 2BS. The frequent involvement of wPt4916 on 2BS and wPt4930 on 6BS in interactions with other significant loci on the same or different chromosomes suggested complex genetic control for adult plant resistance to Ug99 in winter wheat germplasm.     

Association mapping for pre-harvest sprouting resistance in white winter wheat

Association mapping identified quantitative trait loci (QTLs) and the markers linked to pre-harvest sprouting (PHS) resistance in an elite association mapping panel of white winter wheat comprising 198 genotypes. A total of 1,166 marker loci including DArT and SSR markers representing all 21 chromosomes of wheat were used in the analysis. General and mixed linear models were used to analyze PHS data collected over 4 years. Association analysis identified eight QTLs linked with 13 markers mapped on seven chromosomes. A QTL was detected on each arm of chromosome 2B and one each on chromosome arms 1BS, 2DS, 4AL, 6DL, 7BS and 7DS. All except the QTL on 7BS are located in a location similar to previous reports and, if verified, the QTL on 7BS is likely to be novel. Principal components and the kinship matrix were used to account for the presence of population structure but had only a minor effect on the results. Although, none of the QTLs was highly significant across all environments, a QTL on the long arm of chromosome 4A was detected in three different environments and also using the best linear unbiased predictions over years. Although previous reports have identified this as a major QTL, its effects were minor in our biparental mapping populations. The results of this study highlight the benefits of association mapping and the value of using elite material in association mapping for plant breeding programs.     

Molecular Assortment of Lens Species with Different Adaptations to Drought Conditions Using SSR Markers

The success of drought tolerance breeding programs can be enhanced through molecular assortment of germplasm. This study was designed to characterize molecular diversity within and between Lens species with different adaptations to drought stress conditions using SSR markers. Drought stress was applied at seedling stage to study the effects on morpho-physiological traits under controlled condition, where tolerant cultivars and wilds showed 12.8–27.6% and 9.5–23.2% reduction in seed yield per plant respectively. When juxtaposed to field conditions, the tolerant cultivars (PDL-1 and PDL-2) and wild (ILWL-314 and ILWL-436) accessions showed 10.5–26.5% and 7.5%–15.6% reduction in seed yield per plant, respectively under rain-fed conditions. The reductions in seed yield in the two tolerant cultivars and wilds under severe drought condition were 48–49% and 30.5–45.3% respectively. A set of 258 alleles were identified among 278 genotypes using 35 SSR markers. Genetic diversity and polymorphism information contents varied between 0.321–0.854 and 0.299–0.836, with mean value of 0.682 and 0.643, respectively. All the genotypes were clustered into 11 groups based on SSR markers. Tolerant genotypes were grouped in cluster 6 while sensitive ones were mainly grouped into cluster 7. Wild accessions were separated from cultivars on the basis of both population structure and cluster analysis. Cluster analysis has further grouped the wild accessions on the basis of species and sub-species into 5 clusters. Physiological and morphological characters under drought stress were significantly (P = 0.05) different among microsatellite clusters. These findings suggest that drought adaptation is variable among wild and cultivated genotypes. Also, genotypes from contrasting clusters can be selected for hybridization which could help in evolution of better segregants for improving drought tolerance in lentil.     

Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes

Key message

A stable QTL that may be used in marker-assisted selection in wheat breeding programs was detected for yield, yield components and drought tolerance-related traits in spring wheat association mapping panel.

Abstract

Genome-wide association mapping has become a widespread method of quantitative trait locus (QTL) identification for many crop plants including wheat (Triticum aestivum L.). Its benefit over traditional bi-parental mapping approaches depends on the extent of linkage disequilibrium in the mapping population. The objectives of this study were to determine linkage disequilibrium decay rate and population structure in a spring wheat association mapping panel (n = 285–294) and to identify markers associated with yield and yield components, morphological, phenological, and drought tolerance-related traits. The study was conducted under fully irrigated and rain-fed conditions at Greeley, CO, USA and Melkassa, Ethiopia in 2010 and 2011 (five total environments). Genotypic data were generated using diversity array technology markers. Linkage disequilibrium decay rate extended over a longer genetic distance for the D genome (6.8 cM) than for the A and B genomes (1.7 and 2.0 cM, respectively). Seven subpopulations were identified with population structure analysis. A stable QTL was detected for grain yield on chromosome 2DS both under irrigated and rain-fed conditions. A multi-trait region significant for yield and yield components was found on chromosome 5B. Grain yield QTL on chromosome 1BS co-localized with harvest index QTL. Vegetation indices shared QTL with harvest index on chromosome 1AL and 5A. After validation in relevant genetic backgrounds and environments, QTL detected in this study for yield, yield components and drought tolerance-related traits may be used in marker-assisted selection in wheat breeding programs.     

