ASSIGNMENT ON

APPLICATION OF MOLECULAR MARKERS

SUBMITTED BY

PUNESH (2009BS36D) (Ph.D. BIOCHEMISTRY)

CCS HARYANA AGRICULTURE UNIVERSITY HISAR

APPLICATION OF MOLECULAR MARKERS
Crop improvement and breeding have undergone a tremendous shift to feed the growing population in Asian countries. Breeding a new variety by using conventional breeding methods will take about eight and twelve years, although the release of an improved variety cannot be guaranteed. Conventional breeding is both time consuming and dependent on environmental conditions. Novel and innovative methods of biotechnology such as tissue culture, transformation, clonal propagation and molecular markers, have contributed to the development of high yielding, nutrient-rich modified or new varieties. Plant tissue culture played an important role in an improvement of perennial crops. In this article, we have discussed the application of molecular markers in the plant tissue culture, marker assisted selection and breeding. Morphological markers in plants. Marker in lifesciences is defined as a trait or an allele or a DNA sequence or a cytogenetic segment or a chromosome fragment or a protein or an enzyme used as an experimental probe to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene. In general, different types of markers are used in lifesciences. Markers are broadly classified into morphological markers and molecular markers. Morphological markers (or also called phenotypic markers) are those distinguishable traits that are evident to human naked eyes. These markers are selected based on the experience of the breeder to correlate a phenotypic trait with a trait of interest. Morphological markers differ among species, genus and varieties of plants and animals. It is the easiest and quickest way to identify or detect the presence of a morphological marker with that of a trait for improvement. For example: Many morphological markers, originating mainly from mutational studies, have been assigned to specific barley chromosomes. Morphological markers are detected and well established in different members of Poaceae (grass family) family such as different growth, yield and disease resistance traits are related to markers such as purple hull, phenol staining, glabrous leaf and hull, purple node, awned panicle, lazy growth habit, brown pericarp and others. Molecular markers in plants. Molecular markers have wide application in different branches of lifesciences. In genetics, molecular marker is defined as a fragment of DNA sequence that is associated to a part of the genome carrying genes responsible for a trait. It is usually defined as an allele or DNA sequence or chromosome fragment indicating the existence of a metabolism or chemical or physical process. Sometimes this category of markers is referred to as biomarkers or biosignatures or molecular signatures. In medicine, it could be a substance that is introduced into an organism as a means to detect something (Ex: rubidium chloride is used as a radioactive isotope to evaluate perfusion of heart In biology, it could be a substance native to the organism whose detection indicates a particular disease state (Ex: presence of an antibody might indicate an infection). Molecular markers are also sub-divided into Biochemical markers (Eg: Isozymes, Allozymes), DNA based markers (Eg: RFLP, RAPD, SSRs), Physiological markers, Biomarkers and Protein markers (also called biochemical

markers) depending on the biochemistry, physiology and origin of markers within the plant. Molecular markers are phenotypically neutral. This is a significant advantage compared to traditional phenotypic markers. Isozyme is a molecular marker system based on the staining of proteins with identical function, but different electrophoretic mobilities. The weakness of Isozyme markers is that each of the proteins that are being scored might not be expressed in the same tissue and at the same time in development. Therefore several samplings of the genetic population need to be made. Molecular markers have very rapidly complemented the classical strategies. Molecular markers include biochemical constituents (e.g. secondary metabolites in plants) and macromolecules, viz. proteins and deoxyribonucleic acids (DNA). Analysis of secondary metabolites is restricted to those plants that produce a suitable range of metabolites which could be easily analyzed and which could be distinguished between varieties. These metabolites, which are being used as markers should be neutral to environmental effects or management practices. Hence, amongst the molecular markers used, DNA markers are more suitable and ubiquitous to most of the living organisms. The following properties are desirable for ideal DNA markers: • Codominant inheritance (determination of homozygous and heterozygous states of diploid organisms) • Easy access (availability) and Easy exchange of data between laboratories • Easy and fast assay • Frequent occurrence in genome • High reproducibility and Highly polymorphic nature • Selective neutral behaviour (the DNA sequences of any organism are neutral to environmental conditions or management practices) It is extremely difficult to find a molecular marker which would meet all the above criteria. Depending on the type of study to be undertaken, a marker system can be identified that would fulfill atleast a few of the above characteristics. Various types of molecular markers are utilized to evaluate DNA polymorphism and are generally classified as “hybridization based markers” and “polymerase chain reaction (PCR) based markers”. In the former, DNA profiles are visualized by hybridizing the restriction enzyme digested DNA, to a labelled probe, which is a DNA fragment of known origin or sequence. PCR-based markers involve in vitro amplification of particular DNA sequences or loci, with the help of specifically or arbitrarily chosen oligonucleotide sequences (primers) and a thermostable DNA polymerase enzyme. The following are the examples of DNA based molecular markers: • Restriction Fragment Length Polymorphisms (RFLPs) • Random Amplified Polymorphic DNAs (RAPDs) • Amplified Fragment Length Polymorphisms (AFLPs) • Simple Sequence Repeats (SSRs) • Sequence Tagged Microsatellite Sites (STMS) • Sequence Tagged Sites (STS) • Single Nucleotide Polymorphism (SNP)

