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1
Cheon, Kyeong-Seong, Young-Min Jeong, Hyoja Oh, Jun Oh, Do-Yu Kang, Nyunhee Kim, Eungyeong Lee, et al. "Development of 454 New Kompetitive Allele-Specific PCR (KASP) Markers for Temperate japonica Rice Varieties." Plants 9, no. 11 (November 10, 2020): 1531. http://dx.doi.org/10.3390/plants9111531.
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Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.
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Majeed, Uzma, Essam Darwish, Shoaib Ur Rehman, and Xueyong Zhang. "Kompetitive Allele Specific PCR (KASP): A Singleplex Genotyping Platform and Its Application." Journal of Agricultural Science 11, no. 1 (December 15, 2018): 11. http://dx.doi.org/10.5539/jas.v11n1p11.
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Single nucleotide polymorphism (SNP) can be detected by mining sequence databases or by using different singleplex or multiplex SNP genotyping platforms. Development of high-throughput genotyping molecular markers can be instrumental towards maximizing genetic gain. In this review we provide an overview of Kompetitive Allele Specific PCR (KASP) genotyping platform requirements and its application that might be helpful in KASP marker development. This literature further illustrates the possibilities to design KASP primers. Several research institutes routinely using KASP platform, producing in excess of humungous data points yearly for breeding cultivars and as well as for medical and commercial purposes. KASP genotyping technology offers cost effectiveness and high throughput molecular marker development platform. Conventional molecular markers can be converted into more robust and high throughput KASP markers. More than 2000 published references clearly show the popularity of KASP technology among the researchers.
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Radanović, Aleksandra, Yves Sprycha, Milan Jocković, Monja Sundt, Dragana Miladinović, Constantin Jansen, and Renate Horn. "KASP Markers Specific for the Fertility Restorer Locus Rf1 and Application for Genetic Purity Testing in Sunflowers (Helianthus annuus L.)." Genes 13, no. 3 (March 5, 2022): 465. http://dx.doi.org/10.3390/genes13030465.
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Single nucleotide polymorphisms (SNPs) were significantly associated with fertility restoration of cytoplasmic male sterility (CMS) PET1 by the restorer gene Rf1. For these SNPs, four Kompetitive allele-specific PCR (KASP) markers were successfully designed. The KASP markers cover the fertility restorer locus Rf1, spanning about 3 Mb, and clearly differentiate restorer and maintainer lines. For genetic purity testing in sunflower hybrid production, the efficiency for detecting contaminations in samples was simulated using mixtures of hypocotyls or leaves. Contaminations of restorer lines with 1%, 3%, 5%, 10%, and 50% of maintainer lines were screened with all four KASP markers. Contaminations of 10% could be clearly detected in pools of 100 plants. Contaminations below this level require detection on a single plant level. For single plant detections, ethyl methanesulfonate-treated sunflower F1 hybrids, which had been phenotypically evaluated for male sterility (potential mutation in the Rf1 gene) were screened. Nine identified either partially male-sterile or male-sterile plants were analyzed with all four KASP markers and only one proved to be a hybrid with a mutation, seven were male-sterile contaminants in the F1 seeds used (1.6%) and one a recombinant plant. The four KASP markers should be valuable tools for marker-assisted selection (MAS) in sunflower breeding regarding the restorer locus Rf1.
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Swisher Grimm, Kylie D., and Lyndon D. Porter. "Development and Validation of KASP Markers for the Identification of Pea seedborne mosaic virus Pathotype P1 Resistance in Pisum sativum." Plant Disease 104, no. 6 (June 2020): 1824–30. http://dx.doi.org/10.1094/pdis-09-19-1920-re.
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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.
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Sayadi Maazou, Abdoul-Raouf, Melaku Gedil, Victor O. Adetimirin, Silvestro Meseka, Wende Mengesha, Deborah Babalola, Queen Nkem Offornedo, and Abebe Menkir. "Comparative Assessment of Effectiveness of Alternative Genotyping Assays for Characterizing Carotenoids Accumulation in Tropical Maize Inbred Lines." Agronomy 11, no. 10 (October 9, 2021): 2022. http://dx.doi.org/10.3390/agronomy11102022.
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The development of maize varieties with increased concentration of Provitamin A (PVA) is an effective and affordable strategy to combat vitamin A deficiency in developing nations. However, the considerably high cost of carotene analysis poses a major challenge for maize PVA biofortification, prompting the use of marker-assisted selection. Presently, two types of genotyping with PVA trait-linked functional markers have been developed and extensively used in breeding programs. The two systems are low throughput gel-based genotyping and genotyping with Kompetitive Allele-Specific PCR (KASP) single nucleotide polymorphism (SNPs) markers. Although the KASP SNPs genotyping was developed to replace the gel-based genotyping, studies have not been conducted to compare the effectiveness of the KASP SNPs markers with the gel-based markers. This study was conducted to assess the carotenoid content of 64 tropical PVA biofortified maize inbred lines containing temperate germplasm in their genetic backgrounds and screen them with both gel-based and KASP markers of PSY1, LCYE and crtRB1 genes. Many of the 64 inbred lines had PVA concentrations surpassing the 15 µg/g provitamin A breeding target set by the HarvestPlus Challenge Program. Favorable alleles of crtRB1, crtRB1 and the KASP SNPs markers were detected in 25 inbred lines with high PVA concentrations. Inbred lines with the favorable alleles of LCYE had the highest concentrations of non-PVA carotenoids, whereas those with the favorable alleles of crtRB1 had high levels of PVA carotenoids. Data from the sequenced region of LCYE revealed one SNP in the first intron that clearly differentiated the high and low β-carotene maize inbred lines. The results of our study demonstrate that the automated KASP SNPs markers can replace the gel-based genotyping for screening a large number of early generation maize inbred lines for PVA content.
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Anuarbek, Shynar, Saule Abugalieva, and Yerlan Turuspekov. "Validation of Bread Wheat KASP Markers in Durum Wheat Lines in Kazakhstan." Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences. 73, no. 5 (October 1, 2019): 462–65. http://dx.doi.org/10.2478/prolas-2019-0071.
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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.
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Volkova, N. E., H. Slishchuk, V. Sichkar, and O. Zakharova. "Single Nucleotide Polymorphism of the chickpea gene encoding acetohydroxyacid synthase: molecular-genetical analysis." Faktori eksperimental'noi evolucii organizmiv 30 (September 1, 2022): 13–18. http://dx.doi.org/10.7124/feeo.v30.1454.
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Aim. Chickpea AHAS gene molecular-marker analysis. To achieve this aim following tasks were set: chickpea AHAS gene SNP markers research and development, chickpea varieties and samples genotyping by the markers. Methods. CTAB method of DNA isolation and purification, real-time polymerase chain reaction. Results. Chickpea AHAS1 gene KASP gene specificity was found: there were no DNA amplification with lens and maize samples observed. Three Ukrainian chickpea varieties and 28 ICRISAT collection samples KASP genotyping detected only wild-type C-allele. Authors own SNP markers 58 chickpea samples genotyping showed that AHAS1 gene within samples was homozygous, no mutant T allele was detected. Conclusions. 31 chickpea samples contained wild-type C-allele by KASP genotyping, which is associated with imidazoline herbicide susceptibility. 58 chickpea samples contained C-allele by authors own SNP markers genotyping. Mutant T-allele that is associated with tolerance absence indicates researched samples imidazole herbicide tolerance low level.
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Wu, Jianhui, Qilin Wang, Liangsheng Xu, Xianming Chen, Bei Li, Jingmei Mu, Qingdong Zeng, Lili Huang, Dejun Han, and Zhensheng Kang. "Combining Single Nucleotide Polymorphism Genotyping Array with Bulked Segregant Analysis to Map a Gene Controlling Adult Plant Resistance to Stripe Rust in Wheat Line 03031-1-5 H62." Phytopathology® 108, no. 1 (January 2018): 103–13. http://dx.doi.org/10.1094/phyto-04-17-0153-r.