Meta-QTL analysis of the genetic control of ear emergence in elite European winter wheat germplasm

Variation in ear emergence time is critical for the adaptation of wheat (Triticum aestivum L.) to specific environments. The aim of this study was to identify genes controlling ear emergence time in elite European winter wheat germplasm. Four doubled haploid populations derived from the crosses: Avalon × Cadenza, Savannah × Rialto, Spark × Rialto, and Charger × Badger were selected which represent diversity in European winter wheat breeding programmes. Ear emergence time was recorded as the time from 1st May to heading in replicated field trials in the UK, France and Germany. Genetic maps based on simple sequence repeat (SSR) and Diversity Arrays Technology (DArT) markers were constructed for each population. One hundred and twenty-seven significant QTL were identified in the four populations. These effects were condensed into 19 meta-QTL projected onto a consensus SSR map of wheat. These effects are located on chromosomes 1B (2 meta-QTL), 1D, 2A (2 meta-QTL), 3A, 3B (2 meta-QTL), 4B, 4D, 5A (2 meta-QTL), 5B, 6A, 6B 7A (2 meta-QTL), 7B and 7D. The identification of environmentally robust earliness per se effects will facilitate the fine tuning of ear emergence in predictive wheat breeding programmes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Elucidating the genetic basis of biomass accumulation and radiation use efficiency in spring wheat and its role in yield potential

One of the major challenges for plant scientists is increasing wheat (Triticum aestivum) yield potential (YP). A significant bottleneck for increasing YP is achieving increased biomass through optimization of radiation use efficiency (RUE) along the crop cycle. Exotic material such as landraces and synthetic wheat has been incorporated into breeding programmes in an attempt to alleviate this; however, their contribution to YP is still unclear. To understand the genetic basis of biomass accumulation and RUE, we applied genome‐wide association study (GWAS) to a panel of 150 elite spring wheat genotypes including many landrace and synthetically derived lines. The panel was evaluated for 31 traits over 2 years under optimal growing conditions and genotyped using the 35K wheat breeders array. Marker‐trait association identified 94 SNPs significantly associated with yield, agronomic and phenology‐related traits along with RUE and final biomass (BM_PM) at various growth stages that explained 7%–17% of phenotypic variation. Common SNP markers were identified for grain yield, BM_PM and RUE on chromosomes 5A and 7A. Additionally, landrace and synthetic derivative lines showed higher thousand grain weight (TGW), BM_PM and RUE but lower grain number (GM2) and harvest index (HI). Our work demonstrates the use of exotic material as a valuable resource to increase YP. It also provides markers for use in marker‐assisted breeding to systematically increase BM_PM, RUE and TGW and avoid the TGW/GM2 and BM_PM/HI trade‐off. Thus, achieving greater genetic gains in elite germplasm while also highlighting genomic regions and candidate genes for further study.     

Association mapping for quality traits in soft winter wheat

Improvement of end-use quality in bread wheat (Triticum aestivum L.) depends on a thorough understanding of the genetic basis of important quality traits. The main goal of our study was to investigate the genetic basis of 1,000-kernel weight, protein content, sedimentation volume, test weight, and starch concentration using an association mapping approach. We fingerprinted 207 diverse European elite soft winter wheat lines with 115 SSR markers and evaluated the genotypes in multi-environment trials. The principal coordinate analysis revealed absence of a clear population but presence of a family structure. Therefore, we used linear mixed models and marker-based kinship matrices to correct for family structure. In genome-wide scans, we detected main effect QTL for all five traits. In contrast, epistatic QTL were only observed for sedimentation volume and test weight explaining a small proportion of the genotypic variation. Consequently, our findings suggested that integrating epistasis in marker-assisted breeding will not lead to substantially increased selection gain for quality traits in soft winter wheat.     

