Kompetitive Allele Specific PCR Assay Research Articles

Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice

BackgroundThe lack of stable-high yielding and direct-seeded adapted varieties with better germination ability from deeper soil depth and availability of molecular markers are major limitation in achieving the maximum yield potential of rice under water and resource limited conditions. Development of high-throughput and trait-linked markers are of great interest in genomics-assisted breeding. The aim of present study was to develop and validate novel KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving germination and seedling vigor of deep sown direct seeded rice (DSR).ResultsOut of 58 designed KASP assays, four KASP assays did not show any polymorphism in any of the eleven genetic backgrounds considered in the present study. The 54 polymorphic KASP assays were then validated for their robustness and reliability on the F1s plants developed from eight different crosses considered in the present study. The third next validation was carried out on 256 F3:F4 and 713 BC3F2:3 progenies. Finally, the reliability of the KASP assays was accessed on a set of random 50 samples from F3:F4 and 80–100 samples from BC3F2:3 progenies using the 10 random markers. From the 54 polymorphic KASP, based on the false positive rate, false negative rate, KASP utility in different genetic backgrounds and significant differences in the phenotypic values of the positive (desirable) and negative (undesirable) traits, a total of 12 KASP assays have been selected. These 12 KASP include 5 KASP on chromosome 3, 1 on chromosome 4, 3 on chromosome 7 and 3 on chromosome 8. The two SNPs lying in the exon regions of LOC_Os04g34290 and LOC_Os08g32100 led to non-synonymous mutations indicating a possible deleterious effect of the SNP variants on the protein structure.ConclusionThe present research work will provide trait-linked KASP assays, improved breeding material possessing favourable alleles and breeding material in form of expected pre-direct-seeded adapted rice varieties. The marker can be utilized in introgression program during pyramiding of valuable QTLs/genes providing adaptation to rice under DSR. The functional studies of the genes LOC_Os04g34290 and LOC_Os08g32100 possessing two validated SNPs may provide valuable information about these genes.

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

Rapid identification of Meloidogyne hapla by KASP assay

Meloidogyne hapla causes significant economic damage to many cultivated plants including especially vegetables, fruit trees and potatoes. Due to the high number of hosts of this species, it limits the variety of crops to be planted in infested areas. In order to make quarantine measures and management strategies timely against M. hapla, the species need to be identified accurately and quickly. In this study, Kompetitive Allele-Specific PCR (KASP) genotyping assays were developed to identify M. hapla. Two forward primers and one reverse primer were designed as well and performed in the KASP protocols. The designed KASP assays were able to successfully distinguish M. hapla isolates from other root-knot nematodes (RKNs) and plant-parasitic nematodes (PPNs) species. The results showed that even DNA isolated from only one juvenile of M. hapla can be sufficient for molecular identification, and the sensitivity of the assay was very high. The developed KASP assays can be used for accurate and rapid identification of M. hapla.

Automating high-throughput screening for anthracnose resistance in common bean using allele specific PCR

BackgroundCommon beans (Phaseolus vulgaris L.) provide important protein and calories globally. Anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889) is a major disease in common bean and causes significant yield losses in bean production areas. Screening for markers linked to known disease resistance genes provides useful information for plant breeders to develop improved common bean varieties. The Kompetitive Allele Specific PCR (KASP) assay is an affordable genetic screening technique that can be used to accelerate breeding programs, but manual DNA extraction and KASP assay preparation are time-consuming. Several KASP markers have been developed for genes involved in resistance to bean anthracnose, which can reduce yield by up to 100%, but their usefulness is hindered by the labor required to screen a significant number of bean lines. Our research objective was to develop publicly available protocols for DNA extraction and KASP assaying using a liquid handling robot (LHR) which would facilitate high-throughput genetic screening with less active human time required. Anthracnose resistance markers were used to compare manual and automated results.ResultsThe 12 bean anthracnose differential cultivars were screened for four anthracnose KASP markers linked to the resistance genes Co-1, Co-3 and Co-42 both by hand and with the use of an LHR. A protocol was written for DNA extraction and KASP assay thermocycling to implement the LHR. The LHR protocol reduced the active human screening time of 24 samples from 3h44 to 1h23. KASP calls were consistent across replicates but not always accurate for their known linked resistance genes, suggesting more specific markers still need to be developed. Using an LHR, information from KASP assays can be accumulated with little active human time.ConclusionResults suggest that LHRs can be used to expedite time-consuming and tedious lab work such as DNA extraction or PCR plate filling. Notably, LHRs can be used to prepare KASP assays for large sample sizes, facilitating higher throughput use of genetic marker screening tools.

