Kompetitive Allele-specific PCR Markers Research Articles

Development and Validation of KASP Markers for the Identification of Pea seedborne mosaic virus Pathotype P1 Resistance in Pisum sativum.

As pesticides have become heavily relied on for management of insect pests vectoring economically important pathogens of vegetable crops, development of pathogen-resistant germplasm remains a promising alternative to reduce or eliminate costly and timely chemical inputs. Molecular markers can be used to rapidly identify resistant genotypes to aid breeders in advancing germplasm. This study developed two kompetitive allele-specific PCR (KASP) genotyping markers for rapid screening of Pisum sativum genotypes for resistance to Pea seedborne mosaic virus pathotype P1 (PSbMV-P1), the most economically devastating strain worldwide. The KASP markers differentiate two eIF4E PSbMV-P1-resistant allelic variants from susceptible eIF4E variants. A single nucleotide polymorphism (Resistant 1) and a 3-basepair deletion (Resistant 2) present in either of the two resistant alleles were used for marker design. Forty-four P. sativum lines previously characterized for resistance to PSbMV were inoculated with PSbMV-P1 in a greenhouse, observed for visual symptoms, assayed for virus susceptibility by enzyme-linked immunosorbent assay (ELISA), and genotyped by KASP marker analysis. The KASP markers were 100% accurate in characterizing PSbMV-P1-susceptible and PSbMV-P1-resistant genotypes when correlated with the ELISA results. The Resistant 1 marker also correlated with resistance to PSbMV pathotypes P2 and P4 completely, making this marker a new advanced tool for P. sativum breeding programs.

Scale development and utilization of universal PCR-based and high-throughput KASP markers specific for chromosome arms of rye (Secale cereale L.)

BackgroundRye (Secale cereale L., 2n = 2x = 14, RR), a relative of common wheat, is a large gene resource pool for wheat improvement. Accurate and convenient identification of the rye chromatin in wheat background will facilitate the transfer and utilization of elite genes derived from rye in wheat breeding.ResultsIn the present study, five rye cultivars including Imperial, German White, Jingzhouheimai, Baili and Guyuan were sequenced by specific-locus amplified fragment sequencing (SLAF-seq) to develop large-scale rye-specific markers. Based on SLAF-seq and bioinformatics analyses, a total of 404 universal PCR-based and a whole set of Kompetitive allele-specific PCR (KASP) markers specific for the 14 individual rye chromosome arms were developed and validated. Additionally, two KASP markers specific for 1RS and 2RL were successfully applied in the detection of 1RS translocations in a natural population and 2RL chromosome arms in wheat-rye derived progenies that conferred adult resistance to powdery mildew.ConclusionThe 404 PCR-based markers and 14 KASP markers specific for the 14 individual rye chromosome arms developed in this study can enrich the marker densities for gene mapping and accelerate the utilization of rye-derived genes in wheat improvement. Especially, the KASP markers achieved high-throughput and accurate detection of rye chromatin in wheat background, thus can be efficiently used in marker-assisted selection (MAS). Besides, the strategy of rye-specific PCR-based markers converting into KASP markers was high-efficient and low-cost, which will facilitate the tracing of alien genes, and can also be referred for other wheat relatives.

Genome-wide association studies for inflorescence type and remontancy in Hydrangea macrophylla

