Marker assisted selection: a strategy for wheat improvement
Bhakti Rana1, Preeti Rana 2, Manoj K. Yadav1, Sundeep Kumar1
1Department of Biotechnology, Sardar Vallabh Bhai Patel University of Agriculture and Technology, Meerut U.P., INDIA
2BIT, Muzzafarnagar U.P., INDIA
Corresponding author: Bhakti Rana
Department of Biotechnology, Bhai Patel University of Agriculture and Technology, Meerut U.P., INDIA
E-mail: bhaktirana@gmail.com
Abstract
Wheat is most widely grown crop in the world, best adapted to temperate region and is a staple food of about 35% of the world population. Molecular markers have been introduced over last two decades, which has revolutionized the entire scenario of biological sciences. Traditionally, breeders have relied on visible traits to select improved varieties however; MAS rely on identifying marker DNA sequences that are inherited alongside a desired trait during the first few generations. Molecular markers are also considered as useful tools for pyramiding of different resistance genes and developing multi-line cultivars targeting for durable resistance to the disease. With the development of methodologies for the analysis of plant gene structure and function, molecular markers have been utilized for identification of traits to locate the gene(s) for a trait of interest on a plant chromosome and are widely used to study the organization of plant genomes and for the construction of genetic linkage maps. Breeders used molecular markers to increase the precision of selection for best trial combinations. With the development of AFLP and microsatellite marker systems, renewed studies are underway to analyze the genetic basis of many important traits in wheat. In light of the fresh challenges CIMMYT is giving emphasis on molecular breeding, functional genomics, deployment of transgenes for abiotic stresses etc., should get priority to maintain pace with time and growth. In near future, molecular markers can provide simultaneous and sequential selection of agronomically important genes in wheat breeding programs allowing screening for several agronomically important traits at early stages and effectively replace time consuming bioassays in early generation screens.
Introduction
Wheat is most widely grown crop in the world, best adapted to temperate region and is a staple food of about 35% of the world population. Wheat is a major source of energy, protein and dietary fiber in human nutrition since decades. Wheat is a major crop contributing importantly to the nutrient supply of the global population. Since the beginning of agriculture, ten thousand years ago, the importance of varietals improvement is well known. In the ancient time, selection and introduction were commonly used method, since knowledge about use of hybridization, mutation and polyploidy were not in practice. Selections were made on the basis of physical appearance and to fulfill the requirements. Till 15th century, most of the varieties were developed either through primary selection or introduction. Breeders had the advantages of variability until 1970s and due to intensive crossing programmes; green revolution took place during 1967-1968. The development and promotion of modern, high yielding varieties was the most important factor contributing to the enormous success of green revolution. During two generations leading up to the turn of the century the global population grew by 90 percent whilst food production expanded by 115 percent. The global food security is quite fragile, particularly when looking towards the middle of the century because of projected needs for human, animal and industrial uses. Global wheat production is expected to increase from nearly 600 million tons of present production level to around 760 million tons in 2020 with limited expansion of sown area. But now, due to non-existence of variability for the yield trait, it is not possible to develop a new variety either by selection or introduction. Selection based on knowing the location of the genes of interest gives the breeder a significant advantage, particularly for quantitative traits, where classical selection is done on the phenotype as a whole rather than on the underlying genetic determinants. Identification of significant QTL marker associations forms the baseline for MAS of quantitative traits. Furthermore, other breeding methods like hybridization, which reshuffle existing variability in the population and tools like polyploidy and mutation bring challenges for allele in the available traits and are not possible to introduce a new trait from unrelated species. Molecular markers have been introduced over last two decades, which has revolutionized the entire scenario of biological sciences. DNA based molecular markers have acted as versatile tools and have found there position in various fields like taxonomy, physiology, embryology, genetic engineering etc. PCR brought about a new class of DNA profiling markers that facilitated the development of marker based gene tags, map based cloning of agronomically important genes, variability studies, phylogenetic analysis, synteny mapping, marker assisted selection of various genotypes. Molecular markers are identifiable DNA sequences found at specific locations on the chromosomes and transmitted by the standard laws of inheritance from one generation to next and considered as landmarks in the chromosome maps that can be useful to monitor the transfer of specific chromosome segments known to carry useful agronomic traits. Molecular markers have also provided an excellent opportunity to develop saturated genetic maps and to integrate genetic, cytological and molecular maps. Molecular markers are being used to tag specific chromosome segments bearing the desired gene(s) to be transferred into the breeding lines.
Traditionally, breeders have relied on visible traits to select improved varieties however; MAS rely on identifying marker DNA sequences that are inherited alongside a desired trait during the first few generations. Thereafter, plants that carry the traits can be picked out quickly by looking for the marker sequences, allowing multiple rounds of breeding to be run in quick succession (Kumar et al. 2007). Molecular markers make selection possible for breeders to combine desirable alleles at a greater number of loci and at earlier generations than is possible with conventional breeding methodologies. Molecular markers can circumvent more cumbersome, established pedigree breeding strategies and even generate plant genotypes unachievable by conventional methods (Young 1999). Molecular markers are required in a broad spectrum of gene screening approaches, ranging from gene-mapping with traditional ‘forward-genetics’ approaches through QTL identification studies to genotyping and haplotyping studies. Molecular markers are also considered as useful tools for pyramiding of different resistance genes and developing multi-line cultivars targeting for durable resistance to the disease (Xia et al. 2005).
