Search Results (42)

Rye (Secale cereale), a cereal crop with high cold tolerance, serves as an ideal model for investigating plant cold adaptation mechanisms. Despite recent progress in identifying numerous genes and metabolic changes associated with cold tolerance, the detailed regulatory networks and coordinated interactions between metabolic pathways under low-temperature stress in rye remain unclear. In this study, we focused on the winter rye variety “Winter” and systematically explored its metabolic regulatory responses to cold stress through a combination of low-temperature treatments, phenotypic observations, antioxidant enzyme activity assays, and transcriptomic analysis. Four rye varieties (“Winter”, HZHM3, HZHM8, and “Victory”) were compared for cold tolerance, with the results indicating that “Winter” and HZHM3 exhibit superior cold tolerance. Physiological analysis revealed that after 12 h of exposure to −4 °C, the activities of catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) in “Winter” were significantly upregulated, displaying an initial increase followed by a decline over time. Transcriptomic sequencing identified 1643 differentially expressed genes (DEGs), and GO, KEGG, and GSEA enrichment analyses highlighted the critical roles of carbohydrate metabolism (ko00630) and amino acid metabolism (ko00250) pathways in the cold stress response. These pathways are interconnected through key metabolic intermediates such as L-glutamate, collectively regulating cold adaptation. Furthermore, based on the transcriptomic data, we identified and developed molecular markers associated with cold tolerance, detecting 10,846 EST-SSR and 250,116 EST-SNP markers. We successfully developed 13 EST-SSR primer pairs applicable to rye and 7 KASP markers. Notably, the KASP-665 marker effectively distinguishes between winter and spring rye, providing a reliable tool for marker-assisted selection in cold tolerance breeding. This study not only elucidates the metabolic regulatory mechanisms of rye under low-temperature stress but also provides a solid theoretical and technical foundation for future cold-tolerance breeding programs. Full article

Amaranthus retroflexus is a highly invasive annual broadleaf weed in soybean fields, significantly reducing soybean yield and quality. Diphenyl ether herbicides, particularly fomesafen, are extensively applied to control A. retroflexus. Fomesafen resistance of A. retroflexus is emerging in Northeast China, and rapid resistance detection is urgent for managing these resistant weeds. An Arg-128-Gly mutation in the ppo2 gene of A. retroflexus has been shown to confer fomesafen resistance. In current study, we developed a rapid detection method based on Kompetitive Allele-Specific PCR (KASP) technology to detect the Arg-128-Gly mutation in the ppo2 gene of A. retroflexus. Initially, we developed this KASP detection method using cDNA as the template; however, the entire process requires significant costs and considerable operational time. To mitigate these expenses and simplify the workflow, we subsequently optimized this KASP rapid detection method by utilizing genomic DNA as the template. This new resistance detection technique directly utilizes A. retroflexus genomic DNA as the template, and, by adding specific labelled primers, probes, and enzymes, it can determine whether the ppo2 gene harbors an Arg-128-Gly mutation, thereby rapidly identifying fomesafen resistance in A. retroflexus. Furthermore, we compared the detection efficiency of the new KASP assay, whole plant dose–response assay, and DNA sequencing, all of which produced consistent outcomes, supporting the accuracy and reliability of the KASP rapid detection method. Collectively, we established a rapid resistance detection method based on KASP technology, which is of high reliability and time-saving, and will significantly advance precise management of resistant weeds. Full article

Populus deltoides holds significant ecological and economic importance and is a crucial gene donor for the world’s staple poplar varieties. To select and breed P. deltoides with improved agronomic traits, nine growth and leaf traits were examined in 375 different genotypes, assessing their genetic diversity and performing correlation and comprehensive ranking analyses. Phenotyping results were then utilized to screen a total of 2,009,263 SNP (single nucleotide polymorphism) loci significantly associated with the nine phenotypic traits. A total of 45 SNP loci exhibited significant associations with growth traits based on a general linear model (GLM) analysis. By analyzing the Linkage disequilibrium (LD) block of five SNP loci with significant leaf area and height, we identified five candidate genes related to leaf area and height. Three of the five SNP loci were successfully validated using KASP (kompetitive allele-specific PCR) assays. One loci Chr08_16007979 was closely linked with leaf area, and two loci Chr05_12148738, and Chr05_17106547 were closely linked with height. The developed functional KASP markers offer valuable insights for subsequent further marker-assisted breeding and genetic improvement studies in southern-type poplars. Full article