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Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating diseases of wheat worldwide. Growing resistant cultivars is considered the best approach to manage this disease. In order to identify the resistance gene(s) in wheat line 03031-1-5 H62, which displayed high resistance to stripe rust at adult plant stage, a cross was made between 03031-1-5 H62 and susceptible cultivar Avocet S. The mapping population was tested with Chinese P. striiformis f. sp. tritici race CYR32 through artificial inoculation in a field in Yangling, Shaanxi Province and under natural infection in Tianshui, Gansu Province. The segregation ratios indicated that the resistance was conferred by a single dominant gene, temporarily designated as YrH62. A combination of bulked segregant analysis (BSA) with wheat 90K single nucleotide polymorphism (SNP) array was used to identify molecular markers linked to YrH62. A total of 376 polymorphic SNP loci identified from the BSA analysis were located on chromosome 1B, from which 35 kompetitive allele-specific PCR (KASP) markers selected together with 84 simple sequence repeat (SSR) markers on 1B were used to screen polymorphism and a chromosome region associated with rust resistance was identified. To saturate the chromosomal region covering the YrH62 locus, a 660K SNP array was used to identify more SNP markers. To develop tightly linked markers for marker-assisted selection of YrH62 in wheat breeding, 18 SNPs were converted into KASP markers. A final linkage map consisting of 15 KASP and 3 SSR markers was constructed with KASP markers AX-109352427 and AX-109862469 flanking the YrH62 locus in a 1.0 cM interval. YrH62 explained 63.8 and 69.3% of the phenotypic variation for disease severity and infection type, respectively. YrH62 was located near the centromeric region of chromosome 1BS based on the positions of the SSR markers in 1B deletion bins. Based on the origin, responses to P. striiformis f. sp. tritici races, and marker distances, YrH62 is likely different from the other reported stripe rust resistance genes/quantitative trait loci on 1B. The gene and tightly linked KASP markers will be useful for breeding wheat cultivars with resistance to stripe rust.
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Amalova, A., Yu Genievskaya, S. Abugalieva, V. Chudinov, and Ye Turuspekov. "VALIDATION OF MARKER-TRAIT ASSOCIATIONS IN SIX-ROWED BARLEY LINES BRED IN KAZAKHSTAN." Eurasian Journal of Applied Biotechnology, no. 4 (October 5, 2022): 40–48. http://dx.doi.org/10.11134/btp.4.2022.5.
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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.
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Kante, Moctar, Hannele Lindqvist-Kreuze, Leticia Portal, Maria David, and Manuel Gastelo. "Kompetitive Allele Specific PCR (KASP) Markers for Potato: An Effective Tool for Increased Genetic Gains." Agronomy 11, no. 11 (November 16, 2021): 2315. http://dx.doi.org/10.3390/agronomy11112315.
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Potato virus Y (PVY) and Phytophthora infestans (Mont.) de Bary that causes potato late blight (LB), pose serious constraints to cultivated potatoes due to significant yield reduction, and phenotyping for resistance remains challenging. Breeding operations for vegetatively propagated crops can lead to genotype mislabeling that, in turn, reduces genetic gains. Low-density and low-cost molecular marker assessment for phenotype prediction and quality control is a viable option for breeding programs. Here, we report on the development of kompetitive allele specific PCR (KASP) markers for LB and PVY resistance, and for routine quality control assessment of different breeding populations. Two KASP markers for LB resistance and two for PVY Ryadg were validated with an estimated assay power that ranged between 0.65 and 0.88. The developed QC KASP markers demonstrated the capability of discriminating tetraploid calls in breeding materials, including full-sibs and half-sibs. Routine implementation of the developed markers in a breeding program would assist with better allocation of resources and enable precise characterization of breeding material, thereby leading to increased genetic gains.
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Shrestha, Swati, Vincent Njung’e Michael, Yuqing Fu, and Geoffrey Meru. "Genetic Loci Associated with Resistance to Zucchini Yellow Mosaic Virus in Squash." Plants 10, no. 9 (September 17, 2021): 1935. http://dx.doi.org/10.3390/plants10091935.
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Zucchini Yellow Mosaic Virus (ZYMV) is an aphid-transmitted potyvirus that causes severe yield losses in squash (Cucurbita moschata) production worldwide. Development of resistant cultivars using traditional breeding approaches relies on rigorous and resource-intensive phenotypic assays. QTL-seq, a whole genome re-sequencing based bulked segregant analysis, is a powerful tool for mapping quantitative trait loci (QTL) in crop plants. In the current study, the QTL-seq approach was used to identify genetic loci associated with ZYMV resistance in an F2 population (n = 174) derived from a cross between Nigerian Local (resistant) and Butterbush (susceptible). Whole genome re-sequencing of the parents and bulks of resistant and susceptible F2 progeny revealed a mapping rate between 94.04% and 98.76%, and a final effective mapping depth ranging from 81.77 to 101.73 across samples. QTL-seq analysis identified four QTLs significantly (p < 0.05) associated with ZYMV resistance on chromosome 2 (QtlZYMV-C02), 4 (QtlZYMV-C04), 8 (QtlZYMV-C08) and 20 (QtlZYMV-C20). Seven markers within the QTL intervals were tested for association with ZYMV resistance in the entire F2 population. For QtlZYMV-C08, one single nucleotide polymorphism (SNP) marker (KASP-6) was found to be significantly (p < 0.05) associated with ZYMV resistance, while two SNPs (KASP-1 and KASP-3) and an indel (Indel-2) marker were linked to resistance within QtlZYMV-C20. KASP-3 and KASP-6 are non-synonymous SNPs leading to amino acid substitutions in candidate disease resistant gene homologs on chromosomes 20 (CmoCh20G003040.1) and 8 (CmoCh08G007140.1), respectively. Identification of QTL and SNP markers associated with ZYMV resistance will facilitate marker-assisted selection for ZYMV resistance in squash.
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Sejake, Thulo, Nemera Shargie, Riann Christian, Assefa B. Amelework, and Toi J. Tsilo. "Genetic diversity in sorghum (Sorghum bicolor L. Moench) accessions using SNP based Kompetitive allele-specific (KASP) markers." June 2021, no. 15(06):2021 (June 10, 2021): 890–98. http://dx.doi.org/10.21475/ajcs.21.15.06.p3088.
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Genetic diversity analysis is an important component in conventional and marker-assisted breeding. The objective of this study was to assess the level of genetic diversity among 100 sorghum accessions, which were selected randomly from the Sorghum National Germplasm Bank maintained at Agricultural Research Council, South Africa. A total of 136 Kompetitive Allele-Specific PCR (KASP) markers were used in this study. The KASP markers were previously derived from single-nucleotide polymorphic (SNP) analysis of the world-wide sorghum accessions by other research groups. A total of 110 KASP markers were polymorphic and recorded an average polymorphic information content (PIC) value of 0.3, which indicated high level of discrimination of the markers. The markers had an average gene diversity and observed heterozygosity of 0.3 and 0.10, respectively. Analysis of molecular variance revealed a significantly high variation among accessions (83% and 89%) than within accessions (10% and 11%) based on breeding status and geographic origin, respectively. Genetic distance varied from 0.0 between SA0672 and SA0673, SA1282 and SA0670 to 0.57 between Hakika and SA1442 with an average mean of 0.30. The dendrogram and model-based population analysis identified three and four distinct groups in 95 sorghum accessions, respectively. These results imply the presence of genetic diversity and lack of genetic bottleneck within the National Sorghum Germplasm Bank, which could be highly relevant for sorghum breeding and germplasm maintenance
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Wang, Na, Yanzhou Xie, Yingzhuang Li, Shengnan Wu, Shuxian Li, Yu Guo, and Chengshe Wang. "High-Resolution Mapping of the Novel Early Leaf Senescence Gene Els2 in Common Wheat." Plants 9, no. 6 (May 30, 2020): 698. http://dx.doi.org/10.3390/plants9060698.
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Early leaf senescence negatively impacts the grain yield in wheat (Triticum aestivum L.). Induced mutants provide an important resource for mapping and cloning of genes for early leaf senescence. In our previous study, Els2, a single incomplete dominance gene, that caused early leaf senescence phenotype in the wheat mutant LF2099, had been mapped on the long arm of chromosome 2B. The objective of this study was to develop molecular markers tightly linked to the Els2 gene and construct a high-resolution map surrounding the Els2 gene. Three tightly linked single-nucleotide polymorphism (SNP) markers were obtained from the Illumina Wheat 90K iSelect SNP genotyping array and converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers. To saturate the Els2 region, the Axiom® Wheat 660K SNP array was used to screen bulked extreme phenotype DNA pools, and 9 KASP markers were developed. For fine mapping of the Els2 gene, these KASP markers and previously identified polymorphic markers were analyzed in a large F2 population of the LF2099 × Chinese Spring cross. The Els2 gene was located in a 0.24-cM genetic region flanked by the KASP markers AX-111643885 and AX-111128667, which corresponded to a physical interval of 1.61 Mb in the Chinese Spring chromosome 2BL containing 27 predicted genes with high confidence. The study laid a foundation for a map-based clone of the Els2 gene controlling the mutation phenotype and revealing the molecular regulatory mechanism of wheat leaf senescence.
