iSelect SNP Research Articles

Genetic mapping of resistance to the Ug99 race group of Puccinia graminis f. sp. tritici in a spring wheat landrace CItr 4311.

A gene for Ug99 resistance from wheat landrace CItr 4311 was detected on the long arm of chromosome 2B. Wheat landrace CItr 4311 has seedling resistance to stem rust caused by Puccinia graminis f. sp. tritici race TTKSK and field resistance to the Ug99 race group. Parents, F1 seedlings, 121 doubled haploid (DH) lines, and 124 recombinant inbred lines (RILs) developed from a cross between CItr 4311 and the susceptible line LMPG-6 were evaluated for seedling resistance to race TTKSK. Goodness-of-fit tests indicated that a single dominant gene in CItr 4311 conditioned the TTKSK resistance. The 90K wheat iSelect SNP platform was used to genotype parents and the DH population. The seedling resistance locus was mapped to the chromosome arm 2BL. Parents and the DH population were evaluated for field resistance in Kenya. One major QTL for the field resistance was consistently detected in the same region on 2BL as the seedling resistance. Using KASP assays, five linked SNP markers were used to verify the result in the 124 RIL, 35 wheat accessions, 46 DH lines from the LMPG-6/PI 165194 cross and F1 seedlings, and susceptible bulks derived from crosses between six resistant landraces with LMPG-6. Race specificity, mapping results, and haplotype similarity with lines with Sr9h (Gabo 56, Timstein, and PI 670015), support the hypothesis that the Sr gene in CItr 4311 and the landraces is Sr9h. The KASP assays developed in this study will be useful for pyramiding the TTKSK resistance from CItr 4311 with other Sr genes effective against Ug99.

Development and Genetic Characterization of an Advanced Backcross-Nested Association Mapping (AB-NAM) Population of Wild × Cultivated Barley.

The ability to access alleles from unadapted germplasm collections is a long-standing problem for geneticists and breeders. Here we developed, characterized, and demonstrated the utility of a wild barley advanced backcross-nested association mapping (AB-NAM) population. We developed this population by backcrossing 25 wild barley accessions to the six-rowed malting barley cultivar Rasmusson. The 25 wild barley parents were selected from the 318 accession Wild Barley Diversity Collection (WBDC) to maximize allelic diversity. The resulting 796 BC2F4:6 lines were genotyped with 384 SNP markers, and an additional 4022 SNPs and 263,531 sequence variants were imputed onto the population using 9K iSelect SNP genotypes and exome capture sequence of the parents, respectively. On average, 96% of each wild parent was introgressed into the Rasmusson background, and the population exhibited low population structure. While linkage disequilibrium (LD) decay (r(2) = 0.2) was lowest in the WBDC (0.36 cM), the AB-NAM (9.2 cM) exhibited more rapid LD decay than comparable advanced backcross (28.6 cM) and recombinant inbred line (32.3 cM) populations. Three qualitative traits: glossy spike, glossy sheath, and black hull color were mapped with high resolution to loci corresponding to known barley mutants for these traits. Additionally, a total of 10 QTL were identified for grain protein content. The combination of low LD, negligible population structure, and high diversity in an adapted background make the AB-NAM an important tool for high-resolution gene mapping and discovery of novel allelic variation using wild barley germplasm.

Quantitative Trait Loci Associated with Phenological Development, Low-Temperature Tolerance, Grain Quality, and Agronomic Characters in Wheat (Triticum aestivum L.).