The detailed description of development of molecular markers is published by different peer reviewed articles. The comparison of different methods of estimating genetic diversity could define their usefulness in plant breeding and genetic improvement programs. RFLPs have been used the most extensively. RFLP, SSR and AFLP markers are most effective in detection of polymorphism in plants. RFLP markers have several advantages in comparison with the RAPD and Isozyme markers, such as (1) they are codominant and unaffected by the environment, (2) any source DNA could be used for the analysis and (3) mapping markers linked to traits in a population that has not undergone specific stress effects. The primary drawback of RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals. AFLPs and SSRs are more popular than RFLP. RFLP process is laborious. SNP is chosen when sufficient amount of sequence information is available and analysis tool is available to assign gene function.

Application of markers in plant genetics and tissue culture. In commercial industry, molecular markers are used in the confirmation of genetic fidelity in micropropagated tree species, where life span is quite long and performance of micropropagated plants could only be ascertained after their long juvenile stage in field conditions. RFLPs (using nDNA and cpDNA probes) and RAPDs are also used for characterization and identification of genetic resources of perennial crops and to solve problems related to plant genetic diversity conservation. Scientists have used markers in detection of epigenetic variations originated during storage and conservation, (i.e. detect duplications, seed mixtures, inadvertent outcrossing and genetic drift). SSR markers are used

to determine the degree of relatedness among individuals or groups of accessions, to clarify the genetic structure, or partitioning of variation among individuals, accessions, population and species of rice. Markers are used to detect genes for segregation distortion in anther culture derived plants. Genetic diversity Assessment of genetic diversity is important in plant breeding if there is to be improvement by selection. For assessment of genetic diversity, molecular markers have been generally superior to morphological, pedigree, heterosis, and biochemical data (isozymes and chromatography). Genetic diversity commonly is measured by genetic distance (GD) or genetic similarity (GS = 1 - GD), both of which imply that there are either differences or similarities at the genetic level. Published applications of molecular marker-based GD in plant breeding have been limited primarily to maize, but results should apply to other species. Melchinger (1993) reviewed the application of molecular marker-based GD for assigning maize inbred lines to heterotic groups, determining the relation between inbred lines and hybrids, and predicting hybrid performance. Data showed that GS calculated from molecular marker data faithfully separated inbred lines into their heterotic groups. There also seems to be promise of assigning inbreds of unknown pedigree to heterotic groups although a large number of markers (> 100) and well-characterized reference populations may be needed to obtain an accurate assessment of GS. Strong correlations (0.61 to 0.95) between Malecot's coancestry coefficient (f) and GS for related (f > 0) genotypes indicated that pedigree data provide reliable estimates of GS. Genetic similarity estimates based on molecular markers are expected to be superior to estimates of f because of unreliable or incomplete pedigree data and because of the assumptions required to calculate f. Molecular marker-based GD has some potential for predicting hybrid performance of related lines, but in typical hybrid-breeding programs, in which hybrids are produced from unrelated lines from different heterotic groups, molecular marker-based GD has been of no value in predicting hybrid performance. These results suggest the use of molecular marker-based GD for predicting hybrid performance in crops in which either hybrids are being explored, such as wheat (Triticum aestivum [Vill., Host] Mackey), and soybean (Glycine max (L.) Merr.), or hybrid breeding is being practiced, but distinct heterotic groups have not been developed. Molecular marker-based GD also has potential for assessing changes in genetic diversity over time, protection of intellectual property rights, registration of germplasm in countries having ratified the rules of the UPOV convention, and evaluation of new sources of germplasm for their potential to increase genetic diversity. Direct applications to plant breeding, however, have been limited so far to prediction of hybrid performance. But accurate prediction of hybrid performance does not seem likely unless gene action is primarily dominant or overdominant, complementary heterotic groups are established, trait heritability is high, at least 30 to 50% of the markers are linked to QTL, and no more than 20 to 30% of the markers are dispersed randomly. Molecular markers, however, may be useful for early generation testing in hybrid-breeding programs. If individual markers or marker intervals associated with combining ability can be identified when a plant or progeny is crossed onto a given tester, then these markers could be used as a