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Hu, Jinghuang, Jingting Li, Peipei Wu, Yahui Li, Dan Qiu, Yunfeng Qu, Jingzhong Xie, et al. "Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61." International Journal of Molecular Sciences 20, no. 3 (February 11, 2019): 750. http://dx.doi.org/10.3390/ijms20030750.
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The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompetitive Allele Specific PCR (KASP), and six simple sequence repeat (SSR) markers, together with previously identified polymorphic markers, saturated the genetic linkage map for Pm61, especially in the proximal side of the target gene that was short of gene-linked markers. In the newly established genetic linkage map, Pm61 was located in a 0.71 cM genetic interval and can be detected in a high throughput scale by the KASP markers Xicsk8 and Xicsk13 or by the standard PCR-based markers Xicscx497 and Xicsx538. The newly saturated genetic linkage map will be useful in molecular marker assisted-selection of Pm61 in breeding for disease resistant cultivar and in its map-based cloning.
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Yonet, Nilay, Yıldız Aydin, Goksel Evci, and Ahu Altinkut Uncuoglu. "Genomic Evaluation of Sunflower Broomrape (Orobanche Cumana) Germplasm by KASP Assay." Helia 41, no. 68 (July 26, 2018): 57–72. http://dx.doi.org/10.1515/helia-2017-0016.
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AbstractOrobanche cumana Wallr. is a holoparasitic plant for only sunflower, hence it is called as sunflower broomrape. Yield loss created by O. cumana which is generally 50 % can reach to 100 %. In this study, it was planned to perform molecular characterization of O. cumana germplasm as nine locations of Thrace region obtained from Trakya Agricultural Research Institute by using Single Nucleotide Polymorphism (SNP) markers, widely used in plant breeding programs, in Competitive Allele Specific PCR (KASP) assay which is a fluorescent tagged allele specific PCR method based, economic, reliable and easily repeatable genotyping technology. Databases and literature were scanned to spot variations on O. cumana genome which is not known clearly. So far, four SSR (Simple Sequence Repeat) marker (Ocum-197, Ocum-006, Ocum-023 and Ocum-151) regions showing polymorphic pattern were used for searching possible SNPs. Primer pairs were designed for amplification of the regions possibly having SNPs and PCR amplifications with these primer pairs were performed and 1 candidate deletion was detected on the amplicon which was amplified by Ocum-197 SSR marker. Following, the deletion was converted to KASP primers and KASP assay was performed. The deletion marker, Del-197, has grouped the samples from nine locations in the resulting allelic discrimination plot and infestation was performed according to this grouping, As a conclusion, Del-197 is considered as a selective marker for the ability to rapidly assay allelic variation at DNA markers for O. cumana populations that have effects on infestation results were evaluated as races, F, G, H and I in Thrace region.
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Jagtap, Ashok Babadev, Yogesh Vikal, and Gurmukh Singh Johal. "Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize." International Journal of Molecular Sciences 21, no. 19 (October 6, 2020): 7386. http://dx.doi.org/10.3390/ijms21197386.
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Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).
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Kebede, Aida Z., Wubishet A. Bekele, Jennifer W. Mitchell Fetch, Aaron D. Beattie, Shiaoman Chao, Nicholas A. Tinker, Thomas G. Fetch, and Curt A. McCartney. "Localization of the Stem Rust Resistance Gene Pg2 to Linkage Group Mrg20 in Cultivated Oat (Avena sativa)." Phytopathology® 110, no. 10 (October 2020): 1721–26. http://dx.doi.org/10.1094/phyto-03-20-0076-r.
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Stem rust is an important disease of cultivated oat (Avena sativa) caused by Puccinia graminis f. sp. avenae. In North America, host resistance is the primary strategy to control this disease and is conferred by a relatively small number of resistance genes. Pg2 is a widely deployed stem rust resistance gene that originates from cultivated oat. Oat breeders wish to develop cultivars with multiple Pg genes to slow the breakdown of single gene resistance, and often require DNA markers suited for marker-assisted selection. Our objectives were to (i) construct high density linkage maps for a major oat stem rust resistance gene using three biparental mapping populations, (ii) develop Kompetitive allele-specific PCR (KASP) assays for Pg2-linked single-nucleotide polymorphisms (SNPs), and (iii) test the prediction accuracy of those markers with a diverse panel of spring oat lines and cultivars. Genotyping-by-sequencing SNP markers linked to Pg2 were identified in an AC Morgan/CDC Morrison recombinant inbred line (RIL) population. Pg2-linked SNPs were then analyzed in an AC Morgan/RL815 F2 population and an AC Morgan/CDC Dancer RIL population. Linkage analysis identified a common location for Pg2 in all three populations on linkage group Mrg20 of the oat consensus genetic map. The most predictive markers were identified and converted to KASP assays for use in oat breeding programs. When used in combination, the KASP assays for the SNP loci avgbs2_126549.1.46 and avgbs_cluster_23819.1.27 were highly predictive of Pg2 status in panel of 54 oat breeding lines and cultivars.
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Winfield, Mark, Amanda Burridge, Matthew Ordidge, Helen Harper, Paul Wilkinson, Danny Thorogood, Liz Copas, Keith Edwards, and Gary Barker. "Development of a minimal KASP marker panel for distinguishing genotypes in apple collections." PLOS ONE 15, no. 11 (November 30, 2020): e0242940. http://dx.doi.org/10.1371/journal.pone.0242940.
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Accurate identification of named accessions in germplasm collections is extremely important, especially for vegetatively propagated crops which are expensive to maintain. Thus, an inexpensive, reliable, and rapid genotyping method is essential because it avoids the need for laborious and time-consuming morphological comparisons. Single Nucleotide Polymorphism (SNP) marker panels containing large numbers of SNPs have been developed for many crop species, but such panels are much too large for basic cultivar identification. Here, we have identified a minimum set of SNP markers sufficient to distinguish apple cultivars held in the English and Welsh national collections providing a cheaper and automatable alternative to the markers currently used by the community. We show that SNP genotyping with a small set of well selected markers is equally efficient as microsatellites for the identification of apple cultivars and has the added advantage of automation and reduced cost when screening large numbers of samples.
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Jiang, Yunfeng, Luyao Duan, Fangnian Guan, Fangjie Yao, Li Long, Yuqi Wang, Xuyang Zhao, et al. "Exome Sequencing from Bulked Segregant Analysis Identifies a Gene for All-Stage Resistance to Stripe Rust on Chromosome 1AL in Chinese Wheat Landrace ‘Xiaohemai’." Plant Disease 106, no. 4 (April 1, 2022): 1209–15. http://dx.doi.org/10.1094/pdis-08-21-1618-re.
Full textAbstract:
Stripe rust caused by Puccinia striiformis f. sp. tritici is one of the most destructive diseases of wheat. Identifying novel resistance genes applicable for developing disease-resistant cultivars is important for the sustainable control of wheat stripe rust. Chinese wheat landrace ‘Xiaohemai’ (‘XHM’) is an elite germplasm line with all-stage resistance (ASR) effective against predominant Chinese P. striiformis f. sp. tritici races. In this study, we performed a bulked segregant analysis coupled with exome capture sequencing (BSE-seq) to identify a candidate genomic region strongly associated with stripe rust resistance on chromosome 1AL in 173 F2:3 lines derived from the cross ‘XHM’ × ‘Avocet S’. The gene, designated as YrXH-1AL, was validated by a conventional quantitative trait locus analysis using newly developed Kompetitive allele-specific PCR (KASP) markers, explaining up to 48.50% of the phenotypic variance. By testing a secondary mapping population comprising 144 lines from the same cross at the seedling stage with prevalent P. striiformis f. sp. tritici race CYR34, YrXH-1AL was identified as a single Mendelian factor in a 1.5-cM interval flanked by KASP markers KP1A_484.33 and KP1A_490.09. This region corresponded to a 5.76-Mb genomic interval on ‘Chinese Spring’ chromosome 1AL. Furthermore, two cosegregating KASP markers showed high polymorphisms among 130 Chinese wheat cultivars and could be used for marker-assisted selection. Because no other Yr genes for ASR that originated from common wheat have been detected on chromosome 1AL, YrXH-1AL is likely a novel gene that can be incorporated into modern breeding materials to develop wheat cultivars with enhanced stripe rust resistance.
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Afanasenko, Olga, Irina Rozanova, Anastasiia Gofman, Nina Lashina, Fluturë Novakazi, Nina Mironenko, Olga Baranova, and Alexandr Zubkovich. "Validation of Molecular Markers of Barley Net Blotch Resistance Loci on Chromosome 3H for Marker-Assisted Selection." Agriculture 12, no. 4 (March 22, 2022): 439. http://dx.doi.org/10.3390/agriculture12040439.