Inflorescence type and remontancy are two valuable traits in bigleaf hydrangea (Hydrangea macrophylla L.) and both are recessively inherited. Molecular marker-assisted selection (MAS) can greatly reduce the time necessary to breed cultivars with desired traits. In this study, a genome-wide association study (GWAS) using 5803 single-nucleotide polymorphisms (SNPs) was performed using a panel of 82 bigleaf hydrangea cultivars. One SNP locus (Hy_CAPS_Inflo) associated with inflorescence type was identified with general linear model (GLM) and mixed linear model (MLM) methods that explained 65.5% and 36.1% of the phenotypic variations, respectively. Twenty-three SNPs associated with remontancy were detected in GLM whereas no SNP was detected in MLM. The SNP locus (Hy_CAPS_Inflo) was converted to a cleaved amplified polymorphic sequence (CAPS) marker that showed absolute identification accuracy (100%) of inflorescence type in a validation panel consisting of eighteen H. macrophylla cultivars. The SNP was investigated in 341 F1 progenies using genotyping by sequencing (GBS) and co-segregated with inflorescence type (χ2 = 0.12; P = 0.73). The SNP was subsequently used for breeding selection using kompetitive allele specific PCR (KASP) technology. Future directions for the use of genomics and MAS in hydrangea breeding improvement are discussed. The results presented in this study provide insights for further research on understanding genetic mechanisms behind inflorescence type and remontancy in H. macrophylla. The CAPS and KASP markers developed here will be immediately useful for applying MAS to accelerate breeding improvement in hydrangea.

Four Consistent Loci Confer Adult Plant Resistance to Leaf Rust in the Durum Wheat Lines Heller#1 and Dunkler.

The durum wheat lines Heller#1 and Dunkler from the International Maize and Wheat Improvement Center Global Wheat Program showed moderate and stable adult plant resistance to leaf rust under high disease pressure over field environments in northwestern Mexico. Leaf rust phenotyping was performed on two recombinant inbred line (RIL) populations derived from crosses of Heller#1 and Dunkler with the susceptible parent Atred#2, conducted under artificially induced Puccinia triticina epidemics in 2013, 2014, 2015, and 2016. The Atred#2 × Heller#1 and Atred#2 × Dunkler populations were genotyped by single nucleotide polymorphism (SNP) platforms and diversity arrays technology markers, respectively. Four leaf rust resistance quantitative trait loci were detected simultaneously in the two RIL populations: Lr46, QLr.cim-2BC, QLr.cim-5BL, and QLr.cim-6BL based on phenotypic data across all four crop seasons. They explained 11.7 to 46.8%, 7.2 to 26.1%, 8.4 to 24.1%, and 12.4 to 28.5%, respectively, of the phenotypic variation for leaf rust resistance in Atred#2 × Heller#1 and 16.3 to 56.6%, 6.7 to 15.7%, 4.1 to 10.1%, and 5.1 to 20.2% of the variation in the Atred#2 × Dunkler population. Only the resistance allele of QLr.cim-2BC was from the susceptible parent Atred#2, and resistance alleles at other loci came from the resistant parents Heller#1 and Dunkler. The SNP markers closely linked to Lr46 and QLr.cim-2BC were converted to kompetitive allele specific PCR markers for use in marker-assisted selection to improve leaf rust resistance through crosses with Heller#1 and Dunkler sources.

Discovery of two novel and adjacent QTLs on chromosome B02 controlling resistance against bacterial wilt in peanut variety Zhonghua 6

Key messageTwo novel and adjacent genomics and candidate genes for bacterial wilt resistance were identified on chromosome B02 in peanut variety Zhonghua 6 using both traditional QTL mapping and QTL-seq methods.Peanut (Arachis hypogaea) is an important oilseed crop worldwide. Utilization of genetic resistance is the most economic and effective approach to control bacterial wilt, one of the most devastating plant diseases, in peanut production. To accelerate the genetic improvement of bacterial wilt resistance (BWR) in peanut breeding programs, quantitative trait locus (QTL) mapping has been conducted for two resistant varieties. In this context, we deployed linkage mapping as well as sequencing-based mapping approach, QTL-seq, to identify genomic regions and candidate genes for BWR in another highly resistant variety Zhonghua 6. The recombination inbred line population (268 progenies) from the cross Xuhua 13 × Zhonghua 6 was used in BWR evaluation across five environments. QTL mapping using both SSR- and SNP-based genetic maps identified a stable QTL (qBWRB02-1) on chromosome B02 with 37.79–78.86% phenotypic variation explained (PVE) across five environments. The QTL-seq facilitated further dissection of qBWRB02-1 into two adjacent genomic regions, qBWRB02-1-1 (2.81–4.24 Mb) and qBWRB02-1-2 (6.54–8.75 Mb). Mapping of newly developed Kompetitive allele-specific PCR (KASP) markers on the genetic map confirmed their stable expressions across five environments. The effects of qBWRB02-1-1 (49.43–68.86% PVE) were much higher than qBWRB02-1-2 (3.96–6.48% PVE) and other previously reported QTLs. Nineteen putative candidate genes affected by 49 non-synonymous SNPs were identified for qBWRB02-1-1, and ten of them were predicted to code for disease resistance proteins. The major and stable QTL qBWRB02-1-1 and validated KASP markers could be deployed in genomics-assisted breeding (GAB) to develop improved peanut varieties with enhanced BWR.

