Department of Biotechnology. Molecular Markers. In plant breeding. Nitin Swamy

Transcription

1 Department of Biotechnology Molecular Markers Nitin Swamy In plant breeding 17

2 1. Introduction Molecular breeding (MB) may be defined in a broad-sense as the use of genetic manipulation performed at DNA molecular levels to improve characters of interest in plants and animals, including genetic engineering or gene manipulation, molecular marker-assisted selection, genomic selection, etc. More often, however, molecular breeding implies molecular marker-assisted breeding (MAB) and is defined as the application of molecular biotechnologies, specifically molecular markers, in combination with linkage maps and genomics, to alter and improve plant or animal traits on the basis of genotypic assays. This term is used to describe several modern breeding strategies, including marker-assisted selection (MAS), marker-assisted backcrossing (MABC), marker-assisted recurrent selection (MARS), and genome-wide selection (GWS) or genomic selection (GS) (Ribaut et al., 2010). Molecular Marker A molecular marker is a DNA sequence which is readily detected and whose inheritance can easily be monitored. A molecular marker is a DNA sequence in the genome which can be located and identified. As a result of genetic alterations (mutations, insertions, deletions), the base composition at a particular location of the genome may be different in different plants. The use of molecular markers is based on naturally occurring DNA polymorphism, which forms on the basis for designing strategies to exploit for applied purposes. A marker should be polymorphic, that is, it must exist in different forms so that chromosome carrying the mutant gene can be distinguished from the chromosome with the normal gene by a marker it also carries. These differences, collectively called as polymorphisms can be mapped and identified. Plant breeders always prefer to detect the gene as the molecular marker, although this is not always possible. The alternative is to have markers which are closely associated with genes and inherited together. The molecular markers are highly reliable and advantageous in plant breeding programmes: Molecular markers provide a true representation of the genetic makeup at the DNA level. They are consistent and not affected by environmental factors. Molecular markers can be detected much before development of plants occurs. A large number of markers can be generated as per the needs. 2

3 Basic principle of molecular marker detection Let us assume that there are two plants of the same species one with disease sensitivity and the other with disease resistance. If there is DNA marker that can identify these two alleles, then the genome can be extracted, digested by restriction enzymes, and separated by gel electrophoresis. The DNA fragments can be detected by their separation.for instance, the disease resistant plant may have a shorter DNA fragment while the disease sensitive plant may have a longer DNA fragment (Fig. 53.1). Molecular markers are of two types: 1. Based on nucleic acid (DNA) hybridization (non-pcr based approaches). 2. Based on PCR amplification (PCR-based approaches). Markers Based On DNA Hybridization: The DNA piece can be cloned, and allowed to hybridize with the genomic DNA which can be detected. Marker-based DNA hybridization is widely used. The major limitation of this approach is that it requires large quantities of DNA and the use of radioactivity (labeled probes). Restriction fragment length polymorphism (RFLP): RFLP was the very first technology employed for the detection of polymorphism based on the DNA sequence differences. Its application was first shown by Botstein et al., in 1980 for linkage mapping in humans. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and re-combinations of genomic DNA. An outline of the RFLP analysis is given in Fig. 53.2, and schematically depicted in Fig The procedure basically involves the isolation of genomic DNA, its digestion by restriction enzymes, separation by 3

4 electrophoresis, and finally hybridization by incubating with cloned and labeled probes (Fig. 53.2). 4 Based on the presence of restriction sites, DNA fragments of different lengths can be generated by using different restriction enzymes. In the Fig. 53.3, two DNA molecules from two plants (A and B) are shown. In plant A, a mutations has occurred leading to the loss of restriction site that can be digested byecori. The result is that when the DNA molecules are digested by the enzyme Hindlll, there is no difference in the DNA fragments separated. However, with the enzyme EcoRI, plant A DNA molecule is not digested while plant B DNA molecule is digested. This results in a polymorphic pattern of separation. Advantages i. It is a codominant marker. ii. The results are highly reproducible. Disadvantages i. The process is long and tedious. ii. Large amount of DNA is required. iii. Use of radioactively labeled probes. These disadvantages can be overcome using PCR based markers. Markers Based on PCR Amplification: Polymerase chain reaction (PCR) is a novel technique for the amplification of selected regions of DNA.The advantage with PCR is that even a minute quantity of DNA can be amplified. Thus, PCR-based molecular markers require only a small quantity of DNA to start with.

