Allele-specific Polymerase Chain Reaction Markers Research Articles

Identification of Novel Quantitative Trait Loci Associated with Table Grape Fruit Quality Characteristics in a Segregating Population and Transferability of Existing Quality Markers

Grape (Vitis) production and fruit quality traits such as cluster size, berry shape, and timing of fruit development are key aspects when selecting cultivars for commercial production. Molecular markers for some, but not all, of these traits have been identified using biparental or association mapping populations. Previously identified markers were tested for transferability using a small (24 individual) test panel of commercially available grape cultivars. Markers had little to no ability to differentiate grape phenotypes based on the expected characteristics, except the marker for seedlessness. Using a biparental interspecific cross, 43 quantitative trait loci (QTLs) (previously identified and new genomic regions) associated with berry shape, number, size, cluster weight, cluster length, time to flower, veraison, and full color were detected. Kompetitive allele-specific polymerase chain reaction markers designed on newly identified QTLs were tested for transferability using the same panel. Transferability was low when use types were combined, but they were varied when use types were evaluated separately. A comparison of a 4-Mb region at the end of chromosome 18 revealed structural differences among grape species and use types. Table grape cultivars had the highest similarity in structure for this region (>75%) compared with other grape species and commodity types.

Responses of wheat kernel weight to diverse allelic combinations under projected climate change conditions.

In wheat, kernel weight (KW) is a key determinant of grain yield (GY). However, it is often overlooked when improving wheat productivity under climate warming. Moreover, little is known about the complex effects of genetic and climatic factors on KW. Here, we explored the responses of wheat KW to diverse allelic combinations under projected climate warming conditions. To focus on KW, we selected a subset of 81 out of 209 wheat varieties with similar GY, biomass, and kernel number (KN) and focused on their thousand-kernel weight (TKW). We genotyped them at eight kompetitive allele-specific polymerase chain reaction markers closely associated with TKW. Subsequently, we calibrated and evaluated the process-based model known as Agricultural Production Systems Simulator (APSIM-Wheat) based on a unique dataset including phenotyping, genotyping, climate, soil physicochemistry, and on-farm management information. We then used the calibrated APSIM-Wheat model to estimate TKW under eight allelic combinations (81 wheat varieties), seven sowing dates, and the shared socioeconomic pathways (SSPs) designated SSP2-4.5 and SSP5-8.5, driven by climate projections from five General Circulation Models (GCMs) BCC-CSM2-MR, CanESM5, EC-Earth3-Veg, MIROC-ES2L, and UKESM1-0-LL. The APSIM-Wheat model reliably simulated wheat TKW with a root mean square error (RMSE) of < 3.076g TK-1 and R2 of > 0.575 (P < 0.001). The analysis of variance based on the simulation output showed that allelic combination, climate scenario, and sowing date extremely significantly affected TKW (P < 0.001). The impact of the interaction allelic combination × climate scenario on TKW was also significant (P < 0.05). Meanwhile, the variety parameters and their relative importance in the APSIM-Wheat model accorded with the expression of the allelic combinations. Under the projected climate scenarios, the favorable allelic combinations (TaCKX-D1b + Hap-7A-1 + Hap-T + Hap-6A-G + Hap-6B-1 + H1g + A1b for SSP2-4.5 and SSP5-8.5) mitigated the negative effects of climate change on TKW. The present study demonstrated that optimizing favorable allelic combinations can help achieve high wheat TKW. The findings of this study clarify the responses of wheat KW to diverse allelic combinations under projected climate change conditions. Additionally, the present study provides theoretical and practical reference for marker-assisted selection of high TKW in wheat breeding.

Fine mapping and marker development for the wheat leaf rust resistance gene Lr32.

