Molecular markers and their significance for crop improvement
Molecular markers and their significance for crop improvement
The productivity of domestic crop plants has evolved through the collective efforts of plant scientists since the dawn of agriculture and represents mankind’s greatest achievements. From a historical perspective, improved crop yields have been influenced perhaps more by genetic improvement than by any other single factor (Fehr, 1984). Despite the breeding progress already achieved, additional gains in agricultural productivity are demanded at an ever-faster pace by population growth and by changes in agricultural practices, biotic and abiotic environments and consumer preferences.
The main objective of plant breeding is to attain sustainability in agriculture. This can only be achieved by enhancing crop yield, keeping yield stability, and improving the quality by crossing elite cultivars of choice with lines that possess desired new traits. Conventional plant breeding involves crossing of the elite cultivar with the donor followed by selection of superior recombinants. The process involves several crosses and several generations, requiring a careful phenotypic selection. Moreover, the whole process is time consuming and laborious. Additionally, there is a threat of transferring undesirable traits along with the traits of interest. These drawbacks are major hindrances in enhancing agricultural production (Collard et al. 2005). The availability of molecular marker technology provides solutions to problems associated with conventional breeding. Utilization of molecular markers by tagging the desired genes or chromosome regions during breeding makes the process more efficient and faster (Collard et al. 2005). Using molecular tools to select favorable alleles and selection against undesirable background regions also helps the breeder concentrate his/her work on improved populations.
What is a Genetic marker?
A Genetic markers represent genetic differences between individuals of same or different species. Generally, they do not represent the target genes themselves but act as ‘signs’ or ‘flags’. Genetic markers that are located in close proximity to genes (i.e. tightly linked) may be referred to as gene ‘tags’. Like genes all the genetic markers occupy specific genomic positions within chromosomes. There are three major types of genetic markers: (1) morphological (also ‘classical’ or ‘visible’) markers which themselves are phenotypic traits or characters; (2) biochemical markers, which include allelic variants of enzymes called isozymes; and (3) DNA (or molecular) markers, which reveal sites of variation in DNA. Markers help us to determine the location (map) of genes that control important traits .
Morphological markers are usually visually characterized phenotypic characters such as flower colour, seed shape, growth habits or pigmentation. Isozyme markers are differences in enzymes that are detected by electrophoresis and specific staining. The major disadvantages of morphological and biochemical markers are that they may be limited in number and are influenced by environmental factors or the developmental stage of the plant. Both these markers have been used, albeit to a little extent, by the crop breeders.
DNA markers (also referred to as molecular markers) are a piece of the DNA molecule that is associated with a certain trait of an organism. These are most widely used type of marker predominantly due to their abundance. They arise from different classes of DNA mutations such as substitution mutations (point mutations), rearrangements (insertions or deletions) or errors in replication of tandemly repeated DNA. These markers are selectively neutral because they are usually located in non-coding regions of DNA. Unlike morphological and biochemical markers, DNA markers are practically unlimited in number and are not affected by environmental factors and/or the developmental stage of the plant.
Types of molecular markers
DNA markers may be broadly divided into three classes based on the method of their detection: (1) hybridization-based; (2) polymerase chain reaction (PCR)-based and (3) DNA sequence-based
Several types of molecular markers which have been developed and are being used in plant sciences research are restriction fragment length polymorphism (RFLP), sequence tagged sites (STS), expressed sequence tags (ESTs), simple sequence repeats (SSRs) or microsatellites, randomly amplified polymorphic DNA (RAPDs), sequence characterized amplified regions (SCARs), amplified fragment length polymorphism (AFLP), single nucleotide polymorphic (SNP) and Diversity Array (DArT) markers. In addition several other variations of these markers have been developed (Table 1). A brief description of each of these markers is presented below:
I. Southern Hybridization Based markers
i). Restriction fragment length polymorphism (RFLP): These are single or low copy DNA fragments and are simply inherited. These probes could be genomic clones, cDNA clones, or even cloned genes. The RFLP markers show co-dominance and are highly reliable in linkage analysis and breeding. Their detection is based on radio labelling, require large quantities of DNA, are labour intensive and relatively expensive and hazardous. Hence, their large-scale use in practical plant breeding may be restricted.