Plants must respond to environmental cues and schedule their development in order to react to periods of abiotic stress and commit fully to growth and reproduction under favorable conditions. This study was initiated to identify SNP markers for characters expressed from the seedling stage to plant maturity in spring and winter wheat (Triticum aestivum L.) genotypes adapted to western Canada. Three doubled haploid populations with the winter cultivar ‘Norstar’ as a common parent were developed and genotyped with a 90K Illumina iSelect SNP assay and a 2,998.9 cM consensus map with 17,541 markers constructed. High heritability’s reflected large differences among the parents and relatively low genotype by environment interactions for all characters considered. Significant QTL were detected for the 15 traits examined. However, different QTL for days to heading in controlled environments and the field provided a strong reminder that growth and development are being orchestrated by environmental cues and caution should be exercised when extrapolating conclusions from different experiments. A QTL on chromosome 6A for minimum final leaf number, which determines the rate of phenological development in the seedling stage, was closely linked to QTL for low-temperature tolerance, grain quality, and agronomic characters expressed up to the time of maturity. This suggests phenological development plays a critical role in programming subsequent outcomes for many traits. Transgressive segregation was observed for the lines in each population and QTL with additive effects were identified suggesting that genes for desirable traits could be stacked using Marker Assisted Selection. QTL were identified for characters that could be transferred between the largely isolated western Canadian spring and winter wheat gene pools demonstrating the opportunities offered by Marker Assisted Selection to act as bridges in the identification and transfer of useful genes among related genetic islands while minimizing the drag created by less desirable genes.

Molecular mapping and marker development for the Triticum dicoccoides-derived stripe rust resistance gene YrSM139-1B in bread wheat cv. Shaanmai 139.

YrSM139-1B maybe a new gene for effective resistance to stripe rust and useful flanking markers for marker-assisted selection were developed. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important foliar disease of wheat. Two dominant stripe rust resistant genes YrSM139-1B and YrSM139-2D were pyramided in bread wheat cultivar Shaanmai 139; one from wild emmer and the other from Thinopyrum intermedium. Three near-isogenic F7:8 line pairs (contrasting RILs), N122-1013R/S, N122-185R/S, and N122-1812R/S, independently derived from different F2 plants and differing at the YrSM139-1B locus were generated from the cross Shaanmai 139 × Hu 901-19 through marker-assisted selection. A large F2:3 population from cross N122-1013R × N122-1013S tested for stripe rust response and subjected to analysis with markers in the 1BS10-0.5 bin region using SSR expressed sequence tags (EST) and site-specific sequence markers developed from the 90 K Illumina iSelect SNP array. Five EST-STS markers and four allele-specific PCR markers were mapped to the YrSM139-1B region. The 30.5 cM genetic map for YrSM139-1B consisted of nine markers, two of which were closer to YrSM139-1B than Xgwm273, which was used in producing the contrasting RIL pairs. Race response data and allelism tests showed that YrSM139-1B is different from Yr10, Yr15, and Yr24/26/CH42.

Mapping resistance to the Ug99 race group of the stem rust pathogen in a spring wheat landrace.

A new gene for Ug99 resistance from wheat landrace PI 374670 was detected on the long arm of chromosome 7A. Wheat landrace PI 374670 has seedling and field resistance to stem rust caused by Puccinia graminis f. sp tritici Eriks. & E. Henn (Pgt) race TTKSK. To elucidate the inheritance of resistance, 216 BC1F2 families, 192 double haploid (DH) lines, and 185 recombinant inbred lines (RILs) were developed by crossing PI 374670 and the susceptible line LMPG-6. The parents and progeny were evaluated for seedling resistance to Pgt races TTKSK, MCCFC, and TPMKC. The DH lines were tested in field stem rust nurseries in Kenya and Ethiopia. The DH lines were genotyped with the 90K wheat iSelect SNP genotyping platform. Goodness-of-fit tests indicated that a single dominant gene in PI 374670 conditioned seedling resistance to the three Pgt races. The seedling resistance locus mapped to the long arm of chromosome 7A and this result was verified in the RIL population screened with the flanking SNP markers using KASP assays. In the same region, a major QTL for field resistance was detected in a 7.7 cM interval and explained 34-54 and 29-36% of the variation in Kenya and Ethiopia, respectively. Results from tests with specific Pgt races and the csIH81 marker showed that the resistance was not due to Sr22. Thus, a new stem rust resistance gene or allele, either closely linked or allelic to Sr15, is responsible for the seedling and field resistance of PI 374670 to Ug99.