first screen to identify the top 50% of the progenies for field evaluation. Although this procedure would not decrease the time to cultivar development, it would decrease the amount of material tested or permit the evaluation of a wider range of germplasm for the same amount of field resources. An equally important application of molecular marker-based GD may be in the selection of parents to cross in a breeding program. This application deserves serious attention because breeders currently rely primarily on pedigree and performance data for choosing parents in breeding programs. Using molecular markers to select parents has the potential to allow simultaneous maintenance of genetic diversity and performance. Dudley et al. (1992) presented one application of molecular markers for choosing parents, and additional research is needed in this area. Using molecular markers to choose parents likely will require establishment of a relation between GD and genetic variation, and many of the same conditions necessary for predicting hybrid performance may be required for choosing parents. Using molecular markers as a diagnostic tool to survey new or exotic germplasm for novel genetic diversity also may be possible. It is unlikely, however, that this use will be possible with random genomic or cDNA clones because molecular marker-based genetic diversity will not guarantee genetic diversity for the traits of interest. Screening with probes of expressed genes with known function offers the greatest potential in this area. Mapping QTL The vast majority of molecular marker research in quantitative traits has been devoted to mapping QTL. Mapping QTL is really a misnomer, because what is actually being done is the mapping of chromosomal regions containing one or more putative QTL. With current mapping technology, the existence of a single QTL between two flanking markers cannot be resolved clearly. Most studies reported to date have detected, localized, and estimated genetic effects in the same experiment because of resource limitations. Genetic effects of mapped QTL regions are over estimated by this procedure because of sampling errors (Lande and Thompson, 1990; Lande, 1992). Furthermore, few researchers have followed up with the necessary experiments to verify the effect of a chromosomal region on phenotype across mapping populations. Our purpose in this section is not to review QTL mapping studies in detail, but rather to outline the steps taken in QTL mapping experiments, to demonstrate the general results that have been obtained, to outline some of the problems in translating these results into plant improvement, and to show the types of previously unattainable information that these results have contributed. The first step in QTL mapping studies is to detect QTL, while minimizing the occurrence of false positives (Type I errors, that is, declaring an association between a marker and QTL when in fact one does not exist). Two distinct methods are used to detect QTL. The single marker approach, sometimes referred to as the one-way analysis of variance (ANOVA), has been used extensively, especially with isozymes. The second approach, interval mapping, detects QTL by using flanking markers. This approach is more complicated analytically than the ANOVA approach and involves application of the maximum likelihood method, which requires sophisticated computer software. Lander and Botstein (1989) have developed formulae for calculating significance levels appropriate for both methods when the genome size, number of chromosomes, number