Differential response of wild and cultivated wheats to water deficits during grain development: changes in soluble carbohydrates and invertases

Wheat, staple food crop of the world, is sensitive to drought, especially during the grain-filling period. Water soluble carbohydrates (WSCs), stem reserve mobilization and higher invertase activity in the developing grains are important biochemical traits for breeding wheat to enhance tolerance to terminal drought. These traits were studied for three accessions of Triticum dicoccoides(a tetraploid wheat progenitor species) - acc 7054 (EC 171812), acc 7079 (EC 171837) and acc 14004 (G-194-3 M-6 M) selected previously on the basis of grain filling characteristics. Check wheat cultivars- PBW-343 (a popular bread wheat cultivar for irrigated environments) and C-306 (widely adapted variety for rain-fed agriculture) were also included in this set. Analysis of variance revealed significant genotypic differences for the content of water soluble carbohydrates, activity of acid invertase and alkaline invertase. Acc 7079 was found to be a very efficient mobilizer of water soluble carbohydrates (236.43 mg g^-1 peduncle DW) when averaged over irrigated and rain-fed conditions. Acid invertase activity revealed marked genotypic differences between wild and cultivated wheats. Alkaline invertase activity was highest in Acc 7079 when pooled across both the environments. On the whole, acc 7079 qualifies as a suitable donor for enhancing tolerance of bread wheat to terminal drought. The association of physio-biochemical differences observed with grain filling attributes on one hand and molecular markers on the other could be of use in improving wheat for water stress conditions.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

An integrated DArT-SSR linkage map of durum wheat

Genetic mapping in durum wheat (Triticum durum Desf.) is constrained by its large genome and allopolyploid nature. We developed a Diversity Arrays Technology (DArT) platform for durum wheat to enable efficient and cost-effective mapping and molecular breeding applications. Genomic representations from 56 durum accessions were used to assemble a DArT genotyping microarray. Microsatellite (SSR) and DArT markers were mapped on a durum wheat recombinant inbred population (176 lines). The integrated DArT-SSR map included 554 loci (162 SSRs and 392 DArT markers) and spanned 2022 cM (5 cM/marker on average). The DArT markers from durum wheat were positioned in respect to anchor SSRs and hexaploid wheat DArT markers. DArT markers compared favourably to SSRs to evaluate genetic relationships among the durum panel, with 1315 DArT polymorphisms found across the accessions. Combining DArT and SSR platforms provides an efficient and rapid method of generating linkage maps in durum wheat.     

Development and assessment of Diversity Arrays Technology for high-throughput DNA analyses in <Emphasis Type="Italic">Musa</Emphasis>

Diversity Arrays Technology (DArT) is a DNA hybridisation-based molecular marker technique that can detect simultaneously variation at numerous genomic loci without sequence information. This efficiency makes it a potential tool for a quick and powerful assessment of the structure of germplasm collections. This article demonstrates the usefulness of DArT markers for genetic diversity analyses of Musa spp. genotypes. We developed four complexity reduction methods to generate DArT genomic representations and we tested their performance using 48 reference Musa genotypes. For these four complexity reduction methods, DArT markers displayed high polymorphism information content. We selected the two methods which generated the most polymorphic genomic representations (PstI/BstNI 16.8%, PstI/TaqI 16.1%) to analyze a panel of 168 Musa genotypes from two of the most important field collections of Musa in the world: Cirad (Neufchateau, Guadeloupe), and IITA (Ibadan, Nigeria). Since most edible cultivars are derived from two wild species, Musa acuminata (A genome) and Musa balbisiana (B genome), the study is restricted mostly to accessions of these two species and those derived from them. The genomic origin of the markers can help resolving the pedigree of valuable genotypes of unknown origin. A total of 836 markers were identified and used for genotyping. Ten percent of them were specific to the A genome and enabled targeting this genome portion in relatedness analysis among diverse ploidy constitutions. DArT markers revealed genetic relationships among Musa genotype consistent with those provided by the other markers technologies, but at a significantly higher resolution and speed and reduced cost.     

Evaluation of some physiological traits as indicators of drought tolerance in bread wheat genotypes

The objective of this work was to evaluate the ability of some physiological traits to identify drought-tolerant bread wheat genotypes. To this end, twenty bread wheat genotypes were assessed under post anthesis drought stress (rain-fed) and irrigated conditions. The Stress Tolerance Index (STI) was used as a measure of drought tolerance. Relationships between STI and Cell Membrane Stability (CMS), Proline Concentration (PC), Relative Water Content (RWC), Chlorophyll a/b Ratio (Chl a/b), Relative Chlorophyll Content (RCC), Excised Leaf Water Retention (ELWR), and Relative Water Loss (RWL) were determined in order to find out whether these physiological traits could be used as the indicators of drought tolerance. The results showed that ELWR, RWL, and CMS could be considered as reliable indicators in screening wheat genotypes for drought tolerance.     