Implementation of upfront DPYD genotyping with a low-cost and high-throughput assay to guide fluoropyrimidine treatment in cancer patients.

Genetic variants in the dihydropyrimidine dehydrogenase (DPYD) gene are associated with reduced dihydropyrimidine dehydrogenase enzyme activity and can cause severe fluoropyrimidine-related toxicity. We assessed the frequency of the four most common and well-established DPYD variants associated with fluoropyrimidine toxicity and implemented a relatively low-cost and high-throughput genotyping assay for their detection. This study includes 457 patients that were genotyped for the DPYD c.1129-5923C>G, c.1679T>G, c.1905 + 1G>A and c.2846A>T variants, either by Sanger sequencing or kompetitive allele specific PCR (KASP) technology. Of these, 172 patients presented toxicity during treatment with fluoropyrimidines (post-treatment group), and 285 were tested before treatment (pretreatment group). Heterozygous DPYD variants were identified in 7.4% of the entire series of 457 patients, being the c.2846A>T the most frequent variant. In the post-treatment group, 15.7% of the patients presented DPYD variants, whereas only 2.5% of the patients in the pretreatment group presented a variant. The KASP assays designed in this study presented 100% genotype concordance with the results obtained by Sanger sequencing. The combined assessment of the four DPYD variants in our population increases the identification of patients at high risk for developing fluoropyrimidine toxicity, supporting the upfront routine implementation of DPYD variant genotyping. Furthermore, the KASP genotyping assay described in this study presents a rapid turnaround time and relatively low cost, making upfront DPYD screening feasible in clinical practice.

Quizalofop resistance in weedy rice (Oryza sativa L.) is mainly conferred by an Ile1781Leu mutation

Weedy rice (Oryza sativa L.) is an economically important weed species in rice (Oryza sativa L.) cropping systems. Two weedy rice samples (acc7 and acc8) suspected to be resistant to quizalofop-ethyl (quizalofop) were collected in Arkansas. In this research, susceptibility to quizalofop and resistance mechanisms have been explored. Dose-response assays displayed a resistance index of 42- and 58-fold for the acc7 and acc8, respectively. Experiments with metabolism inhibitors demonstrated that NBD-Cl (4-chloro-7-nitrobenzofurazan) increased quizalofop efficacy slightly in acc8, whereas malathion did not improve effectiveness in resistant samples. Sequencing of the ACCase gene displayed an Ile1781Leu substitution in the resistant samples, like the mutation present in Provisia™ rice. In addition, an allele-specific PCR was developed to genotype the Ile1781Leu mutation. The gene copy number of ACCase showed similar values among samples. In the resistant plants, a KASP (Kompetitive Allele Specific PCR) assay to detect the ALSS653D (acetolactate synthase) and HIS1 (HPPD Inhibitor Sensitive 1) traits revealed that 37.5% of plants carried the ALSS653D trait, whereas 25% showed the HIS1 allele. In summary, a target-site mutation is the main resistance mechanism to quizalofop in weedy rice. Results also suggest the presence of herbicide metabolism (a non-target site resistance mechanism) mediated by glutathione-S-transferases (GSTs) in one resistant sample.