Background/Objectives: Hybridity authentication is an important component of quality assurance and control (QA/QC) in breeding programs. Here, we introduce HybridQC v1.0, a QA/QC software program specially designed for parental purity and hybridity determination. HybridQC rapidly detects molecular marker polymorphism between parents of a cross and utilizes only the informative markers for hybridity authentication. Methods: HybridQC is written in Python and designed with a graphical user interface (GUI) compatible with Windows operating systems. We demonstrated the QA/QC analysis workflow and functionality of HybridQC using Kompetitive allele-specific PCR (KASP) SNP genotype data for cowpea (Vigna unguiculata). Its performance was validated in other crop data, including sorghum (Sorghum bicolor) and maize (Zea mays). Results: The application efficiently analyzed low-density SNP data from multiple cowpea bi-parental crosses embedded in a single Microsoft Excel file. HybridQC is optimized for the auto-generation of key summary statistics and visualization patterns for marker polymorphism, parental heterozygosity, non-parental alleles, missing data, and F₁ hybridity. An added graphical interface correctly depicted marker efficiency and the proportions of true F₁ versus self-fertilized progenies in the data sets used. The output of HybridQC was consistent with the results of manual hybridity discernment in sorghum and maize data sets. Conclusions: This application uses QA/QC SNP markers to rapidly verify true F₁ progeny. It eliminates the extensive time often required to manually curate and process QA/QC data. This tool will enhance the optimization efforts in breeding programs, contributing to increased genetic gain. Full article

A validated marker system is crucial to running an effective genomics-assisted breeding program. We used 36 Kompetitive Allele-Specific PCR (KASP) markers to genotype 376 clones from the biofortified cassava pipeline, and fingerprinted 93 of these clones with DArTseq markers to characterize breeding materials and evaluate their relationships. The discriminating ability of the 36-quality control (QC) KASP and 6602 DArTseq markers was assessed using 92 clones genotyped in both assays. In addition, trait-specific markers were used to determine the presence or absence of target genomic regions. Hierarchical clustering identified two major groups, and the clusters were consistent with the breeding program origins. There was moderate genetic differentiation and a low degree of variation between the identified groups. The general structure of the population was similar using both assays. Nevertheless, KASP markers had poor resolution when it came to differentiating the genotypes by seed sources and overestimated the prevalence of duplicates. The trait-linked markers did not achieve optimal performance as all markers displayed variable levels of false positive and/or false negative. These findings represent the initial step in the application of genomics-assisted breeding for the biofortified cassava pipeline, and will guide the use of genomic selection in the future. Full article

Barley is one of the most important cereal crops in the world, and its value as a food is constantly being revealed, so the research into and the use of barley germplasm are very important for global food security. Although a large number of barley germplasm samples have been collected globally, their specific genetic compositions are not well understood, and in many cases their origins are even disputed. In this study, 183 barley germplasm samples from the Shanghai Agricultural Gene Bank were genotyped using genotyping-by-sequencing (GBS) technology, SNPs were identified and their genetic parameters were estimated, principal component analysis (PCA) was preformed, and the phylogenetic tree and population structure of the samples were also analyzed. In addition, a genome-wide association study (GWAS) was carried out for the hulled/naked grain trait, and a KASP marker was developed using an associated SNP. The results showed that a total of 181,906 SNPs were identified, and these barley germplasm samples could be roughly divided into three categories according to the phylogenetic analysis, which was generally consistent with the classification of the traits of row type and hulled/naked grain. Population structure analysis showed that the whole barley population could be divided into four sub-populations (SPs), the main difference from previous classifications being that the two-rowed and the hulled genotypes were sub-divided into two SPs. The GWAS analysis of the hulled/naked trait showed that many associated loci were unrelated to the Nud/nud locus, indicating that there might be new loci controlling the trait. A KASP marker was developed for one exon-type SNP on chromosome 7. Genotyping based on the KASP assay was consistent with that based on SNPs, indicating that the gene of this locus might be associated with the hulled/naked trait. The above work not only lays a good foundation for the future utilization of this barley germplasm population but it provides new loci and candidate genes for the hulled/naked trait. Full article