of marker intervals, and the overall false positive rate desired are given. Several statistical procedures have been developed for the application of both ANOVA and interval mapping. When the same false positive rates are used, there are few reasons to suspect that the two methods would detect substantially different QTL. Stuber et al. (1992) compared the two methods and found that they identified basically the same QTL. Those researchers reported, however, some advantages to using the interval mapping approach. Because of the increased power associated with using flanking markers, the method gives the most likely location of the QTL under the assumption of a single QTL in the interval, and the interval mapping approach allows ambiguous or missing data. Once QTL are detected, the next step is to estimate the genotypic effect of the QTL and to localize the QTL to a precise genomic region. The interval mapping approach seems superior to the ANOVA approach for both estimation of effects and localization of the QTL. The success of both methods depends on the linkage between marker(s) and QTL, the number and type of progeny evaluated, the heritability of the trait, and the magnitude of the effects at QTL that one desires to detect. Several methods and genetic designs have been suggested for detecting, estimating effects, and localizing QTL. Manipulating traits controlled by a few major loci The manipulation of traits controlled by a few major loci (loci that can be studied using Mendelian genetics) may offer the greatest promise in the short term for application to plant breeding. The primary applications of this technique will be for traits controlled by a single gene (monogenic) or those controlled by at most two or three loci (oligogenic). The most successful applications will be in those species with well developed molecular marker maps. These applications will be immediately useful for "defensive breeding," that is, when a desirable genotype is available but lacks resistance to important insects and diseases. Other applications, not fitting into the category of defensive breeding may include seed modifications controlled by a few genes, restorer genes for cytoplasmic male sterility, dwarfing genes for shorter plant height, and maturity genes for adaptability. The first requirement of using molecular markers in this context is to develop a precise molecular marker linkage map and then to use these markers to map gene(s) controlling the trait of interest. Many methods for mapping genes of interest are available, including a variety of applications suitable to most species with polymorphic markers. The final step is to use marker-facilitated selection to transfer the gene(s) to the genotypes desired. Two methods are available, both of which begin with inbred lines, backcross selection, and pedigree selection. If the cost of molecular-marker technology is ignored, the primary factor affecting the design and the success of marker-facilitated selection is how tightly linked a single marker is to the gene or how tightly bracketed the gene is by two markers. The idea is to obtain a marker or a set of flanking markers linked tightly enough to the gene so that a recombination event does not occur between the marker and the gene during backcrossing or pedigree selection. Melchinger (1990) presented extensive theoretical and numerical results for the backcross method concerning the optimal family size and the number of plants per family that must be genotyped with molecular markers. The results are complicated and will have to be assessed on a case by case basis. The economics of marker assisted backcrossing will be a function of the cost of marker assays, the cost of direct

screening, and the value of accelerating the backcrossing program. Results regarding sample sizes required for pedigree selection are unavailable, although marker-gene linkage is the primary consideration. There are many practical applications of molecular markers to traits controlled by few loci. Most of the applications involve situations in which either screening for the trait is difficult or scoring of the trait occurs late in plant development. These applications may include pests for which natural inoculum is unreliable or artificial inoculation procedures are undeveloped or unreliable. Examples include nematodes or Aspergillus, both of which have broad host ranges and unreliable natural and artificial inoculation. Diseases in which resistance is influenced strongly by the environment also would be good candidates for marker facilitated selection. Unfortunately, the very situations favoring marker-facilitated selection also make it difficult to map the resistance genes precisely. Marker-facilitated selection has been advantageous for backcrossing recessive genes, when progeny tests are needed after every backcross generation to identify heterozygotes or when resistance can be determined only after flowering. Markers in these situations could greatly reduce workload and backcrossing time. Other examples include pyramiding resistance genes, developing multilines in which many race-specific resistance genes are involved that are sometimes difficult to distinguish, and selecting for resistance to exotic or quarantined pathogens. One of the primary advantages of marker-facilitated backcrossing has been in increasing the speed of recovery of the recurrent parent genome. In addition to having a tight marker-gene linkage, one or more neutral polymorphic markers will be required per chromosome arm. The idea is to screen for plants having the resistance gene and to identify those plants with the greatest proportion of markers homozygous for the recurrent parent. A possible limitation of this procedure is that there may be an increase in the number of plants needing to be assayed with markers. The procedure could be applied, however, to subsequent backcross generations to ensure the recovery of unlinked segments of recurrent parental genome. Manipulating traits controlled by many loci The molecular marker manipulation of traits controlled by many loci (from a plant breeding perspective many is generally greater than five) is of great interest to plant breeders and represents one of the fields greatest challenges. Plant breeders concentrate effort on breeding for quantitative traits, and breeding for qualitative traits is generally a trivial, albeit time consuming, process. The matter is further complicated because breeders usually evaluate simultaneously in many populations -- four or more complexly inherited traits. Obviously, any technology enhancing the breeders' ability to select for these traits would be adopted rapidly.The molecular marker manipulation of qualitative traits is feasible precisely because so much is known about the biology of these traits. The number of loci is known; there are no questions about what a major locus is -- in fact, many of these loci have been cloned; the gene action is known precisely; epistatic interactions, if any, are relatively easy to decipher; and interactions with environment are easy to determine. In short, the amount that can be known about qualitative traits is limited only by one's desire to know. As pointed out earlier, for quantitative traits, the answers to these questions are based only upon averages over an unknown number of loci. At the outset, the manipulation of quantitative traits by molecular