Use of genetic markers for the detection of off-types for DUS phenotypic traits in the inbreeding crop, barley

Detection of crop off-types is of interest for multiple uses, including the assessment of uniformity for new plant variety applications during distinctness, uniformity and stability (DUS) testing for the awarding of plant breeders’ rights (PBR). Here, we investigate whether genetic markers, in this case Kompetitive Allele-Specific PCR (KASP), can be used for the identification off-types for phenotypes assessed for DUS in the inbreeding cereal crop, barley (Hordeum vulgare). To demonstrate proof of principle, KASP markers diagnostic for phenotypic expression of nine DUS phenotypes, and DNA from two barley varieties (‘Pelican’ and ‘Felicie’) carrying contrasting alleles at each marker were used. We found that for the majority of markers, it was possible to robustly call alleles down to template DNA concentrations of 2 ng, but not ≤ 0.2 ng. When used in mixtures of DNA consisting of ‘Felicie’ DNA spiked with different concentrations of ‘Pelican’ DNA, robust allele calling was possible in DNA mixtures down to 18 ng:2 ng. Collectively, this demonstrates that where diagnostic markers are available, molecular identification of a single off-type for a given DUS trait within a bulk of ten individuals should be possible. We validated this assumption, with all of the diagnostic genetic markers investigated found to robustly detect DUS off-types at a frequency of 10% in DNA extracted from tissue collected from pools of 10 individuals. Ultimately, this work demonstrates that, where diagnostic polymorphisms are known for DUS traits, KASP markers should be able to robustly detect off-types or cross-contamination within DNA samples from a diploid inbred species down to 10%. While just two varieties that contrasted for the eight DUS targeted were investigated in this study, as the markers used are diagnostic for their relevant phenotype (or a proportion of the variation observed for that phenotype), in theory the approach should be valid for any variety studied—although the introduction of novel alleles via spontaneous mutation or more exotic germplasm pools may mean that marker sets would need to be periodically added to or updated. However, we nevertheless demonstrate the principle that, for a subset of DUS traits, molecular markers can now be robustly used as a tool towards determining all three components of the DUS testing process in barley. These results are relevant for the assessment of varietal uniformity by crop breeders, crop testing authorities and germplasm maintenance, as well as highlighting the potential use of bulk samples rather than individual plant samples for assessment of distinctness by molecular methods.

QTL mapping for grain yield-related traits in bread wheat via SNP-based selective genotyping.