II. Polymerase chain reaction (PCR) based markers
i). Sequence tagged sites (STS):The RFLP probes, linked to desirable traits can be converted to polymerase chain reaction (PCR) based markers. In this the RFLP probes are end-sequenced and complementary primers are synthesized. These primers (generally 20 mers) are then used for amplifying specific genomic sequences using PCR. For example, STS markers have been developed for RFLP markers linked with bacterial blight resistance genes xa5, xa13 and Xa21, powdery mildew and stem rust resistance gene in barley etc. One major limitation of these markers is the reduced polymorphism than the corresponding RFLP marker.
ii). Expressed sequence tags (EST): These markers are developed by end sequencing of random cDNA clones. The cDNA markers are first mapped as RFLP markers and then partially sequenced to convert them into PCR based markers. Thus, these are like STS markers. These can be used for synteny mapping and cloning of specific genes. Most of these could be functional genes. Large number of EST markers have been identified in rice (more than 1450) (Harushima et al., 1998) and Arabidopsis.
iii). Simple sequence repeats (SSRs): Also called as microsatellites, these are short tandem repeats dispersed throughout the genome. These are generally di-to-tetra- nucleotide repeats and are highly hyper variable. These are flanked with unique sequences that are highly conserved. The flanking unique sequences are analyzed and their
Table: An over view of the molecular markers
| Marker | Abbrev. | Description |
| Restriction fragment length polymorphism | RFLP | The occurrence of variation in the length of DNA fragments that are produced after cleavage with a restriction enzyme. There are differences in DNA lengths because of the presence or absence of recognition site(s) for that particular restriction enzyme. |
| Variable number tandem repeat or minisatellites | VNTR | A short DNA sequence that is present as tandem repeats [motifs of 10-100 bases] and in highly variable copy number. |
| Simple sequence length polymorphism | SSLP | The sequence of unique DNA that flanks microsatellites [i.e.tandemly repeated motifs of 1-6 bases] is used to construct oligonucleotide primers. Variation is based on size differences of the microsatellite. SSLP, SSR, STR, STMS and microsatellite are synonyms |
| Simple sequence repeats | SSR | |
| Short tandem repeats | STR | |
| Sequence tagged microsatellite site | STMS | |
| Inter-simple sequence repeat. | ISSR | The stretches of unique DNA in between the SSRs are amplified. SSRs such as [GATA] 4 are used to prime PCR on genomic |
| Single primer amplification reaction. | SPAR | DNA.Tetranucleotide repeats perform better than di-or tri-nucleotide repeats |
| Cleaved amplified polymorphic sequences | CAPS | PCR amplified DNA that is not yet polymorphic is digested with restriction endonucleases to reveal polymorphism. |
| Amplified fragment length polymorphism | AFLP | AFLPs are DNA fragments [80-500bp] obtained from endonuclease restriction, followed by ligation of oligonucleotide adaptors to the fragments and selective amplification by PCR.The PCR-primers consist of a core sequence [part of the adaptor], a restriction enzyme –specific sequence and 1-3 selective nucleotides. The AFLP fragments are separated by gel-electrophoresis and generally scored as a dominant marker. |
iv). Randomly amplified polymorphic DNAs (RAPDs): Williams et al. (1990) originally developed the technique. In this arbitrary decamer sequences are used as primers for amplification. These markers are dominant markers because the polymorphism is due to presence or absence of a particular amplified fragment. One major advantage of these markers is that this does not need any prior sequence information. These markers have been used for constructing linkage maps in several species and also for tagging genes of economic importance. One major limitation of these markers is lack of repeatability in certain cases.
v). Sequence characterized amplified regions (SCARs): These markers overcome the limitation of RAPDs. In this, the RAPD fragments that are linked to a gene of interest are cloned and their termini sequenced. Based on the terminal sequences, longer primers (20 mers) are designed. These SCAR primers lead to a more specific amplification of particular locus. These are similar to STS markers in construction and application. The presence or absence of the band indicates variation in sequences. The SCAR markers thus are dominant markers. These however, can be converted to codominant markers in certain cases by digesting the amplified fragment with tetra cutting restriction enzymes. The RAPD primers linked to gene of importance have been converted to SCAR markers in several cases (see Joshi et al., 1998).
vi). Amplified fragment length polymorphism (AFLP): This technique was developed in Vos et al. (1995). In this technique restriction fragments generated by a frequent (4 base) and a rare (6-base) cutter are anchored with oligonucleotide adapters of a few oligonucleotide bases. This method generates a large number of restriction fragments facilitating the detection of polymorphism. The number of DNA fragments amplified can be controlled, by choosing different base numbers and composition of nucleotides in the adapters. This technique is more reliable since stringent reaction conditions are used for primer annealing. This technique thus shows an ingenious combination of RFLP and PCR techniques and is extremely useful in detection of polymorphism even between closely related genotypes. AFLP maps have been constructed in several species and integrated into already existing RFLP maps (for example in tomato) (Haanstra et al., 1999).