Molecular diagnosis and differentiation of Meloidogyne arenaria, Meloidogyne javanica and Meloidogyne incognita using SNP-based KASP assays

Root-knot nematodes (RKNs) are one of the most economically important plant-parasitic nematodes in many crops. Meloidogyne arenaria, M. incognita and M. javanica are the most common species attacking tomato, pepper, eggplant, cucumber, melon, watermelon and zucchini, which have economic importance in vegetable growing areas. Thus, a rapid and accurate identification system is required for RKNs. In this study, Kompetitive Allele-Specific PCR (KASP) genotyping assays were developed to identify three common RKNs. The novel technique developed in this study utilized only two primers: one allele-specific forward primer and a reverse common primer. Meloidogyne arenaria, M. incognita and M. javanica were separated easily from each other and other RKN species. This is the first report on three species identified by the KASP assay. The results showed that the KASP assays are suitable for the high-throughput identification of these species. The developed system can be used to identify three RKN species in research, routine and quarantine laboratories.

Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis.

The currently used conventional susceptibility testing for drug-resistant Mycobacterium tuberculosis (M.TB) is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP). A total of 300 clinical samples were collected, and DNA extraction was performed using the "isoChip®" Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples. Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1×101 copies/µL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored rpoB gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had katG gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had embB gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method. The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in M. tuberculosis. It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.

Screening Indels from the whole genome to identify the candidates and their association with economic traits in several goat breeds

In the present study, the re-sequencing of our previous whole-genome sequencing (WGS) for selected individuals of Dazu-black goat (DBG) and Inner-Mongolia Cashmere goat (IMCG) breeds was used to detect and compare the differentiation in Indels depending on the reference genome of goat. Then, three selected candidate Indels rs668795676, rs657996810, and rs669452874 of the three genes SUFU, SYCP2Land GLIPR1L1, respectively, have been chosen, based on the results of prior GWAS across the genome, and examined for their association with body weight and dimensions (body height, body length, heart girth, chest width, cannon circumference, and chest depth) by kompetitive allele specific PCR assay for 342 goats from the three studied goat breeds (DBG, n = 203; ♂99, ♀104), IMCG (n = 65; 15♂, 50♀), and Hechuan white goat (HWG, n = 74; 34♂, 40♀) breeds. The analysis of 192.747Gb WGS revealed an average 334,151 Indels in the whole genome of DBG and IMCG breeds. Chromosome 1 had a maximum number of mutations (Indels) of 58,497 and 55,527 for IMCG and DBG, respectively, while chromosome 25 had the least number of mutations of 15,680 and 16,103 for IMCG and DBG, respectively. The majority of Indels were either Ins or Del of short fragments of 1-5bp, which covered 79.06 and 71.78% of the total number of Indels mutations in IMCG and DBG, respectively. Comparing the differences of Indels between the studied goat breeds revealed 100 and 110 unique Indels for IMCG and DBG, respectively. The Indels loci in the intron region were unique for both studied goat breeds which were related to 30 and 38 candidate genes in IMCG and DBG, respectively, including SUFU, SYCP2L, and GLIPR1L1 genes. Concerning rs669452874 locus, body height and body length of Del/Del genotype in DBG were significantly higher (P < 0.05) than that of Ins/Del genotype, while body height and body length of Del/Del genotype in IMCG were significantly higher (P < 0.01) than those of Ins/Ins genotype, whereas body height and body length and heart girth of Del/Del genotype in HWG were significantly higher (P < 0.01) than those of the Ins/Del and Ins/Ins genotypes. Thus, Del/Del genotype of rs669452874 locus can be used as a candidate molecular marker related to the body dimensions in the studied goat breeds.