markers has a serious disadvantage. Even with these limitations, there still may be many applications of marker-QTL associations. But very little theoretical or applied research has been published in this area. The most-cited application has been marker-assisted selection (MAS) although the context of application often has been ignored. In plant breeding, there are two distinct but related applications: germplasm enhancement (recurrent selection) and cultivar or hybrid development. These two applications are separated because recurrent selection usually is applied to random mating populations possibly at or near linkage equilibrium, whereas cultivar or hybrid development typically begins with populations derived by crossing elite inbred lines at or near maximum linkage disequilibrium . Clearly, two different approaches are needed for these breeding schemes. Lande and Thompson (1990) and Lande (1992) investigated the efficiency of MAS for both individual and mass selection in random-mating populations. There are three approaches to applying MAS to plant breeding: (1) selection on markers alone with no measurement of phenotype; (2) simultaneous selection on markers and phenotype; and (3) two-stage selection, the first stage involving use of markers to select among seedlings and the second involving phenotypic selection among surviving adults. On individuals, the efficiency of MAS relative to that of phenotypic selection of the same intensity is (p/h2)1/2, where p is the proportion of the additive genetic variance accounted for by markers, and h2 is the heritability. Selection on markers alone will be more efficient than phenotypic selection only when the proportion of the genetic variance explained by markers exceeds the heritability of the trait. Therefore, selection on markers alone will be most useful for traits with low heritabilities when large proportions of their variability have been explained by markers. Lande and Thompson (1990) concluded that molecular marker loci can be used to enhance the efficiency of artificial selection for quantitative traits. The potential efficiency of MAS depends upon the heritability of the trait, the proportion of the genetic variance explained by the markers, and the selection method. A major practical problem in using MAS is that recombination will reduce linkage disequilibrium between the markers and QTL, thus diminishing selection effectiveness. The successful application of MAS will require very tight linkages between markers and QTL. Understanding the genetic architecture of quantitative traits Although there have been few direct applications of molecular markers in plant breeding, published research has begun to provide information on the genetic architecture of quantitative traits. Molecular marker-mapping data from several species now suggest that genetic variation for quantitative traits is controlled by a few loci with large effects and many loci with progressively smaller effects (Lande, 1992). Beavis et al. (1991) found 14 genomic regions associated with plant height in four F2 maize populations, but few of these regions were in common across populations. All 14 regions, however, were associated with known qualitative genetic loci. Thus, circumstantial evidence supports Robertson's (1985) hypothesis that QTL have alleles with a range of effects, alleles with large effects causing recognition of the locus as qualitative, and alleles with small effects causing recognition of the locus as quantitative. The results from QTL-mapping studies regarding gene action and epistasis are, so far, difficult to interpret. Stuber et al. (1992) reported that, with one exception, all the QTL mapped for grain yield in maize showed heterozygote superiority. Either overdominance or