Conventionally, plant breeding depends upon morphological/phenotypic markers for the identification of agronomic traits. With the development of methodologies for the analysis of plant gene structure and function, molecular markers have been utilized for identification of traits to locate the gene(s) for a trait of interest on a plant chromosome and are widely used to study the organization of plant genomes and for the construction of genetic linkage maps. Molecular markers are independent from environmental variables and can be scored at any stage in the life cycle of a plant. Over the last several years, there has thus been marked increase in the application of molecular markers in the breeding programmes of various crop plants. Molecular markers not only facilitate the development of new varieties by reducing the time required for the detection of specific traits in progeny plants, but also fasten the identification of desired genes and their corresponding molecular markers, thus accelerating efficient breeding of resistance traits into wheat cultivars by marker assisted selection (MAS).
Marker assisted selection
MAS is a breakthrough technology that changes the process of variety cultivation from traditional field based format to a laboratory format. It is the use of molecular markers to track the location of genes of interest in a breeding programme. MAS is a form of indirect selection and most widely used application of DNA markers. Once traits are mapped a closely linked marker may be used to screen large number of samples for rapid identification of progeny that carry desirable characteristics. MAS is one of the most widely used applications of molecular marker technologies and one that plant breeders have been quick to embrace. Biotechnology have provided additional tools that do not require the use of transgenic crops to revolutionize plant breeding progress in molecular genetics has resulted in the development of DNA tags and marker assisted selection strategies for cultivar development. Several molecular marker types are available and they each have their advantages and disadvantages. Restriction fragment length polymorphisms (RFLPs) were the first to be developed (some 15 years) and have been widely and successfully used to construct linkage maps of various species, including wheat. With the development of the polymerase chain reaction (PCR) technology, several marker types emerged. The first of those were random amplified polymorphic DNA (RAPD), which quickly gained popularity over RFLPs due to the simplicity and decreased costs of the assay. However, most researchers now realize the weaknesses of RAPDs and use them with much less frequency. Microsatellite markers or simple sequence repeats (SSRs) combine the power of RFLPs (codominant markers, reliable, specific genome location) with the ease of RAPDs and have the advantage of detecting higher levels of polymorphism. The amplified fragment length polymorphism (AFLP) approach takes advantage of the PCR technique to selectively amplify DNA fragments previously digested with one or two restriction enzymes (Hosington et al., FAO Document Repository). Later, microsatellite markers or SSRs (Simple Sequence Repeats) were developed, which took advantage over RAPD and RFLP. Playing with the number of selective bases of the primers and considering the number of amplification products per primer pair, this approach is certainly the most powerful in terms of polymorphisms identified per reaction.
The essential requirements for MAS in a plant-breeding program are as follows (Mohan et al. 1997):
(a) Marker(s) should co-segregate or be closely linked (1 cM or less) with the desired trait.
(b) An efficient means of screening large populations for the molecular markers should be available. A relatively easy analysis based on PCR technology is the best option.
(c) The screening technology should have high reproducibility across laboratories, be economical to use and user friendly.
RFLPs, RAPDs and AFLPs do not fit the first requirement. However, techniques are available to turn them into user-friendly markers. RFLP clones can be sequenced and primers designed to amplify the DNA fragments are shown by hybridization to be polymorphic. However, the resulting STS or SCAR does not always turn out to be polymorphic and further manipulations are needed if this is the case. The amplified fragment is usually digested with one or two restriction endonucleases to detect small length differences, or the fragment from two or more cultivars is cloned and sequenced again to create ASAs. ASAs are usually based on single nucleotide differences. RAPD and AFLP fragments can be isolated from the gel, cloned and sequenced to generate STSs or SCARs. Attempts to generate such markers for wheat are neither always successful nor easily achieved. SSRs, on the other hand, if tightly linked to genes of interest are probably the most attractive markers since no further manipulations are needed for implementation. Despite the large number of markers for wheat genes listed in Table 2 few of those markers are close enough to the genes of interest to be useful in breeding applications.
Breeders used molecular markers to increase the precision of selection for best trial combinations. Variety developed by MAS are not considered genetically modified organisms (GMOs) and accepted by local and international market. Molecular marker aided selection methods have resulted in significant improvement in breeding efficiency by reducing trial and error aspect of breeding process and by allowing for time and cost savings. Molecular marker systems will benefit from the constant increase in the integration of biotechnological production of segregating populations such as homozygous double haploids in wheat breeding cycles since, the major requirement of being co-dominant for molecular markers will disappear. Marker assisted selection (MAS) offers an opportunity to select desirable lines based on genotype rather than phenotype. Marker assisted selection is an invaluable tool for gene pyramiding (Bringing genes from different individuals together in one individual) and has been fairly successful for combining single gene traits.