Full textAbstract:
The most widespread and harmful disease of barley is net form of net blotch caused by the ascomycete Pyrenophora teres f. teres Drechsler (Ptt). A cost effective and environmentally sustainable strategy for barley protection against Ptt is to develop barley cultivars possessing genetic resistance. In previous GWA analysis, we identified SNP-markers associated with a resistance locus on chromosome 3H in the interval of 45.82–54.53 cM. These SNPs have been described previously in the literature to be located within the same region of chromosome 3H. The aim of the study was to validate QTL markers controlling resistance to Ptt on chromosome 3H in this region by KASP genotyping in four F2 populations of crosses between the resistant cultivars, Morex, Fox, and Zolo, and the accession, Local k-21578, with the susceptible barley cv. Gesine and in a doubled haploid (DH) population of Canadian Lake Shore (CLS)/Harrington. Eleven of fifteen studied markers showed high efficacy (97.5–100%) for co-segregation with resistance to Ptt in the DH population, CLS/Harrington. Three of these markers located at 54.53 cM and one at 51.27 cM were effective in two F2 populations of crosses of Morex and Fox with susceptible cv. Gesine. These markers are also located close to each other on the physical map (442,203,921–443,119,491 bp). Apparently, in cultivars, CLS, Morex, and Fox, resistance to Ptt is determined by the same locus. Markers JHI-Hv50k-2016-166392 (47.1 cM, 112,536,071 bp), Clone ID 3255462_1 (51.63 cM, 363,531,898 bp), and Clone ID 3255462_2 (51.63 cM, 363,531,871 bp) showed high efficacy in the DH population and in the F2 population, Local k-21578/Gesine. Apparently, at least two loci controlling Ptt resistance exist in the chromosome region of 47.0–54.3 cM: one at 46.0–48.44 cM and another at 51.27–54.8 cM. These regions were found to harbor several genes involved in important plant functions, including disease response and signaling pathways. Allele-specific PCR markers were developed based on the KASP assay data and tested on six resistant, two moderately resistant, and two susceptible barley genotypes. Four markers were found to be effective to differentiate susceptible and resistant barley genotypes. The KASP and allele-specific PCR markers associated with Ptt resistance on chromosome 3H will be useful for pyramiding resistance QTLs in barley marker-assisted selection.
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Fan, Kai, Jie Zhang, Min Wang, Wenjun Qian, Litao Sun, Jiazhi Shen, Zhaotang Ding, and Yu Wang. "Development and Application of SNP-KASP Markers Based on Genes Related to Nitrogen Uptake, Assimilation and Allocation in Tea Plant (Camellia sinensis L.)." Agronomy 12, no. 10 (October 17, 2022): 2534. http://dx.doi.org/10.3390/agronomy12102534.
Full textAbstract:
Nitrogen is essential for the growth and quality formation of tea plants. Excessive and inefficient use of nitrogen fertilizer leads to cost increases and ecosystem pollution. It is important to improve the nitrogen use efficiency (NUE) for tea plantation. Breeding high-NUE varieties by marker-assisted selection using NUE-associated genes is a viable approach. However, few molecular markers related to nitrogen uptake and utilization have been identified in tea plants. In this study, a total of 2554 SNP loci within NUE-related genes were identified in a database. Of the non-synonymous SNPs, 46 were successfully converted to KASP markers. These markers were deployed on 35 tea germplasms to assess their suitability and accuracy in genetic analysis. The results show that 42 markers exhibited polymorphisms and the PIC values ranged from 0.05 to 0.38. The clustering results of the phylogenetic tree was basically consistent with the phenotype, showing that tea germplasms with high nitrogen accumulation and large biomass were grouped into one cluster. Using these markers, the fingerprints of these germplasms were constructed. The preliminary association analysis showed that there were two SNPs (CsSNP07 and CsSNP11) within CsNRT2.4 (CSS0001304) that were significantly associated with nitrogen accumulation (F = 4.631, P = 0.039 and F = 3.054, p = 0.047) and one SNP (CsSNP40) within CsAAP6 (CSS0035405) that was significantly associated with biomass (F = 3.842, p = 0.032). These functional SNP-KASP markers will be valuable for the early evaluation of tea germplasms and could accelerate the breeding of high-NUE varieties.
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Wu, Jianhui, Qilin Wang, Zhensheng Kang, Shengjie Liu, Haiyang Li, Jingmei Mu, Miaofei Dai, Dejun Han, Qingdong Zeng, and Xianming Chen. "Development and Validation of KASP-SNP Markers for QTL Underlying Resistance to Stripe Rust in Common Wheat Cultivar P10057." Plant Disease 101, no. 12 (December 2017): 2079–87. http://dx.doi.org/10.1094/pdis-04-17-0468-re.
Full textAbstract:
Stripe rust (Puccinia striiformis f. sp. tritici) is among the most important diseases of wheat (Triticum aestivum L.) globally. Utilization of adult plant resistance (APR) constitutes a key tool for maintaining protection against this disease. The CIMMYT wheat cultivar P10057 displayed a high level of APR to stripe rust in germplasm evaluation in field environments. To clarify the genetic basis and identify quantitative trait loci (QTLs) involved in stripe rust resistance in P10057, three wheat populations were used: 150 F5:6 recombinant inbred lines (RILs) derived from the cross Mingxian 169 × P10057, and 161 and 140 F2:3 lines from Avocet S × P10057 and Zhengmai 9023 × P10057, respectively. These three populations were evaluated for infection type (IT) and disease severity (DS) in Shaanxi, Gansu, and Sichuan during the 2014–15 and 2015–16 cropping seasons. Genotyping was performed with Kompetitive Allelic Specific PCR (KASP) and simple sequence repeat (SSR) markers linked to the resistance loci. Using QTL analysis, two genomic regions associated with resistance were found on chromosome arms 2BS and 3BS, respectively. These two stable QTLs, designated Qyrlov.nwafu-2BS and Qyrlov.nwafu-3BS, were detected across all environments and explained average 22.6 to 31.6% and 21.3 to 32.3% of stripe rust severity phenotypic variation, respectively. Qyrlov.nwafu-2BS may be the resistance allele derived from CIMMYT germplasm and Qyrlov.nwafu-3BS likely corresponds to the locus Sr2/Lr27/Yr30/Pbc. The KASP markers IWA5377, IWA2674, and IWA5830 linked to QYrlov.nwafu-2BS and IWB57990 and IWB6491 linked to Qyrlov.nwafu-3BS were reliable for marker-assisted selection (MAS) in the Zhengmai 9023 × P10057 population. These QTLs with KASP markers are expected to contribute in developing wheat cultivars with improved stripe rust resistance.
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Driskill, Mandie, Katie Pardee, Kim E. Hummer, Jason D. Zurn, Keenan Amundsen, Annette Wiles, Claudia Wiedow, Josef Patzak, John A. Henning, and Nahla V. Bassil. "Two fingerprinting sets for Humulus lupulus based on KASP and microsatellite markers." PLOS ONE 17, no. 4 (April 14, 2022): e0257746. http://dx.doi.org/10.1371/journal.pone.0257746.
Full textAbstract:
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).
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Offornedo, Queen, Abebe Menkir, Deborah Babalola, and Melaku Gedil. "Developing and deploying an efficient genotyping workflow for accelerating maize improvement in developing countries." Gates Open Research 6 (August 3, 2022): 3. http://dx.doi.org/10.12688/gatesopenres.13338.3.
Full textAbstract:
Background: Molecular breeding is an essential tool for accelerating genetic gain in crop improvement towards meeting the need to feed an ever-growing world population. Establishing low-cost, flexible genotyping platforms in small, public and regional laboratories can stimulate the application of molecular breeding in developing countries. These laboratories can serve plant breeding projects requiring low- to medium-density markers for marker-assisted selection (MAS) and quality control (QC) activities. Methods: We performed two QC and MAS experiments consisting of 637 maize lines, using an optimised genotyping workflow involving an in-house competitive allele-specific PCR (KASP) genotyping system with an optimised sample collection, preparation, and DNA extraction and quantitation process. A smaller volume of leaf-disc size plant samples was collected directly in 96-well plates for DNA extraction, using a slightly modified CTAB-based DArT DNA extraction protocol. DNA quality and quantity analyses were performed using a microplate reader, and the KASP genotyping and data analysis was performed in our laboratory. Results: Applying the optimized genotyping workflow expedited the QC and MAS experiments from over five weeks (when outsourcing) to two weeks and eliminated the shipping cost. Using a set of 28 KASP single nucleotide polymorphisms (SNPs) validated for maize, the QC experiment revealed the genetic identity of four maize varieties taken from five seed sources. Another set of 10 KASP SNPs was sufficient in verifying the parentage of 390 F1 lines. The KASP-based MAS was successfully applied to a maize pro-vitamin A (PVA) breeding program and for introgressing the aflatoxin resistance gene into elite tropical maize lines. Conclusion: This improved workflow has helped accelerate maize improvement activities of IITA's Maize Improvement Program and facilitated DNA fingerprinting for tracking improved crop varieties. National Agricultural Research Systems (NARS) in developing countries can adopt this workflow to fast-track molecular marker-based genotyping for crop improvement.