II. Polymerase chain reaction (PCR) based markers
Single Nucleotide polymorphisms (SNPs)
Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in a genome sequence is changed. Most SNPs (actually two out every three- SNPs) involve the replacement of cytocine (c) with Thrymine (T). These occur every 100 to 300 bases along the genome. SNPs are generally pronounced as ‘snips’. Although SNPs have only two alleles and are less informative than typical multiallele simple sequence length polymorphism (SSLPs) but this disadvantage can be offset by using greater density of SNPs A genome scan with 1000 well spaced SNPs for example will extract about the same linkage information as the current standard of 400 well spaced SSLPs.
There are two types of nucleotide base substitutions resulting in SNPs:
- A transition substitution occurs between purines (A, G) or between pyrimidines (C, T). This type of substitution constitutes two thirds of all SNPs.
- A transversion substitution occurs between a purine and a pyrimidine.
Table: Comparison of marker techniques commonly used in plant research
| Feature | Technique | ||||
| RFLPs | RAPDs | AFLP | SSRs | SNPs | |
| DNA required (µg) | 10 | 0.02 | 0.5-1.0 | 0.05 | 0.05 |
| DNA quality | High | High | Moderate | Moderate | High |
| PCR based | No | Yes | Yes | Yes | Yes |
| No. of polymorphic loci analyzed | 1.0-3.0 | 1.5-50 | 20-100 | 1.0-3.0 | 1.0 |
| Ease to use | Not easy | Easy | Easy | Easy | Easy |
| Amenable to automation | Low | Moderate | Moderate | High | High |
| Reproducibility | High | Unreliable | High | High | High |
| Development cost | Low | Low | Moderate | High | High |
| Cost for analysis | High | Low | Moderate | Low | Low |
Development of saturated maps
In the past genetic maps were based mainly on morphological and isozyme markers. But these markers are limited and are influenced by environment and developmental stage. Molecular markers on the other hand are large in number and are not influenced by the environment and developmental stage. Saturated linkage maps are a pre-requisite for gene tagging, marker assisted selection and map based gene cloning. Saturated linkage maps have been developed in several crop plants like maize, rice, tomato, wheat, potato, barley, cotton, Brassica etc. For example, rice has more than 2000 RFLP markers (Harushima et al., 1998,), several thousand SSR markers (McCouch et al. 2002, IRGSP 2005); tomato has more than 1500 markers (Haanstra et al., 1999). D-genome of wheat has more than 550 mapped RFLP markers (Boyko et al., 1999) and several thousand SSR markers (Somers et al. 2004, Song et al. 2005). In rice centromere positions in all the 12 linkage groups have been defined (Singh et al., 1996). The markers used for developing saturated maps include RFLPs, RAPDs, microsatellites AFLPs SNPs, DArT and a combination of these (Semagn et al 2006).
DNA fingerprinting for varietal identification
Smith and Smith (1992) have presented comprehensive review on the need for fingerprinting of crop varieties. DNA fingerprinting can be used for varietal identification as well as for ascertaining variability in the germplasm. Although any type of marker can be used but RAPDs, microsatellites and AFLPs are the markers of choice for the purpose because all these are PCR based and does not require any prior information on nucleotides. The fingerprinting information is useful for quantification of genetic diversity, characterization of accessions in plant germplasm collections and for protection of proprietary germplasm especially the cms lines. These markers have been used to differentiate even closely related cultivars (Melchinger et al., 1991). Paull et al. (1998) analyzed 124 Australian major wheat varieties and important lines. They were able to distinguish even closely related lines and classified these into four groups. One of the most recent applications of molecular markers has been shown in sex identification of dioecious plants (Parasnis et al., 1999). Here, microsatellite markers (GATA)4is found to reveal sex-specific differences. This can be used as diagnostic markers for identifying male and female plants, right at seedling stage.
Phylogenetic and evolutionary studies
An important use of genetic markers has been to attempt to discern evolutionary relationships within and between species, genera or larger taxonomic groupings. Such studies involve studying similarities and differences among taxa using numerous genetic markers (Paterson et al., 1991). Although phylogenetic trees have previously been established for many species on the basis of visible and isozyme markers and chromosome homology, the DNA markers have recently added to length and breadth of phylogenetic information available for a number of species.
Germplasm evaluation
- Differentiating cultivars and constructing heterotic groups
- Identifying germplasm redundancy, under represented alleles, and genetic gaps in germplasm collections
- Monitoring genetic shifts that occur during germplasm storage, regeneration, domestication, and breeding
- Screening germplasm for novel/ superior genes (alleles)
- Constructing a representative subset or core collection
Molecular markers in heterosis breeding
One of the earliest conceived ideas about the use of molecular markers was its use in heterosis breeding. Earlier results of Lee et al. (1989) in corn suggested that RFLP analysis might provide an alternative to field-testing when attempting to assign maize inbred lines to heterotic groups. Since then several attempts were made to correlate heterosis with variability at molecular level. Melchinger et al. (1991) analyzed 32 maize inbred lines for molecular marker diversity. It was concluded that molecular markers were useful for assigning maize inbred lines to establish heterotic groups and investigating relationships among the inbred lines. However, degree of heterozygosity at RFLP loci was not associated with heterosis for yield for crosses among unrelated lines. Zhang et al. (1995), on the other hand observed a high correlation between specific heterozygosity and mid parent heterosis in rice.