Marker assisted selection (MAS) is based on the identification and use of markers, which are linked to the gene(s) controlling the trait of interest. By virtue of linkage, selection may be applied to the marker itself. The advantage consists in the opportunity of speeding up the application of the selection procedure. For instance, a character which is expressed only at the mature plant stage may be selected at the plantlet stage, if selection is applied to a molecular marker. Selection may be applied simultaneously to more than one character. Selection for a resistance gene may be carried out without needing to expose the plant to the pest, pathogen or deleterious agent. If linkage exists between a molecular marker and a quantitative trait locus (QTL), selection may become more efficient and rapid. The construction of detailed molecular and genetic maps of the genome of the species of interest is a prerequisite for most forms of MAS. However, the current cost of the application of these techniques is significant, and the choice of one cost technique rather than others may be dictated by factors. There are few examples of crop varieties in farmer’s fields, which have developed through MAS.
Genetic resistance in wheat against diseases like leaf rust, stripe rust, stem rust, spot blotch, hill bunt etc., is generally governed by one, two or three genes and these genes can be tagged with any of the above DNA markers, specially which are based on PCR technology. In wheat, RFLPs have been used to map seed storage protein loci, loci associated with protein flour colour, cultivar identification, vernalization and frost resistance gene, intrachromosomal mapping of genes for dwarfing and vernalization, resistance to preharvest sprouting, quantitative trait loci (QTL) controlling tissue culture response, nematode resistance and milling yield. PCR based markers have been useful for characterization of genes for resistance against common bunt, powdery mildew, leaf rust resistance against hessian fly and Russsian wheat aphid. RFLP, DNA sequencing, and a number of PCR-based markers are being used extensively for reconstructing phylogenies of various species. The techniques are speculated to provide path breaking information regarding the fine time scale on which closely related species have diverged and what sort of genetic variations are associated with species formation. Efforts are being made for studying the genetic variation in plants to understand their evolution from wild progenitors and to classify them into appropriate groups (Jeffrey 1995). RFLP markers have proved their importance as markers for gene tagging locating and manipulating quantitative trait loci (QTL), in evolutionary studies for deducing the relationship between the hexaploid genome of bread wheat and its ancestors (Gill 1991). Specific markers like STMS (Sequence-tagged microsatellite markers) ALPs (Amplicon length polymorphisms) or STS markers have proved to be extremely valuable in the analysis of gene pool variation of crops during the process of cultivar development and classification of germplasm.
Wheat biotechnological research have been relatively slow, due to its ploidy level, the size and complexity of its genome, the very high percentage of repetitive sequences and low level of polymorphism (Table 1). Lack of genetic polymorphism in crops like wheat and soybeans and the consequent problems to identify molecular markers have been a major limitation to the impact of marker assisted selection (MAS) in wheat breeding. However, the identification of a high number of polymorphism in Single Sequence Repeats (SSR) should therefore, greatly enhance the potential to find molecular markers in wheat.
RAPDs emerged as a convenient and effective technique for tracing alien chromosome segments in translocation lines (Williams et al. 1990). RAPD markers provide a useful alternative to RFLP analysis for screening markers linked to a single trait within near isogenic lines and bulked segregants. He et al. (1992) reported the development of a DNA polymorphism detection method by combining RAPD with DGGE (denaturing gradient gel elecrophoresis) for pedigree analysis and fingerprinting of wheat cultivars. RAPD markers can be converted to more user-friendly Sequence Characterized Amplified Region (SCAR) markers, which display a less complex banding pattern. SCAR markers linked to resistance genes against fungal pathogens have been characterized in combination with RAPD and RFLP (Procunier et al.1997; Myburg et al. 1998; Liu et al. 1999). In recent years, RAPD and other PCR based markers like Sequence Characterized Amplified Regions (SCAR), Sequence Tagged Sites (STS) and Differential Display Reverse Transcriptase PCR (DDRT-PCR) are increasingly being used for identification of desirable traits in wheat and related genera. These markers have been used in particular for disease resistance against viral and fungal pathogens and also for insect and nematode pests and have the potential of pyramiding of resistance genes for effective breeding programs. PCR based markers have been extensively characterized for genes of resistance against common bunt, Tilletia tritici (Demeke et al. 1996), powdery mildew, Erysiphe graminis (Hartl et al.; 1995; Qi et al. 1996), leaf rust, Puccinia recondita (Dedryver et al. 1996; Feuillet et al. 1995; Seyfarth et al. 1999), resistance against Hessian fly, Mayetiola destructor (Dweikat et al. 1994) and Russian wheat aphid, Diuraphis noxia (Myburg et al. 1998; Venter and Botha, 2000). SSRs or Microsatellites are more promising molecular markers for the identification and differentiation of genotypes within a species. The high level of polymorphism and easy handling has made microsatellites extremely useful for different applications in wheat breeding (Devos et al. 1995; Roder et al. 1995; Bryan et al. 1997; Korzun et al. 1997; Roy et al. 1999.). Microsatellites have also been used to identify resistance genes like Pm6 from Triticum timopheevii (Tao et al. 1999) and Yr15 from breadwheat (Chague et al. 1999) (Table 2).