pseudo-overdominance, therefore, likely was important in the mapping population. These results cannot separate the two causes of heterozygote superiority, primarily because the number of QTL residing in a marker interval cannot be resolved. Although not mentioned by the authors, heterozygote superiority also could result from overestimation of effects as pointed out by Lande and Thompson (1990) and Lande (1992). Stuber et al. (1992) and Paterson et al. (1991) found little evidence for epistasis in maize and tomato (Lycopesicon esculentum [E]), respectively. Paterson et al. (1991) concluded that molecular markermapping studies conducted to date may identify preferentially QTL that function independently of unlinked genetic factors. Those researchers suggested that the role of epistasis in quantitative inheritance needs to be studied in larger populations, with more closely spaced markers, and/or with specially constructed genetic stocks carrying particular QTL. Stuber et al. (1992) found limited evidence for G x E interaction even though a standard analysis of the data revealed significant G x E interaction for many of the traits. Thus it may be possible to detect QTL with large effects in relatively few environments. Paterson et al. (1991) reported that individual QTL showed a range of sensitivities to environments, some QTL beingn detected in all environments and some being detected in only one environment . Application of molecular markers, micro-level tests and interclass hybridizations in improving wheat grain quality Hard and soft wheats are two distinct classes of wheat, and generally crosses are made within class because of distinct quality goals. For example, hard wheats with strong gluten and high protein content are preferred for bread and chapati making and soft wheat with weak gluten and low protein content for biscuit, cakes, pretzels and white salted noodles. This has restricted combinations of desirable alleles from both the classes and hence reduced the genetic base. Analysis of large numbers of wheat varieties released in India during the last 100 years showed the predominance of hard wheats with null mutation in puroindoline A. This shows that within class (hard) hybridizations had been attempted in majority of crosses in India in wheat breeding. Moreover, during the past two decades wheat breeders have used the short arm of rye chromosome 1R as source of genes for disease and pest resistance and improved agronomic performance. However, reduced gluten strength and loaf volume and increased dough stickiness have been reported associated with 1B/1R translocation. Therefore, combining desirable alleles using interclass hybridizations would expand genetic bases and improve the chances of wheat improvement for yield and quality traits. This can be accomplished by utilizing the existing knowledge of genetic basis of major grain quality traits such as grain hardness, protein content and gluten strength at molecular level as well as their phenotypic assessment using micro-level tests in early segregating generations. Application of molecular markers to aquaculture and brood stock management with special emphasis on microsatellite DNA The use of molecular genetic markers to address questions related to aquaculture management has steadily grown over the last two decades. These markers can provide valuable information for various aspects of aquaculture practice, such as: (i) genetic identification and discrimination of aquaculture stocks; (i¡) monitoring of inbreeding or other

changes in the genetic composition of the stocks that may result from such phenomena as breeding programmes, founder events and genetic drift; (iii) comparisons between hatchery and wild stocks; (¡v) assessment of the impact on natural populations of escaped or released cultured fish; (v) assignment of progeny to parents through genetic tags, so that animals from different families can be reared together in breeding programmes; (vi) identification of marker genetic loci associated with quantitative trait loci (QTL) and use of these markers in selection programmes (marker assisted selection); and (vii) assessment of successful implementation of genetic manipulations such as induction of polyploidy and gynogenesis. Allozymes were the first molecular genetic tools useidn aquaculture management. Mitochondrial DNA (mtDNA) provided another valuable marker, because of increased resolution and discrimination power in studies of population structure. Nuclear DNA markers were more recently introduced in aquaculture studies and their main attractiveneslasy in their abundance in the genome, their Mendelian inheritance, and their potential to detect high levels of polymorphism. Especially microsatellite DNA analysis revolutionized the use of molecular genetic markers in aquaculture and seems to be the marker destined to dominatteh is type of studies in the coming years. Application of molecular markers (RAPD, AFLP and Microsatellites) to Iberian pig genotype characterization Use of molecular markers is specially interesting for the genetic study of the Iberian pig, whose morphologic and physiologic variability prevents the knowledge of his populational structure. Crossbreeding, mainly with Duroc, is a common practice to increase the carcass performance which leads to the attainment of worse quality cured products. Molecular analysis techniques allow the estimation of genetic variability and divergence between species and populations, and thus they can be used for phylogenetic studies, conservation programs and control of the genetic origin of products. This work presents the results obtained with the application of three different techniques of DNA polymorphisms detection to samples from different populations of Iberian pigs. The RAPD technique was used for the identification of diagnostic markers which allowed the detection of Duroc genes in Iberian pig samples. AFLP technique was used for the attainment of specific markers of one Iberian pig population, with a high inbreeding level, in order to study its possible utilization in a conservation program. Finally, seven populations have been analysed with 19 microsatellite markers. The analysis of these polymorphic loci genotypes enabled the estimation of different parameters of genetic diversity (heterozygosity, genetic distances). MOLECULAR MARKERS AND THEIR APPLICATIONS IN CEREALS BREEDING The development of molecular techniques for genetic analysis has led to a great increas in our knowledge of cereal genetics and our understanding of the structure and behaviour of cereal genomes. These molecular techniques, in particular the use of molecular markers, have been used to monitor DNA sequence variation in and among the species and create new sources of genetic variation by introducing new and favourable traits from landraces and related grass species. Improvements in marker detection systems and in the techniques used to identify markers linked to useful traits, has enabled great advances to be made in recent years. While RFLP markers have been the basis for most work in crop plants,