We identified four chromosome regions harboring QTL for grain yield-related traits, and breeder-friendly KASP markers were developed and validated for marker-assisted selection. Identification of major stable quantitative trait loci (QTL) for grain yield-related traits is important for yield potential improvement in wheat breeding. In the present study, 266 recombinant inbred lines (RILs) derived from a cross between Zhongmai 871 (ZM871) and its sister line Zhongmai 895 (ZM895) were evaluated for thousand grain weight (TGW), grain length (GL), grain width (GW), and grain number per spike (GNS) in 10 environments and for grain filling rate in six environments. Sixty RILs, with 30 higher and 30 lower TGW, respectively, were genotyped using the wheat 660K SNP array for preliminary QTL mapping. Four genetic regions on chromosomes 1AL, 2BS, 3AL, and 5B were identified to have a significant effect on TGW-related traits. A set of Kompetitive Allele Specific PCR markers were converted from the SNP markers on the above target chromosomes and used to genotype all 266 RILs. The mapping results confirmed the QTL named Qgw.caas-1AL, Qgl.caas-3AL, Qtgw.caas-5B, and Qgl.caas-5BS on the targeted chromosomes, explaining 5.0-20.6%, 5.7-15.7%, 5.5-17.3%, and 12.5-20.5% of the phenotypic variation for GW, GL, TGW, and GL, respectively. A novel major QTL for GNS on chromosome 5BS, explaining 5.2-15.2% of the phenotypic variation, was identified across eight environments. These QTL were further validated using BC1F4 populations derived from backcrosses ZM871/ZM895//ZM871 (121 lines) and ZM871/ZM895//ZM895 (175 lines) and 186 advanced breeding lines. Collectively, selective genotyping is a simple, economic, and effective approach for rapid QTL mapping and can be generally applied to genetic mapping studies for important agronomic traits.

Mapping of QTL for partial resistance to powdery mildew in two Chinese common wheat cultivars

The increasing severity and prevalence of powdery mildew aided by extensive use of semi-dwarf cultivars and high levels of nitrogenous fertilizers are causing significant yield losses in wheat. Resistant cultivars are the most cost-effective and environmentally friendly approach to manage the disease. The objective of this study was to identify quantitative trait loci (QTL) for powdery mildew resistance in a doubled haploid (DH) population from a cross between leading Chinese cultivars, Yangmai 16 and Zhongmai 895. A high-density genetic map comprising of 14,480 non-redundant markers (equal to 148,179 SNPs) in 21 wheat chromosomes was constructed by genotyping the population with the Wheat 660 K SNP array. The DH population was phenotyped for powdery mildew resistance at the adult plant stage in multiple field trials, including four environments in the 2016–2017 cropping season and two environments in 2017–2018. Composite interval mapping detected six stable QTL explaining 3.8–23.6% of the phenotypic variance across environments. QPmyz.caas-5DS, QPmyz.caas-6BL and QPmyz.caas-7BS, are probably new QTL for powdery mildew resistance. One SNP marker closely linked to QPmyz.caas-6BL, the most stable QTL, was converted into a Kompetitive Allele-Specific PCR marker (K_AX-94973433) and validated on 103 commercial wheat cultivars. Significantly lower maximum disease severities of cultivars with the resistance-associated allele than those with the susceptibility-associated allele at QPmyz.caas-6BL in some environments indicated partial effectiveness of the marker. The novel QTL and their closely linked markers identified in the present study should facilitate development of cultivars with improved powdery mildew resistance.

A recombination bin-map identified a major QTL for resistance to Tomato Spotted Wilt Virus in peanut (Arachis hypogaea)

Tomato spotted wilt virus (TSWV) is a devastating disease to peanut growers in the South-eastern region of the United States. Newly released peanut cultivars in recent years are crucial as they have some levels of resistance to TSWV. One mapping population of recombinant inbred line (RIL) used in this study was derived from peanut lines of SunOleic 97R and NC94022. A whole genome re-sequencing approach was used to sequence these two parents and 140 RILs. A recombination bin-based genetic map was constructed, with 5,816 bins and 20 linkage groups covering a total length of 2004 cM. Using this map, we identified three QTLs which were colocalized on chromosome A01. One QTL had the largest effect of 36.51% to the phenotypic variation and encompassed 89.5 Kb genomic region. This genome region had a cluster of genes, which code for chitinases, strictosidine synthase-like, and NBS-LRR proteins. SNPs linked to this QTL were used to develop Kompetitive allele specific PCR (KASP) markers, and the validated KASP markers showed expected segregation of alleles coming from resistant and susceptible parents within the population. Therefore, this bin-map and QTL associated with TSWV resistance made it possible for functional gene mapping, map-based cloning, and marker-assisted breeding. This study identified the highest number of SNP makers and demonstrated recombination bin-based map for QTL identification in peanut. The chitinase gene clusters and NBS-LRR disease resistance genes in this region suggest the possible involvement in peanut resistance to TSWV.