Wheat leaf rust is caused by the fungal pathogen Puccinia triticina and is one of the wheat diseases of concern globally. Among the known leaf rust resistance genes (Lr) genes, Lr32 is a broadly effective gene derived from the diploid species Aegilops tauschii coss. accession RL5497-1 and has been genetically mapped to chromosome arm 3DS. However, Lr32 resistance has not been utilized in current cultivars in part due to the lack of modern, predictive DNA markers. The goals of this study were to fine map the Lr32 region and develop SNP-based kompetitive allele-specific polymerase chain reaction markers. The genomic analysis was conducted by using doubled haploid and F2-derived mapping populations. For marker development, a 90K wheat chip array, 35K and 820K Axiom R SNPs, A. tauschii pseudomolecules v4.0 and International Wheat Genome Sequencing Consortium ReqSeq v2.1 reference genomes were used. Total 28 kompetitive allele-specific polymerase chain reaction and 2 simple sequence repeat markers were developed. The Lr32 region was fine mapped between kompetitive allele-specific polymerase chain reaction markers Kwh142 and Kwh355 that flanked 34-35 Mb of the diploid and hexaploid reference genomes. Leaf rust resistance mapped as a Mendelian trait that cosegregated with 20 markers, recombination restriction limited the further resolution of the Lr32 region. A total of 10-11 candidate genes associated with disease resistance were identified between the flanking regions on both reference genomes, with the majority belonging to the nucleotide-binding domain and leucine-rich repeat gene family. The validation analysis selected 2 kompetitive allele-specific polymerase chain reaction markers, Kwh147 and Kwh722, for marker-assisted selection. The presence of Lr32 along with other Lr genes such as Lr67 and Lr34 would increase the resistance in future wheat breeding lines and have a high impact on controlling wheat leaf rust.

Identification and validation of genetic locus Rfk1 for wheat fertility restoration in the presence of Aegilops kotschyi cytoplasm.

Major fertility restorer locus for Aegilops kotschyi cytoplasm in wheat, Rfk1,was mapped to chromosome arm 1BS. Most likely candidate gene is TraesCS1B02G197400LC, which is predicted to encode a pectinesterase/pectinesterase inhibitor. Cytoplasmic male sterility (CMS) is widely used for heterosis and hybrid seed production in wheat. Genes related to male fertility restoration in the presence of Aegilops kotschyi cytoplasm have been reported, but the fertility restoration-associated gene loci have not been investigated systematically. In this study, a BC1F1 population derived from a backcross between KTP116A, its maintainer line TP116B, and its restorer line LK783 was employed to map fertility restoration by bulked segregant RNA-Seq (BSR-Seq). A major fertility allele restorer locus for Ae. kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm1BS, and it was contributed by LK783. Morphological and cytological studies showed that male fertility restoration occurred mainly after the late uninucleate stage. Based on simple sequence repeat and single-nucleotide polymorphism genotyping, the gene locus was located between Xnwafu_6 and Xbarc137 on chromosome arm 1BS. To further isolate the specific region, six Kompetitive allele-specific polymerase chain reaction markers derived from BSR-Seq were developed to delimit Rfk1 within physical intervals of 26.0Mb. After searching for differentially expressed genes within the candidate interval in the anthers and sequencing analysis, TraesCS1B02G197400LC was identified as a candidate gene for Rfk1 and it was predicted to encode a pectinesterase/pectinesterase inhibitor. Expression analysis also confirmed that it was specifically expressed in the anthers, and its expression level was higher in fertile lines compared with sterile lines. Thus, TraesCS1B02G197400LC was identified as the most likely candidate gene for Rfk1, thereby providing insights into the fertility restoration mechanism for K-type CMS in wheat.

Genetic Characterization of Leaf and Stripe Rust Resistance in the Brazilian Wheat Cultivar Toropi.