Use of molecular markers in wheat improvement programme: conservation of genetic resources
Loss of genetic diversity has become a problem not only of the natural plant and animal population but also agriculturally important species. Ancient cultivars or landraces and wild relatives of domesticated species are being lost as modern varieties become adopted by farmers. This has led to calls for genetic conservation of crop germplasm (Frankel and Benett 1970). Microsatellites are commonly used to study genetic relationships among genotypes within species because of their high level of polymorphism (Devos et al. 1995; Roder et al. 1995; Korzun et al. 1997). Microsatellites markers are currently used to identify genotypes Quantitative trait loci (QTLs) and genetic diversity (Medini et al. 2005). Knowledge of genetic diversity of wheat varieties is a prerequisite for the successful management of conservation programs. The first microsatellite in wheat possessed 279 microsatellites (Roder et al. 1998) by now a total of 1235 microsatellite loci were developed and mapped (Somers et al. 2004). Recently 101 microsatellite markers derived from expressed sequence tags EST-SSRs were added into a set of microsatellites (Gao et al. 2004).
Resistance to biotic stress: gene pyramiding for multiple disease resistance
Development of resistant varieties for single disease is not enough to save plant product and to feed growing population especially in developing countries. In India, there are number of constraints in the successful production of wheat like leaf and yellow rust, powdery mildew and spot blotch. These all diseases together toll heavy yield losses and put us in the situation to redesign our experiments to develop multiple pest resistant varieties in wheat. The grown varieties on the market do carry some known disease resistance genes against powdery mildew and rusts and low levels of quantitative resistance against leaf spot diseases. However, most of the resistance genes against rusts and powdery mildew are already broken down. In the sustainable agriculture, which is economical both for the farmer and nature, multiple disease resistance is an essential tool against pathogens attack beside cultural practices like crop rotation. With the biotrophic fungi like rusts and powdery mildew, the only solution is the durable disease resistance.
In late 1980’s Fusarium head blight (FHB) become a world wide problem. Earlier, it was sporadic and localized to China, South America and a few European countries. Varieties resistant to FHB such as Wuhan 1, 2 and 3 and Shangai 7 and 8 and Suzhoe wheats were the product of generous collaboration between various Chinese institutions. Fusarium head blight is a serious disease of wheat (Triticum aestivum L) in humid and semi humid areas of the world. Evaluation of Fusarium head blight resistance is time consuming, laborious and costly as the inheritance of resistance is complex phenomenon. The most recent biotic threat to global wheat production is Ug 99, a virulent race of stem rust which has emerged from Uganda and has been confirmed at widely distributed testing locations in Kenya and Ethiopia. Yield losses of up to 71 percent have been recorded under experimental conditions (Dixon 2003).
Resistance to abiotic stress
Traditional approaches at transferring resistance to crop plants are limited by the complexity of stress tolerance traits, as most of these are quantitatively linked traits (QTLs). The direct introduction of a small number of genes offers convenient alternative and a rapid approach for the improvement of stress tolerance. Although, present engineering strategies rely on the transfer of one or several genes that encode either biochemical pathways or endpoints of signalling pathways, these gene products provide some protection either directly or indirectly against environmental stresses. Drought is a major abiotic factor that limits crop productivity, thereby causing enormous loss.
Low temperature (LT) tolerance is a complex quantitative character that is expressed in anticipation of and during exposure of plants to temperatures that approach freezing. This environmentally reduced character is determined by a highly integrated system of structural and developmental genes that are regulated by environmentally responsive complex pathways. The superior LT-tolerance genes have been tagged using molecular markers that allow plant breeders to select hardy genotypes without having to wait for a test frost in the field (Fowler and Limin 2007).
Quality traits
Other than reporter genes, perhaps the most targetted trait for genetic engineering in wheat is quality. Seed storage proteins (SSP) are contained in the seed of higher plants. These proteins have been classified as albumins, globulins and glutenins on the basis of their solubility in solvents. The high molecular weight glutenin subunits (HMW-GS) genes in wheat are located on the long arm of the homeologous chromosomes 1A, 1B and 1D. Bread-making properties are particularly associated with variation at the Glu-D1 and Glu-A1 loci. The HMW-GS 1Ax1, 1Ax2, 1Dx5 and 1Dx10 have been shown to be associated with stronger dough, better elasticity and, hence, improved bread-making quality. Many elite wheat varieties lack the desired studies have demonstrated that the introduction of one or two HMW-GS genes results in a stepwise increase in dough elasticity. The transgenic lines produced so far have also demonstrated a very high level of expression and stability over several generations. This may imply that native genes are more tolerated by a plant genome.subunits and, thus, many research groups are attempting to introduce these via genetic engineering (Shewry et al. 1995; Altpeter et al. 1996; Barro et al. 1997; Vasil and Anderson 1997). In addition to increasing the bread-making quality, altered amino acid composition of the SSP is feasible and could result in improved nutritional properties. For example the insertion of genes for proteins such as zeins or albumins, could lead to an increase in the desired amino acid. Other approaches are also being considered such as reducing the level of anti-nutritional factors and modifying starch and oil composition and content.