Resistance to Heterodera filipjevi and H. avenae in Winter Wheat is Conferred by Different QTL.

The coexistence of cereal cyst nematode (CCN) species Heterodera avenae and H. filipjevi, often involving multiple pathotypes, is a limiting factor for wheat production in China. Some of the known genes for resistance to CCN are not effective against both nematode species, hence complicating breeding efforts to develop CCN-resistant wheat cultivars. Here, we demonstrate that the CCN resistance in wheat cultivar Madsen to both Heterodera spp. is controlled by different genetic loci, both of which originated from Aegilops ventricosa. A new quantitative trait locus (QTL), QCre-ma7D, was identified and localized in a 3.77-Mb genomic region on chromosome arm 7DL, which confers resistance to H. filipjevi. QCre-ma2A on chromosome arm 2AS corresponds to CCN resistance gene Cre5 and confers resistance to H. avenae. This QTL is a new locus on chromosome arm 7DL and is designated Cre9. Three Kompetitive allele-specific PCR markers (BS00150072, BS00021745, and BS00154302) were developed for molecular marker-assisted selection of Cre9 and locally adapted wheat lines with resistance to both nematode species were developed. QCre-ma2A on chromosome arm 2AS corresponds to CCN resistance gene Cre5 and confers resistance to H. avenae. The identification of different loci underlying resistance to H. filipjevi and H. avenae and the development of adapted resistant entries will facilitate breeding of wheat cultivars that are resistant to these devastating nematodes in China.

Development and validation of a robust KASP marker for zt2 locus in faba bean (Vicia faba)

AbstractFaba bean has the potential to become a key food and feed protein crop in many areas of the world. The presence of tannins in its seed coat has limited the deployment of this crop as feed and food. The expression of either of the two recessive genes, zt1 and zt2, causes a great reduction of tannins from the seed coat and results in a white flower phenotype. Molecular markers linked to these loci are fundamental tools for speeding up the breeding of low‐tannin varieties. The main aim of this study was to develop and validate a robust molecular marker linked to the zt2 locus. We used 176 recombinant inbred lines of the Disco/2 × ILB 938/2 cross at F6 and genotyped those using 257 SNP (single nucleotide polymorphism) markers. An SNP marker associated with zt2 locus was found on faba bean chromosome 3 and was used to develop a high‐throughput low‐cost KASP (kompetitive allele‐specific PCR) marker. The KASP marker can successfully discriminate low‐tannin faba beans carrying zt2 from those carrying zt1 and wild‐type alleles.

Identification of quantitative trait loci for kernel traits in a wheat cultivar Chuannong16

BackgroundKernel length (KL), kernel width (KW) and thousand-kernel weight (TKW) are key agronomic traits in wheat breeding. Chuannong16 (‘CN16’) is a commercial cultivar with significantly longer kernels than the line ‘20828’. To identify and characterize potential alleles from CN16 controlling KL, the previously developed recombinant inbred line (RIL) population derived from the cross ‘20828’ × ‘CN16’ and the genetic map constructed by the Wheat55K SNP array and SSR markers were used to perform quantitative trait locus/loci (QTL) analyses for kernel traits.ResultsA total of 11 putative QTL associated with kernel traits were identified and they were located on chromosomes 1A (2 QTL), 2B (2 QTL), 2D (3 QTL), 3D, 4A, 6A, and 7A, respectively. Among them, three major QTL, QKL.sicau-2D, QKW.sicau-2D and QTKW.sicau-2D, controlling KL, KW and TKW, respectively, were detected in three different environments. Respectively, they explained 10.88–18.85%, 17.21–21.49% and 10.01–23.20% of the phenotypic variance. Further, they were genetically mapped in the same interval on chromosome 2DS. A previously developed kompetitive allele-specific PCR (KASP) marker KASP-AX-94721936 was integrated in the genetic map and QTL re-mapping finally located the three major QTL in a 1- cM region flanked by AX-111096297 and KASP-AX-94721936. Another two co-located QTL intervals for KL and TKW were also identified. A few predicted genes involved in regulation of kernel growth and development were identified in the intervals of these identified QTL. Significant relationships between kernel traits and spikelet number per spike and anthesis date were detected and discussed.ConclusionsThree major and stably expressed QTL associated with KL, KW, and TKW were identified. A KASP marker tightly linked to these three major QTL was integrated. These findings provide information for subsequent fine mapping and cloning the three co-localized major QTL for kernel traits.