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Offornedo, Queen, Abebe Menkir, Deborah Babalola, and Melaku Gedil. "Developing and deploying an efficient genotyping workflow for accelerating maize improvement in developing countries." Gates Open Research 6 (January 17, 2022): 3. http://dx.doi.org/10.12688/gatesopenres.13338.1.
Full textAbstract:
Background: Molecular breeding is an essential tool for accelerating genetic gain in crop improvement, towards meeting the need to feed an ever-growing world population. Establishing low-cost, flexible genotyping platforms in small, public and regional laboratories can stimulate the application of molecular breeding in developing countries. These laboratories can serve plant breeding projects requiring low- to medium-density markers for marker-assisted selection (MAS) and quality control (QC) activities. Methods: We performed two QC and MAS experiments consisting of 637 maize lines, using an optimised genotyping workflow involving an in-house competitive allele-specific PCR (KASP) genotyping system with an optimised sample collection, preparation, and DNA extraction and quantitation process. A smaller volume of leaf-disc size plant samples was collected directly in 96-well plates for DNA extraction, using a slightly modified CTAB-based DArT DNA extraction protocol. DNA quality and quantity analyses were performed using a microplate reader, and the KASP genotyping and data analysis was performed in our laboratory. Results: Applying the optimized genotyping workflow expedited the QC and MAS experiments from over five weeks (when outsourcing) to two weeks and eliminated the shipping cost. Using a set of 28 KASP single nucleotide polymorphisms (SNPs) validated for maize, the QC experiment revealed the genetic identity of four maize varieties taken from five seed sources. Another set of 10 KASP SNPs was sufficient in verifying the parentage of 390 F1 lines. The KASP-based MAS was successfully applied to a maize pro-vitamin A (PVA) breeding program and for introgressing the aflatoxin resistance gene into elite tropical maize lines. Conclusion: This improved workflow has helped accelerate maize improvement activities of IITA's Maize Improvement Program and facilitated DNA fingerprinting for tracking improved crop varieties. National Agricultural Research Systems (NARS) in developing countries can adopt this workflow to fast-track molecular marker-based genotyping for crop improvement.
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Kim, Me-Sun, Ju-Young Yang, Ju-Kyung Yu, Yi Lee, Yong-Jin Park, Kwon-Kyoo Kang, and Yong-Gu Cho. "Breeding of High Cooking and Eating Quality in Rice by Marker-Assisted Backcrossing (MABc) Using KASP Markers." Plants 10, no. 4 (April 19, 2021): 804. http://dx.doi.org/10.3390/plants10040804.
Full textAbstract:
The primary goals of rice breeding programs are grain quality and yield potential improvement. With the high demand for rice varieties of premium cooking and eating quality, we developed low-amylose content breeding lines crossed with Samgwang and Milkyqueen through the marker-assisted backcross (MABc) breeding program. Trait markers of the SSIIIa gene referring to low-amylose content were identified through an SNP mapping activity, and the markers were applied to select favorable lines for a foreground selection. To rapidly recover the genetic background of Samgwang (recurrent parent genome, RPG), 386 genome-wide markers were used to select BC1F1 and BC2F1 individuals. Seven BC2F1 lines with targeted traits were selected, and the genetic background recovery range varied within 97.4–99.1% of RPG. The amylose content of the selected BC2F2 grains ranged from 12.4–16.8%. We demonstrated the MABc using a trait and genome-wide markers, allowing us to efficiently select lines of a target trait and reduce the breeding cycle effectively. In addition, the BC2F2 lines confirmed by molecular markers in this study can be utilized as parental lines for subsequent breeding programs of high-quality rice for cooking and eating.
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Vinarao, Ricky, Christopher Proud, Peter Snell, Shu Fukai, and Jaquie Mitchell. "QTL Validation and Development of SNP-Based High Throughput Molecular Markers Targeting a Genomic Region Conferring Narrow Root Cone Angle in Aerobic Rice Production Systems." Plants 10, no. 10 (October 3, 2021): 2099. http://dx.doi.org/10.3390/plants10102099.
Full textAbstract:
Aerobic rice production (AP) provides potential solutions to the global water crisis by consuming less water than traditional permanent water culture. Narrow root cone angle (RCA), development of deeper rooting and associated genomic regions are key for AP adaptation. However, their usefulness depends on validation across genetic backgrounds and development of linked markers. Using three F2 populations derived from IRAT109, qRCA4 was shown to be effective in multiple backgrounds, explaining 9.3–17.3% of the genotypic variation and introgression of the favourable allele resulted in 11.7–15.1° narrower RCA. Novel kompetitive allele specific PCR (KASP) markers were developed targeting narrow RCA and revealed robust quality metrics. Candidate genes related with plant response to abiotic stress and root development were identified along with 178 potential donors across rice subpopulations. This study validated qRCA4’s effect in multiple genetic backgrounds further strengthening its value in rice improvement for AP adaptation. Furthermore, the development of novel KASP markers ensured the opportunity for its seamless introgression across pertinent breeding programs. This work provides the tools and opportunity to accelerate development of genotypes with narrow RCA through marker assisted selection in breeding programs targeting AP, which may ultimately contribute to more sustainable rice production where water availability is limited.
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Bomireddy, Deekshitha, Sunil S. Gangurde, Murali T. Variath, Pasupuleti Janila, Surendra S. Manohar, Vinay Sharma, Sejal Parmar, et al. "Discovery of Major Quantitative Trait Loci and Candidate Genes for Fresh Seed Dormancy in Groundnut." Agronomy 12, no. 2 (February 6, 2022): 404. http://dx.doi.org/10.3390/agronomy12020404.
Full textAbstract:
Spanish bunch groundnut varieties occupy most of the cultivated area in Asia and Africa, and these varieties lack required 2-3 weeks of fresh seed dormancy (FSD) hampering kernel quality. Genomic breeding can help to improve commercial groundnut cultivars for FSD in a shorter time with greater precision. In this regard, a recombinant inbred line (RIL) population from the cross ICGV 02266 (non-dormant) × ICGV 97045 (dormant) was developed and genotyped with a 5 K mid-density genotyping assay. A linkage map was constructed with 325 SNP loci spanning a total map length of 2335.3 cM and five major QTLs were identified on chromosomes Ah01, Ah11, Ah06, Ah16 and Ah17. Based on differential gene expression using transcriptomic information from dormant (Tifrunner) and non-dormant (ICGV 91114) genotypes, histone deacetylases, histone-lysine N-methyltransferase, cytochrome P450, protein kinases, and ethylene-responsive transcription factor were identified as key regulators involved in the hormonal regulation of dormancy. Six Kompetitive Allele Specific PCR (KASP) markers were successfully validated in the diverse panel including selected RILs of the same population and germplasm lines. These validated KASP markers could facilitate faster breeding of new varieties with desired dormancy using marker-assisted early generation selection.
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Netpakdee, Chaiwat, Sittiwut Mathasiripakorn, Arthit Sribunrueang, Sompong Chankaew, Tidarat Monkham, Siwaret Arikit, and Jirawat Sanitchon. "QTL-Seq Approach Identified Pi63 Conferring Blast Resistance at the Seedling and Tillering Stages of Thai Indigenous Rice Variety “Phaladum”." Agriculture 12, no. 8 (August 5, 2022): 1166. http://dx.doi.org/10.3390/agriculture12081166.
