Search

Chinese cabbage is a popular leaf vegetable, and many cultivars have been developed for different regions and growing seasons. For the identification of the cultivars, many marker systems have been applied, but they usually require a large number of markers and expensive equipment. Therefore, it is necessary to develop an efficient and economical method for identifying Chinese cabbage cultivars. In this study, we aimed to develop a marker system with the minimum number of markers using simple PCR and gel electrophoresis. A total of 48 simple sequence repeats (SSRs) in a previous study were screened based on their chromosomal location and applied to 105 Chinese cabbage cultivars. The minimum number of markers was selected based on their genomic location, polymorphic information content, and allele frequency. To validate the cultivar identification capability of selected SSRs, they were applied to genetically similar cultivar pairs from a previous study. Eleven SSRs were finally selected, and they successfully identified cultivars with high genetic similarity, as well as all 105 Chinese cabbage cultivars tested. The proposed SSRs require only 11 primer sets, simple PCR, and gel electrophoresis, which need less time and resources compared to previous ones. These SSRs can be used not only in small seed companies and laboratories but also in large-scale seed companies. [ABSTRACT FROM AUTHOR]

Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCRamplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon, Cenchrus, Solanum, and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus, Cynodon, Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species’ introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified. [ABSTRACT FROM AUTHOR]

Radish is a root vegetable crop, and many F1 cultivars have been developed by exploiting heterosis. Considerable time and resources are required to test the performance and combining ability of F1 cultivars and parental lines; therefore, economical methods to identify them are needed before field tests. This study was conducted to determine the minimum number of markers needed to identify cultivars and elite lines. PCR-based markers were applied to radish cultivars and elite lines and selected based on chromosomal location, polymorphic information content, and allele frequency. The power of selected marker sets to detect genetically similar cultivars or elite lines was confirmed using cultivars with high genetic similarity and elite lines with the same parents. Eleven markers successfully identified not only all 105 radish cultivars tested but also cultivars with high genetic similarity reported in previous studies. Fourteen markers, including the nine among 11 markers for cultivar identification, also successfully distinguished all 115 elite lines tested, including ones with the same parents and separated at F2–F6 generations. The markers developed for the identification of radish cultivars and elite lines require only 11 and 14 markers, respectively, and simple PCR and gel electrophoresis instruments. This method is very simple and can be easily applied to other crops. [ABSTRACT FROM AUTHOR]

Chinese cabbage is a popular leaf vegetable, and many cultivars have been developed for different regions and growing seasons. For the identification of the cultivars, many marker systems have been applied, but they usually require a large number of markers and expensive equipment. Therefore, it is necessary to develop an efficient and economical method for identifying Chinese cabbage cultivars. In this study, we aimed to develop a marker system with the minimum number of markers using simple PCR and gel electrophoresis. A total of 48 simple sequence repeats (SSRs) in a previous study were screened based on their chromosomal location and applied to 105 Chinese cabbage cultivars. The minimum number of markers was selected based on their genomic location, polymorphic information content, and allele frequency. To validate the cultivar identification capability of selected SSRs, they were applied to genetically similar cultivar pairs from a previous study. Eleven SSRs were finally selected, and they successfully identified cultivars with high genetic similarity, as well as all 105 Chinese cabbage cultivars tested. The proposed SSRs require only 11 primer sets, simple PCR, and gel electrophoresis, which need less time and resources compared to previous ones. These SSRs can be used not only in small seed companies and laboratories but also in large-scale seed companies.

Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCR-amplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon, Cenchrus, Solanum, and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus, Cynodon, Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species’ introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified.