Genome-wide association analysis and validation with KASP markers for nut and shell traits in almond (Prunus dulcis [Mill.] D.A.Webb)

Almond shell hardness is an economically important quality trait that protects the integrity of the kernel. It is directly related with shelling or crack-out percent. In the U.S. and Australian markets, soft to semi-hard shells varieties have been used in the industry, while in the European market, hard shells are dominantly sold and distributed. The origin of a hard shell is from wild almond species. Soft shells in the USA and Australia have been selected for a higher crack-out percent or larger kernel and ease of processing. Here, we examined breeding populations totaling 264 trees that were genotyped with genotyping-by-sequencing (GBS) where shell hardness was measured with a texture analyzer, and shell weight, in-shell weights and shell thickness were measured and crack-out percent was calculated. In a genome-wide association, seven genetic loci were found to be associated with shell hardness, crack-out percent, shell thickness and shell weight. Of these seven, one was shared between multiple traits. Kompetitive allele-specific PCR (KASP) assays were developed and tested in unrelated breeding lines from Australia, Spain, France and Italy. One KASP assay was found to be highly predictive of shell hardness in this diverse panel and can be applied for use in marker-assisted breeding.

Development of SNP molecular markers associated with resistance to reniform nematode in soybean using KASP genotyping

Reniform nematode (Rotylenchulus reniformis, Linford and Oliveira) is a sedentary, semi-endoparasite that infects a wide range of plant hosts and is one of the top three nematode pathogens affecting soybean in the southeastern United States. Previous studies have linked resistance to reniform nematode in soybean to two quantitative trait loci on chromosomes 11 and 18. A Kompetitive Allele-Specific PCR (KASP) assay was designed using SNP markers within these two regions to distinguish reniform nematode-resistant soybean based on genotype. A collection of 44 soybean plant introductions with resistant phenotype to reniform nematode and 40 susceptible soybean lines were genotyped at the two target loci to validate the KASP assay design. Of the 44 observed resistant lines, two carried the susceptible genotype; PI 438489B at the locus on chromosome 18 and PI 495017C on chromosome 11. Of the 40 observed susceptible soybean lines, only 25 had the expected susceptible genotype at the loci on chromosome 18 and 13 on chromosome 11. Our KASP assay was 68% accurate in predicting the phenotype of 84 soybean accessions based on their genotype at the SNP marker on chromosome 18 and 83% accurate at chromosome 11. These results indicate a moderate correlation of soybean SNP markers GlyREN18_46 and GlyREN11_190 with reniform nematode resistance. Further research is required to improve the accuracy of KASP assays to predict soybean response to reniform nematode, particularly host susceptibility.

Ppe.CR.1 DNA test for predicting chilling requirement in peach

Chilling requirement (CR) is an important agronomic trait controlling the floral bud break for proper flowering in peach. Even though it has been widely researched and several peach CR quantitative trait loci (QTLs) have been identified, no diagnostic DNA tests validated in the U.S. peach breeding germplasm are available for this trait. Breeders and growers need a simple DNA test to predict the CR of peach cultivars for their particular environment. Therefore, we developed a quick and reliable Kompetitive Allele Specific PCR (KASP) DNA test using haplotype information from 9K IPSC genotype data of the U.S. peach germplasm integrating four CR-associated SNP markers from the previously reported CR QTL region on linkage group 1. Four KASP assays (Ppe.CR.1-1 to -4) were developed and validated on 77 peach cultivars, and nine accessions from two F2 populations, with 96 and 74% accuracy in determining expected CR genotype (compared to SNP array) and predicting phenotype, respectively. Furthermore, the Ppe.CR.1 showed 80% accuracy in predicting the precise CR phenotype in the Clemson University peach breeding material. Only one Ppe.CR.1 KASP assay (Ppe.CR.1-1) is needed to distinguish between haplotypes with CR lower and higher than 800 chilling hours, and two Ppe.CR.1 assays (Pp.CR.1-1 and -4), are capable of distinguishing low, moderate, and high CR alleles. Coupled with the crude DNA extraction, the Ppe.CR.1 DNA test provides a low-cost option for breeders and growers to predict CR in peach material with more than 70% accuracy.