Validation of Bread Wheat KASP Markers in Durum Wheat Lines in Kazakhstan

Abstract Development of efficient DNA markers plays an important role in modern breeding projects of many crops, including cultivated hexaploid bread wheat (BW) and tetraploid durum wheat (DW). Findings of genome-wide association studies on major polyploid crops, such as BW, may also help in molecular breeding studies in relative cultivated species with a similar genetic background, including DW. Therefore, the validation of identified quantitative trait loci or marker-trait associations is an important preliminary step in marker-assisted selection (MAS) oriented projects. In this study, thirty-two SNP (single nucleotide polymorphism) markers of six agronomic traits identified in BW, harvested in Kazakhstan, were converted to KASP (Kompetitive Allele-Specific PCR) as-says. Generated 32 KASP assays were used in the analysis of 29 DW accessions from Kazakhstan. Firstly, the group of DW accessions was tested using replicated and randomised one-metre blocks in field conditions of southeast Kazakhstan and evaluated for main agronomic traits. The analysis showed that 14 KASP assays were polymorphic in the scoring of 29 DW accessions. The t-test suggested that the segregation in eight KASP assays was significantly associated with five agronomic traits. The study confirms robustness of KASP assays in MAS of DW breeding projects for the improvement of yield potential.

Identification of the quantitative trait loci controlling spike-related traits in hexaploid wheat (Triticum aestivum L.).

Totally, 48 loci responsible for six spike-related traits were identified in wheat, and a major locus QGl-4A for grain length was mapped and validated for marker-assisted selection in breeding. Wheat yield is determined by the number of spikes, number of grains per spike (GN), and one-thousand kernel weight (TKW), among which GN and TKW are greatly related to the spike development and thus the spike-related traits, including spike length (SL), number of spikelet per spike (SN), grain length (GL) and grain width (GW). To identify the key loci governing the spike-related traits (SL, SN, GN, TKW, GL and GW), we conducted the quantitative trait loci (QTL) analysis combined with wheat 660K SNP chip and Kompetitive allele-specific PCR (KASP) assay, using the F2 and F2:3 populations derived from Luohan6 (LH6) with big spike and grain and Zhengmai366 with small spike and grain, and identified a total of 48 QTLs on 18 chromosomes. Moreover, a major stable QTL for GL on chromosome 4A, designated as QGl-4A, was mapped into a 0.37cM interval between KASP markers Xib4A-10 and Xib4A-12, corresponding to 20Mb physical region in the Chinese Spring genome. This QTL explained 17.30% and 5.12% of the phenotypic variation for GL in the F2 and F2:3 populations. Further association analysis of flanking markers Xib4A-10 and Xib4A-12 in 192 wheat varieties showed that these two markers could be used for marker-assisted selection in breeding. These results provide valuable information for map-based cloning of the target genes involved in the regulation of spike-related traits in common wheat.

Genome-Wide Linkage Mapping Reveals Stripe Rust Resistance in Common Wheat (Triticum aestivum) Xinong1376.

