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Successful release of new and better crop varieties increasingly requires genomics and molecular biology. This volume presents basic information on plant molecular marker techniques from marker location up to gene cloning. The text includes a description of technical approaches in genome analysis such as comparison of marker systems, positional cloning, and array techniques in 19 crop plants.
This activity started with low-throughput restriction fragment length polymorphisms and culminated in recent years with single nucleotide polymorphisms SNPs , which are abundant and uniformly distributed. Although the latter became the markers of choice for many, their discovery needed previous sequence information. However, with the availability of microarrays, SNP platforms have been developed, which allow genotyping of thousands of markers in parallel.
Besides SNPs, some other novel marker systems, including single feature polymorphisms, diversity array technology and restriction site-associated DNA markers, have also been developed, where array-based assays have been utilized to provide for the desired ultra-high throughput and low cost.
These microarray-based markers are the markers of choice for the future and are already being used for construction of high-density maps, quantitative trait loci QTL mapping including expression QTLs and genetic diversity analysis with a limited expense in terms of time and money. In this study, we briefly describe the characteristics of these array-based marker systems and review the work that has already been done involving development and use of these markers, not only in simple eukaryotes like yeast, but also in a variety of seed plants with simple or complex genomes.
A study of DNA polymorphism has become an active area of research in all important crops and several model plant species like Arabidopsis thaliana and Brachypodium distachyon.
This involves development and use of molecular markers, which have proved useful not only for marker-assisted selection during plant breeding, but also for understanding crop domestication and plant evolution.
This was, however, unimaginable during the s and still remains cost ineffective, therefore DNA-based molecular markers for example, restriction fragment length polymorphisms, random amplified polymorphic DNAs, simple sequence repeat SSRs and amplified fragment length polymorphisms AFLPs have largely been employed for the study of DNA polymorphism Collard et al.
More recently, however, single nucleotide polymorphisms SNPs , whose discovery was largely based on sequence information, became the markers of choice due to their abundance and uniform distribution throughout a genome.
Once discovered, SNP genotyping can be done using any of the dozens of available methods. However, this appeared inadequate and there has been an increasing demand to develop ultra-high-throughput low-cost assays for a variety of novel marker systems including SNPs. These new methods will allow ultra-high-throughput genotyping of either one or few individuals for hundreds of thousands of markers, or that of thousands of individuals for one or few markers.
High-density oligonucleotide arrays, which are now becoming available in several crops, provide a means for achieving this goal of low-cost ultra-high-throughput genotyping. These arrays may also be custom made according to specific needs and, therefore, also allowed for the development of novel marker systems like single feature polymorphisms SFPs including gene-specific hybridization polymorphisms and gene expression markers , diversity array technology DArT and restriction site-associated DNA RAD markers, which have now become the markers of choice.
Technologies have also been developed, which make use of tag arrays for detection of the products of genotyping reactions. These novel array- or chip-based markers are useful for a variety of purposes including genome-wide association studies, population studies, bulk segregant analysis, quantitative trait loci QTL interval mapping, whole genome profiling and background screening and so on Steinmetz et al.
A brief account of the development and use of these high-throughput array-based molecular markers in plants is presented in this study. Once SNPs are discovered, genotyping for these markers can be done using any one of more than 30 different available methods, although only a few of these methods are microarray based, providing the desired ultra-high throughput for reviews, see Khlestkina and Salina, ; Kim and Misra, The choice of genotyping method largely depends upon the nature of study.
For instance, whereas in some cases, we need to scan one or more individuals for SNPs ranging in number from dozens to several thousands, in other cases, we may need to allelotype thousands of individuals for a specific locus. In either case, ultra-high throughput and low-cost techniques are needed.
Several high-density platforms are now available for genotyping one or more genomic DNA samples for dozens to thousands of SNPs in parallel Table 1. Therefore, only an overview of these platforms is included. Each assay involves a multiplexed SNP genotyping reaction, involving use of two allele-specific oligonucleotides and a locus-specific oligonucleotide for each SNP, the locus-specific oligonucleotide carrying an anti-tag sequence for detection by the BeadArray.
At a particular level of multiplexing pooling , 96 or BeadArrays each bead carrying a tag oligo for detection of the product of SNP genotyping reaction are arranged in a matrix, called Sentrix Arrays Matrix, so that up to samples can be processed in one reaction, thus permitting genotyping of each of these samples for as many as SNPs, simultaneously. Multiple pools can also be used to increase the number of SNPs further for details with illustration, consult Fan et al.
However, beyond a certain level of multiplexing, Infinium assay that is discussed later in this review provides a better alternative. GoldenGate technology is now being used for several crops. For instance, at Southern California Genotyping Consortium, Oligonucleotide Pool Assays are being developed for several plant systems including Arabidopsis , barley, wheat and maize, which will be used in the future for high-throughput SNP genotyping in these plant systems.
In soybean, recently a custom-made SNP GoldenGate assay was successfully designed for genotyping of three RIL mapping populations; the above SNPs were discovered through resequencing of five diverse accessions involved as parents of the above three RIL mapping populations and were selected such that each of these SNPs segregated in at least one mapping population Hyten et al. The above genotyping activity may certainly be extended further through the use of Solexa's ultra-high throughput and low-cost resequencing technology for reviews, on ultra-high-throughput DNA sequencing, see Gupta, ; Mardis, Illumina's BeadChip-based Infinium assay, which is considered to be a more global approach for genotyping, also utilizes BeadArray technology and is a direct approach allowing parallel detection of most SNPs in a genome.
More importantly, it eliminates the multiplexing bottleneck in sample preparation needed in GoldenGate , making assay scalability mainly dependent on array-feature density Fan et al. Like GoldenGate assay, this genotyping system of Beckman Coulter combines solid-phase primer extension assay and universal tags for SNP genotyping. DNA samples are used for either 12 or 48 multiplex PCR in a plate using tagged extension primers that are extended using single fluorescence-labelled nucleotide terminator reactions.
The PCR-amplified fragments are resolved by hybridization to the complementary tags available on SNPware Tag Array plate having tag arrays in well microplate format, each well with 16 or 52 unique tags that are complementary to the tags of the 12 or 48 extension primers, plus four controls to ensure accuracy.
An individual SNP associated with a PCR-amplified fragment is identified by the position of the hybridizing tag in the well. This allows genotyping of samples for either 12 or 48 SNPs per array, as against genotyping of relatively fewer samples for one thousand to more than a million SNPs in other high-throughput microarray-based SNP genotyping systems Figure 1.
Comparison of SNP multiplexing levels and number of samples analyzed per array in microarray-based SNP genotyping systems reproduced with permission from ref. It allows tens of thousands of genotyping reactions in each of four reaction tubes that are used for each assay.
Possible addition of a single specific nucleotide is allowed in each of the four reaction tubes by adding in each tube only one of the four dNTPs, which differ for the four tubes.
Furthermore, MIP uses a single circularizable probe called padlock probe , whereas in GoldenGate assay, both upstream and downstream probes are separate Nilsson et al. A technology making use of modified padlock probes like those used in MegAllele system has been recently utilized in bread wheat Reid et al.
Tiling arrays developed by Affymetrix GeneChip platform on the basis of known sequences in several organisms have also been used for SNP discovery and detection. These tiling arrays may be either designed for resequencing sequencing by hybridization or SBH of specific genomic regions for SNP genotyping or may be designed for interrogating every individual nucleotide in a template genomic sequence by multiple probes available on the array.
In the latter case, probes are available in the form of probesets, so that for each SNP allele, there is one probeset with multiple probe pairs each pair with a perfect match and a mismatch , the probes of the two alleles at a locus differing only at a specified position.
The hybridization patterns are used for inferring SNP genotypes. Tiling arrays have already been used in some crops for genome-wide discovery and detection of SNPs. These SNPs are being currently validated Collard, personal communication , and the collection of these rice SNPs is being extended through use of machine-learning ML -based techniques.
The ML approach was used earlier in Arabidopsis , where in one study 20 diverse strains of this weed were genotyped for more than a million non-redundant SNPs for analyzing the patterns of DNA polymorphism Clark et al. During marker-assisted selection in plant breeding, one may be interested in microarray-based genotyping of thousands of plants for a specific gene of interest. This can be done by arraying PCR products from all segregating individual plants on a glass, followed by hybridization of this array with labelled probes representing alternative alleles of the gene.
The utility of this technique described as tagged microarray marker approach has been demonstrated for humans and pea Flavell et al. However, Affymetrix GeneChips can also be used for SNP genotyping of a number of samples for one or more genes of interest as done in Arabidopsis , where the array AT was used for the study of variation in several genes for example, Eds16 Cho et al. These technologies, however, do not fall within the scope of the present discussion on microarray-based markers.
Efforts are also being made for microarray-based genome-wide capturing of exons for selective resequencing that may allow SNP genotyping Hodges et al.
When labelled genomic DNA from two or more genotypes is separately hybridized to the same high-density oligonucleotide array, SFPs are detected as significant differences in hybridization signals among the genotypes used Figure 2. Schematic representation of a the procedure used for detecting SFPs and ELPs using conventional oligonucleotide expression GeneChip, and b difference in i hybridization intensities of the reference and the genotype under investigation due to deletion upper panel , SFP lower panel and ii pattern of hybridization between the reference and the genotype under investigation due to ELP.
Also to ensure that only a single locus will hybridize to each feature, sequences that are likely to hybridize to multiple loci are eliminated although repeat sequences and multicopy genes have also been used for SFP detection. In an Affymetrix GeneChip often used for SFP analysis, each of a large number of genes is represented by a variable number of probes covering the entire gene sequence, thus constituting a probeset the entire GeneChip having a number of probesets, which is equal to the number of genes to be sampled.
Such a strategy provides an opportunity to assay multiple loci within each gene for detecting polymorphisms that is, SFPs. While in an ELP, all probes representing a probeset will give same hybridization affinity with a single cRNA sample and the hybridization intensity will differ only with different cRNA samples probeset level polymorphism , but in case of SFPs, the affinity of a particular probe will differ from that of all other probes in a probeset for the same cRNA sample probe level polymorphism; see Figure 2.
In the very first study involving SFPs, Winzeler et al. Subsequently, SFPs in yeast were put to a variety of uses including fine mapping and positional cloning of a QTL for high-temperature growth Steinmetz et al.
In the initial phase of SFP development, it was thought that the technique is suitable for only small genomes, as an increase in genome size leads to significant reduction in signal-to-noise ratio.
Consequently, the technique was later used in a number of seed plants including those with moderately complex genomes for example, Arabidopsis and rice and also those with large and highly complex genomes for example, maize, soybean, tomato, lettuce, barley and wheat.
The results of SFP analysis in these plant species with complex genomes are summarized in Table 2. However, there are some limitations that make this technology less competitive with some of the recently developed ultra-high-throughput SNP technologies discussed earlier in this review see later for details.
As evident from the information presented in Table 2 , SFPs have been used for a variety of purposes including detection of marker-trait associations, which sometimes involved construction of a molecular map followed by QTL interval mapping.
For instance, in Arabidopsis , bulk segregant analysis has been used for mapping of circadian and developmental genes Hazen et al. Similarly, in tomato, 17 SFPs were identified, which were tightly linked to a disease resistance locus T Zhu, unpublished data; reported by Zhu and Salmeron, It has been argued that for crop plants with large genomes carrying high proportion of repetitive DNA, SNP genotyping for association studies at the whole genome level becomes prohibitive.
For instance, in maize, an estimated one million SNP markers are needed for genome-wide association studies Gore et al. This seems unnecessary because large proportion of these SNPs would belong to the non-coding regions. Under these circumstances, an attractive alternative is provided by SFP technology, which also allows coupling of genotyping with gene expression analysis. However, to make SFP technology useful in crops with complex genomes, a complexity reduction and gene enrichment method needs to be developed for the preparation of target DNA that is used for hybridization.
Several other limitations of using SFP technology in crops with complex genomes that have been recognized during the use of this technology include the following: First, while using cRNA as surrogate for genomic DNA, extensive replications with samples from multiple tissues are needed, thus increasing the cost per data point. Third, in an array-hybridization experiment in maize, the detection of probeset polymorphism ELPs was found to be more effective than the detection of probe polymorphism SFP , thus limiting the resolution to gene level rather than to nucleotide level Gore et al.
Fourth, SFP technology often fails to detect polymorphisms due to SNPs occurring at the edges of the oligonucleotide probes and mainly detects only those polymorphisms that are due to internal SNPs at positions 6—15, as observed in maize and barley.
As mentioned above, complexity reduction and gene enrichment of the target DNA is one approach to make SFP technology suitable for complex genomes. This has often been achieved through the use of cRNAs, as done in Arabidopsis and several crop plants, including barley, rice, maize and wheat. This allows simultaneous acquisition of data for SFP genotyping and expression analysis, and thus facilitates development of two different types of markers, SFPs and gene expression markers; the latter recorded as large differences in transcript levels between the parents of a mapping population.
The other complexity reduction and gene-enrichment methods include methylation filtration, C 0 t filtration and AFLP, but these methods offered only a modest improvement in power to detect SFPs, when used with maize genome cf.
Gore et al. This method has not yet been widely tested, but its utility in maize suggested that it may also prove useful for other crops with large and complex genomes. For instance, the use of robustified projection pursuit involved differentiation of signal intensities between two genotypes, first at the level of probeset and then at the level of individual probes.
Several other statistical tools and softwares are available, which would lead to improvement in SFP detection. Methods have also been suggested to reduce the number of false positives and false negatives observed during SFP studies.
The false positives are believed to result due to alternative splicing or polyadenylation, gene duplications, chance alignments with RNA from another region, gene expression markers resulting from polymorphism s at trans -acting regulators and secondary structures in target DNA or SNPs that occur immediately adjacent to the position of a 25mer probe cf.
Luo et al. The number of these false positives can be reduced by i sampling more than one tissues per genotype Rostoks et al. Similarly, false negatives can be attributed to position in nucleotides of known SNP s on the corresponding array feature, and can be reduced by increasing feature density per gene Rostoks et al.
Diversity array technology is a high-throughput microarray hybridization-based technique that allows simultaneous typing of several hundred polymorphic loci spread over a genome without any previous sequence information about these loci Jaccoud et al.
The process of developing new crop varieties can take almost 25 years. Now, however, biotechnology has considerably shortened the time to years for new crop varieties to be brought to the market. One of the tools which can make it easier and faster for scientists to select plant traits is marker-assisted selection MAS. DNA is packaged in chromosome pairs strands of genetic material , one coming from each parent. Some traits, like flower color, may be controlled by only one gene. Other more complex characteristics, however, like crop yield or starch content, may be influenced by many genes. Traditionally, plant breeders have selected plants based on their visible or measurable traits, called the phenotype.
Next generation sequencing platforms and high-throughput genotyping assays have remarkably expedited the pace of development of genomic tools and resources for several crops. Complementing the technological developments, conceptual shifts have also been witnessed in designing experimental populations. Availability of second generation mapping populations encompassing multiple alleles, multiple traits, and extensive recombination events is radically changing the phenomenon of classical QTL mapping.
Despite strong interest over many years, the usage of quantitative trait loci in plant breeding has often failed to live up to expectations. Most reports of markers used for MAS focus on markers derived from the mapping population. There are very few studies that examine the reliability of these markers in other genetic backgrounds, and critically, no metrics exist to describe and quantify this reliability.
Plant breeding with marker-assisted selection in Brazil. Private seed companies increasingly use marker-assisted selection, especially for the species of great importance to the seed market, e. In the Brazilian public institutions few breeding programs use it efficiently. The possible reasons are: lack of know-how, lack of appropriate laboratories, few validated markers, high cost, and lack of urgency in obtaining cultivars. In this article we analyze the use and the constraints of marker-assisted selection in plant breeding programs of Brazilian public institutes. Key words: Molecular markers, indirect selection, MAS. The possibility of using molecular markers in plant breeding was presented in the 80's by Beckmann and Soller and Paterson et al.
It seems that you're in Germany. We have a dedicated site for Germany. Successful release of new and better crop varieties increasingly requires genomics and molecular biology. This volume presents basic information on plant molecular marker techniques from marker location up to gene cloning. The text includes a description of technical approaches in genome analysis such as comparison of marker systems, positional cloning, and array techniques in 19 crop plants. A special section focuses on converting this knowledge into general and specific breeding strategies, particularly in relation to biotic stress.
Regret for the inconvenience: we are taking measures to prevent fraudulent form submissions by extractors and page crawlers. Received: April 20, Published: June 1, Molecular mapping and breeding of physiological traits. Adv Plants Agric Res. DOI: Download PDF.
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