Radish is a root vegetable crop, and many F1 cultivars have been developed by exploiting heterosis. Considerable time and resources are required to test the performance and combining ability of F1 cultivars and parental lines; therefore, economical methods to identify them are needed before field tests. This study was conducted to determine the minimum number of markers needed to identify cultivars and elite lines. PCR-based markers were applied to radish cultivars and elite lines and selected based on chromosomal location, polymorphic information content, and allele frequency. The power of selected marker sets to detect genetically similar cultivars or elite lines was confirmed using cultivars with high genetic similarity and elite lines with the same parents. Eleven markers successfully identified not only all 105 radish cultivars tested but also cultivars with high genetic similarity reported in previous studies. Fourteen markers, including the nine among 11 markers for cultivar identification, also successfully distinguished all 115 elite lines tested, including ones with the same parents and separated at F2–F6 generations. The markers developed for the identification of radish cultivars and elite lines require only 11 and 14 markers, respectively, and simple PCR and gel electrophoresis instruments. This method is very simple and can be easily applied to other crops.

Fingerprinting of crops is gaining importance in plant breeding due to its applications for variety protection, dispute resolution, and forensic science studies. Before the development of genomic and proteomic technologies, varieties were differentiated based on morphological markers using distinctiveness, uniformity, and stability (DUS). However, in the mid-20th century, protein-based markers were discovered and were used for fingerprinting and genetic diversity analysis of crops. Now in the genomics era, deoxyribonucleic acid (DNA) markers are frequently used for fingerprinting of crops. The journey of crop fingerprinting using DNA markers started from RFLPs, which were non-PCR based markers, and progress through to polymerase chain reaction (PCR) based markers randomly emplified polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPS), simple sequence repeat markers (SSRs), inter simple sequence repeats (ISSRs), single nucleotide polymorphism (SNPs), diversity array technology (DArT). The latest technological advancements in the field of genomics and bioinformatics have shifted focus towards whole genome sequencing of crops rather than using different marker systems. The future of crop fingerprinting is linked with the development of cost-effective whole genome sequencing techniques. Such technologies would allow differentiation of highly similar varieties, mutants, some clones and vegetatively propagated crops. In this review a brief overview of different marker systems is given for their effectiveness in DNA fingerprinting of crops and their putative applications, with recommendations for future developments. [ABSTRACT FROM AUTHOR]

Maize is the only major cereal crop that can naturally accumulate appreciable levels of carotenoids which are the source of provitamin A. The association of the total carotenoid content in maize kernels with allelic variation PSY1 InDel1 was estimated. We used a collection of 54 maize genotypes of different ecogeographical origins, methods of PCR analysis, spectrophotometry, and statistical analysis in this study. The total carotenoid content of genotypes with a favorable allele of InDel1 is 0.60 mg/100 g, which is significantly higher than that of the samples with an unfavorable allele (0.43 mg/100 g). Thus, the use of PCR-based markers for PSY1 InDell polymorphism is a reliable method for identifying genotypes with high carotenoid accumulation in maize kernels. [ABSTRACT FROM AUTHOR]

Rye (Secale cereale L.) 4R chromosome contains elite genes that are applicable for wheat (Triticum aestivum L.) cultivar improvement. PCR-based 4R-specific markers can benefit the detection of elite genes on 4R in wheat backgrounds. In this study, a new fluorescence in situ hybridization (FISH) map of the 4RKu chromosome of rye Kustro has been constructed. A set of 4RKu dissection lines was obtained and 301 new 4RKu-specific markers were developed using specific length amplified fragment sequencing (SLAF-seq) technology. These markers were combined with the 99 4RKu-specific markers previously developed, and were physically mapped to 4RKu chromosome using the new FISH map and the 4RKu dissection lines. A total of 338 of the 400 markers have been successfully mapped to six regions of 4RKu chromosome. Additionally, the powdery mildew resistance gene(s) on the 4RLKu arm was located to the segment between L.4 and L.8, the same region where 115 4RLKu-specific markers were mapped. The markers developed in this study can be used to identify a specific segment of 4R chromatin in wheat backgrounds, help construct a high-density physical map of 4R chromosome, and facilitate the utilization of elite genes on 4R chromosome in wheat breeding programs.