Full textAbstract:
Rice blast (BL) caused by Magnaporthe oryzae is a fungal disease causing significant yield losses in rice production worldwide. To overcome the breakdown of resistance by the rapid adaptation of pathogens, identifying resistance (R) genes or QTLs in indigenous rice, which harbors the R genes that co-evolved with the local pathogen race, is necessary. In this study, a recombinant inbred line (RIL) population derived from a cross between RD6 and Phaladum (PLD) was used to map quantitative trait loci (QTL) for BL resistance through a QTL-seq approach. A single QTL (qBLchr4) associated with BL resistance at the seedling and maximum tillering stages was mapped on the long arm of chromosome 4. Five genes, LOC_Os04g0616600, LOC_Os04g0617900 (OsGLP4-1), LOC_Os04g0619600 (OsRLCK161), LOC_Os04g0620800 (Pi63), and LOC_Os04g0621500, were considered the candidate genes representing qBLchr4. Subsequently, the Kompetitive Allele-Specific PCR (KASP) markers specific for the SNP variant and position of each gene were designed for validation in the mapping population. These markers showed the high phenotypic variance explained (PVE) values in all testing methods and/or environments, signifying the major effect of qBLchr4. Among these markers, the Pi63-KASP marker explained the highest and most stable phenotypic variation across all testing methods and/or environments, with 84.18%, 80.34%, and 23.43% in the upland short row (USR) method, Sila environment, and Mueang environment, respectively. Therefore, Pi63 was suggested to be the strongest candidate gene. These results represent the potential utility of future BL resistance breeding and/or pyramiding using marker-assisted selection (MAS).
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Rosso, M. Luciana, Chao Shang, Qijian Song, Diana Escamilla, Jay Gillenwater, and Bo Zhang. "Development of Breeder-Friendly KASP Markers for Low Concentration of Kunitz Trypsin Inhibitor in Soybean Seeds." International Journal of Molecular Sciences 22, no. 5 (March 6, 2021): 2675. http://dx.doi.org/10.3390/ijms22052675.
Full textAbstract:
Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals’ protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F3:5 lines derived from PI 547656 (low KTI) X Glenn (normal KTI) were genotyped using the BARCSoySNP6K_v2 Beadchip. F3:4 and F3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.
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Ige, Adenike D., Bunmi Olasanmi, Edwige Gaby Nkouaya Mbanjo, Ismail S. Kayondo, Elizabeth Y. Parkes, Peter Kulakow, Chiedozie Egesi, et al. "Conversion and Validation of Uniplex SNP Markers for Selection of Resistance to Cassava Mosaic Disease in Cassava Breeding Programs." Agronomy 11, no. 3 (February 25, 2021): 420. http://dx.doi.org/10.3390/agronomy11030420.
Full textAbstract:
Cassava mosaic disease (CMD) is a major viral disease adversely affecting cassava production in Africa and Asia. Genomic regions conferring resistance to the disease have been mapped in African cassava germplasm through biparental quantitative trait loci (QTL) mapping and genome-wide association studies. To facilitate the utilization of these markers in breeding pipelines to support selections, proof-of-concept technical and biological validation research was carried out using independent pre-breeding and breeding populations. Kompetitive Allele-Specific Polymerase Chain Reaction (KASP) assays were designed from three single nucleotide polymorphism (SNP) markers linked to a major resistance locus on chromosome 12 (S12_7926132, S12_7926163) and a minor locus on chromosome 14 (S14_4626854). The designed assays were robust and easy to score with >99% genotype call rate. The overall predictive accuracy (proportion of true positives and true negatives) of the markers (S12_7926132 and S14_4626854) was 0.80 and 0.78 in the pre-breeding and breeding population, respectively. On average, genotypes that carried at least one copy of the resistant allele at the major CMD2 locus had a significantly higher yield advantage. Nevertheless, variation was observed in prediction accuracies for the major locus (S12_7926132) among sub-families from the two populations, suggesting the need for context-specific utilization, for example, by screening for co-segregation of favorable SNP alleles with resistance in the parents being used for crosses. Availability of these validated SNP markers on the uniplex KASP genotyping platform represents an important step in translational genetics toward marker-assisted selection to accelerate introgression of favorable resistant alleles in breeding populations.
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Mu, Jingmei, Jianhui Wu, Shengjie Liu, Miaofei Dai, Daojie Sun, Shuo Huang, Qilin Wang, et al. "Genome-Wide Linkage Mapping Reveals Stripe Rust Resistance in Common Wheat (Triticum aestivum) Xinong1376." Plant Disease 103, no. 11 (November 2019): 2742–50. http://dx.doi.org/10.1094/pdis-12-18-2264-re.
Full textAbstract:
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.
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Qureshi, N., H. S. Bariana, P. Zhang, R. McIntosh, U. K. Bansal, D. Wong, M. J. Hayden, J. Dubcovsky, and M. Shankar. "Genetic Relationship of Stripe Rust Resistance Genes Yr34 and Yr48 in Wheat and Identification of Linked KASP Markers." Plant Disease 102, no. 2 (February 2018): 413–20. http://dx.doi.org/10.1094/pdis-08-17-1144-re.
Full textAbstract:
The Australian continent was free from wheat stripe rust caused by Puccinia striiformis f. sp. tritici until exotic incursions occurred in 1979 and 2002. The 2002 incursion enabled the identification of a new stripe rust resistance gene (Yr34) in the advanced breeding line WAWHT2046. In this study, we developed and validated markers closely linked with Yr34, which is located in the distal region in the long arm of chromosome 5A. Four kompetitive allele-specific polymerase chain reaction (KASP) and three sequence-tagged site (STS) markers derived from the International Wheat Genome Sequencing Consortium RefSeq v1.0 scaffold-77836 cosegregated with Yr34. Markers sun711, sun712, sun725, sunKASP_109, and sunKASP_112 were shown to be suitable for marker-assisted selection in a validation panel of 71 Australian spring wheat genotypes, with the exception of cultivar Orion that carried the Yr34-linked alleles for sunKASP_109 and sunKASP_112. Markers previously reported to be linked with adult plant stripe rust resistance gene Yr48 also cosegregated with Yr34. Wheat genotypes carrying Yr34 and Yr48 produced identical haplotypes for the Yr34-linked markers identified in this study and those previously reported to be linked with Yr48. Phenotypic testing of genotypes carrying Yr34 and Yr48 showed that both genes conferred similar seedling responses to pre-2002 and post-2002 P. striiformis f. sp. tritici pathotypes. Further testing of 600 F2 plants from a cross between WAWHT2046 and RIL143 (Yr48) with P. striiformis f. sp. tritici pathotype 134 E16A+Yr17+Yr27+ failed to reveal any susceptible segregants. Our results strongly suggest that Yr34 and Yr48 are the same gene, and that Yr48 should be considered a synonym of Yr34.
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Xiao, Zhiliang, Congcong Kong, Fengqing Han, Limei Yang, Mu Zhuang, Yangyong Zhang, Yong Wang, et al. "Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea." Agronomy 11, no. 5 (May 14, 2021): 982. http://dx.doi.org/10.3390/agronomy11050982.
Full textAbstract:
Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.
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Alsamman, Alsamman M., Shafik D. Ibrahim, and Aladdin Hamwieh. "KASPspoon: an in vitro and in silico PCR analysis tool for high-throughput SNP genotyping." Bioinformatics 35, no. 17 (January 8, 2019): 3187–90. http://dx.doi.org/10.1093/bioinformatics/btz004.
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Abstract Motivation Fine mapping becomes a routine trial following quantitative trait loci (QTL) mapping studies to shrink the size of genomic segments underlying causal variants. The availability of whole genome sequences can facilitate the development of high marker density and predict gene content in genomic segments of interest. Correlations between genetic and physical positions of these loci require handling of different experimental genetic data types, and ultimately converting them into positioning markers using a routine and efficient tool. Results To convert classical QTL markers into KASP assay primers, KASPspoon simulates a PCR by running an approximate-match searching analysis on user-entered primer pairs against the provided sequences, and then comparing in vitro and in silico PCR results. KASPspoon reports amplimers close to or adjoining genes/SNPs/simple sequence repeats and those that are shared between in vitro and in silico PCR results to select the most appropriate amplimers for gene discovery. KASPspoon compares physical and genetic maps, and reports the primer set genome coverage for PCR-walking. KASPspoon could be used to design KASP assay primers to convert QTL acquired by classical molecular markers into high-throughput genotyping assays and to provide major SNP resource for the dissection of genotypic and phenotypic variation. In addition to human-readable output files, KASPspoon creates Circos configurations that illustrate different in silico and in vitro results. Availability and implementation Code available under GNU GPL at (http://www.ageri.sci.eg/index.php/facilities-services/ageri-softwares/kaspspoon). Supplementary information Supplementary data are available at Bioinformatics online.
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Fang, Tilin, Lei Lei, Genqiao Li, Carol Powers, Robert M. Hunger, Brett F. Carver, and Liuling Yan. "Development and deployment of KASP markers for multiple alleles of Lr34 in wheat." Theoretical and Applied Genetics 133, no. 7 (April 12, 2020): 2183–95. http://dx.doi.org/10.1007/s00122-020-03589-x.