valuable markers have been generated from RAPDs and AFLPs. Simple sequence repeats (SSR) or microsatellite markers have been developed more recently for major crop plants and this marker system is predicted to lead to even more rapid advances in both marker development and implementation in breeding programs. Identification of markers linked to useful traits has been based on complete linkage maps and bulked segregant analysis. However, alternative methods, such as the construction of partial maps and combination of pedigree and marker information, have also proved useful in identifying marker/trait associations. Genotype identification and genetic diversity It is vital for plant breeding programmes to have sufficient diversity available to allow for the production of new varieties that are aimed towards the improvement of crop productivity and able to withstand damage from biotic and abiotic factors. In this respect, efforts have also been made to predict the prospects of developing superior genotypes from a cross by the measurement of genetic similarity (GS) or genetic distance (CD) between the parents, since the later can be used as an estimation of expected genetic variances in different sets of segregating progenies derived, from different crosses. DNA fingerprinting of cereals species and cultivated varieties has a long scientific history. When DNA profiling technology first came into use, restriction fragment length polymorphism (RFLP) were considered state-of-art. RFLP technology was followed random amplification of polymorphic DNA (RAPD), followed by amplified length polymorphism (AFLP) and most recently use microsatellite markers or single sequence repeat (SSR). Advantage of SSR markers are: - the method is relatively simple and can be automated; - most of the markers are monolocus and show Mendelian inheritance; - SSR markers are high informative; - a high number of public SSR primer pairs are available; - effective cost per genotype and primer (similar to that for RAPD). Trait tagging and marker-assisted selection A large number of cereal studies have used markers as a tool to identify major genes, QTLs, or to introduce new characters in elite germplasm. In wheat, for example, molecular markers have been identified that are associated with around 40 traits of economic importance. Knowing the location of these genes/traits and specific alleles offers the possibility to apply marker-assisted selection (MAS) in cereals, because one of the main objectives of plant breeding is the introgression of one or more favourable genes from a donor parent into the background of an elite variety. Marker-assisted selection allows plant selection at the juvenile stage from an early generation. For simply inherited traits, conventional PCR, which requires a small amount of DNA, is becoming very useful for screening large populations of segregating progenies. Unfavourable alleles can be eliminated or greatly reduced during the early stages of plant development through MAS, focusing the selection in the field on reduced numbers of mature plants. Application of molecular markers in breeding for resistance to Barle yellow mosaic virus.

