"Frontmatter". In: Plant Genomics and Proteomics
Download 1.13 Mb. Pdf ko'rish
|
Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
M
OLECULAR M APS As mentioned in previous chapters there are a number of different types of molecular maps. The genetic map includes the loci ordered with respect to the frequency with which they recombine. A physical map is a linear order of the genomic sequence in some form whether it is as a set of ordered BACs or as a complete genome sequence. The most useful type of molecular map will be the result of integrating the genetic and the physical maps. When the genetic markers are used to anchor the physical contigs any locus that is genetically mapped can then be placed in a specific physical region of the genome. The ultimate example in plants of an integrated genetic and phy- sical map is Arabidopsis, for which the complete genome is available. However, other examples that have large, but as yet incomplete, genomic resources are maize (http://www.maizemap.org/iMapDB/iMap.html) and soybean (http://hbz7.tamu.edu/homelinks/phymap/soybean/ soytool_4.htm). DNA-based markers have revolutionized the whole process of generat- ing genetic maps because, for the first time, a large number of loci can be followed in a single segregating population. The range of genetic markers that are available includes restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNAs (RAPD) (Williams et al., 1990), amplified fragment length polymorphisms (AFLP) (Vos et al., 1995), single-nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs) or microsatellites (Senior and Heun, 1993). The usefulness or neces- sity of each of the marker systems depends largely on the genetic resources available for the species under consideration. M ARKER S YSTEMS RFLP S These were the first generation of markers and are generally detected by the hybridization of a probe to restriction-digested genomic DNA. Many of the M A R K E R S Y S T E M S 1 4 9 probes are derived from single-copy sequences, with EST sequences being one rich source of potential probes. One current application of RFLPs is the generation of a high-density map in wheat. In this example, a series of chro- mosome deletion lines are subjected to hybridization with labeled cDNAs as probes (http://wheat.pw.usda.gov/NSF/). Despite there being multiple copies of the genome present in hexaploid wheat, the different members of the homologous groups can be identified by the disappearance of a band in the deletion line even if there were no polymorphisms available for that par- ticular gene (Figure 8.1). Thus deletion lines are exceptionally useful for mapping regions that are polymorphism poor. Unfortunately, this type of deletion resource is not available for many plant species, although a set of maize-oat addition lines that can be used for mapping maize genes has been constructed (Okagaki et al., 2001). 1 5 0 8. I D E N T I F I C AT I O N A N D M A N I P U L AT I O N O F C O M P L E X T R A I T S 1 2 3 4 5 6 a b c d FIGURE 8.1. Using deletion lines to map chromosomal locations of ESTs. Lanes a–d represent restriction-digested DNA from a hexaploid plant (containing 6 chromo- somes designated 1A, 1A, 2B, 2B, 3C, 3C) and where the DNA was extracted from: a) Plants that have all the chromosomes; b) plants that are 2B, 2B, 3C, 3C; c) plants that are 1A, 1A, 3C, 3C; d) plants that are 1A, 1A, 2B, 2B. The Southern blot was hybridized with an EST sequence that showed no polymorphisms in any of the acces- sions and so could not be mapped. However, from the use of deletion stocks it is pos- sible to place the specific EST: bands 1 and 2 are on chromosome 3, bands 4 and 5 are on chromosome 2, and bands 3 and 6 are on chromosome 1. Deletion stocks that have less than a whole pair of chromosomes missing can be used to place the ESTs more accurately on the chromosomes. AFLP S These markers are RFLPs detected by PCR amplification. The polymor- phic fragments are observed against the background of all of the possible sized restriction fragments that can be amplified. Adaptors are added to the ends of restriction fragments, and these adaptors are then used as primers in a PCR reaction. Every possible band should be amplified, and the complex mixture of bands is separated on gels or through automated sequencers. The polymorphisms can be cloned and sequenced to generate sequence-tagged sites (STSs). Potential epigenetic effects resulting from hyper- or hypomethylated regions of the genome can be investigated by using methylation-sensitive and -insensitive restriction enzyme isoschizo- mers. However, as mentioned in Chapter 1 species with large amounts of DNA (>20 pg per 1C) can be problematic when studying genetic diversity with AFLP techniques. RAPD S Statistically a sequence of 10 bp should appear once every 10 6 nucleotides. PCR amplification using genomic DNA as the target and a series of single random 10-base primers has been very successful in generating large numbers of polymorphisms (Williams et al., 1990). The methodology can be used when little other genomic information is known. Unfortunately, the technique appears to suffer from irreproducibility between laboratories and sources of thermostable enzyme, although, within a laboratory, reproducible results can be achieved (Jones et al., 1997). M ICROSATELLITES AND SSR S Microsatellites or SSRs are genetic markers that are derived from short (usually <6 bp) tandemly repeated sequences such as (GA) n , (AAT) n , (GT) n . The terms are often used interchangeably, although microsatellites are gen- erally longer than the 2- to 3-bp unit of the SSRs. This type of sequence is widely dispersed through most animal and plant genomes and polymor- phisms are due to the variability in the number of repeats at a given site. SSRs can be isolated from genomic libraries or enriched genomic libraries (Panaud et al., 1995) or generated from an analysis of cDNA sequences. They can be converted into STSs with primers designed in unique regions sur- rounding the repeat and have become an important source of genetic markers for many eukaryotic genomes (Panaud et al., 1995), especially when the primers are designed in a conserved region of a transcribed sequence, making them applicable over a wide range of taxa. M A R K E R S Y S T E M S 1 5 1 S INGLE -N UCLEOTIDE P OLYMORPHISMS Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence differs between two individual DNA samples. For example, a SNP might change the DNA sequence AGGATTCA to AGGATTTA. SNPs can occur in both coding (gene) and noncoding regions of the genome. Many SNPs have no effect on cell function because they may not change protein structure (in fact, any SNP that occurs at the third position in the amino acid codon will have no effect if it does not change the amino acid sequence of the resulting protein). Their high frequency (perhaps as high as 2–3% in plant DNAs) means that they can be particularly useful in linkage mapping (Kristensen et al., 2001; Lai, 2001). They must be derived from sequence information, and that information must be obtained from two or more individuals. Informat- ics tools can be used to compare the sequences and identify variations, but the raw data in the form of trace files may be important in deciding which polymorphisms may be real. In the building of unigene sets, these differ- ences are eliminated in the formation of the consensus sequence and need to be retrieved. Because there is no a priori way of differentiating between a true SNP and sequencing errors, each potential SNP must be validated. Even at a frequency of 1% these polymorphisms would generate an exceptionally large number of haplotypes if every polymorphism could be inherited inde- pendently. However, relatively few haplotypes are observed, indicating that perhaps the rate of SNP production is similar to the rate at which recombi- nation occurs across the regions of the genome making up the haplotype blocks. Therefore, SNPs are most likely to be useful for defining haplotypes, rather than for their information individually, and so the use of SNPs is likely to involve linkage disequilibrium studies using the haplotype rather than the use of specific SNPs as individual molecular markers. 6> Download 1.13 Mb. Do'stlaringiz bilan baham: |
Ma'lumotlar bazasi mualliflik huquqi bilan himoyalangan ©fayllar.org 2024
ma'muriyatiga murojaat qiling
ma'muriyatiga murojaat qiling