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Tariq, Huma. "A novel and high throughput wheat (Triticum aestivum L.) genotyping using Kompetitive Allele Specific PCR assay for genes underpinning major economic attributes." Pakistan Journal of Agricultural Sciences 58, no. 06 (December 1, 2021): 1799–807. http://dx.doi.org/10.21162/pakjas/21.900.
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Wheat breeding programs are increasingly using molecular tools to improve efficiency and speed of developing productive cultivars. Molecular markers such as Kompetitive Allele Specific PCR (KASP) have now become a principal approach to study genetic relationship among various wheat species. The objective of our research study was to identify and distinguished the wheat parents and their hybrid for genes controlling major agronomical attributes. Germplasm comprised of fifteen genotypes were genotyped for gluten strength (Glu A1, Glu D1), height reduction (Rht 1), starch quality (Waxy), vernalization (Vrn A1, Vrn B1), photoperiod sensitivity (Ppd 1), Tan spot disease (Tns-1) and leaf rust disease (Lr34). Statistical analysis with endpoint genotyping, graphical display and principal coordinate revealed significant results for KASP assay. Most variable crosses were S3 X I1, S3 X I3, S2 X I1, S2 X I2 and S1 X I1 with maximum segregants. Whereas, variation was observed for Glu-D1, Tsn1, Glu-A1 followed by Lr34 alleles. Therefore, the objective was achieved successfully with the most reliable KASP markers. The diverse attributes dissected, and key hybrids identified in this study would provide guidance for the improvement of the economically important traits and provide understanding of genetic variability in distinct segregating populations. Besides we have identified significantly important partial mutants in the germplasm for Waxy allele. These findings could provide the end user with wheat products with improved quality such as with or without amylose content.
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Schulz, Dietmar, Marcus Linde, and Thomas Debener. "Detection of Reproducible Major Effect QTL for Petal Traits in Garden Roses." Plants 10, no. 5 (April 29, 2021): 897. http://dx.doi.org/10.3390/plants10050897.
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The detection of QTL by association genetics depends on the genetic architecture of the trait under study, the size and structure of the investigated population and the availability of phenotypic and marker data of sufficient quality and quantity. In roses, we previously demonstrated that major QTL could already be detected in small association panels. In this study, we analyzed petal number, petal size and fragrance in a small panel of 95 mostly tetraploid garden rose genotypes. After genotyping the panel with the 68 K Axiom WagRhSNP chip we detected major QTL for all three traits. Each trait was significantly influenced by several genomic regions. Some of the QTL span genomic regions that comprise several candidate genes. Selected markers from some of these regions were converted into KASP markers and were validated in independent populations of up to 282 garden rose genotypes. These markers demonstrate the robustness of the detected effects independent of the set of genotypes analyzed. Furthermore, the markers can serve as tools for marker-assisted breeding in garden roses. Over an extended timeframe, they may be used as a starting point for the isolation of the genes underlying the QTL.
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Jang, Woojong, Hyun Oh Lee, Jang-Uk Kim, Jung-Woo Lee, Chi-Eun Hong, Kyong-Hwan Bang, Jong-Wook Chung, and Ick-Hyun Jo. "Complete Mitochondrial Genome and a Set of 10 Novel Kompetitive Allele-Specific PCR Markers in Ginseng (Panax ginseng C. A. Mey.)." Agronomy 10, no. 12 (November 26, 2020): 1868. http://dx.doi.org/10.3390/agronomy10121868.
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Panax ginseng C. A. Mey., a perennial herb belonging to the family Araliaceae, is a valuable medicinal plant with distinctive biological characteristics. However, comprehensive analyses of the mitochondrial genome (mitogenome) are lacking. In this study, we sequenced the complete mitogenome of ginseng based on long-read data from the Nanopore sequencing platform. The mitogenome was assembled into a “master circle” form of 464,705 bp and contained 72 unique genes. The genome had three large repeat regions, and 10.42% of the sequences were mitogenome sequences of plastid origin (MTPTs). In total, 278 variants (213 SNPs and 65 InDels) were discovered, most of which were identified in intergenic regions. The MTPT regions were mutational hotspots, harboring 74.5% of the variants. The ginseng mitogenome showed a higher mutation rate than that of the chloroplast genome, and this pattern is uncommon in plants. In addition, 10 Kompetitive allele-specific PCR (KASP) markers were developed from 10 SNPs, excluding those in MTPT regions. These markers accurately identified the genotypes of 59 Korean ginseng accessions and elucidated mitogenome diversity. These results provide insight into organellar genomes and genetic diversity in ginseng. Moreover, the complete mitogenome sequence and 10 KASP markers will be useful for ginseng research and breeding.
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Seo, Jeong-Hyun, Sanjeev Kumar Dhungana, Beom-Kyu Kang, In-Youl Baek, Jung-Sook Sung, Jee-Yeon Ko, Chan-Sik Jung, Ki-Seung Kim, and Tae-Hwan Jun. "Development and Validation of SNP and InDel Markers for Pod-Shattering Tolerance in Soybean." International Journal of Molecular Sciences 23, no. 4 (February 21, 2022): 2382. http://dx.doi.org/10.3390/ijms23042382.
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Pod-shattering causes a significant yield loss in many soybean cultivars. Shattering-tolerant cultivars provide the most effective approach to minimizing this loss. We developed molecular markers for pod-shattering and validated them in soybeans with diverse genetic backgrounds. The genes Glyma.16g141200, Glyma.16g141500, and Glyma.16g076600, identified in our previous study by quantitative trait locus (QTL) mapping and whole-genome resequencing, were selected for marker development. The whole-genome resequencing of three parental lines (one shattering-tolerant and two shattering-susceptible) identified single nucleotide polymorphism (SNP) and/or insertion/deletion (InDel) regions within or near the selected genes. Two SNPs and one InDel were converted to Kompetitive Allele-Specific PCR (KASP) and InDel markers, respectively. The accuracy of the markers was examined in the two recombinant inbred line populations used for the QTL mapping, as well as the 120 varieties and elite lines, through allelic discrimination and phenotyping by the oven-drying method. Both types of markers successfully discriminated the pod shattering-tolerant and shattering-susceptible genotypes. The prediction accuracy, which was as high as 90.9% for the RILs and was 100% for the varieties and elite lines, also supported the accuracy and usefulness of these markers. Thus, the markers can be used effectively for genetic and genomic studies and the marker-assisted selection for pod-shattering tolerance in soybean.
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Tsehay, Sewalem, Rodomiro Ortiz, Eva Johansson, Endashaw Bekele, Kassahun Tesfaye, Cecilia Hammenhag, and Mulatu Geleta. "New Transcriptome-Based SNP Markers for Noug (Guizotia abyssinica) and Their Conversion to KASP Markers for Population Genetics Analyses." Genes 11, no. 11 (November 20, 2020): 1373. http://dx.doi.org/10.3390/genes11111373.
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The development and use of genomic resources are essential for understanding the population genetics of crops for their efficient conservation and enhancement. Noug (Guizotia abyssinica) is an economically important oilseed crop in Ethiopia and India. The present study sought to develop new DNA markers for this crop. Transcriptome sequencing was conducted on two genotypes and 628 transcript sequences containing 959 single nucleotide polymorphisms (SNPs) were developed. A competitive allele-specific PCR (KASP) assay was developed for the SNPs and used for genotyping of 24 accessions. A total of 554 loci were successfully genotyped across the accessions, and 202 polymorphic loci were used for population genetics analyses. Polymorphism information content (PIC) of the loci varied from 0.01 to 0.37 with a mean of 0.24, and about 49% of the loci showed significant deviation from the Hardy-Weinberg equilibrium. The mean expected heterozygosity was 0.27 suggesting moderately high genetic variation within accessions. Low but significant differentiation existed among accessions (FST = 0.045, p < 0.0001). Landrace populations from isolated areas may have useful mutations and should be conserved and used in breeding this crop. The genomic resources developed in this study were shown to be useful for population genetics research and can also be used in, e.g., association genetics.
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Patil, Gunvant, Juhi Chaudhary, Tri D. Vuong, Brian Jenkins, Dan Qiu, Suhas Kadam, Grover J. Shannon, and Henry T. Nguyen. "Development of SNP Genotyping Assays for Seed Composition Traits in Soybean." International Journal of Plant Genomics 2017 (May 25, 2017): 1–12. http://dx.doi.org/10.1155/2017/6572969.