Challenges and future strategies in India
India’s population of more than a billion is growing at a rate of around 1.8% per year, almost going parallel with the annual growth rat of cereals. Therefore, the estimated demand of wheat production for the year 2020 is around 109 million tones, which is 30 million tones more than the record production of 75 million tones harvested in the crop season 1999-2000. Since then, India is struggling to achieve the impressive figure of its record production. However, the ever increasing population has alarmed food security in India and attempts have been initiated to integrate modern technology tools in conventional breeding to improve the most important crops such as rice, wheat and legumes. There is little doubt that wheat has been a difficult species for the application of molecular genetics. The low level of polymorphism between elite varieties coupled with the hexaploid nature of the crop provides significant challenges for those attempting to develop molecular markers and to use them in genetic studies. With the development of AFLP and microsatellite marker systems, renewed studies are underway to analyze the genetic basis of many important traits in wheat. Future challenges include developing strategies for reducing the cost per assay, acquire more desirable markers to further complement the efforts of wheat breeders as well as evaluating emerging technologies to increase throughput with reduced cost. Integrating molecular breeding efforts in a few target national program partners also is considered an important challenge. One of the major concerns of wheat researchers is to make Indian wheat globally competitive by reducing the cost of cultivation and increasing farmer’s profitability. With the availability of detailed information regarding the location and function of gene(s) encoding for useful traits, scientists in future will be well equipped for efficiently creating varieties with exact combinations of desirable traits. However, genetic transformation will remain a significantly important tool for understanding gene functions and for testing the utility of new sequences. In near future, crop varieties could be tailor-made to meet both local consumer preferences and the demands of particular environment or niche. CIMMYT, Mexico has played an important role in strengthening the Indian wheat programme since the advent of Green Revolution. In light of the fresh challenges CIMMYT is giving emphasis on molecular breeding, functional genomics, deployment of transgenes for abiotic stresses etc., should get priority to maintain pace with time and growth. In near future, molecular markers can provide simultaneous and sequential selection of agronomically important genes in wheat breeding programs allowing screening for several agronomically important traits at early stages and effectively replace time consuming bioassays in early generation screens. Although, the potential of biotechnology has often been exaggerated, a high level of optimism is clearly justified for its use in the improvement of wheat. Undoubtedly, functional genomics, as it is now termed, will revolutionize the way in which plant breeding is undertaken in the future. Basic research is leading to an improved understanding of the genetic mechanisms operating within a plant in response to the diverse stresses that it is exposed to, as well as the overall production of biomass and grain. The challenge for developing countries is to tap as much of this emerging technology as possible.
References
Allen GJ, Jones RGW, Leigh RA (1995). Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits. Plant Cell Environ 18: 105-115.
Anderson JA, Sorrells ME, Tanksley SD (1993). RFLP analysis of genomic regions associated with resistance to preharvest sprouting in wheat. Crop Science 33: 453-459.
Anderson JA, Waldron BL, Moreno-Se-villa B, Stack RW, Frohberg RC (1998) Detection of Fusarium head blight resistance QTL in wheat using AFLPs and RFLPs. In: Slinkard AE (ed) Proc 9th Int Wheat Genetics Symp, Saskatoon, Saskatchewan, Canada, 2-7 Aug 1998, vol. 1, pp.135-137.
Autrique E, Singh RP, Tanksley SD, Sorrells ME (1995) Molecular markers for four leaf rust resistance genes introgressed into wheat from wild relatives. Genome 38: 75-83.
Barro F, Rooke L, Bekes F, Gras P, Tatham ST, Fido R, Lazzari PA, Shewry PR, Barcelo, P (1997) Transformation of wheat with high molecular weight genes results in improved functional properties. Nature Biotech15: 1295-1299.
Blanco A, Degiovanni C, Laddomada B, Sciancalepore A, Simeone R, Devos KM, Gale MD (1996) Quantitative trait loci influencing grain protein content in tetraploid wheats. Plant Breed 115: 310-316.
Bryan GJ, Collins AJ, Stephenson P, Orry A, Smith JB, Gale MD (1997) Isolation and characterisation of microsatellites from hexaploid bread wheat. Theor Appl Genet 94: 557-563.
Chague V, Fahima T, Dahan A, Sun GL, Korol AB, Ronin YI, Grama A, Roder MS, Nevo E (1999) Isolation of microsatellite and RAPD markers flanking the Yr15 gene of wheat using NILs and bulked segregant analysis. Genome 42: 1050-1056.
Chao S, Sharp PJ, Worland AJ, Warham EJ, Koebner RMD, Gale MD (1989) RFLP-based genetic maps of wheat homoeologous group 7 chromosomes. Theor Appl Genet 78: 495-504
Cushman JC, Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Current Opinion in Plant Biology 3: 117-124.
D’Ovidio R, Anderson OD (1994) PCR analysis to distinguish between alleles of a member of a multigene family correlated with wheat bread-making quality. Theor Appl Genet 88: 759-763.