Leaf and stripe rust are major threats to wheat production worldwide. The effective, multiple rust resistances present in the Brazilian cultivar Toropi makes it an excellent choice for a genetic study of rust resistance. Testing of DNA from different seed lots of Toropi with 2,194 polymorphic 90K iSelect single nucleotide polymorphism markers identified significant genetic divergence, with as much as 35% dissimilarity between seed lots. As a result, further work was conducted with a single plant line derived from Toropi variant Toropi-6.4. A double haploid population with 168 lines derived from the cross Toropi-6.4 × Thatcher was phenotyped over multiple years and locations in Canada, New Zealand, and Kenya, with a total of seven field trials undertaken for leaf rust and nine for stripe rust. Genotyping with the 90K iSelect array, simple sequence repeat and Kompetitive allele-specific polymerase chain reaction markers resulted in a genetic map of 3,043 cM, containing 1,208 nonredundant markers. Significant quantitative trait loci (QTL) derived from Toropi-6.4 were identified in multiple environments on chromosomes 1B (QLr.crc-1BL/QYr.crc-1BL), 3B (QLr.crc-3BS), 4B (QYr.crc-4BL), 5A (QLr.crc-5AL and QYr.crc-5AL), and 5D (QLr.crc-5DS). The QTL QLr.crc-1BL/QYr.crc-1BL colocated with the multi-rust resistance locus Lr46/Yr29, while the QTL QLr.crc-5DS located to the Lr78 locus previously found in a wheat backcross population derived from Toropi. Comparisons of QTL combinations showed QLr.crc-1BL to contribute a significantly enhanced leaf rust resistance when combined with QLr.crc-5AL or QLr.crc-5DS, more so than when QLr.crc-5AL and QLr.crc-5DS were combined. A strong additive effect was also seen when the stripe rust resistance QTL QYr.crc-1BL and QYr.crc-5AL were combined.

Functional assessment and SNP-based molecular marker development of two 1Sl-encoded HMW glutenin subunits in Aegilops longissima L.

We investigated the functional properties of two 1Sl-encoded high molecular weight glutenin subunits (HMW-GS), 1Slx2.3* and 1Sly16*, from Aegilops longissima L., and developed single-nucleotide polymorphism (SNP)-based molecular markers. Two HMW-GS near-isogenic lines (NILs), HMW-NIL1 (1Ax1, 1Slx2.3* + 1Sly16*, and 1Dx2 + 1Dy12) and HMW-NIL2 (1Ax1, 1Bx17 + 1By18, and 1Dx2 + 1Dy12), were developed by crossing the ‘Chinese Spring’ (CS)-Ae. longissima 1Sl/1B substitution line (CS-1Sl/1B) with the spring wheat cultivar CB037A, and consecutive selfing combined with selection and identification. Quality analysis of the two NILs showed that the 1Slx2.3* + 1Sly16* subunits conferred better dough rheological properties and breadmaking quality than the 1Bx17 + 1By18 subunits; in particular, they significantly improved dough strength, loaf volume and texture, and final bread score. Allele-specific polymerase chain reaction markers for both genes were developed based on SNP variations and successfully amplified 290-bp and 283-bp specific fragments from the 1Slx2.3* and 1Sly16* genes, respectively. Both SNP-based molecular markers were further validated using 38 wheat varieties, two recombination inbred lines, and four F2 generations and showed high specificity and accuracy. Thus, these markers can be used for molecular marker-assisted selection in the early generations of wheat quality improvement programs.

Utilization of the Genomewide Wheat 55K SNP Array for Genetic Analysis of Stripe Rust Resistance in Common Wheat Line P9936

Breeding for resistance to stripe rust (caused by Puccinia striiformis f. tritici) is essential for reducing losses in yield and quality in wheat. To identify genes for use in breeding, a biparental population of 186 recombinant inbred lines (RILs) from a cross of the Chinese landrace Mingxian 169 and CIMMYT-derived line P9936 was evaluated in field nurseries either artificially or naturally inoculated in two crop seasons. Each of the RILs and parents was genotyped with the wheat 55K single-nucleotide polymorphism (SNP) 'Breeders' array and a genetic linkage map with 8,225 polymorphic SNP markers spanning 3,593.37 centimorgans was constructed. Two major quantitative trait loci (QTL) and two minor QTL were identified. The major QTL QYr.nwafu-3BS.2 and QYr.nwafu-7BL on chromosomes arms 3BS and 7BL were detected in all field locations and explained an average 20.4 and 38.9% of phenotypic variation stripe rust severity, respectively. QYr.nwafu-3BS.2 likely corresponds to the locus Yr30/Sr2 and QYr.nwafu-7BL may be a resistance allele identified previously in CIMMYT germplasm. The other minor QTL had limited individual effects but increased resistance when in combinations with other QTL. Markers linked to QYr.nwafu-7BL were converted to kompetitive allele-specific polymerase chain reaction markers and validated in a panel of wheat accessions. Wheat accessions carrying the same haplotype as P9936 at the identified SNP loci had lower average stripe rust severity than the average severity of all other haplotypes.