The rye (Secale cereale L.) 5R chromosome contains some elite genes that can be used to improve wheat cultivars. In this study, a set of 5RKu dissection lines was obtained, and 111 new PCR-based and 5RKu-specific markers were developed using the specific length amplified fragment sequencing (SLAF-seq) method. The 111 markers were combined with the 52 5RKu-specific markers previously reported, and 65 S. cereale Lo7 scaffolds were physically mapped to six regions of the 5RKu chromosome using the 5RKu dissection lines. Additionally, the 5RLKu arm carried stripe rust resistance gene(s) and it was located to the region L2, the same region where 22 5RKu-specific markers and 11 S. cereale Lo7 scaffolds were mapped. The stripe rust resistance gene(s) located in the 5RLKu arm might be new one(s) because its source and location are different from the previously reported ones, and it enriches the resistance source of stripe rust for wheat breeding programs. The markers and the S. cereale Lo7 scaffolds that were mapped to the six regions of the 5RKu chromosome can facilitate the utilization of elite genes on the 5R chromosome in the improvement of wheat cultivars.

Background: Single nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci (QTL) studies. Marker validation is essential for QTL studies, but the cost and workload are considerable when large numbers of markers need to be verified. Marker systems with low development costs would be most suitable for this task. Results: We have tested allele specific polymerase chain reaction (PCR), tetra markers and a genotyping tool based on the single strand specific nuclease CEL-I to verify randomly selected SNP markers identified previously either with a SNP array or by genotyping by sequencing in rice and mungbean, respectively. The genotyping capacity of allele-specific PCR and tetra markers was affected by the sequence context surrounding the SNP; SNPs located in repeated sequences and in GC-rich stretches could not be correctly identified. In contrast, CEL-I digestion of mixed fragments produced from test and reference DNA reliably pinpointed the correct genotypes, yet scoring of the genotypes became complicated when multiple SNPs were present in the PCR fragments. A cost analysis showed that as long the sample number remains small, CEL-I genotyping is more cost-effective than tetra markers. Conclusions: CEL-I genotyping performed better in terms of genotyping accuracy and costs than tetra markers. The method is highly useful for validating SNPs in small to medium size germplasm panels. [ABSTRACT FROM AUTHOR]

Investigating the origin and dispersal pathways is instrumental to mitigate threats and economic and environmental consequences of invasive crop pathogens. In the case of Puccinia striiformis causing yellow rust on wheat, a number of economically important invasions have been reported, e.g., the spreading of two aggressive and high temperature adapted strains to three continents since 2000. The combination of sequence-characterized amplified region ( SCAR) markers, which were developed from two specific AFLP fragments, differentiated the two invasive strains, PstS1 and PstS2 from all other P. striiformis strains investigated at a worldwide level. The application of the SCAR markers on 566 isolates showed that PstS1 was present in East Africa in the early 1980s and then detected in the Americas in 2000 and in Australia in 2002. PstS2 which evolved from PstS1 became widespread in the Middle East and Central Asia. In 2000, PstS2 was detected in Europe, where it never became prevalent. Additional SSR genotyping and virulence phenotyping revealed 10 and six variants, respectively, within PstS1 and PstS2, demonstrating the evolutionary potential of the pathogen. Overall, the results suggested East Africa as the most plausible origin of the two invasive strains. The SCAR markers developed in the present study provide a rapid, inexpensive, and efficient tool to track the distribution of P. striiformis invasive strains, PstS1 and PstS2. [ABSTRACT FROM AUTHOR]

Common bean ( Phaseolus vulgaris ) is the most important grain legume for direct human consumption in the world, particularly in developing countries where it constitutes the main source of protein. Unfortunately, common bean yield stability is constrained by a number of pests and diseases. As use of resistant genotypes is the most economic and ecologically safe means for controlling plant diseases, efforts have been made to genetically characterize resistance genes ( R genes) in common bean. Despite its agronomic importance, genomic resources available in common bean were limited until the recent sequencing of common bean genome (Andean genotype G19833). Besides allowing the annotation of Nucleotide Binding-Leucine Rich Repeat (NB-LRR) encoding gene family, which is the prevalent class of disease R genes in plants, access to the whole genome sequence of common bean can be of great help for intense selection to increase the overall efficiency of crop improvement programs using marker-assisted selection (MAS). This review presents the state of the art of common bean NB-LRR gene clusters, their peculiar location in subtelomeres and correlation with genetically characterized monogenic R genes, as well as how the availability of the whole genome sequence can boost the development of molecular markers for MAS. [ABSTRACT FROM AUTHOR]