D’Ovidio R, Porceddu E (1996) PCR-based assay for detecting 1B-genes for low molecular weight glutenin subunits related to gluten quality properties in durum wheat. Plant Breed 115: 413-415.
de la Pena RC, Murray TD, Jones SS (1997) Identification of an RFLP interval containing Pch2 on chromosome 7AL in wheat. Genome 40: 249-252.
Dedryver F, Jubier MF, Thouvenin J, Goyeau H (1996) Molecular markers linked to the leaf rust resistance gene Lr24 in different wheat cultivars. Genome 39: 830-835.
Demeke T, Laroche A, Gaudet DA (1996) A DNA marker for the Bt-10 common bunt resistance gene in wheat. Genome 39: 51-55.
Devos KM, Bryan GJ, Collins AJ, Stephenson P, Gale MD (1995) Application of two microsatellite sequences in wheat storage proteins as molecular markers. Theor Appl Genet 90: 247-252.
Dixon J (2007) The Economics of Wheat. Developments in Plant Breeding 12: 9-22.
Dubcovsky J, Santa Maria G, Epstein E, Luo MC, Dvorak J (1996) Mapping of the K+/Na+ discrimination locus Kna1 in wheat. Theor Appl Genet 92: 448-454.
Dweikat I, Ohm H, Mackenzie S, Patterson F, Cambron S, Ratcliffe R (1994) Association of a DNA marker with Hessian fly resistance gene H9 in wheat. Theor Appl Genet 89: 964-968.
Dweikat I, Ohm H, Patterson F, Cambron S (1997) Identification of RAPD markers for 11 Hessian fly resistance genes in wheat. Theor Appl Genet 94: 419-423.
Eastwood RF, Lagudah ES, Appels R (1994) A directed search for DNA sequence tightly linked to cereal cyst nematode resistance genes in Triticum tauschii. Genome 37: 311-319.
Faris JD, Anderson JA, Francl LJ, Jordahl JG (1997) RFLP mapping of resistance to chlorosis induction by Pyrenophora tritici-repentis in wheat. Theor Appl Genet 94: 98-103.
Feuillet C, Messmer M, Schachermayr G, Keller B (1995) Genetic and physical characterization of the LR1 leaf rust resistance locus in wheat (Triticum aestivum L.). Mol Gen Genet 248: 553-562.
Fowler DB, Limin AE (2007) Progress in Breeding wheat with tolerance to low temperature in different phenological developmental stages. Development in Plant Breeding 12: 301-312.
Frankel OH, Bennet E (1970) Genetic resources in plants-their exploration and conservation. Davis FA. Philadelphia, USA.
Galiba G, Quarrie SA, Sutka J, Morgounov A, Snape JW (1995). RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat. Theor Appl Genet 90: 1174-1179.
Gao LF, Jing RL, Huo NX, Li Y, Li XP, Zhou RH, Chang XP, Tang JF, Ma ZY, Jia JZ (2004) One hundred and one new microsatellite loci derived from ESTs (EST-SSRs) in bread wheat. Theor Appl Genet 108:1392-1400.
Gill KS, Lubbers EL, Gill BS, Raupp WJ, Cox TS (1991) Genome 34: 362-374.
Gill KS, Gill BS (1996) A PCR-based screening assay of Ph1, the chromosome pairing regulator gene of wheat. Crop Sci 36: 719-722.
Hartl L, Weiss H, Stephan U, Zeller FJ, Jahoor A (1995) Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L). Theor Appl Genet 90: 601-606.
He S, Ohm H, Mackenzie S (1992). Detection of DNA sequence polymorphisms among wheat varieties. Theor Appl Genet 84: 573-578.
Hoisington D, Bohorova N, Fennell S, Khairallah M, Pellegrineschi A, Ribaut MJ. The application of biotechnology to wheat improvement. FAO Document Repository.
Hu XY, Ohm HW, Dweikat I (1997) Identification of RAPD markers linked to the gene Pm1 for resistance to powdery mildew in wheat. Theor Appl Genet 94: 832-840.
Humphreys DG, Procunier JD, Mauthe W, Howes NK, Brown PD, Mac-Kenzie RIH (1998) Marker-assisted selection for high protein concentration in wheat. In: Fowler DB, Ged-des WE, Johnston AM, Preston KR (eds.) Wheat Protein, Production and Marketing. Proc Wheat Protein Symp, Saskatoon, Saskatchewan, Canada, 9-10 Mar. (1998), pp. 255-258.
Jeffrey AJ, Wilson V, Thein SL (1985) Nature 314: 67-73.
Jia J, Devos KM, Chao S, Miller TE, Reader SM, Gale MD (1996) RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat. Theor Appl Genet 92: 559-565.
Johnston SJ, Sharp PJ, McIntosh RA, Guillen-Andrade H, Singh RP, Khairallah M (1998) Molecular markers for the Sr2 stem rust resistance gene In: Slinkard AE (ed) Proc 9th Int Wheat Genet Symp, Saskatoon, Sas-katchewan, Canada, 2-7 Aug 1998, vol. 3, pp. 117-119.