Gene tagging
Gene tagging refers to mapping of genes of economic importance close to known markers. Thus, a molecular marker very closely linked to a gene can act as a `tag' that can be used for indirect selection of gene in breeding programmes. With the construction of molecular map, especially the RFLP maps, several genes of economic importance like disease resistance, stress tolerance, insect resistance, fertility restoration genes, yield attributing traits etc. have been tagged (Table 2). Gene tagging is a pre-requisite for marker-assisted selection and map based gene cloning. In addition gene mapping is throwing more light on evolution of several genes. For example, in Brassica napus, two different restorers, Rfn and Rfp restore two cms sources, nap and pol. Mapping studies showed that both the genes map to same chromosomal region (Li et al., 1998).
Table: Selected examples of desired genes in selected crop plants tagged with molecular markers
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Crop Trait/geneb
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Rice - Blast resistance genes Pi-2(t) Pi-4(t), Pi-10(t)
- Bacterial blight resistance genes xa1, xa2, Xa3, Xa4, xa5, xa8, xa13, Xa21, Xa26, Xa27, Xa29, Xa30
Wheat - Cereal cyst nematode
- Leaf rust resistance genes Lr9, Lr10, Lr19, Lr21, Lr24, Lr37
Rye - Fertility restoration gene Rfg-1
Maize - Leaf blight resistance gene rhm
- Northern corn blight resistance gene Htn-1
- apomixis locus from Trypsacum dactyloides
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Sources: Mohan et al. (1997), Brar and Dhaliwal (1996).
Marker assisted selection
Plant breeders have relied heavily on generating new gene combinations and selecting these new gene combinations empirically. The response in the improvement had been tremendous so far and its efficiency can further be improved by marker-assisted selection. The essential requirements for marker-assisted selection in a plant breeding programme are:
a) Marker(s) should co-segregate or be closely linked (less than 1cM) with the desired trait.
b) An efficient means of screening large populations for molecular marker(s) should be available. PCR based technique to some extent fulfil this.
c) The screening technique should have high reproducibility across laboratories, be economical to use and should be user friendly.
Marker assisted selection can be practiced more efficiently for characters whose phenotypic selection is difficult. For example, transferring a fertility restorer gene from one line to another line through backcrossing needs test crossing before subsequent backcrossing. If such genes are tagged with molecular markers, desirable plants with fertility restorer gene (in heterozygous condition), can be identified and backcrossed. Similarly, screening for abiotic stresses is very difficult. If desirable genes conferring tolerance to abiotic stresses are tagged, these can be selected easily in segregating generations. Also genetic markers can be assayed in non-target areas such as growth chamber, green houses or off-season nurseries, thus permitting more rapid progress. The efficiencies of scale and time accorded by DNA markers are valuable in breeding most species but are of special value in breeding species that have large stature or long generation time (such species as orchard or forest trees) where fewer individuals might save several hectares and fewer generations may save several decades (Paterson et al., 1991)
Marker assisted selection (MAS) is being used efficiently in the following areas:
i. Gene pyramiding.
ii. Marker assisted alien introgression.
iii. Simultaneous identification and pyramiding of QTLs from primitive cultivars and alien species.
Orthologous gene mapping
Molecular markers are being used extensively for studying the divergence and evolution of crop plants. "Comparative mapping" is the name given to this approach. In this marker clones especially cDNA clones of one crop plant are being mapped onto the linkage maps of other crops. This approach of comparative mapping is useful in several ways:
i. More saturated maps can be generated by mapping marker clones of one crop onto the linkage map of other crops.
ii. By cross mapping divergence and evolutionary history of various crop plants can be revealed.
iii. It can make gene cloning from complex organisms comparatively easier.
The first comparative maps in plants were generated by the group lead by S.D.Tanksley in tomato, pepper and potato genomes (Tanksley et al. 1988).
Map-based gene cloning
It refers to the isolation of a gene corresponding to a target trait using molecular maps. Saturated molecular maps offer opportunity for isolating genes whose biochemical products are not known. Map-based cloning consists of four major steps:
a). Development of a high-resolution molecular linkage maps in the region of interest.
b). Physical mapping of the region of interest. This can be achieved through generation of yeast artificial chromosome (YAC) or Bacterial artificial chromosome (BAC) contigs.
c). Identification of appropriate YAC or BAC clones for isolating putative clones harbouring the gene of interest.
d). Verification through transformation that the target gene is isolated.
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