Sequence variations involving DNA insertions and deletions within introns provide suitable targets for the development of PCR-based markers. In this study, 37861 potential introns were identified in sorghum and primers were designed to develop intron length polymorphism (ILP) markers. Drastic variation was observed in the number of ILP markers on each chromosome with a range of 1498 (chr. 5) - 7290 (chr. 1), which was also reflected in the fluctuations in their density. About 200 ILP markers were assessed for their potential as PCR-based markers in 24 sorghum genotypes representing different races. Of these, 172 gave clear and robust amplification without multiple amplicons. Forty-eight of these markers were polymorphic yielding 122 alleles with an average of 2.5 alleles per marker. The number of alleles ranged from 2 to 6 while the PIC value ranged from 0.14 to 0.69. Cluster analysis grouped the genotypes into two major clusters. These markers would form an important addition to the existing DNA marker resources in sorghum and could be highly useful for genetic diversity analysis, construction of linkage maps and comparative genomics studies. [ABSTRACT FROM AUTHOR]

Microsatellite markers targeting (GATA) motifs are known to be highly polymorphic. Genome-wide development of such markers has not been reported in sorghum. The main objective of this study was to identify Class I microsatellites with (GATA) motifs in the sorghum genome through in silico analysis and assess their potential as molecular markers. The study identified a total of 128 such motifs, of which 14, 16 and 98 motifs were present in the genic, upstream and non-genic regions, respectively. The majority of the (GATA) motifs were found in the non-genic regions of the genome while 23.44 % of them were found within the genes and upstream of genes. About 110 PCR-based markers were developed targeting these microsatellites and 50 of them distributed across the genome were validated in 24 diverse sorghum genotypes representing different racial groups. Thirty-eight markers were polymorphic, with average polymorphism information content value of 0.69, and the sorghum genotypes could be grouped into two major clusters. These markers with robust amplification combined with good allelic diversity represent a new set of microsatellite markers in sorghum reported for the first time that will be highly useful for various genetics and molecular breeding applications. [ABSTRACT FROM AUTHOR]

The nuclear male sterility gene ms8 is expected to facilitate the production of sweet pepper ( Capsicum annuum L.) hybrids as it provides means for hybridization without the labor-intensive hand emasculation of female inbred lines. The development of molecular markers linked to ms8 locus will help the breeding practice for the selection of hybrid parental lines. In this study, F population resulting from a cross between the sweet pepper male sterile line 320 and the male fertile variety Elf was used to identify DNA markers linked to the ms8 locus. With the use of RAPD-BSA technique, seven markers linked to the ms8 locus were found. Four of them were converted into SCAR markers. In addition, two COSII/CAPS markers linked to the ms8 locus were identified. Comparative mapping with reference pepper maps indicated that the ms8 locus is located on the lower arm of the pepper chromosome P4. Identified markers are useful for molecular breeding, however, at present markers tightly linked to ms8 locus are still lacking. Identification of molecular markers linked to the ms8 locus and determination of its chromosomal localization are useful for fine mapping and also provide the perspective for ms8 gene cloning. [ABSTRACT FROM AUTHOR]