Genetic effects of Rht-B1b and Rht-D1b dwarfing alleles on plant height and agronomic traits of bread wheat (Triticum aestivum L.) breeding lines

Plant height is an important agronomic trait related to plant architecture and grain yield in wheat. We investigated the effects of the gibberellic acid (GA)-insensitive Rht-B1b and Rht-D1b semi-dwarfing alleles in a set of 599 F4:5 spring wheat breeding lines grown in rainfed and supplemental irrigation conditions at Setif semi-arid high plateaus region (Algeria). Genotyping of diagnostic markers was performed using Kompetitive Allele Specific PCR assays. The Rht-B1b mutant allele was present in 66% of the evaluated lines, while the Rht-D1b allele was found only in 8% of the lines. Relative to wild-type, Rht-B1b or Rht-D1b alone reduced plant height by 16.0 and 16.3% in rainfed, and by 20.7 and 21.8% in irrigated environments, respectively. The two dwarfing alleles when combined decreased height by 39.3 and 56.6% in rainfed and irrigated conditions, respectively. The semi-dwarf breeding lines generally outperformed the tall genotypes for yield and yield components including the number of grains per spike and harvest index irrespective of environment. Variation in heading date under the influence of dwarfing alleles has not been found. In addition, above-ground biomass was reduced in most of the short statured lines, compared to their tall counter parts across environments. We also demonstrate clear yield and yield components penalty for Rht-B1b + Rht-D1b double dwarf backgrounds.

Modifications of Kompetitive Allele-Specific PCR (KASP) Genotyping for Detection of Rare Alleles.

KASP is commonly used to genotype bi-allelic SNPs and In/Dels, and the standard protocol works well when both alleles are nearly equally prevalent in the DNA template. To detect rare alleles in bulked samples or to distinguish more than three genotypes, such as tri-allelic loci or mutations across orthologous genes in polyploids, adjustments to the protocol and/or data analysis are required. In this chapter, we present modified protocols for these non-traditional applications, including reaction conditions that enhance the fluorophore signal from rare alleles, resulting in increased KASP assay sensitivity. We also describe alternative KASP data analysis approaches that increase statistical certainty of genotyping calls. Furthermore, this increased assay sensitivity enables high-throughput genotyping using KASP, as samples can be pooled and tested in a single reaction. For example, rare alleles can be detected in mixed seed pools when present in ratios as low as 1 in 200. The assay modifications presented here expand the options available for complex genotyping, and retain KASP's advantages of being cheap, fast, and accurate.

VALIDATION OF MARKER-TRAIT ASSOCIATIONS IN SIX-ROWED BARLEY LINES BRED IN KAZAKHSTAN

Barley is an important cereal crop in Kazakhstan, mostly used for animal feeding, malting, and the food industry. The success of barley production is dependent on the genetic resources (cultivars) available, and local breeding programs that focus on the development of competitive cultivars. One way to develop new high-yielding cultivars and improve the efficiency of breeding programs is the application of modern molecular genetic and genomic tools. One such technology is genome-wide association study (GWAS), which has been successfully applied to identify the quantitative trait loci (QTL) associated with the valuable traits. The identified single nucleotide polymorphisms (SNPs) based on GWAS can be converted to flexible and cost-effective KASP (Kompetitive Allele Specific PCR) assays and validated for use in future marker-assisted breeding projects. The purpose of this study was to genotype eleven promising six-rowed barley lines using twenty-one KASP assays associated with agronomic traits reported in previous GWAS. The genotyping results suggested that only seven out of twenty-one KASP markers were polymorphic in this group of barley accessions. The t-test output suggested that six out of nine agronomic traits were significantly associated with seven KASPs. Notably, two assays (ipbb_hv_6, ipbb_hv_108) affected both vegetation period (VP) and yield per m2 (YM2) in conditions of Northern Kazakhstan, where barley is growing in more than 80% of total crop sowing areas of the country. The application of these highly informative KASP markers can help enhance the efficiency of local breeding projects in barley.