Genetic Diversity of Registered Wheat Varieties in Henan Province Based on Pedigree and Single-Nucleotide Polymorphism

To understand the genetic diversity of wheat varieties in Henan Province at whole genome level, we selected 96 regis- tered wheat varieties in the past decade for SNP genotyping using the Illumina 90k iSelect SNP chip. The results showed that 47.39% of the SNPs (38 661 out of 81 587) were polymorphism between varieties. The genome distribution of polymorphic SNPs showed a tendency of B > A > D. The genetic similarity of the 96 varieties ranged from 0.552 to 0.998 with an average value of 0.719. However, genetic similarity of 0.652 to 0.812 was found for 94.3% of the tested varieties, indicating highly similarity for most of the varieties in Henan province. The 96 wheat varieties could be classified as seven groups by UPGMA analyses. Based on pedigree and SNP analyses, very narrow genetic background was observed for the registered varieties in the past decade in Henan province. It is an urgent task for wheat breeders to explore and introduce diversified germplasm resources into wheat breeding program to increase the yield potential and stress tolerance in the future.

Efficiently tracking selection in a multiparental population: the case of earliness in wheat.

Multiparental populations are innovative tools for fine mapping large numbers of loci. Here we explored the application of a wheat Multiparent Advanced Generation Inter-Cross (MAGIC) population for QTL mapping. This population was created by 12 generations of free recombination among 60 founder lines, following modification of the mating system from strict selfing to strict outcrossing using the ms1b nuclear male sterility gene. Available parents and a subset of 380 SSD lines of the resulting MAGIC population were phenotyped for earliness and genotyped with the 9K i-Select SNP array and additional markers in candidate genes controlling heading date. We demonstrated that 12 generations of strict outcrossing rapidly and drastically reduced linkage disequilibrium to very low levels even at short map distances and also greatly reduced the population structure exhibited among the parents. We developed a Bayesian method, based on allelic frequency, to estimate the contribution of each parent in the evolved population. To detect loci under selection and estimate selective pressure, we also developed a new method comparing shifts in allelic frequency between the initial and the evolved populations due to both selection and genetic drift with expectations under drift only. This evolutionary approach allowed us to identify 26 genomic areas under selection. Using association tests between flowering time and polymorphisms, 6 of these genomic areas appeared to carry flowering time QTL, 1 of which corresponds to Ppd-D1, a major gene involved in the photoperiod sensitivity. Frequency shifts at 4 of 6 areas were consistent with earlier flowering of the evolved population relative to the initial population. The use of this new outcrossing wheat population, mixing numerous initial parental lines through multiple generations of panmixia, is discussed in terms of power to detect genes under selection and association mapping. Furthermore we provide new statistical methods for use in future analyses of multiparental populations.

Ultra-dense genetic map of durum wheat × wild emmer wheat developed using the 90K iSelect SNP genotyping assay

Wild emmer wheat (T. turgidum ssp. dicoccoides, genome BBAA) gene pool is an important source for wheat research and improvement. To utilize this resource, we hybridized wild emmer wheat (subpopulation judaicum, accession Zavitan) with durum wheat (T. turgidum ssp. durum, cv. Svevo) and developed an F6 recombinant inbred line (RIL) population. The wheat 90K iSelect SNP genotyping assay was used for genotyping of the RILs, detecting segregation for 16,387 polymorphic markers. The genetic map was constructed based on the genotypic data of 140 RILs and included a total of 14,088 markers grouped into 2,296 genetic loci in 14 linkage groups, corresponding to the 14 chromosomes of tetraploid wheat. The map was 2,110 cM long with an average distance of 0.92 cM between adjacent markers. The B genome was slightly more polymorphic (57 %) for co-dominant SNP markers than the A genome. The map included 1,012 null allele markers, in which only one SNP allele was detected, and the frequency of these markers in the B genome of wild emmer greatly exceeded that of the A genome (69 and 31 %, respectively), which may reflect a greater rate of genomic changes in the B genome. Comparison of our mapped SNP sequences with the barley genome revealed that most of the markers (92.4 %) were syntenic. This ultra-dense SNP-based genetic map with a high level of synteny to barley provides a useful framework for genetic analyses of important traits, positional cloning and marker-assisted selection, as well as for comparative genomics and genome organization studies in wheat and other cereals.

A genome wide association scan for (1,3;1,4)-β-glucan content in the grain of contemporary 2-row Spring and Winter barleys.