Due to their haploid nature, uniparental inheritance and apparent absence of recombination, organellar DNA markers are ideal tools for studying both intraspecific and interspecific phylogenies. Although 'universal' cytoplasmic DNA primers have become available for both red and green seaweeds, they are not transferable to brown seaweeds. Thus, based on the sequence of mitochondrial genome of the kelp Laminaria digitata, we developed universal-type primers for seven intergenic spacer loci and one mononucleotide tandem repeat locus. We investigated the conservation and sequence variation of these loci among nine Laminarialean and three fucoid species, as well as intraspecific polymorphism within five of these species. Six loci were conserved in at least four of the five brown algal families tested and five were polymorphic within at least one species. Although overall levels of divergence were relatively homogenous (π = 0.314 ± 0.031), a hierarchical analysis suggests there is variation in the rate of evolutionary change among the six intergenic spacers. Due to their high degree of transferability, versatility and to non-coding nature, we argue that these intergenic spacer loci are highly efficient markers for within-family phylogenetic studies and for intra-specific phylogeographic studies in brown seaweeds. [ABSTRACT FROM AUTHOR]

Bulb color in onions (Allium cepa) is an important trait whose complex inheritance mechanism involves epistatic interactions among major color-related loci. Recent studies revealed that inactivation of dihydroflavonol 4-reductase (DFR) in the anthocyanin synthesis pathway was responsible for the color differences between yellow and red onions, and two recessive alleles of the anthocyanidin synthase (ANS) gene were responsible for a pink bulb color. Based on mutations in the recessive alleles of these two genes, PCR-based markers for allelic selection were developed. In this study, genotype analysis of onions from segregating populations was carried out using these PCR-based markers. Segregating populations were derived from the cross between yellow and red onions. Five yellow and thirteen pink bulbs from one segregating breeding line were genotyped for the two genes. Four pink bulbs were heterozygous for the DFR gene, which explains the continuous segregation of yellow and pink colors in this line. Most pink onions were homozygous recessive for the ANS gene, except for two heterozygotes. This finding indicated that the homozygous recessive ANS gene was primarily responsible for the pink color in this line. The two pink onions, heterozygous for the ANS gene, were also heterozygous for the DFR gene, which indicated that the pink color was produced by incomplete dominance of a red color gene over that of yellow. One pink line and six other segregating breeding lines were also analyzed. The genotyping results matched perfectly with phenotypic color segregation. [ABSTRACT FROM AUTHOR]

Rice starch consists of amylopectin and amylose, the latter component being controlled by the Waxy (Wx) gene. Two alleles Wxa and Wxb were found to regulate the quantitative level of the Wx protein as well as the amylose content. A comparison of the genomic sequences of rice Wx genes responsible for the synthesis of grain amylose was made between two pairs of glutinous and non-glutinous reverse mutants. By comparing their 5′ splice codons in the first intron, the low-amylose and glutinous rices were characterized as the Wxb allele based on the G-to-T base substitution, which caused an aberrant splicing. When the entire genomic sequences of Wxb were compared, a unique duplication of 23 bp was found to be present only in the glutinous mutants. This 23 bp insertion was truly unique for tropical glutinous rice because of its similarity between the 800 bp sequences around the duplicated sequences of 24 glutinous varieties and two annual diploid wild rice accessions. This in-frame duplication created a premature translation stop codon at 78 bp downstream. The insertion was unique and none of the structure was identical to any transposon or retrotransposon ever reported in rice before. This unique duplicated sequence opens up opportunities to develop PCR-based markers useful for classifying grain amylose in rice. [Copyright &y& Elsevier]

PCR-based markers were developed from mononucleotide simple-sequence repeats in the chloroplast genome of Nicotiana tabacum and applied to the analysis of genetic diversity. These markers were found to detect high levels of polymorphism at three taxonomic levels in Solanaceous plants. Of 36 chloroplast loci examined, 26 show some degree of polymorphism among potato accessions. Among a set of 30 tetraploid potato cultivars it is apparent that a single chloroplast haplotype is prevalent, presumably a result of the widespread use as a female parent of the imported US cultivar Rough Purple Chili in the latter half of the 19th century. Nonetheless, there is considerable chloroplast diversity in the cultivated potato, and it is clear that a large proportion of this variability has arisen through the use of wild or primitive cultivated species of potato in introgression programmes. This variability should be used in future breeding programmes. An examination of single accessions from 24 potato species, as well as representatives from tobacco and other members of the Solanaceae, reveals high levels of inter-specific chloroplast DNA variation. These data, and the ease of use and potential for multiplexing of these markers, suggest that cpSSRs will be of great utility in population genetics, germplasm management, evolutionary and phylogenetic studies as well as in, the analysis of material from introgression and somatic-fusion experiments. Interestingly, the polymorphism arising from one of the more-polymorphic chloroplast loci examined, does not originate solely from the SSR, and is due to variation in the copy number of two tandemly arrayed sequence elements. [ABSTRACT FROM AUTHOR]