Validation of KASP assays associated with barley adaptation and productivity traits

Barley (Hordeum vulgare L.) is an important cereal crop traditionally used in animal feed, malting, and food production. In Kazakhstan, barley is the second most cultivated cereal grain. However, despite the long history of barley cultivation in Kazakhstan, traditional breeding methods prevail here. The introduction of marker-assisted selection (MAS) in the breeding process may improve the adaptation and productivity of local cultivars, as well as help in the development of new ones. For that, validation of 23 kompetitive allele-specific PCR (KASP) assays for adaptation and productivity traits developed using previous GWAS results was performed. The collection of 35 two-rowed barley promising lines was grown at the Kazakh research institute of agriculture and plant growing (KRIAPG, Almaty region, Kazakhstan) in 2021 and studied for 5 adaptation-related (heading time, heading-maturity time, plant height, peduncle length, and spike length) and 5 productivity-related (number of kernels per spike, the weight of kernels per spike, weight of kernels per plant, thousand kernels weight, and yield per m2) traits. The same collection was genotyped using 23 KASP assays. As a result, 21 KASPs demonstrated a good level of polymorphism (MAF &gt; 0.10 and I &gt; 0.36) in the studied barley collection. Six KASP assays confirmed their associations with adaptation and productivity traits (P &lt; 0.05); nine KASPs were associated with other agronomic traits (P &lt; 0.05). Nine KASP assays were identified for adaptation traits, one assay was detected for productivity traits and six KASPs were found to be associated with both types of traits. Four KASP assays (ipbb_hv_9, ipbb_hv_101, ipbb_hv_109, and ipbb_hv_110) confirmed significant (P &lt; 0.05) effect of shorter heading-maturity time on higher productivity traits under stress conditions of south-east Kazakhstan. Thus, in this study fifteen out of the studied twenty-one KASP assays were validated for their associations with adaptation and productivity traits and potentially can be included in the barley breeding projects.

Reliable DNA Markers for a Previously Unidentified, Yet Broadly Deployed Hessian Fly Resistance Gene on Chromosome 6B in Pacific Northwest Spring Wheat Varieties.

Hessian fly [Mayetiola destructor (Say)] is a major pest of wheat (Triticum aestivum L.) throughout the United States and in several other countries. A highly effective and economically feasible way to control Hessian fly is with resistant cultivars. To date, over 37 Hessian fly resistance genes have been discovered and their approximate locations mapped. Resistance breeding is still limited, though, by the genes’ effectiveness against predominant Hessian fly biotypes in a given production area, genetic markers that are developed for low-throughput marker systems, poorly adapted donor germplasm, and/or the inadequacy of closely linked DNA markers to track effective resistance genes in diverse genetic backgrounds. The purposes of this study were to determine the location of the Hessian fly resistance gene in the cultivar “Kelse” (PI 653842) and to develop and validate Kompetitive Allele Specific PCR (KASP) markers for the resistance locus. A mapping population was genotyped and screened for Hessian fly resistance. The resulting linkage map created from 2,089 Single Nucleotide Polymorphism SNP markers placed the resistance locus on the chromosome 6B short arm, near where H34 has been reported. Three flanking SNPs near the resistance locus were converted to KASP assays which were then validated by fine-mapping and testing a large panel of breeding lines from hard and soft wheat germplasm adapted to the Pacific Northwest. The KASP markers presented here are tightly linked to the resistance locus and can be used for marker-assisted selection by breeders working on Hessian fly resistance and allow confirmation of this Hessian fly resistance gene in diverse germplasm.

Two fingerprinting sets for Humulus lupulus based on KASP and microsatellite markers.