Wheat (Triticum aestivum L.) is one of the most important food crops worldwide and provides the staple food for 40% of the world’s population. Increasing wheat production has become an important goal to ensure global food security. The grain yield of wheat is a complex trait that is usually influenced by multiple agronomically important traits. Thus, the genetic dissection and discovery of quantitative trait loci (QTL) of wheat-yield-related traits are very important to develop high-yield cultivars to improve wheat production. To analyze the genetic basis and discover genes controlling important agronomic traits in wheat, a recombinant inbred lines (RILs) population consisting of 180 RILs derived from a cross between Xinong822 (XN822) and Yannong999 (YN999), two well-adapted cultivars, was used to map QTL for plant height (PH), spike number per spike (SNS), spike length (SL), grain number per spike (GNS), spike number per plant (SN), 1000- grain weight (TGW), grain length (GL), grain width (GW), length/width of grain (GL/GW), perimeter of grain (Peri), and surface area of grains (Sur) in three environments. A total of 64 QTL were detected and distributed on all wheat chromosomes except 3A and 5A. The identified QTL individually explained 2.24–38.24% of the phenotypic variation, with LOD scores ranging from 2.5 to 29. Nine of these QTL were detected in multiple environments, and seven QTL were associated with more than one trait. Additionally, Kompetitive Allele Specific PCR (KASP) assays for five major QTL QSns-1A.2 (PVE = 6.82), QPh-2D.1 (PVE = 37.81), QSl-2D (PVE = 38.24), QTgw-4B (PVE = 8.78), and QGns-4D (PVE = 13.54) were developed and validated in the population. The identified QTL and linked markers are highly valuable in improving wheat yield through marker-assisted breeding, and the large-effect QTL can be fine-mapped for further QTL cloning of yield-related traits in wheat. Full article

Cowpea (Vigna unguiculata L. Walp) is an important grain legume crop of the subtropics, particularly in West Africa, where it contributes to the livelihoods of small-scale farmers. Despite being a drought-resilient crop, cowpea production is hampered by insect pests, diseases, parasitic weeds, and various abiotic stresses. Genetic improvement can help overcome these limitations, and exploring diverse cowpea genetic resources is crucial for cowpea breeding. This study evaluated the genetic diversity of 361 cowpea accessions from the USDA core collection for the species using 102 Kompetitive Allele Specific PCR (KASP) single nucleotide polymorphism (SNP) markers. A total of 102 KASP-SNP was validated in the germplasm panel, and 72 showed polymorphism across the germplasm panel. The polymorphism information content (PIC) of all SNPs ranged from 0.1 to 0.37, with an average of 0.29, while the mean observed heterozygosity was 0.52. The population structure revealed three distinct populations that clustered into two major groups after phylogenetic analysis. Analysis of molecular variance (AMOVA) indicated greater genetic variation within populations than among populations. Although cowpea generally has a narrow genetic diversity, the accessions used in this study exhibited considerable variation across geographical regions, sub-species, and improvement status. These results indicated that the selected KASP genotyping assay can provide robust and accurate genotyping data for application in the selection and management of cowpea germplasm in breeding programs and genebanks. Full article