The rye (Secale cereale L.) 5R chromosome contains some elite genes that can be used to improve wheat cultivars. In this study, a set of 5RKu dissection lines was obtained, and 111 new PCR-based and 5RKu-specific markers were developed using the specific length amplified fragment sequencing (SLAF-seq) method. The 111 markers were combined with the 52 5RKu-specific markers previously reported, and 65 S. cereale Lo7 scaffolds were physically mapped to six regions of the 5RKu chromosome using the 5RKu dissection lines. Additionally, the 5RLKu arm carried stripe rust resistance gene(s) and it was located to the region L2, the same region where 22 5RKu-specific markers and 11 S. cereale Lo7 scaffolds were mapped. The stripe rust resistance gene(s) located in the 5RLKu arm might be new one(s) because its source and location are different from the previously reported ones, and it enriches the resistance source of stripe rust for wheat breeding programs. The markers and the S. cereale Lo7 scaffolds that were mapped to the six regions of the 5RKu chromosome can facilitate the utilization of elite genes on the 5R chromosome in the improvement of wheat cultivars. [ABSTRACT FROM AUTHOR]

Rye ( Secale cereale L.) 4R chromosome contains elite genes that are applicable for wheat ( Triticum aestivum L.) cultivar improvement. PCR-based 4R-specific markers can benefit the detection of elite genes on 4R in wheat backgrounds. In this study, a new fluorescence in situ hybridization (FISH) map of the 4R Ku chromosome of rye Kustro has been constructed. A set of 4R Ku dissection lines was obtained and 301 new 4R Ku -specific markers were developed using specific length amplified fragment sequencing (SLAF-seq) technology. These markers were combined with the 99 4R Ku -specific markers previously developed, and were physically mapped to 4R Ku chromosome using the new FISH map and the 4R Ku dissection lines. A total of 338 of the 400 markers have been successfully mapped to six regions of 4R Ku chromosome. Additionally, the powdery mildew resistance gene(s) on the 4RL Ku arm was located to the segment between L.4 and L.8, the same region where 115 4RL Ku -specific markers were mapped. The markers developed in this study can be used to identify a specific segment of 4R chromatin in wheat backgrounds, help construct a high-density physical map of 4R chromosome, and facilitate the utilization of elite genes on 4R chromosome in wheat breeding programs.

Background: Single nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci (QTL) studies. Marker validation is essential for QTL studies, but the cost and workload are considerable when large numbers of markers need to be verified. Marker systems with low development costs would be most suitable for this task., Results: We have tested allele specific polymerase chain reaction (PCR), tetra markers and a genotyping tool based on the single strand specific nuclease CEL-I to verify randomly selected SNP markers identified previously either with a SNP array or by genotyping by sequencing in rice and mungbean, respectively. The genotyping capacity of allele-specific PCR and tetra markers was affected by the sequence context surrounding the SNP; SNPs located in repeated sequences and in GC-rich stretches could not be correctly identified. In contrast, CEL-I digestion of mixed fragments produced from test and reference DNA reliably pinpointed the correct genotypes, yet scoring of the genotypes became complicated when multiple SNPs were present in the PCR fragments. A cost analysis showed that as long the sample number remains small, CEL-I genotyping is more cost-effective than tetra markers., Conclusions: CEL-I genotyping performed better in terms of genotyping accuracy and costs than tetra markers. The method is highly useful for validating SNPs in small to medium size germplasm panels.