Verification of clonal identity of hop (Humulus lupulus L.) cultivars within breeding programs and germplasm collections is vital to conserving genetic resources. Accurate and economic DNA-based tools are needed in dioecious hop to confirm identity and parentage, neither of which can be reliably determined from morphological observations. In this study, we developed two fingerprinting sets for hop: a 9-SSR fingerprinting set containing high-core repeats that can be run in a single PCR reaction and a kompetitive allele specific PCR (KASP) assay of 25 single nucleotide polymorphisms (SNPs). The SSR set contains a sex-linked primer pair, HI-AGA7, that was used to genotype 629 hop accessions from the US Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR), the USDA Forage Seed and Cereal Research (FSCR), and the University of Nebraska-Lincoln (UNL) collections. The SSR set identified unique genotypes except for 89 sets of synonymous samples. These synonyms included: cultivars with different designations, the same cultivars from different sources, heat-treated clones, and clonal variants. Population structure analysis clustered accessions into wild North American (WNA) and cultivated groups. Diversity was slightly higher in the cultivated samples due to larger sample size. Parentage and sib-ship analyses were used to identify true-to-type cultivars. The HI-AGA7 marker generated two male- and nine female-specific alleles among the cultivated and WNA samples. The SSR and KASP fingerprinting sets were compared in 190 samples consisting of cultivated and WNA accession for their ability to confirm identity and assess diversity and population structure. The SSR fingerprinting set distinguished cultivars, selections and WNA accessions while the KASP assays were unable to distinguish the WNA samples and had lower diversity estimates than the SSR set. Both fingerprinting sets are valuable tools for identity confirmation and parentage analysis in hop for different purposes. The 9-SSR assay is cost efficient when genotyping a small number of wild and cultivated hop samples (<96) while the KASP assay is easy to interpret and cost efficient for genotyping a large number of cultivated samples (multiples of 96).

Molecular sexual determinants in Pistacia genus by KASP assay.

Pistacia is a genus of dioecious plant species whose trees can take 4-5years to reach the economically valuable fruit-bearing stage. The fruits have great importance as raw material in the food, healthcare, and baking industries. For that reason, the identification of individual plants in the early juvenile period for the pollination and positioning of trees is crucial for growers. The objective of this study is to develop markers for each Pistacia species that can help in screening the sex of plant seedlings before they reach the reproductive stage, without waiting for morphological characteristics to appear. Within this context, by using the power of the kompetitive allele-specific PCR (KASP) assay technology as a marker screening system, we successfully discriminated seven out of eight Pistacia species: P. atlantica, P. integerrima, P. khinjuk, P. mutica, P. terebinthus, P. vera, and P. lentiscus. We used a high-throughput DNA sequence read archive (SRA) to assemble a reference genome that was employed in our studies as a de novo bioinformatics method. Four genomic regions from SRA and three single-nucleotide polymorphism (SNP) positions from Kafkas et al. BMC Genomics 16:98, 2015) were selected and sequenced with collected plant material from predominantly the Antepfıstıgı Research Institute Collection Garden, and eight species were aligned intraspecifically for SNP mining. In total, 12 SNP markers were converted to KASP markers, and 5 of them (SNP-PIS-133396, SNP-PIS-167992, P-ATL-91951-565, P-INT-91951-256, P-KHI-91951-115) showed clear allelic discrimination between male and female plants. SNP-PIS-167992 and P-ATL-91951-565 were identified as the best marker assays because they showed allelic frequency differences for all individuals and for both homozygous and heterozygous characters. These markers could be the most comprehensive ones for the whole genus because they showed discriminative power for several species. This study is the first one to use the KASP assay for sex discrimination in Pistacia species, and it can be regarded as a precursor study for sex discrimination by KASP for plants in general.

Development and validation of a novel core set of KASP markers for the traits improving grain yield and adaptability of rice under direct-seeded cultivation conditions

The development and utilization of molecular-markers play an important role in genomics-assisted breeding during pyramiding of valuable genes. The aim of present study was to develop and validate a novel core-set of KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving rice grain yield and adaptability under direct-seeded cultivation conditions. The 110 phenotypically validated KASP assays out of 171 designed KASP, include assays for biotic-resistance genes, anaerobic germination, root-traits, grain yield, lodging resistance and early-uniform emergence. The KASP assays were validated for their robustness and reliability at five different levels using diverse germplasm, segregating and advanced population, comparison with SSR markers and on F1s. The present research work will provide (i) breeding material in form of anticipated pre-direct-seeded adapted rice varieties (ii) single improved breeding line with many useful genes and (iii) KASP assay information for the useful QTL/genes providing grain yield and adaptability to rice under direct-seeded cultivation conditions.