Stripe rust, also known as yellow rust, is a significant threat to wheat yield worldwide. Adult plant resistance (APR) is the preferred way to obtain durable protection. Chinese winter wheat cultivar Xinong1376 has maintained acceptable APR to stripe rust in field environments. To characterize APR in this cultivar, 190 F10 recombinant inbred lines (RILs) developed from Xiaoyan81 × Xinong1376 were evaluated for infection type and disease severity in fields either artificially or naturally inoculated. The population along with parents were genotyped using the Illumina 90K single-nucleotide polymorphism arrays. Six quantitative trait loci (QTL) were detected using the inclusive composite interval mapping method. QYr.nwafu-4AL and QYr.nwafu-6BL.3 conferred stable resistance in all environments, and likely corresponded to a gene-rich region on the long arm of chromosomes 4A and 6B. QYr.nwafu-5AL, QYr.nwafu-5BL, QYr.nwafu-3BL.1, and QYr.nwafu-3BL.2 were detected only in some environments but enhanced the level of resistance conferred by QYr.nwafu-4AL and QYr.nwafu-6BL.3. Kompetitive allele-specific PCR (KASP) markers developed for QYr.nwafu-4AL and QYr.nwafu-6BL.3 were confirmed in a subset of RILs and 133 wheat genotypes. The QTL on 4AL and 6BL with their linked KASP markers would be useful for marker-assisted selection to improve stripe rust resistance in breeding programs.

Thatcher wheat line RL6149 carries Lr64 and a second leaf rust resistance gene on chromosome 1DS.

The leaf rust resistance gene Lr64 in the Thatcher wheat RL6149 was mapped to chromosome 6AL with SNP and KASP markers and a second leaf rust resistance gene was mapped to chromosome 1DS. RL6149, a near-isogenic line of Thatcher wheat, carries leaf rust resistance gene Lr64 on chromosome arm 6AL. The objective of this study was to develop molecular markers that can be easily used to select wheat lines with Lr64. RL6149 was crossed with Thatcher and F2 plants derived from a single F1 plant were advanced to F6 lines by single seed descent. The 100 F7 recombinant inbred lines (RIL) were inoculated with two races of P.triticina that differed widely for virulence in order to identify resistant and susceptible RIL. Thirty RIL that differed for resistance and the parental lines were genotyped with the 90K Infinium iSelect single nucleotide polymorphism (SNP) array to find closely linked markers with Lr64. Seven linked SNPs on chromosome arm 6AL were converted into Kompetitive Allele Specific PCR (KASP) markers that were genotyped on the 100 RIL. A genetic linkage map for the seven KASP markers spanned 19.1cM on chromosome arm 6AL. KASP marker K-IWB59855 was tightly linked to Lr64. A second unexpected gene for leaf rust resistance also segregated in the F7 lines. Four KASP markers that spanned 18.6cM located the gene on chromosome 1DS. The KASP marker K-IWB38437 was tightly linked to the second leaf rust resistance gene.

Rapid Development of KASP Markers for Disease Resistance Genes Using Pooled Whole-Genome Resequencing

Marker-assisted selection (MAS) is becoming increasingly common in commercial potato breeding programmes and can provide information to breeders on potential resistance to diseases and pests early in the breeding cycle when phenotypic screens are impractical. In recent years, a wealth of information on molecular markers for genes involved in resistance to biotic stresses of potato has been revealed. However, markers come in many different forms and ideally a breeding programme would routinely perform annual genotyping on a single unified platform. In this study, we present an approach to consolidate various types of molecular markers to a single SNP-based platform using whole-genome sequencing of carefully constructed pools of DNA using, as examples, molecular markers for the H1 nematode and R2 blight resistance genes. The approach led to the identification of a number of candidate nucleotide polymorphisms which allowed successful design and deployment of kompetitive allele-specific PCR (KASP) assays. We further show how the sequence data generated can be used to aid design of KASP assays for diagnostic SNPs for other traits mined from the literature.