Genetics of pre-harvest sprouting resistance in a cross of Canadian adapted durum wheat genotypes

Severe losses attributable to pre-harvest sprouting (PHS) have been reported in Canada in recent years. The genetics of PHS resistance have been more extensively studied in hexaploid wheat and generally not using combinations of elite agronomic parents. The objective of our research was to understand the genetic nature of PHS resistance in an elite durum cross. A doubled haploid (DH) population and checks were phenotyped in replicated trials for grain yield and PHS traits over 3 years in western Canada. The response of intact spikes to sprouting conditions, sampled over two development time points, was measured in a rain simulation chamber. The DH population was genotyped with simple sequence repeat and Diversity Arrays Technology markers. Genotypes were a significant source of variation for grain yield and PHS resistance traits in each tested environment. Transgressive segregant DH genotypes were identified for grain yield and PHS resistance measurements. Low or no correlation was detected between grain yield and PHS, while correlation between PHS resistance measurements was moderate. The heritability of PHS resistance was moderate and higher than grain yield. Significant quantitative trait loci with small effect were detected on chromosomes 1A, 1B, 5B, 7A and 7B. Both parents contributed to the PHS resistance. Promising DH genotypes with high and stable grain yield as well as PHS resistance were identified, suggesting that grain yield and PHS can be improved simultaneously in elite genetic materials, and that these DH genotypes will be useful parental material for durum breeding programs.     

Genomewide Association Studies for 50 Agronomic Traits in Peanut Using the ‘Reference Set’ Comprising 300 Genotypes from 48 Countries of the Semi-Arid Tropics of the World

Peanut is an important and nutritious agricultural commodity and a livelihood of many small-holder farmers in the semi-arid tropics (SAT) of world which are facing serious production threats. Integration of genomics tools with on-going genetic improvement approaches is expected to facilitate accelerated development of improved cultivars. Therefore, high-resolution genotyping and multiple season phenotyping data for 50 important agronomic, disease and quality traits were generated on the ‘reference set’ of peanut. This study reports comprehensive analyses of allelic diversity, population structure, linkage disequilibrium (LD) decay and marker-trait association (MTA) in peanut. Distinctness of all the genotypes can be established by using either an unique allele detected by a single SSR or a combination of unique alleles by two or more than two SSR markers. As expected, DArT features (2.0 alleles/locus, 0.125 PIC) showed lower allele frequency and polymorphic information content (PIC) than SSRs (22.21 alleles /locus, 0.715 PIC). Both marker types clearly differentiated the genotypes of diploids from tetraploids. Multi-allelic SSRs identified three sub-groups (K = 3) while the LD simulation trend line based on squared-allele frequency correlations (r²) predicted LD decay of 15–20 cM in peanut genome. Detailed analysis identified a total of 524 highly significant MTAs (pvalue >2.1×10–6) with wide phenotypic variance (PV) range (5.81–90.09%) for 36 traits. These MTAs after validation may be deployed in improving biotic resistance, oil/ seed/ nutritional quality, drought tolerance related traits, and yield/ yield components.     

Association mapping and gene-gene interaction for stem rust resistance in CIMMYT spring wheat germplasm

The recent emergence of wheat stem rust Ug99 and evolution of new races within the lineage threatens global wheat production because they overcome widely deployed stem rust resistance (Sr) genes that had been effective for many years. To identify loci conferring adult plant resistance to races of Ug99 in wheat, we employed an association mapping approach for 276 current spring wheat breeding lines from the International Maize and Wheat Improvement Center (CIMMYT). Breeding lines were genotyped with Diversity Array Technology (DArT) and microsatellite markers. Phenotypic data was collected on these lines for stem rust race Ug99 resistance at the adult plant stage in the stem rust resistance screening nursery in Njoro, Kenya in seasons 2008, 2009 and 2010. Fifteen marker loci were found to be significantly associated with stem rust resistance. Several markers appeared to be linked to known Sr genes, while other significant markers were located in chromosome regions where no Sr genes have been previously reported. Most of these new loci colocalized with QTLs identified recently in different biparental populations. Using the same data and Q?+?K covariate matrices, we investigated the interactions among marker loci using linear regression models to calculate P values for pairwise marker interactions. Resistance marker loci including the Sr2 locus on 3BS and the wPt1859 locus on 7DL had significant interaction effects with other loci in the same chromosome arm and with markers on chromosome 6B. Other resistance marker loci had significant pairwise interactions with markers on different chromosomes. Based on these results, we propose that a complex network of gene-gene interactions is, in part, responsible for resistance to Ug99. Further investigation may provide insight for understanding mechanisms that contribute to this resistance gene network.     