This work aimed to assess the variability of casein genes in a population of 153 bucks and 825 lactating does of the Sarda breed, and to perform association analysis between polymorphic sites and milk yield and composition traits. To genotype the casein genes, we chose an SNP panel including 44 SNPs mapping to the four casein genes CSN1S1, CSN2, CSN1S2, and CSN3. Genotyping (made by KASP™ genotyping assay, based on competitive allele-specific PCR) revealed the high variability of the Sarda goat, and haplotype analysis revealed linkage disequilibrium (LD) between CSN1S1 and CSN2 genes, in addition to two LD blocks within the CSN1S2 and two LD blocks within the CSN3 gene, in bucks and does. Association analysis revealed that variability at all four casein genes was associated with milk protein content, total solids, and milk energy. The three Ca-sensitive casein genes were associated with lipid content, and CSN1S2 showed a unique pattern, with intron variants associated with milk yield, in addition to milk pH, NaCl, and SCS (Somatic Cell Score). This information might prove useful in selection schemes and in future investigations aiming to better understand the biology of lactation, and the direct link between genotype and phenotype. Full article

Protein content is one of the main factors determining the end-use quality of wheat. NO APICAL MERISTEM-B1 (NAM-B1) is a major gene regulating wheat grain protein content. The present study aimed to identify new genetic resources using the wild-type NAM-B1 allele to breed high-protein-content wheat cultivars. We genotyped the HIGH GRAIN PROTEIN CONTENT-B1 (GPC-B1) locus and NAM-B1 allele in 165 wheat cultivars. A kompetitive allele-specific polymerase chain reaction (KASP) marker was designed for functional NAM-B1 allele screening. The results revealed that 41 out of 165 cultivars carried the GPC-B1 locus. Among the 41 GPC-B1-carrying cultivars, the wild-type NAM-B1 allele was identified in only 3 cultivars, none of which were Korean. The remaining 38 cultivars showed a 1-bp insertion in NAM-B1, resulting in a stop codon in the middle of the gene, rendering it nonfunctional. Overall, this study reveals that the utilization of the three selected cultivars possessing the wild-type NAM-B1 gene, in conjunction with the developed KASP assay, could increase the protein content in Korean wheat cultivars. Full article

The individual risk of an unfavorable cardiovascular outcome is determined by genetic factors in addition to lifestyle factors. This study was aimed at analyzing possible associations of several genetic factors with the risk of myocardial infarction (MI). For our study, we selected genes that have been significantly associated with MI in meta-analyses: the chromosomal region 9p21.3, the CETP gene, and the APOE gene. In total, 2286 randomly selected patients were included. Rs708272 and rs429358 and rs7412 were analyzed using RT-PCR via the TaqMan principle, and rs1333049 vas analyzed via a commercial KASP assay. In our sample, the frequencies of alleles and genotypes were consistent with frequencies in comparable populations of Eastern and Western Europe. Allele C of rs1333049 was significantly associated with MI among males (p = 0.027) and in the whole study sample (p = 0.008). We also revealed a significant association of the ɛ2/ɛ4 genotype of APOE with MI among males (p < 0.0001) and in the whole study sample (p < 0.0001). Thus, among the tested polymorphisms, some genotypes of rs1333049 and rs429358 and rs7412 are the most strongly associated with MI and can be recommended for inclusion into a genetic risk score. Full article

Although the potato chip industry is booming, and distinct chip-processing clones have been released over the past 60 years, the genetic architecture of their chip-processing characteristics remains largely unknown. Case-control genome-wide association studies (GWAS) with SolCAP SNP array data for chip-processing clones versus all other market classes in the 393-line potato diversity panel were performed using the GWASpoly R package, enabling detection of significant signals on chromosome 10. Our results were replicated using internal replication of a strata-corrected 190-line panel. Furthermore, the genomic scans employing selective sweep approaches such as the cross-population composite likelihood ratio method (XP-CLR) and PCAdapt redetected the same signals as those in our GWAS. Through applications of four selective sweep approaches, various genetic variants were found across the genome that had been differentially selected. These genomic regions under selection along with transcriptomic data analysis are involved in carbohydrate metabolism-related genes or loci and transcription factors, indicating to be associated with the improvement of chip-processing performance of potato cultivars. Kompetitive allele-specific PCR (KASP) assays were designed for the causal SNPs to use in validating the chip-processing clones. The results could have implications for genomics-assisted breeding of the promising chip-processing cultivars in potato. Full article