5 PCR-based markers may be divided into two types: 1. Locus non-specific markers e.g. random amplified polymorphic DNA (RAPD); amplified fragment length polymorphism (AFLP). 2. Locus specific markers e.g. simple sequence repeats (SSR); single nucleotide polymorphism (SNP). Random amplified polymorphic DNA (RAPD) markers: RAPD is a molecular marker based on PCR amplification. It was first shown by Welsh and McClelland in An outline of RAPD is depicted in Fig The DNA isolated from the genome is denatured the template molecules are annealed with primers, and amplified by PCR. particular species. Single short oligonucleotide primers (usually a 10-base primer) can be arbitrarily selected and used for the amplification DNA segments of the genome (which may be in distributed throughout the genome). The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bends on gel electrophoresis. Genomic DNA from two different plants often results in different amplification patterns i.e. RAPDs. This is based on the fact that a particular fragment of DNA may be generated from one individual, and not from others. This represents polymorphism and can be used as a molecular marker of a Advantages i. The method is quick and easy. ii. Amount of DNA required is very less. iii. No prior sequence information is needed. iv. Many polymorphic markers can be generated with a single primer. 5

6 Disadvantages i. Reproducibility is low as the results vary greatly with different reaction conditions because the conditions used for PCR, like annealing temperatures, random primers and concentration of reagents etc. is not stringent. ii. They are dominant markers. Amplified fragment length polymorphism (AFLP): AFLP is a novel technique involving a combination of RFLP and RAPD. AFLP is based on the principle of generation of DNA fragments using restriction enzymes and oligonucleotide adaptors (or linkers), and their amplification by PCR. Thus, this technique combines the usefulness of restriction digestion and PCR. This method was first shown by Vos et al., in The DNA of the genome is extracted. It is subjected to restriction digestion by two enzymes (a rare cutter e.g. Msel; a frequent cutter e.g. EcoRI). The cut ends on both sides are then ligated to known sequences of oligonucleotides (Fig. 53.5). PCR is now performed for the pre-selection of a fragment of DNA which has a single specific nucleotide. By this approach of preselective amplification, the pool of fragments can be reduced from the original mixture. In the second round of amplification by PCR, three nucleotide sequences are amplified. This further reduces the pool of DNA fragments to a manageable level (< 100). Autoradiography can be performed for the detection of DNA fragments. Use of radiolabeled primers and fluorescently labeled fragments quickens AFLP. 6

7 AFLP analysis is tedious and requires the involvement of skilled technical personnel. Hence some people are not in favour of this technique. In recent years, commercial kits are made available for AFLP analysis. AFLP is very sensitive and reproducible. It does not require prior knowledge of sequence information. By AFLP, a large number of polymorphic bands can be produced and detected. Advantages i. A large number of polymorphic fragments can be obtained in a single experiment. Disadvantages ii. iii. The process is complex as compared to the more advanced techniques of marker development (discussed ahead) The fragments are mostly scored as dominant markers. Sequence tagged sites (STS): Sequence tagged sites represent unique simple copy segments of genomes, whose DNA sequences are known, and which can be amplified by using PCR. STS markers are based on the polymorphism of simple nucleotide repeats e.g. (GA)n, (GT)n, (CAA)n etc. on the genome. STS have been recently developed in plants. When the STS loci contain simple sequence length polymorphisms (SSLPs), they are highly valuable as molecular markers. STS loci have been analysed and studied in a number of plant species. Microsatellites or Simple sequence repeat or short tandem repeats (SSRs) These are ideal genetic markers for detecting differences between and within species of genes of all eukaryotes (Farooq and Azam, 2002). It consist of tandemly repeated 2-7 base pair units arranged in repeats of mono-, di-, tri-, tetra and penta-nucleotides (A,T, AT, GA, AGG, AAAG etc) with different lengths of repeat motifs. These repeats are widely distributed throughout the plants and animal genomes that display high level of genetic variation based on differences in the number of tandemly repeating units of a locus. The variation in the number of tandemly repeated units results in highly polymorphic banding pattern (Farooq and Azam, 2002) which are detected by PCR, using locus specific flanking region primers where they are known. Some of the prominent features of these markers are that they are dominant fingerprinting markers and codominant sequence tagged microsatellites (STMS) markers (Joshi et al, 2011). 7