Barley yellow mosaic virus disease – caused by Barley mild mosaic virus (BaMMV) and Barley yellow mosaic virus (BaYMV) – has to be considered as one of the most important diseases of winter barley in Europe and East Asia. Because of the transmission by the soilborne fungus Polymyxa graminis, chemical treatments to control the disease are neither efficient nor economic. Therefore, breeding for resistance to this disease is of special importance. However, field selection for resistance genotypes is often difficult to perform because of unpredictable environmental conditions. Consequently, the application of closely linked PCR-based markers for the transmission of resistance gene(s) against barley yellow mosaic virus is now successful and efficient. Application of molecular markers in breeding for resistance to Fusarium head blight in wheat Fusarium head blight is a serious disease of wheat (Triticum aestivum L.) in humid and semi humid areas of the word. In Central Europe, severe natural epidemics of Fusarium head blight (FHB) occur once or twice in a decade and can sharply reduce yield and quality of susceptible genotypes. Deoxynivalenol (DON) are harmful to humans, because they are highly heat stable and cannot be eliminated totally once they entered the food chain. Evaluation of Fusarium head blight resistance is time consuming, laborious and costly because the inheritance of resistance is complex and phenotypic expression is significantly affected by environmental factors. Molecular markers closely linked to the major QTL involved in FHB resistance have recently been found and raise the possibility of using MAS for introducing resistance alleles into elite wheat varieties as have been confirmed also by us. However, do to the multifactorial nature of FHB resistance, the combination of MAS on the major QTL during seedling stage with phenotypic selection on the particular plants after flowering stage could be at the moment more sufficient and safety strategy in breeding of a new varieties combining a high level of yield performance and high level of resistance to Fusarium head blight. Conclusion In recent times many DNA markers have been developed and are powerful tools for successful cereal breeding. The promise of marker-assisted selection in crop breeding still remains but achieving practical benefits is taking longer than expected. The main reasons for this delay are the insufficient quality of markers (regarding their predictive and/or diagnostic value), inadequate experimental design, high costs and complexity of quantitative traits. Molecular markers may complement plant breeding in three general ways. First, molecular markers provide a reliable genetic-diversity measure that can be used for determining relations among inbred lines and cultivars, assessing changes in genetic diversity over time, protecting intellectual property rights, registering germplasm in countries that have ratified rules of the UPOV convention, evaluating new germplasm for its potential to increase genetic diversity, and selecting parents to hybridize in a breeding program. Second, molecular markers through their linkage with alleles with large effects (qualitative traits) and alleles with small effects (quantitative traits) may improve screens for many traits. Third, molecular markers will provide the first understanding of the biology and the architecture of many traits, particularly of quantitative traits.

Adaptation and application of molecular markers to plant improvement will be unique for each species and breeding program. Theoretically, many of the proposed applications of molecular markers are viable. The question is whether they will improve the efficiency and the cost effectiveness of a breeding program. This question can be answered only on a caseby-case basis. Factors such as cost of the molecular marker technology, turnaround time in the lab, cost of measuring a trait, etc. all will determine if and how markers are used in breeding programs. Species in which traits are measured by processing through a commercial factory or species with very long generation times clearly will benefit from applications of molecular-marker technology. For species, such as annual grains and cereals, the situation is ambiguous. One of the primary contributions of molecular markers will be an expansion of our knowledge of genetics and of genome organization. This type of knowledge obviously will improve our scientific understanding of many plant breeding problems, but the direct effect on plant improvement will be intangible and difficult to measure. Other important applications of molecular markers are as follows: • Assessment of genetic variability and fingerprinting of genotypes • Mapping of monogenic and qualitative trait loci (QTL) of economically important traits • Estimation of genetic distance or degree of relatedness between population, inbreds and breeding materials or among groups of accessions in germplasm • Identification of sequences for candidates genes and economic breeding traits • Molecular breeding for stress tolerance – abiotic and biotic • Marker assisted selection for crop improvement in tissue cultured plant species • Genetic purity testing in micropropagation - Micropropagated tree species • Exs itu and in situ conservation of plant genetic resources (PGRs) and genetic diversity conservation • Characterization and evaluation of plant germplasm – self incompatibility and male sterility, female sterility of economically important species • Characterization and identification of genetic resources of perennial crops • To investigate mechanisms that underlie somaclonal variation in the nuclear, mitochondrial and chloroplast genomes • To determine the origin of regenerants such as microspore or anther culture, protoplast fusion • Clonal propagation and stability – to avoid “segregation distortion” in anther culture derived plants, inturn increasing survival of anther culture clones • Detection of epigenetic changes in storage and conservation (i.e. detect duplications, seed mixtures, inadvertent outcrossing and genetic drift) • Study of “Anther culture” developed (double haploid) plants for gene mapping • Screening transgenic plants for resistance genes using linked molecular markers • Cloning and analysis of cDNA encoding a specific synthesis, thus understanding the nature of basic control mechanisms • Simulations and models on the detection of gene and REFERENCES

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