A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Perović, Jelena; Silvar, Cristina; Koenig, Janine; Stein, Nils; Perovic, Dragan; Ordon, Frank

doi:10.1007/s11032-013-9852-x

Cited by 14 publications

(6 citation statements)

References 28 publications

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“…Markers, for which polymorphisms were based on presence or absence of polymerase chain reaction (PCR) fragments between parental lines, were directly mapped. In turn, SNPs were transformed to cleaved amplified polymorphic sequence (CAPS) markers (Perovic et al, 2013b) or pyrosequencing markers using a biotin‐labeled M13 primer (Silvar et al, 2011b). Linkage analyses were performed with JoinMap 4.0 (Van Ooijen, 2006).…”

Section: Methodsmentioning

confidence: 99%

Assessing the Barley Genome Zipper and Genomic Resources for Breeding Purposes

et al. 2015

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The aim of this study was to estimate the accuracy and convergence of newly developed barley (Hordeum vulgare L.) genomic resources, primarily genome zipper (GZ) and population sequencing (POPSEQ), at the genome-wide level and to assess their usefulness in applied barley breeding by analyzing seven known loci. Comparison of barley GZ and POPSEQ maps to a newly developed consensus genetic map constructed with data from 13 individual linkage maps yielded an accuracy of 97.8% (GZ) and 99.3% (POPSEQ), respectively, regarding the chromosome assignment. The percentage of agreement in marker position indicates that on average only 3.7% GZ and 0.7% POPSEQ positions are not in accordance with their centimorgan coordinates in the consensus map. The fine-scale comparison involved seven genetic regions on chromosomes 1H, 2H, 4H, 6H, and 7H, harboring major genes and quantitative trait loci (QTL) for disease resistance. In total, 179 GZ loci were analyzed and 64 polymorphic markers were developed. Entirely, 89.1% of these were allocated within the targeted intervals and 84.2% followed the predicted order. Forty-four markers showed a match to a POPSEQ-anchored contig, the percentage of collinearity being 93.2%, on average. Forty-four markers allowed the identification of twenty-five fingerprinted contigs (FPCs) and a more clear delimitation of the physical regions containing the traits of interest. Our results demonstrate that an increase in marker density of barley maps by using new genomic data significantly improves the accuracy of GZ. In addition, the combination of different barley genomic resources can be considered as a powerful tool to accelerate barley breeding.

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Section: Methodsmentioning

confidence: 99%

Assessing the Barley Genome Zipper and Genomic Resources for Breeding Purposes

et al. 2015

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“…Various codominant marker platforms have been used effectively to map resistance genes in crop plants. Simple sequence repeat (SSR) markers or microsatellites are highly polymorphic and reproducible; however, they are not amenable for high throughput even in the case of modified capillary systems (Perovic et al 2013a ) nor as abundant as single-nucleotide polymorphism (SNP). Due to the property of abundance and high throughput, SNP markers have become the most amenable for gene mapping and breeding (Silvar et al 2011 ; Rasheed et al 2017 ; Lu et al 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Delineating the elusive BaMMV resistance gene rym15 in barley by medium-resolution mapping

et al. 2021

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Barley mild mosaic virus (BaMMV), transmitted by the soil-borne protist Polymyxa graminis, has a serious impact on winter barley production. Previously, the BaMMV resistance gene rym15 was mapped on chromosome 6HS, but the order of flanking markers was non-collinear between different maps. To resolve the position of the flanking markers and to enable map-based cloning of rym15, two medium-resolution mapping populations Igri (susceptible) × Chikurin Ibaraki 1 (resistant) (I × C) and Chikurin Ibaraki 1 × Uschi (susceptible) (C × U), consisting of 342 and 180 F2 plants, respectively, were developed. Efficiency of the mechanical inoculation of susceptible standards varied from 87.5 to 100% and in F2 populations from 90.56 to 93.23%. Phenotyping of F2 plants and corresponding F3 families revealed segregation ratios of 250 s:92r (I × C, χ2 = 0.659) and 140 s:40r (C × U, χ2 = 0.741), suggesting the presence of a single recessive resistance gene. After screening the parents with the 50 K Infinium chip and anchoring corresponding SNPs to the barley reference genome, 8 KASP assays were developed and used to remap the gene. Newly constructed maps revealed a collinear order of markers, thereby allowing the identification of high throughput flanking markers. This study demonstrates how construction of medium-resolution mapping populations in combination with robust phenotyping can efficiently resolve conflicting marker ordering and reduce the size of the target interval. In the reference genome era and genome-wide genotyping era, medium-resolution mapping will help accelerate candidate gene identification for traits where phenotyping is difficult.