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Seed composition is one of the most important determinants of the economic values in soybean. The quality and quantity of different seed components, such as oil, protein, and carbohydrates, are crucial ingredients in food, feed, and numerous industrial products. Soybean researchers have successfully developed and utilized a diverse set of molecular markers for seed trait improvement in soybean breeding programs. It is imperative to design and develop molecular assays that are accurate, robust, high-throughput, cost-effective, and available on a common genotyping platform. In the present study, we developed and validated KASP (Kompetitive allele-specific polymerase chain reaction) genotyping assays based on previously known functional mutant alleles for the seed composition traits, including fatty acids, oligosaccharides, trypsin inhibitor, and lipoxygenase. These assays were validated on mutant sources as well as mapping populations and precisely distinguish the homozygotes and heterozygotes of the mutant genes. With the obvious advantages, newly developed KASP assays in this study can substitute the genotyping assays that were previously developed for marker-assisted selection (MAS). The functional gene-based assay resource developed using common genotyping platform will be helpful to accelerate efforts to improve soybean seed composition traits.
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Tan, Chor-Tee, Silvano Assanga, Guorong Zhang, Jackie C. Rudd, Scott D. Haley, Qingwu Xue, Amir Ibrahim, et al. "Development and Validation of KASP Markers for Wheat Streak Mosaic Virus Resistance Gene Wsm2." Crop Science 57, no. 1 (November 1, 2016): 340–49. http://dx.doi.org/10.2135/cropsci2016.04.0234.
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KÖSOĞLU, Kevser, Sevcan YUMUK, Yıldız AYDIN, Göksel EVCİ, and Ahu ALTINKUT UNCUOĞLU. "Use of SNP markers by KASP assay for MAS studies in sunflower againstPlasmopara halstedii." TURKISH JOURNAL OF AGRICULTURE AND FORESTRY 41 (2017): 480–89. http://dx.doi.org/10.3906/tar-1707-53.
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Li, Peirong, Tongbing Su, Huiping Wang, Xiuyun Zhao, Weihong Wang, Yangjun Yu, Deshuang Zhang, Changlong Wen, Shuancang Yu, and Fenglan Zhang. "Development of a core set of KASP markers for assaying genetic diversity inBrassica rapasubsp.chinensisMakino." Plant Breeding 138, no. 3 (March 5, 2019): 309–24. http://dx.doi.org/10.1111/pbr.12686.
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Liu, Ling-Ling, Chao Fang, Hai-Yu Ma, Xi Yu, Shi-Peng Lv, and Wu-Jun Liu. "Development and validation of KASP markers for the milk traits genes in Kazakh horse." Journal of Applied Animal Research 48, no. 1 (January 1, 2020): 293–99. http://dx.doi.org/10.1080/09712119.2020.1782218.
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Kim, Me-Sun, Ju-Kyung Yu, Seo-Rin Ko, Ki-Jo Kim, Hyeonso Ji, Kwon-Kyoo Kang, and Yong-Gu Cho. "Marker-Assisted Backcrossing (MABc) to Improve Eating Quality with Thin Seed Coat and Aleurone Layer of Non-Glutinous Japonica Variety in Rice." Genes 13, no. 2 (January 24, 2022): 210. http://dx.doi.org/10.3390/genes13020210.
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Brown rice is composed of rice bran, pericarp, seed coat, and aleurone layers, and the rice bran layer contains a large number of substances useful for the human body, such as dietary fiber, α-tocopherol, α-tocotrienol, and vitamins. However, more than 90% of these substances are removed when polished, and white rice has the disadvantage of losing food-related ingredients, such as umami-related amino acids, when compared to the unpolished group. In this study, we tried to develop new breeding lines with a thinner seed coat and aleurone layer to provide high eating quality with softer chewing characteristics and processability in rice grain. We detected an SNP for foreground selection for the backcross population by comparing genome sequences between Samgwang and Seolgaeng and developed high eating quality brown rice breeding lines by applying marker-assisted backcrossing (MABC) breeding programs to backcross populations between Samgwang and Seolgaeng using KASP markers. SNP markers for foreground selection were identified to improve eating and processability through SNP mapping of Samgwang and Seolgaeng with SSIIa as a target gene in this study. Line selection according to genotype of KASP markers was successful in BC1F1 and BC2F1 generations, with the recurrent parent genome recovery ratio ranging from 91.22% to 98.65%. In BC2F1 seeds of the selected lines, thickness of the aleurone layer was found to range from 13.82 to 21.67 μm, which is much thinner than the 30.91 μm of the wild type, suggesting that selection by MABc could be used as an additional breeding material for the development of highly processed rice varieties. These lines will be useful to develop new brown rice varieties with softer chewing characteristics and processability in rice grain.
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Van de Wouw, Angela P., Yueqi Zhang, Nur Shuhadah Mohd Saad, Hua Yang, Elizabeth Sheedy, Candace E. Elliott, and Jacqueline Batley. "Molecular Markers for Identifying Resistance Genes in Brassica napus." Agronomy 12, no. 5 (April 20, 2022): 985. http://dx.doi.org/10.3390/agronomy12050985.
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Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, is the most devastating disease of canola (Brassica napus, oilseed rape) worldwide. Breeding for genetic resistance is the most widely used tool for controlling this disease and minimizing the impact on yield. To date, five resistance genes (Rlm2, LepR3, Rlm4, Rlm7, Rlm9) have been cloned from B. napus, representing alleles of two different gene loci, Rlm2-LepR3 and Rlm4-7-9. We report on the development and validation of Kompetitive Allele-Specific PCR (KASP) markers that can discriminate between the resistant and susceptible alleles of each resistance gene. These markers will provide valuable tools for both researchers and industry through the ability to characterize resistance genes without phenotyping.
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Nissan, Nour, Julia Hooker, Arezo Pattang, Martin Charette, Malcolm Morrison, Kangfu Yu, Anfu Hou, et al. "Novel QTL for Low Seed Cadmium Accumulation in Soybean." Plants 11, no. 9 (April 24, 2022): 1146. http://dx.doi.org/10.3390/plants11091146.
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Soybean is a valuable crop, used in animal feed and for human consumption. Selecting soybean cultivars with low seed cadmium (Cd) concentration is important for the purpose of minimizing the transfer of Cd into the human body. To ensure international trade, farmers need to produce soybean that meets the European Union (EU) Cd limit of 0.2 mg kg−1. In this study, we evaluated two populations of recombinant inbred lines (RILs), X5154 and X4050, for seed Cd accumulation. Linkage maps were constructed with 325 and 280 polymorphic simple sequence repeat (SSR) markers, respectively, and used to identify a novel minor quantitative trait locus (QTL) on chromosome 13 in the X4050 population between SSR markers Satt522 and Satt218. Based on a gene ontology search within the QTL region, seven genes were identified as candidates responsible for low seed Cd accumulation, including Glyma.13G308700 and Glyma.13G309100. In addition, we confirmed the known major gene, Cda1, in the X5154 population and developed KASP and CAPS/dCAPS allele-specific markers for efficient marker-assisted breeding for Cda1.
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Esuma, Williams, Oscar Eyoo, Francisca Gwandu, Settumba Mukasa, Titus Alicai, Alfred Ozimati, Ephraim Nuwamanya, Ismail Rabbi, and Robert Kawuki. "Validation of KASP markers associated with cassava mosaic disease resistance, storage root dry matter and provitamin A carotenoid contents in Ugandan cassava germplasm." Frontiers in Plant Science 13 (November 23, 2022). http://dx.doi.org/10.3389/fpls.2022.1017275.
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IntroductionThe intrinsic high heterozygosity of cassava makes conventional breeding ineffective for rapid genetic improvement. However, recent advances in next generation sequencing technologies have enabled the use of high-density markers for genome-wide association studies, aimed at identifying single nucleotide polymorphisms (SNPs) linked to major traits such as cassava mosaic disease (CMD) resistance, dry matter content (DMC) and total carotenoids content (TCC). A number of these trait-linked SNPs have been converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for downstream application of marker assisted selection.MethodsWe assayed 13 KASP markers to evaluate their effectiveness in selecting for CMD, DMC and TCC in 1,677 diverse cassava genotypes representing two independent breeding populations in Uganda.ResultsFive KASP markers had significant co-segregation with phenotypes; CMD resistance (2), DMC (1) and TCC (2), with each marker accounting for at least 30% of the phenotypic variation. Markers located within the chromosomal regions for which strong marker-trait association loci have been characterised (chromosome 12 markers for CMD, chromosome 1 markers for DMC and TCC) had consistently superior ability to discriminate the respective phenotypes.DiscussionThe results indicate varying discriminatory abilities of the KASP markers assayed and the need for their context-based use for MAS, with PSY2_572 particularly effective in selecting for high TCC. Availing the effective KASP markers on cost-effective genotyping platforms could facilitate practical implementation of marker-assisted cassava breeding for accelerated genetic gains for CMD, DMC and provitamin A carotenoids.