Background(1,3;1,4)-β-Glucan is an important component of the cell walls of barley grain as it affects processability during the production of alcoholic beverages and has significant human health benefits when consumed above recommended threshold levels. This leads to diametrically opposed quality requirements for different applications as low levels of (1,3;1,4)-β-glucan are required for brewing and distilling and high levels for positive impacts on human health.ResultsWe quantified grain (1,3;1,4)-β-glucan content in a collection of 399 2-row Spring-type, and 204 2-row Winter-type elite barley cultivars originating mainly from north western Europe. We combined these data with genotypic information derived using a 9 K Illumina iSelect SNP platform and subsequently carried out a Genome Wide Association Scan (GWAS). Statistical analysis accounting for residual genetic structure within the germplasm collection allowed us to identify significant associations between molecular markers and the phenotypic data. By anchoring the regions that contain these associations to the barley genome assembly we catalogued genes underlying the associations. Based on gene annotations and transcript abundance data we identified candidate genes.ConclusionsWe show that a region of the genome on chromosome 2 containing a cluster of CELLULOSE SYNTHASE-LIKE (Csl) genes, including CslF3, CslF4, CslF8, CslF10, CslF12 and CslH, as well as a region on chromosome 1H containing CslF9, are associated with the phenotype in this germplasm. We also observed that several regions identified by GWAS contain glycoside hydrolases that are possibly involved in (1,3;1,4)-β-glucan breakdown, together with other genes that might participate in (1,3;1,4)-β-glucan synthesis, re-modelling or regulation. This analysis provides new opportunities for understanding the genes related to the regulation of (1,3;1,4)-β-glucan content in cereal grains.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-907) contains supplementary material, which is available to authorized users.

Cloning of a rice male sterility gene by a modified MutMap method

A pollen defective male sterile rice mutant, osms55, was isolated from an elite indica cultivar HHZ using EMS (ethyl methanesulfonate) mutagenesis strategy. Genetic analysis showed that osms55 was controlled by a single recessive gene. Genome-wide SNP analysis using the high-throughput Illumina Infinium iSelect SNP (50 K) microarray technology indicated that the genetic makeup of osms55 is the same as wild type (WT) HHZ. Using a modified MutMap method, we successfully identified a mutation in the LOC_Os02g40450 (MER3) gene that is co-segregated with the male sterility phenotype. The mutation is located at the intron splice-recognition site, leading to a 15 nucleotide deletion in the fifth exon. Different from the published MutMap method that aligns the mutant pool DNA sequence with the assembled WT genome, the method used in this study was to align the re-sequencing data of the mutant pool DNA and WT HHZ with the Nipponbare reference genome. The resulting SNPs of mutant/Nipponbare and WT HHZ/Nipponbare were further compared to determine the candidate mutant gene. This modified method does not need an assembled WT genome as reference and thus is more cost-effective and widely applicable.

High-density mapping of a resistance gene to Ug99 from the Iranian landrace PI 626573

Managing wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is imperative for the preservation of global food security. The most effective strategy is pyramiding several resistance genes into adapted wheat cultivars. A search for new resistance sources to Pgt race TTKSK resistance identified a spring wheat landrace, accession PI 626573, as a potentially novel source of resistance. A cross was made between LMPG-6, a susceptible spring wheat line, and PI 626573 and used to develop a recombinant inbred population to map the resistance. Bulk segregant analysis (BSA) of LMPG-6/PI 626573 F2 progeny determined resistance was conferred by a single dominant gene given the provisional designation SrWLR. The BSA identified nine microsatellite (SSR) markers on the long arm of chromosome 2B associated with the resistant phenotype. Fifteen polymorphic SSRs, including the nine identified in the BSA, were used to produce a linkage map of chromosome 2B, positioning SrWLR in an 8.8 cM region between the SSRs GWM47 and WMC332. This region has been reported to contain the wheat stem rust resistance genes Sr9 and SrWeb, the latter conferring resistance to Pgt race TTKSK. The 9,000 marker Illumina Infinium iSelect SNP assay was used to further saturate the SrWLR region. The cosegregating SNP markers IWA6121, IWA6122, IWA7620, IWA8295, and IWA8362 further delimited the SrWLR region distally to a 1.9 cM region. The present study demonstrates the iSelect assay to be an efficient tool to delimit the region of a mapping population and establish syntenic relationships between closely related species.