Development and validation of a user-friendly KASP marker for the Sw-5 locus in tomato

Tomato spotted wilt virus (TSWV) is a serious pathogen of tomato. The most efficient strategy for control of TSWV is the breeding of varieties resistant to the virus. In tomatoes, the Sw-5 locus controls resistance to TSWV. Molecular markers based on single nucleotide polymorphisms (SNPs) are widely used for screening in plant breeding. In the present study, we developed primers specific to the Sw-5 locus for use in Kompetitive Allele-Specific PCR (KASP) which is a novel method based on SNPs. These primers were validated in a number of tomato genotypes with different genetic backgrounds, showing that this KASP assay can be used successfully for the selection of TSWV-resistant plants in tomato breeding.

Identification and assessment of two major QTLs for dwarf bunt resistance in winter wheat line 'IDO835'.

Two major dwarf bunt resistance QTLs were mapped to a known Bt9 locus and a novel locus. The associated KASP markers were developed and validated in other two populations. Dwarf bunt (DB), caused by Tilletia controversa J.G. Kühn, and common bunt (CB), caused by T. caries and T. foetida, are two destructive diseases that reduce grain yield and quality in wheat. Breeding for bunt-resistant cultivars is important in many wheat production areas, especially where organic wheat is grown. However, few molecular markers have been used in selection of bunt resistance. In the present study, a doubled haploid (DH) population derived from the bunt-resistant line 'IDO835' and the susceptible cultivar 'Moreland' was evaluated for DB resistance in a field nursery in Logan, Utah, for four growing seasons. The population was genotyped with the Illumina 90K SNP iSelect marker platform. Two major QTLs were consistently identified on chromosomes 6DL (Q.DB.ui-6DL) and 7AL (Q.DB.ui-7AL), explaining up to 53% and 38% of the phenotypic variation, respectively. Comparative study suggested that Q.DB.ui-6DL was located in the same region as the CB resistance gene Bt9, and Q.DB.ui-7AL was located at a novel locus for bunt resistance. Based on Chinese Spring reference sequence and annotations (IWGSC RefSeq v1.1), both resistance QTLs were mapped to disease resistance gene-rich (NBS-LRR and kinase genes) regions. To validate the identified QTL and design user-friendly markers for MAS, five SNPs were converted to Kompetitive Allele-Specific PCR (KASP) markers and used to genotype two validation panels, including a DH population and a diverse winter wheat population from USDA-ARS National Small Grain Collection, as well as a Bt gene investigation panel, consisting of 15 bunt differential lines and 11 resistant lines.

Two QTLs controlling Clubroot resistance identified from Bulked Segregant Sequencing in Pakchoi (Brassica campestris ssp. chinensis Makino)

Clubroot, caused by Plasmodiophora Brassicae, is a serious soil-borne disease in worldwide. In recent years, progression of clubroot is rapid and serious in Shanghai, China. In this study, The inheritance of clubroot resistance (CR) were determined in pakchoi using F2 segregation population that were developed by crossing highly resistant line ‘CR38’ and susceptible line ‘CS22’. Two novel QTLs, qBrCR38-1 and qBrCR38-2, was identified by BSA-seq (Bulked Segregant Sequencing) resistant to P. brassicae physiological race 7. Two significant peak qBrCR38-1 and qBrCR38-2 were observed by three statistical methods between interval of 19.7–20.6 Mb in chromosome A07 and 20.0–20.6 Mb in chromosome A08, respectively. In addition, Polymorphic SNPs identified within target regions were converted to kompetitive allele-specific PCR (KASP) assays. In target regions of qBrCR38-1 and qBrCR38-2, there were twenty SNP sites identified, eleven KASP markers of which are significantly associated to CR (P < 0.05). Seven candidate genes were identified and found to be involved in disease resistance (TIR-NBS-LRR proteins), defense responses of bacterium and fungi and biotic/abiotic stress response in the target regions harboring the two QTLs. Two novel QTLs and candidate genes identified from the present study provide insights into the genetic mechanism of CR in B.rapa, and the associated SNPs can be effectively used for marker-assisted breeding.