Kato K, Miura H, Akiyama M, Kuroshima M, Sawada S (1998) RFLP mapping of the three major genes, Vrn1, Q and B1, on the long arm of chromosome 5A of wheat. Euphytica 101: 91-95.
Knox RE, Howes NK (1994) A mono-clonal antibody chromosome marker analysis used to locate a loose smut resistance gene in wheat chromosome 6A. Theor Appl Genet 89: 787-793.
Korzun V, Roder M, Worland AJ, Borner A (1997) Intrachromosomal mapping of genes for dwarfing (Rht12) and vernalization response (Vrn1) in wheat by using RFLP and microsatellite markers. Plant Breeding 116: 227-232.
Kumar S, Kumar M, Kumar, Mukesh YK, Manoj, Kumar, Rajendra (2007) Integration of conventional and non-conventional breeding approaches for seed spices improvement. In: Proc National Workshop on Spices and Aromatic Plants, Feb 27-28, 2007 pp 14-20.
Li S, Zhang Z, Wang B, Zhong Z, Yao J (1995) Tagging the Pm4a gene in NILs by RAPD analysis. Acta Genet Sin 22: 103-108.
Liu Z, Sun Q, Ni Z, Yang T (1999) Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat. Plant Breeding 118: 215-219.
Luo MC, Dvorak J (1996). Molecular mapping of an aluminium tolerance locus on chromosome 4D of Chinese Spring wheat. Euphytica 91: 31-35.
Ma ZQ, Sorrells ME, Tanksley SD (1994). RFLP markers linked to powdery mildew resistance genes Pm1, Pm2, Pm3 and Pm4 in wheat. Genome 37: 871-875.
Ma ZQ, Gill BS, Sorrells ME, Tanksley SD (1993) RFLP markers linked to two Hessian fly-resistance genes in wheat (Triticum aestivum L.) from Triticum tauschii (Coss.) Schmal. Theor Appl Genet 85: 750-754.
Mohan M, Nair S, Bhagwat A, Krishna TG, Yano M, Bhatia CR, Sasaki T (1997) Genome mapping, molecular markers and assisted selection in crop plants. Mol Breeding 3: 87-103.
Mohler V, Jahoor A (1996) Allele specific amplification of polymorphic sites for the detection of powdery mildew resistance loci in cereals. Theor Appl Genet 93: 1078-1082.
Medini M, Hamza S, Rebai A, Baum M (2005) Analysis of genetic diversity in Tunisian durum wheat cultivars and related wild species by SSR and AFLP markers. Genet Resour Crop Evol 52: 21-31.
Myburg AA, Cawood M, Wingfield, BD, Botha AM (1998) Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat. Theor Appl Genet 96: 1162-1169
Nelson JC, Singh RP, Autrique JE, Sorrells ME (1997). Mapping genes conferring and suppressing leaf rust resistance in wheat. Crop Sci 37: 1928-1935.
Nelson JC, Sorrells ME, Van Deynze AE, Lu, YH, Atkinson M, Bernard M, Leroy P, Faris JD, Anderson JA (1995a) Molecular mapping of wheat: major genes and rearrangements in homoeologous groups 4, 5, and 7. Genetics 141: 721-731.
Parker GD, Chalmers KJ, Rathjen AJ, Langridge P (1998) Mapping loci associated with flour colour in wheat (Triticum aestivum L.). Theor Appl Genet 97: 238-245.
Parker GD, Chalmers KJ, Rathjen AJ, Langridge P (1998) Mapping loci associated with flour colour in wheat (Triticum aestivum L.) Theor Appl Genet 97: 238-245.
Paull JG, Chalmers KJ, Karakousis A, Kretschmer JM, Manning S, Langridge P (1998) Genetic diversity in Australian wheat varieties and breeding material based on RFLP data. Theor Appl Genet 96: 435-446.
Paull JG, Pallotta MA, Langridge P, The TT (1995) RFLP markers associated with Sr22 and recombination between chromosome 7A of bread wheat and the diploid species Triticum boeoticum. Theor Appl Genet 89: 1039-1045.
Procunier JD, Knox RE, Bernier AM, Gray MA, Howes NK (1997) DNA markers linked to a T10 loose smut resistance gene in wheat (Triticum aestivum L.). Genome 40: 176-179.
Qi L, Cao M, Chen P, Li W, Liu D (1996) Identification, mapping and application of polymorphic DNA associated with resistance gene Pm21 of wheat. Genome 39: 191-197.
Quarrie SA, Gulli M, Calestani C, Steed A, Marmiroli N (1994) Location of a gene regulating drought-induced abscisic acid production on the long arm of chromosome 5A of wheat. Theor Appl Genet 89: 794-800.
Ren SX, McIntosh RA, Sharp PJ, The TT (1996) A storage-protein marker associated with the suppressor of Pm8 for powdery mildew resistance in wheat. Theor Appl Genet 93: 1054-1060.
Riede CR, Anderson JA (1996) Linkage of RFLP markers to an aluminium tolerance gene in wheat. Crop Sci 36: 905-909.