Association mapping for soilborne pathogen resistance in synthetic hexaploid wheat

Soilborne pathogens such as cereal cyst nematode (CCN; Heterodera avenae) and root lesion nematode (Pratylenchus neglectus; PN) cause substantial yield losses in the major cereal-growing regions of the world. Incorporating resistance into wheat cultivars and breeding lines is considered the most cost-effective control measure for reducing nematode populations. To identify loci with molecular markers linked to genes conferring resistance to these pathogens, we employed a genome-wide association approach in which 332 synthetic hexaploid wheat lines previously screened for resistance to CCN and PN were genotyped with 660 Diversity Arrays Technology (DArT) markers. Two sequence-tagged site markers reportedly linked to genes known to confer resistance to CCN were also included in the analysis. Using the mixed linear model corrected for population structure and familial relatedness (Q+K matrices), we were able to confirm previously reported quantitative trait loci (QTL) for resistance to CCN and PN in bi-parental crosses. In addition, we identified other significant markers located in chromosome regions where no CCN and PN resistance genes have been reported. Seventeen DArT marker loci were found to be significantly associated with CCN and twelve to PN resistance. The novel QTL on chromosomes 1D, 4D, 5B, 5D and 7D for resistance to CCN and 4A, 5B and 7B for resistance to PN are suggested to represent new sources of genes which could be deployed in further wheat improvement against these two important root diseases of wheat.     

Genome-wide association studies of agronomic and quality traits in a set of German winter barley (Hordeum vulgare L.) cultivars using Diversity Arrays Technology (DArT)

A set of about 100 winter barley (Hordeum vulgare L.) cultivars, comprising diverse and economically important German barley elite germplasm released during the last six decades, was previously genotypically characterized by single nucleotide polymorphism (SNP) markers using the Illumina GoldenGate BeadArray Technology to detect associations with phenotypic data estimated in three-year field trials at 12 locations. In order to identify further associations and to obtain information on whether the marker type influences the outcome of association genetics studies, the set of winter barley cultivars was re-analyzed using Diversity Arrays Technology (DArT) markers. As with the analysis of the SNPs, only polymorphic markers present at an allele frequency >5 % were included to detect associations in a mixed linear model (MLM) approach using the TASSEL software (P?≤?0.001). The population structure and kinship matrix were estimated on 72 simple sequence repeats (SSRs) covering the whole barley genome. The respective average linkage disequilibrium (LD) analyzed with DArT markers was estimated at 5.73 cM. A total of 52 markers gave significant associations with at least one of the traits estimated which, therefore, may be suitable for marker-assisted breeding. In addition, by comparing the results to those generated using the Illumina GoldenGate BeadArray Technology, it turned out that a different number of associations for respective traits is detected, depending on the marker system. However, as only a few of the respective DArT and Illumina markers are present in a common map, no comprehensive comparison of the detected associations was feasible, but some were probably detected in the same chromosomal regions. Because of the identification of additional marker–trait associations, it may be recommended to use both marker techniques in genome-wide association studies.     

Marker assisted detection of gene (1Dx5) and translocation (1B/1R) in wheat genotypes

Detection of 1Dx5 gene and presence of 1B/1R wheat rye translocation were studied in nineteen elite Indian wheat genotypes using AS-PCR and STS markers, respectively. Fifteen genotypes had 1B/1R translocation whereas ten showed presence of 1Dx5 gene. More than 50 per cent of the genotypes tested were found positive for both 1Dx5 and 1B/1R translocation. The results are in conformity with HMW glutenin SDS-PAGE profile for 1Dx5 and cytological observations for 1B/1R translocation.