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“…Based on these five DH-lines four crosses were conducted, namely DH3/74 (S) × DH3/6 (R), DH3/74 (S) × DH3/127 (R), DH3/6 (R) × DH3/9 (S) and DH3/62 (S) × DH3/127 (R) (Table 1). In order to identify recombinants, F 2 plants were analyzed using two flanking co-dominant SSRs, i.e., QBS94 (distal) and QBS113 (proximal) (Perovic et al, 2013). Respective markers were analyzed by capillary electrophoresis at the genetic analyzer ABI PRISM ® 3100 (Applied Biosystems, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Molecular markers used may be divided in five types as follows: six SSRs based markers from the pyrosequencing assay (Silvar et al, 2011), three dominant present/absent markers, four size polymorphism markers [insertion/deletion polymorphisms (InDels)], 19 KASP markers and 24 Cleaved Amplified Polymorphic Sequences (CAPS) markers. Size polymorphisms markers and SSRs were amplified in a total volume of 10 μl, according to Perovic et al (2013) and detected either using fluorescently labeled primers (M13) by capillary electrophoresis on the ABI Genetic Analyzer (ABI sequencer, ABI Perkin Elmer, Weiterstadt, Germany), or directly separated on a 1.5% agarose gel. For ABI analysis, 0.1 μl of M13 primer (10.0 pmol/μl)(5′-CACGACGTTGTAAAACGAC-3′) labeled with fluorescent dye was added to the reaction mix.…”

Section: Methodsmentioning

confidence: 99%

High Resolution Mapping of RphMBR1012 Conferring Resistance to Puccinia hordei in Barley (Hordeum vulgare L.)

et al. 2019

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Isolation of disease resistance genes in barley was hampered by the large genome size, but has become easy due to the availability of the reference genome sequence. During the last years, many genomic resources, e.g., the Illumina 9K iSelect, the 50K Infinium arrays, the Barley Genome Zipper, POPSEQ, and genotyping by sequencing (GBS), were developed that enable enhanced gene isolation in combination with the barley genome sequence. In the present study, we developed a fine map of the barley leaf rust resistance gene Rph MBR 1012 . 537 segmental homozygous recombinant inbred lines (RILs) derived from 4775 F 2 -plants were used to construct a high-resolution mapping population (HRMP). The Barley Genome Zipper, the 9K iSelect chip, the 50K Infinium chip and GBS were used to develop 56 molecular markers located in the target interval of 8 cM. This interval was narrowed down to about 0.07 cM corresponding to 0.44 Mb of the barley reference genome. Eleven low-confidence and 18 high-confidence genes were identified in this interval. Five of these are putative disease resistance genes and were subjected to allele-specific sequencing. In addition, comparison of the genetic map and the reference genome revealed an inversion of 1.34 Mb located distally to the resistance locus. In conclusion, the barley reference sequence and the respective gene annotation delivered detailed information about the physical size of the target interval, the genes located in the target interval and facilitated the efficient development of molecular markers for marker-assisted selection for Rph MBR1012.

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A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Cited by 14 publications

References 28 publications

Assessing the Barley Genome Zipper and Genomic Resources for Breeding Purposes

Assessing the Barley Genome Zipper and Genomic Resources for Breeding Purposes

Delineating the elusive BaMMV resistance gene rym15 in barley by medium-resolution mapping

High Resolution Mapping of RphMBR1012 Conferring Resistance to Puccinia hordei in Barley (Hordeum vulgare L.)

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