Quantitative Trait Loci for Slow-Rusting Resistance to Leaf Rust in Doubled-Haploid Wheat Population CI13227 × Lakin.

CI13227 is a U.S. winter wheat line with adult-plant slow-rusting resistance that has been the subject of several studies on the characteristics and components of slow rusting. Previous genetic studies used different populations and approaches and came to different conclusions about the genetic basis of resistance in CI13227. To clarify the situation, a new doubled-haploid (DH) population of CI13227 × Lakin was produced and a linkage map was constructed using 5,570 single-nucleotide polymorphism (SNP) markers derived from wheat 90K SNP assays and 84 simple sequence repeat markers. Three quantitative trait loci (QTL) were identified for three slow-rusting traits on chromosome arms 2DS, 7AL, and 7BL from CI13227. A fourth QTL mapped on chromosome 3BS was from Lakin. The QTL on 2DS, designated QLr.hwwg-2DS, explained 11.2 to 25.6% of the phenotypic variation. It was found in the same position as a slow-rusting QTL in the CI13227 × Suwon 92 population in a previous study and, thus, verified the 2DS QTL. The QTL on chromosome 7BL explained 8.1 and 19.3% of the phenotypic variation and is likely to be Lr68. The other two QTL showed a minor effect on some of the traits evaluated in a single experiment. Flanking SNP closely linked to all QTL were converted to Kompetitive allele-specific polymerase chain reaction markers that can be used in marker-assisted selection to transfer these QTL into adapted wheat cultivars.

PCR-based assays for validation of single nucleotide polymorphism markers in rice and mungbean

BackgroundSingle nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci (QTL) studies. Marker validation is essential for QTL studies, but the cost and workload are considerable when large numbers of markers need to be verified. Marker systems with low development costs would be most suitable for this task.ResultsWe have tested allele specific polymerase chain reaction (PCR), tetra markers and a genotyping tool based on the single strand specific nuclease CEL-I to verify randomly selected SNP markers identified previously either with a SNP array or by genotyping by sequencing in rice and mungbean, respectively. The genotyping capacity of allele-specific PCR and tetra markers was affected by the sequence context surrounding the SNP; SNPs located in repeated sequences and in GC-rich stretches could not be correctly identified. In contrast, CEL-I digestion of mixed fragments produced from test and reference DNA reliably pinpointed the correct genotypes, yet scoring of the genotypes became complicated when multiple SNPs were present in the PCR fragments. A cost analysis showed that as long the sample number remains small, CEL-I genotyping is more cost-effective than tetra markers.ConclusionsCEL-I genotyping performed better in terms of genotyping accuracy and costs than tetra markers. The method is highly useful for validating SNPs in small to medium size germplasm panels.

Identification of QTLs for Rust Resistance in the Peanut Wild Species Arachis magna and the Development of KASP Markers for Marker-Assisted Selection.

Rust is a major pathogen of the peanut crop. Development and adoption of rust-resistant cultivars is the most cost efficient and effective way to control the spread of the disease and reduce yield losses. Some cultivated peanut germplasm accessions have a degree of resistance, but the secondary gene pool is a source of much stronger resistance alleles. Wild species, however, have undesirable agronomic traits that are a disincentive to their use in breeding. The identification of genomic regions that harbor disease resistance in wild species is the first step in the implementation of marker-assisted selection that can speed the introgression of wild disease resistances and the elimination of linkage drag. In this work, we identify genome regions that control different components of rust resistance in a recombinant inbred line population developed from a cross between two Arachis species, the susceptible most probable B genome ancestor of cultivated peanut, Arachis ipaënsis, and an accession of its closest relative, Arachis magna, which is resistant to rust. Quantitative trait loci for several components of resistance were placed in the same position on linkage group B08. Single-nucleotide polymorphism Kompetitive allele-specific polymerase chain reaction markers for rust resistance region were designed and validated for marker function in both diploid and tetraploid contexts.

Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences

Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers.