Hybridization-Based Markers

Abstract

Qualitative traits have long been used for genetic studies, including the preparation of linkage maps. They were also used as markers to identify genomic regions involved in the control of quantitative traits. Their usefulness as markers prompted the search for other more abundant, easily detectable and stable characteristics leading to the development of protein-based and, finally, DNA-based markers. In the current parlance, morphological and protein-based markers are called classical markers, while DNA markers are referred to as molecular markers. DNA-based markers represent variation in genomic DNA sequences of different individuals. Restriction fragment length polymorphism (RFLP) was the first DNA marker, but it was technically demanding and required considerable time for marker genotyping. Therefore, efforts were made to develop more user-friendly marker systems; as a result, several different marker systems like RAPDs, DAF, AFLP, SCAR, SSR, etc. were developed. DNA marker systems have been classified on the basis of different criteria, such as chronology of their development, genomic location and functional significance, and the method of marker detection. On the basis of detection method, the DNA markers are classified into three groups, viz., hybridization-based, PCR-based, and sequence-based markers. The marker systems RFLP, diversity array technology (DArT), variable number of tandem repeats (VNTRs), single-feature polymorphism (SFP), and restriction-site-associated DNA (RAD) markers are detected by DNA:DNA hybridization. This chapter is devoted to the discussion of these hybridization-based marker systems, their salient features, as well as the advantages and limitations of these marker systems.

Appendices

Appendices

1.1 Appendix 2.1: Isolation and Purification of DNA from Plants

The plant DNA isolation and purification procedures can be grouped into three categories, viz., (1) CTAB method, (2) rapid DNA extraction methods, and (3) commercial DNA isolation kits. These procedures usually consist of three steps: (1) rupture and lysis of cells to obtain cell extract (tissue maceration), (2) purification of DNA, and (3) concentration of DNA. Plant tissues (fresh, freeze-dried, or frozen in liquid nitrogen) are usually ruptured by mechanical force. In general, DNAs isolated from fresh and frozen plant tissues are comparable in both quality and quantity. The particular method used for tissue grinding/maceration will mainly depend on the scale of work and the facilities available to the worker. On a small scale, mortar and pestle are widely used, but one may use a multi-pestle, a mixer mill or some other similar equipment on a moderate to large scale. The use of a mill would not only reduce the total time required for tissue maceration, but it may also improve DNA yield.

1.1.1 The CTAB Method

The CTAB procedure of Murray and Thompson (1980) is regarded as the standard method of DNA extraction. It is used to purify high-molecular-weight (50–100 kb) plant genomic DNA without the use of expensive equipment and time-consuming procedures. The powdered tissue is dispersed in an extraction buffer containing CTAB detergent and incubated at 50–60 °C for ~30 min. The suspension is then extracted with chloroform/octanol to remove cell wall debris, denatured proteins, etc. The extract is treated twice with chloroform/octanol, then CTAB is added, and the NaCl concentration is reduced so that CTAB–nucleic acid precipitate is formed. This precipitate is recovered through centrifugation and resuspended in 1 M CsCl, which is later removed by dialysis. In later modifications of the procedure, the precipitate is resuspended in 1 M NaCl or in TE (Tris–HCl and EDTA) buffer. The solution may be treated with RNase, and the DNA concentration can be increased by ethanol precipitation. The CTAB method has been modified by various workers to suit various needs. In one miniprep modification, CTAB is used in the homogenization buffer; the homogenate is extracted once with chloroform, followed by one ethanol precipitation and resuspension of the pellet in water. This method is rapid so that one person can process 100–200 samples per day, and it yields adequately pure DNA for PCR. In general, this method yields ~5 times more DNA per unit weight of tissue sample than the other methods, and the DNA can be stored for long periods. However, the research workers are exposed to hazardous chemicals like CTAB, chloroform, and ß-mercaptoethanol.

1.1.2 Rapid DNA Extraction Methods

Several methods for rapid extraction of plant DNA have been developed (Hill-Ambroz et al. 2002; Bagge and Lübberstedt 2008). These methods have been dubbed as “quick and dirty” DNA extraction methods since the purity of DNA preparations is usually poor. In a rapid DNA extraction procedure for wheat, the tissue is placed in 0.25 M NaOH at 95 °C in a water bath for 1 min and macerated using a mortar and pestle, a 96-solid-pin replicator, or a Matrix Mill. Now 0.1 M Tris–HCl (pH 8.0) is added, the suspension is centrifuged, the supernatant is recovered, and the DNA is precipitated with 3 M sodium acetate and 100 % isopropanol. The DNA samples are then placed at −80 °C for 1 h, and the DNA is pelleted by centrifugation. The pellet is washed with ethanol, and the ethanol is removed by centrifugation. The DNA is then resuspended in TE buffer (pH 8.0) and stored at −20 °C for 30 days. Approximately 1 μg of genomic DNA was isolated from 10 mg leaf tissue at a cost of about US $ 0.10. One person can process nearly 1,000 samples per day (Hill-Ambroz et al. 2002). In a simplification of this procedure, developed for DNA isolation from rice, the leaf tissue is ground in 0.5 M NaOH, and then 0.1 M Tris (pH 8.0) solution is added to the macerate. The suspension is mixed well and centrifuged, and ultimately the supernatant containing the DNA is recovered by pouring off into a fresh tube and stored at −20 °C. The amount and the quality of DNA is enough for PCR analysis, but it cannot be stored for long periods and may not be suitable for SNP assays. Leaf tissue and endosperm tissue drilled out of dry barley seeds or excised from soaked maize seeds have been used for DNA extraction.

1.1.3 DNA Extraction Kits

A variety of plant DNA extraction and purification kits are commercially available. Some examples of such kits are DNeasy Mini and Maxi kits from QIAGEN, NucleoSpin Plant kits from Clontech, PureLink® Genomic Plant DNA Purification Kit from Life Technologies, PowerPlant® DNA Isolation Kit from MO BIO Laboratories, MasterPure™ Plant Leaf DNA Purification Kit from Epicentre, etc. Most of these kits are generic and can be used for DNA isolation from many plant species, but some manufacturers offer kits for specific plant species. The kits include all the buffers, reagents, plasticware, etc., required for DNA extraction and purification after the plant material has been macerated. The manufacturers provide clear-cut directions for the extraction and purification procedures, which may take 40 min to 2 h, depending on the kit and the number of samples processed. Almost all manufacturers offer Mini kits in single sample format, but some of them also provide 96-well format and/or Midi/Maxi kits in single sample format. For example, QIAGEN offers DNeasy Plant Mini Kit for isolation of up to 30 μg DNA per sample, DNeasy Plant Maxi Kit for isolation of up to 260 μg DNA per sample, and the 96-well plate format DNeasy 96 Plant Kit with typical yield of 1–15 μg of high-quality DNA per well. The NucleoSpin Plant II kit from Clontech, advertised as a next-generation kit, has improved silica membrane and affords rapid isolation of more genomic DNA of higher quality. The typical DNA yields from <100 mg of plant tissue (fresh weight) range from 1 to 30 μg DNA suitable for PCR, Southern blotting, and restriction analysis. On the other hand, the NucleoSpin Plant Midi and Maxi kits yield 20–80 μg and 60–260 μg DNA, depending on the size and source of the tissue sample.

It may be clarified that the inclusion of a manufacturer’s products, procedures, services, and/or equipment for description here or elsewhere in this book is only for the purposes of illustration, and it does not in any way imply their appreciation/recommendation/endorsement. The descriptions of such products, procedures, services, or equipment are often based on the information available from the manufacturers, but other materials have also been used.

1.1.4 Determination of Quantity and Quality of the Isolated DNA

The quantity and quality of the isolated DNA may be determined by a comparison of aliquots of the extracted DNA with a standard DNA of known concentration by either gel electrophoresis or spectrophotometry. The spectrophotometric method also reveals DNA purity. The absorbance or optical density (OD) for each DNA sample is recorded at 260 nm and 280 nm. If the ratio of absorbance at 260 nm to that at 280 nm for a sample is between 1.8 and 2.0, it is regarded as pure DNA. Whenever this ratio is outside the above range, the DNA sample should be subjected to further purification by ethanol precipitation. Further, an OD of 1 at 260 nm corresponds to about 50 μg/ml DNA (Sambrook et al. 1989). In the electrophoretic method, 10-μl samples of the isolated DNAs along with the gel loading dye are loaded carefully in separate wells of an agarose gel. The gel is impregnated with the intercalating dye ethidium bromide for visualization of the DNA bands containing as little as 0.05 μg DNA per band. Similarly, 1 μg of uncut lambda DNA along with the loading dye is loaded in a separate well. After 2 h of electrophoresis, the bands for the DNA samples are compared with that for lambda DNA. The quantity of DNA is determined by comparing the width of the bands and the intensity of fluorescence under UV light using the software of a gel documentation and analysis system. A high-molecular-weight DNA preparation gives rise to a single dark band close to the loading well, while a fragmented DNA sample yields a smear (Sambrook et al. 1989). Thus, both spectrophotometric and electrophoretic methods permit estimation of DNA concentration. But DNA purity is revealed by spectrophotometry and DNA quality (high-/low-molecular-weight preparation) is visualized by electrophoresis.

1.2 Appendix 2.2: Genomic and cDNA Libraries

A genomic library is a collection of plasmid clones or phage lysates containing recombinant DNA molecules so that the sum total of DNA inserts in this collection, ideally, represents the entire genome of the concerned organism. For the preparation of a genomic library, total genomic DNA of the organism is extracted and subjected to partial digestion with a suitable restriction enzyme (Singh 2012b). Fragments of suitable size are separated, integrated into a suitable vector, and cloned in a host like Escherichia coli. A genomic library may be enriched in unique sequences by using a methylation-sensitive restriction enzyme like PstI. Since the repeated sequences do not contain many genes, they are far more likely to be methylated than unique sequences. As a result, the repeated sequences would be cut into much larger fragments that are not suitable for cloning. In some species like tomato, the frequency of unique sequences in PstI-derived genomic library is almost comparable to that in a cDNA library and about three times as much as in a EcoRI-derived genomic library. In contrast, in species like rice and lentil, the frequency of unique sequences is only slightly higher in a PstI-derived library than that in EcoRI-derived library.

Similarly, a cDNA library is a population of bacterial transformants or phage lysates, in which each mRNA isolated from an organism or tissue is represented as its cDNA insert in the recombinant DNAs present in this population. Construction of a cDNA library involves isolation and purification of mRNA using a suitable procedure, production of cDNA from this mRNA by reverse transcription catalyzed by the enzyme reverse transcriptase, integration of the cDNAs into a suitable vector (usually, a phage insertion vector), and cloning of the recombinant DNAs in a host like E. coli. cDNA library preparation is demanding, and considerable care needs to be exercised. A cDNA library would represent only those structural genes that are transcribed in the concerned tissue/organ during the given developmental stage. It is also likely to be enriched for abundant mRNA species. In addition, when RNA transcripts of a gene are alternatively spliced, two or more variant forms of such a single gene would be represented in the cDNA library. The genomic and cDNA libraries differ for several features (Table 2.6).

Table 2.6 A comparison between cDNA and genomic libraries

A genomic/cDNA library will consist of thousands of clones, and it is unlikely that all of them would be useful as probes. Therefore, the clones have to be screened for identification of those clones that are suitable for use as probes (de Vienne 2003). Some clones would fail to detect polymorphism, some may produce many bands or a complex pattern of bands, while some others may not generate any band; all such clones are rejected from the probe library. The clones forming complex band patterns would represent highly repeated DNA sequences. Many clones will yield one (in the case of homozygous individuals) or two (in the case of heterozygous individuals) bands; these clones represent unique DNA sequences and are used as probes. Some probes would give rise to more than two scorable bands; these probes most likely detect multiple RFLP loci and may be useful in some studies. The proportion of clones that detect polymorphism depends largely on the species. For example, only 5–10 % of the probes revealed polymorphism among the cultivated varieties of tomato when their DNAs were digested with three different enzymes, and the average number of alleles detected per locus was two. For this reason, it became necessary to use interspecific hybrids for preparation of RFLP maps of tomato. On the other hand, 95 % of the probes detected polymorphism among the DNAs from lines of only the dent group of maize when they were digested with three different enzymes, and the mean number of alleles per locus was more than six.

1.3 Appendix 2.3: Microarrays

An array is an orderly arrangement of data or items. A microarray is a glass slide or thin wafer of silicon glass, onto which a very large number of probes are immobilized as microdots. A probe is a DNA sequence representing a part or whole of a gene/cDNA single-stranded molecule. Microarrays are used for hybridization with a mixture of labeled test DNA molecules to detect the presence of sequences complementary to the probes spotted on the microarray (Singh 2012b; Winzeler et al. 1998). Thus, microarray strategy is the exact opposite of dot blot assay, in which a series of test DNA/RNA molecules are immobilized onto a solid support and a labeled probe is hybridized with them to identify the blots having DNA/RNA molecules complementary to the probe. Each of the probes immobilized onto a microarray is a pure preparation, while the test DNA is a mixture of fluorescence-labeled DNA/cDNA fragments. The results of hybridization are visualized by confocal microscopy. A single assay using, say, a gene expression microarray permits identification of all the genes expressed in a given tissue of an organism at a given time under the given environment. Microarrays were first used in the case of yeast that has less than 7,000 genes. Every yeast gene was obtained as an individual clone, and a single-stranded sample of each gene was spotted onto a glass slide in arrays of 80 × 80 spots. In order to identify the genes expressed in yeast cells under a set of given conditions, mRNA is extracted from these cells, is converted into cDNA by reverse transcription, and is fluorescently labeled. The labeled cDNA is hybridized with the microarray, and the identity of the spots showing fluorescence, i.e., hybridization, is determined by confocal microscopy. The spots showing fluorescence represent the genes that were expressed in the cells from which the mRNA was isolated.

Microarrays are basically of two types, viz., DNA microarrays and antibody microarrays. DNA microarrays, in turn, are of the following two types: (1) spotted microarrays and (2) oligonucleotide microarrays. In the case of spotted microarrays, DNA fragments representing different genes of an organism are obtained from genomic and/or cDNA library of the concerned species or a related species and spotted onto a suitable solid support. On the other hand, oligonucleotide microarrays or DNA chips are produced by synthesizing oligonucleotides at a very high density (up to one million oligonucleotides/cm²) directly on thin wafers of silicon glass. Each oligonucleotide has the sequence of a different gene, is located at a precise position on the microarray, and is synthesized by photolithographic solid-phase DNA synthesis. The DNA chips are inverted onto a controlled temperature hybridization chamber, into which fluorescently labeled test DNA, e.g., cDNA, preparation is injected and allowed to hybridize with the oligonucleotides. Laser excitation enters through the back of the glass support focused at the interface of the array surface and the hybridization solution. Fluorescence emission is collected by a lens and passed onto a sensitive detector, and a quantitative assay of hybridization intensity is obtained.

Microarrays are used for the following types of studies: (1) analysis of gene expression pattern in an organism as affected by the stage of development and/or environment, (2) identification of common regulatory elements by analysis of co-regulated genes, (3) analysis of already identified SNPs (these microarrays are often called SNP chips), (4) detection of genetic diseases, and (5) discovery and analysis of certain types of molecular markers, e.g., DArT, SFP, and RAD markers. In addition, specialized microarrays can be designed for specific purposes. For example, (6) arrays made up of probes that span across exon junctions allow detection and quantification of mRNA isoforms produced by alternative splicing, and (7) genomic tiling microarrays permit a very high-resolution mapping of the transcribed genomic regions. A genomic tiling microarray comprises a set of overlapping oligonucleotide probes that together represent a subset of the genome of a species at very high resolution. Analyses based on microarrays are highly sensitive and very fast, and all the genes present in the genome are analyzed in a single assay. These assays also generate quantitative data on gene expression, and the use of multiple labels of different colors may allow the use of a single microarray for assaying multiple test samples. But the construction of microarrays is expensive and requires genome sequence information. Further, there may be cross-hybridization leading to high background noise, and comparison of expression levels across experiments is often difficult.