8 The reproducibility of microsatellites is such that they can be used efficiently by different Sequence characterized amplified regions (SCARs): SCARs are the modified forms of STS markers.scars are DNA fragments amplified by the PCR using specific bp primers, designed from nucleotide sequences established from cloned RAPD fragments linked to a trait of interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. Obtaining a codominant marker may be an additional advantage of converting RAPDs into SCARs, although SCARs may exhibit dominance when one or both primers partially overlap the site of sequence variation. Length polymorphisms are detected by gel electrophoresis. SCARs are locus specific and have been applied in gene mapping studies and marker assisted selection. Advantages The main advantage of SCARs is that they are quick and easy to use. In addition, SCARs have a high reproducibility and are locus-specific. Due to the use of PCR, only low quantities of template DNA are required. Disadvantages Disadvantages include the need for sequence data to design the PCR primers. Quantitative Trait Loci: These are many characteristics controlled by several genes in a complex manner. Some good examples are growth habit, yield, adaptability to environment, and disease resistance. These are referred to as quantitative traits. The locations on the chromosomes for these genes are regarded as quantitative trait loci (QTL). The major problem, the plant breeder faces is how to improve the complex character controlled by many genes. It is not an easy job to manipulate multiple genes in genetic engineering. Therefore, it is a very difficult and time consuming process. For instance, development of Golden Rice (with enriched pro-vitamin A) involving the insertion of just three genes took about seven years. Linkage analysis: Linkage analysis basically deals with studies to correlate the link between the molecular marker and a desired trait. This is an important aspect of molecular breeding programmes. Linkage analysis has to be carried out among the populations of several generations to establish the appropriate linkage. In the earlier years, linkage analysis was carried out by use of isoenzymes and the associated polymorphisms. Molecular markers are now being used. The techniques employed for this purpose have already been described. 8

9 Molecular Marker Assisted Selection: Selection of the desired traits and improvement of crops has been a part of the conventional breeding programmes. This is predominantly based on the identification of phenotypes. It is now an accepted fact that the phenotypes do not necessarily represent the genotypes. Many a times the environment may mark the genotype. Thus, the plant s genetic potential is not truly reflected in the phenotypic expression for various reasons. The molecular marker assisted selection is based on the identification of DNA markers that link/ represent the plant traits. These traits include resistance to pathogens and insects, tolerance to abiotic stresses, and various other qualitative and quantitative traits. The advantage with a molecular marker is that a plant breeder can select a suitable marker for the desired trait which can be detected well in advance. Accordingly, breeding programmes can be planned. The following are the major requirements for the molecular marked selection in plant breeding: i. The marker should be closely linked with the desired trait. ii. The marker screening methods must be efficient, reproducible and easy to carry out. iii. The analysis should be economical. Molecular Breeding: With rapid progress in molecular biology and genetic engineering, there is now a possibility of improving the crop plants with respect to yield and quality. The term molecular breeding is frequently used to represent the breeding methods that are coupled with genetic engineering techniques. Improved agriculture to meet the food demands of the world is a high priority area. For several years, the conventional plant breeding programmes (although time consuming) have certainly helped to improve grain yield and cereal production. The development of dwarf and semi-dwarf varieties of rice and wheat have been responsible for the Green Revolution, which has helped to feed millions of poverty-stricken people around the world. Many developments on the agriculture front are expected in the coming years as a result of molecular breeding. 9