Rice blast, caused by Pyricularia oryzae, is one of the main rice diseases worldwide. The pyramiding of blast-resistance (Pi) genes, coupled to Marker-Assisted BackCrossing (MABC), provides broad-spectrum and potentially durable resistance while limiting the donor genome in the background of an elite cultivar. In this work, MABC coupled to foreground and background selections based on KASP marker assays has been applied to introgress four Pi genes (Piz, Pib, Pita, and Pik) in a renowned japonica Italian rice variety, highly susceptible to blast. Molecular analyses on the backcross (BC) lines highlighted the presence of an additional blast-resistance gene, the Pita-linked Pita2/Ptr gene, therefore increasing the number of blast-resistance introgressed genes to five. The recurrent genome was recovered up to 95.65%. Several lines carrying four (including Pita2) Pi genes with high recovery percentage levels were also obtained. Phenotypic evaluations confirmed the effectiveness of the pyramided lines against multivirulent strains, which also had broad patterns of resistance in comparison to those expected based on the pyramided Pi genes. The developed blast-resistant japonica lines represent useful donors of multiple blast-resistance genes for future rice-breeding programs related to the japonica group. Full article

Bacterial panicle blight (BPB) and sheath blight (SB) are major diseases of rice and few cultivars have shown a high level of resistance to these diseases. A recombinant inbred line (RIL) population developed from the U.S. cultivars Jupiter (moderately resistant) and Trenasse (susceptible) was investigated to identify loci associated with the partial disease resistance to BPB and SB. Disease phenotypes in BPB and SB, as well as the days-to-heading (DTH) trait, were evaluated in the field. DTH was correlated to BPB and SB diseases, while BPB was positively correlated to SB in the field trials with this RIL population. Genotyping was performed using Kompetitive Allele Specific PCR (KASP) assays and whole-genome sequence (WGS) analyses. Quantitative trait locus (QTL) mapping and bulk segregant analysis using a set of WGS data (QTL-seq) detected a major QTL on the upper arm of chromosome 3 for BPB, SB, and DTH traits within the 1.0–1.9 Mb position. Additional QTLs associated with BPB and SB were also identified from other chromosomes by the QTL-seq analysis. The QTLs identified in this study contain at least nine candidate genes that are predicted to have biological functions in defense or flowering. These findings provide an insight into the complex nature of the quantitative resistance to BPB and SB, which may also be closely linked to the flowering trait. Full article

Small ruminant lentiviruses (SRLVs) affect sheep and goats worldwide. The major gene related to SRLV infections is the Transmembrane Protein Gene 154 (TMEM154). We estimated the haplotype frequencies of TMEM154 in the USA (USDA-ARS) and Brazil (Embrapa) Gene Banks by using two different SNP genotyping methodologies, Fluidigm^TM and KASP^TM. We also genotyped the ZNF389_ss748775100 deletion variant in Brazilian flocks. A total of 1040 blood samples and 112 semen samples from 15 Brazilian breeds were genotyped with Fluidigm for the SNP ZNF389_ss748775100 and 12 TMEM154 SNPs. A total of 484 blood samples from the Santa Inês breed and 188 semen samples from 14 North American sheep breeds were genotyped with KASP for 6 TMEM154 SNPs. All the Brazilian samples had the “I/I” genotype for the ZNF389_ss748775100 mutation. There were 25 TMEM154 haplotypes distributed across the Brazilian breeds, and 4 haplotypes in the US breeds. Haplotypes associated with susceptibility were present in almost all breeds, which suggests that genetic testing can help to improve herd health and productivity by selecting non-susceptible animals as founders of the next generations. Fluidigm and KASP are reliable assays when compared with Beadchip arrays. Further studies are necessary to understand the unknown role of TMEM154 mutations, host–pathogen interaction and new genes associated with the clinical condition. Full article