Roder MS, Plaschke J, Konig SU, Borner A, Sorrells ME, Tanksley SD, Ganal MW (1995) Abundance, variability and chromosomal location of microsatellites in wheat. Mol Gen Genet 246: 327-333.
Roder MS, Korzun V, Wendehake K, Plaschke J, Tixier MH, Leroy P, Ganal MW (1998) A microsatellite map of wheat. Genetics 149: 2007-2023.
Roy JK, Prasad M, Varshney RK, Balyan HS, Blake TK, Dhaliwal HS, Singh H, Edwards KJ, Gupta PK (1999) Identification of a microsatellite on chromosomes 6B and a STS on 7D of bread wheat showing an association with preharvest sprouting tolerance. Theor Appl Genet 99: 336-340.
Schachermayr GM, Messmer MM, Feuillet C, Winzeler H, Winzeler M, Keller B (1995) Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat. Theor Appl Genet 90: 982-990.
Schachermayr GM, Siedler H, Gale MD, Winzeler H, Winzeler M, Keller B (1994) Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat. Theor Appl Genet 88:110-115.
Schachermayr G, Feuillet C, Keller B (1997) Molecular markers for the detection of the wheat leaf rust resistance gene Lr10 in diverse genetic backgrounds. Mol Breed 3: 65-74.
Seyfarth R, Feuillet C, Keller B (1998) Development and characterization of molecular markers for the adult leaf rust resistance genes Lr13 and Lr35 in wheat. In: Slinkard AE (ed) Proc. 9th Int. Wheat Genetics Symp., Saskatoon, Saskatchewan, Canada, 2-7 Aug. 1998, vol. 3, pp. 154-155.
Seyfarth R, Feuillet C, Schachermayr G, Winzeler M, Keller B (1999) Development of a molecular marker for the adult plant leaf rust resistance gene Lr35 in wheat. Theor Appl Genet 99: 554-560.
Seo YW, Johnson JW, Jarret RL (1997) A molecular marker associated with the H21 Hessian fly resistance gene in wheat. Mol Breed 3: 177-181.
Shewry P, Tatham A, Barro F, Barcelo P, Lazzeri P (1995) Biotechnology of bread making: unraveling and manipulating the multi-protein gluten complex. Bio/Technology 13: 1185-1190.
Shi AN, Leath S, Murphy JP (1998) A major gene for powdery mildew resistance transferred to common wheat from wild einkorn wheat. Phytopathology 88: 144-147.
Somers DJ, Issac P, Edwards K (2004) A high density microsatellite consensus map for bread wheat (Triticum aestivum L.) Theor Appl Genet 109: 1105-1114.
Sourdille P, Perretant MR, Charmet G, Leroy P, Gautier MF, Joudrier P, Nelson JC, Sorrells ME, Bernard M (1996) Linkage between RFLP markers and genes affecting kernel hard-ness in wheat. Theor Appl Genet 93: 580-586.
Sun GL, Fahima T, Korol AB, Turpein-en T, Grama A, Ronin YI, Nevo E (1997) Identification of molecular mark-ers linked to the Yr15 stripe rust resistance gene of wheat originated in wild emmer wheat, Triticum dicoccoides. Theor Appl Genet 95: 622-628.
Talbert LE, Bruckner PL, Smith LY, Sears R, Martin TJ (1996). Development of PCR markers linked to resistance to wheat streak mosaic virus in wheat. Theor Appl Genet 93: 463-467.
Tao WJ, Liu JY, Liu DJ, Chen PD (1999) Identification of molecular markers linked to the Pm6 gene conferring powdery mildew resistance in wheat. I Chuan Hsueh Pao (in Chinese) 26: 649-656.
Vasil IK, Anderson O (1997) Genetic engineering of wheat gluten. Trends Plant Sci 2: 292-297.
Venter E, Botha AM (2000) Development of markers linked to Diuraphis noxia resistance in wheat using a novel PCR-RFLP approach. Theor Appl Genet 100: 965-970.
William HM, Hoisington D, Singh RP, Gonzalez-de-Leon D (1997) Detection of quantitative trait loci associated with leaf rust resistance in bread wheat. Genome 40: 253-260.
Williams KJ, Fisher JM, Langridge P (1994) Identification of RFLP markers linked to the cereal cyst nematode resistance gene (Cre) in wheat. Theor Appl Genet 83: 919-924.
Williams KJ, Fisher JM, Langridge P (1996) Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat. Genome 39: 798-801.
Worland AJ, Borner A, Korzun V, Li WM, Petrovic S, Sayers EJ (1997). The influence of photoperiod genes on the adaptability of European winter wheats. In: Braun HJ (ed) Wheat: prospects for global improvement. Dordrecht, Netherlands, Kluwer Academic Press, pp. 517-526.
Yamamori M (1994) An N-band marker for gene Lr18 for resistance to leaf rust in wheat. Theor Appl Genet 89: 643-646.
Young ND (1999) A cautiously optimistic vision for marker assisted selection. Mol Breed. 5: 505-510.