10 Application of molecular markers in plant genome analysis and breeding- Molecular markers have been look upon as atools for a large number of applications ranging fromlocalization of a gene to improvement of plant varietiesby marker-assisted selection, called genome analysiswhich has generated a vast amount of information and anumber of databases are being generated to preserveand popularize it (Joshi et al, 2011). a) Application of MAS in vegetative propagated crops The first generation of DNA markers analysis of vegetative propagated crops at IITA was focused ongermplasm characterization, construction of preliminarylinkage maps and development of disease diagnosticsin plantain/banana, cassava and yam (Ortiz, 2004). a.plantain / Banana At IITA planta in improvement was nominated asthe model system for developing molecular breedingsystems within this Institute (Crouch and Tenkoune,1999). Parthenocarpy (ability to develop fruit in theabsence of seed development) was chosen as an idealcharacter for the initiation of a marker assisted selection. b. Cowpea Cow pea, a legume crop grown in the semi-aridtropics is attached by insect pests. Thus, in cowpea, thedevelopment of markers for resistance to thrips,bruchids, maruca and pod borer is considered of greatpriority. In the long term, markers for resistance toparasitic weed (striga) and markers for genescontributing to drought resistance are considered a highpriority intervention (Morales et al, 2000) program. At present SSRs are the most widely usedmarkers by maize researchers due to their availability inlarge numbers in the public domain including their simplicity and effectiveness. These PCR-based, genetically co-dominate marker are robust, reproducible, hypervariable, abundant, and uniformly dispersed in plant genomes (Powell et al, 1996). Also both SSRs and SNPscan be reliably applied on a large scale and thereforeoffer significant advantages for genetic and breeding purposes. SSR markers have been successfully used for DNA finger printing and analysis of genetic diversity inchina, India, Indonesia and Thailand (Prassana and Pixley, 2010). Following the first report on QTLs for yield related traits in maize (Stuber et al, 1987), maize researchers worldwide have generated numerous reports of molecular markers tagging genes/qlts for diverse traits of agronomic and scientific interest (Prasanna and Pixley, 2010). QLTs for several imp ortant traits affecting maize such as plant height, downy mildew resistance, Maize dwarf Mosaic Virus resistance, head smut resistance, drought stress tolerance, water logging, nutrient components under low nitrogen and high-oil content. 10

11 Further, significant progress has been made worldwide in optimizing MAS for improvement of both qualitative and quantitative inherited traits using maize as a model system. One successful example of MAS for maize development and of particular use is the utilization of opaque 2-specific SSR markers in conversion of maize lines in quality protein maize (QPM) lines with enhanced nutritional quality (Buba et al, 2005). A MAS-derived QPM hybrid is the Vivek QPM hybrid 9, recently released in Almora, India, which was developed through marker-assisted transfer of the 02 gene and phenotypic selection for endosperm modifiers in the parental lines (Buba et al, 2005).Using MAS Scientist at IARI have pyramided major genes /QTLS for resistance toturicum leaf blight and Polysora rust in five elite Indian lines (Prassana et al, 2009) and these are CM 137, CM138, CM139, CM150 and CM151 which are parents of three single-cross hybrids. Marker-based breeding and conventional breeding: Marker-assisted breeding became a new member in the family of plant breeding as various types of molecular markers in crop plants were developed during the 1980s and 1990s. The extensive use of molecular markers in various fields of plant science, e.g. germplasm evaluation, genetic mapping, map-based gene discovery, characterization of traits and crop improvement, has proven that molecular technology is a powerful and reliable tool in genetic manipulation of agronomically important traits in crop plants. Compared with conventional breeding methods, MAB has significant advantages: a. MAB can allow selection for all kinds of traits to be carried out at seedling stage and thus reduce the time required before the phenotype of an individual plant is known. For the traits that are expressed at later developmental stages, undesirable genotypes can be quickly eliminated by MAS. This feature is particularly important and useful for some breeding schemes such as backcrossing and recurrent selection, in which crossing with or between selected individuals is required. b. MAB can be not affected by environment, thus allowing the selection to be performed under any environmental conditions (e.g. greenhouse and off-season nurseries). This is very helpful for improvement of some traits (e.g. disease/pest resistance and stress tolerance) that are expressed only when favorable environmental conditions present. For lowheritability traits that are easily affected by environments, MAS based on reliable markers tightly linked to the QTLs for traits of interest can be more effective and produce greater progress than phenotypic selection. c. MAB using co-dominance markers (e.g. SSR and SNP) can allow effective selection of recessive alleles of desired traits in the heterozygous status. No selfing or test crossing is needed to detect the traits controlled by recessive alleles, thus saving time and accelerating breeding progress. d. For the traits controlled by multiple genes/qtls, individual genes/qtls can be identified and selected in MAB at the same time and in the same individuals, and thus MAB is particularly suitable for gene pyramiding. In traditional phenotypic selection, however, to distinguish individual genes/loci is problematic as one gene may mask the effect of additional genes. 11

12 e. Genotypic assays based on molecular markers may be faster, cheaper and more accurate than conventional phenotypic assays, depending on the traits and conditions, and thus MAB may result in higher effectiveness and higher efficiency in terms of time, resources and efforts saved. The research and use of MAB in plants has continued to increase in the public and private sectors, particularly since 2000s. Reference i. ii. iii. Review : The Importance of Molecular Markers in PlantBreeding ProgrammesBy P.M Jonah, L. L. Bello, O. Lucky, A. Midau, S. M. MoruppaAdamawa State University, Nigeria 12