Issues Affecting Wheat Cultivation In Pakistan Biology Essay

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Pakistan is an agricultural country with vast fertile land and other related resources. Due to the great diversity of land and climate, Pakistan is producing many crops and vegetables. Wheat is the most important among them and is the backbone of its agriculture based economy. In Pakistan wheat is cultivated on about on 70 million hectares and in 2006-2007 and produced 23.7 million tons of wheat which is more than domestic needs (Economic Survey of Pakistan, 2008).

Pakistan has been divided into ten production zones because of great agro ecological areas where wheat is cultivated. The zoning is mainly based on cropping pattern, disease prevalence and climatic factors. Wheat accounts for 37.1 % of the crop area, 65 % of the food grain acreage, and 70 % of the production. 

In Pakistan, wheat is grown in different cropping systems, such as; cotton - wheat, rice - wheat, sugarcane - wheat, maize - wheat, fallow - wheat. Of these, Cotton-Wheat and Rice-Wheat systems together account about 60% of the total wheat area whereas rain-fed wheat covers more than 1.50 m ha area. Rotations with Maize-Sugarcane, Pulses and fallow are also important

Botanically, wheat is a member of the grass family to which rice, barley, corn and several other cereal grain crops also belong. It ranks first in world crop production and is the national food staple of 43 countries. Wheat is best adapted to a cool dry climate. Wheat is one of the major agricultural crop species in the world. The importance of the wheat makes it inevitable to develop the most productive and profitable wheat varieties by the use of the conventional and modern scientific methods. In this regards the use of molecular biology techniques for the development of the new improved varieties is the most important aspect of the modern scientific advancements. Common or bread wheat, Triticum aestivum, is hexaploid with a large genome estimated at ~17,000 Mb that is attributable to the polyploidy nature of wheat and the high content of repetitive elements within the wheat genome. A number of genomic resources have been developed or are being developed for wheat. These include a collection of >500,000 wheat ESTs, bacterial artificial chromosome clones, and >5,000 bin-mapped EST markers.

The development of DNA (or molecular) markers has irreversibly changed the disciplines of plant genetics and plant breeding. While there are several applications of DNA markers in breeding, the most promising for cultivar development is called marker assisted selection (MAS). MAS refers to the use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening. By determining the allele of a DNA marker, plants that possess particular genes or quantitative trait loci (QTLs) may be identified based on their genotype rather than their phenotype.

Wheat production in Pakistan is not encouraging. Pakistan has low per acre production as compared to the wheat production in the advanced countries. There are several factors responsible for the low productivity. One of the reasons for this miserable plight of wheat yield in Pakistan is the lack of the use of modern techniques for the development of new wheat varieties. This accounts for the low per acre yield of the wheat, its vulnerability to various biotic and a biotic stresses. One of the most formidable biotic stresses is the diseases caused by different pathogens which not only undermine the wheat productivity but spoil the efforts of the farmers as well. Among these pathogenic diseases wheat rusts are the most destructive and unleash tremendous wheat productivity losses annually in Pakistan. One of them is the yellow rust caused by a fungus, of wheat and which is the topic of this research work.

In the wake of this scenario it is the need of the hour to undertake the strategies based on the latest technology and research in order to minimize the losses perpetrated by the disease. Management and use of the existing resistance source among different wheat varieties to defend the crop against this disease is best solution. This can be done by bringing rust resistance genes into the wheat varieties which are known for higher yield and which are prone to the disease.

In order to bring resistant genes in a certain variety it is necessary to know which rust resistance gene are carried by a particular wheat variety. This can be done by exploiting molecular markers of the rust resistance genes. Different types of the molecular markers can be used for this purpose detecting the presence of the linked rust resistance gene in a variety and for studying the diversity among the different wheat varieties. Simple sequence repeats (SSR) markers are the most suitable for such purpose.

More than thirty different yellow rust resistance genes have been detected and SSR markers linked to many of these genes have been discovered. Primers of many of these markers have been published in the literature.

In this study presence of the yellow rust resistance gene Yr-9 has been searched out by using its SSR marker Xgwm 488 in the following Pakistani wheat varieties:

1- Bhakkar

2- 0031034

3- Inquilab-91

4- Uqab

5- Sehar

6- Shafaq

7- Kohistan

8- 4112

Aims and Objectives

This study was undertaken with the following objectives:

Growing the seeds of the proposed wheat varieties

DNA extraction

Quality and quantity measuring of the DNA

PCR

Gel electrophoresis

Chapter 2

REVIEW OF LITERATURE

Scientific literature is replete with the discoveries of invaluable genetic components in the naturally occurring plant and animal varieties. The study in which different varieties of a crop are screened for the presence or absence of these precious genomic segments is highly valuable. As a result of such efforts not only we come to know which are the valuable genetic characters possessed by a certain variety but we also become able to develop new and more productive varieties using this knowledge and information. This study has focused on the detecting yellow rust resistant genes in the selected wheat varieties of Pakistan.

Wheat rust diseases

Wheat rust diseases are an apt example of the fact how plant pathogenic fungi have been perpetrating the incredible destruction of the agricultural economy all over the world. The diseases which are studied under the rubric of wheat rusts, the most common these days is called leaf or brown rust and is caused by P. triticina Eriks. This malignant disease traverses the leaf blades, but it also invades the leaf sheaths particularly when it inflicts in the highly favorable environmental conditions which entail high inoculums densities and tremendously susceptible cultivars. In such circumstances most commonly the disease lacks the abundant teliospore production of stem rust at the end of the season. As a consequence of the situation brown leaf lesions are produced rather than black stem lesions that are the characteristics of the stem rust. Leaf rust teliospores are produced at the lower surfaces of the leaves and they are originated from the telia. The other peculiarity associated with them is that epidermal cells keep on covering them. At a temperature 10° to 30°C the disease prevails very rapidly and disease exits anywhere the wheat is cultivated all over the world. Losses in grain yield are primarily attributed to reduced floret set and grain shriveling. The disease inflicts injury to the florets, tillers and whole plants in extremely susceptible genotypes. These losses can be interpreted in the economic terms ranging between 10 to 30 percent.

An other leaf rust disease is caused by . recondita complex (P. triticiduri V. Bourgin) in the Mediterranean Sea and infects both durum and bread wheat (Ezzahiri et al., 1992). The pathogen seldom produces urediniospores. These urediniospores are generally on the lower surface of the leaf. Epidemics produced by this disease are mostly local because urediniospores are lacking here. A lot of telia are given rise in a ring around the initial uredinium.

An other rust disease known as stem rust, is caused by P. graminis Pers. f. sp. tritici Eriks. & E. Henn. This disease is also known as black rust or summer rust as it produces a larger number of shiny black teliospores. These teliospores are produced in the uredinium at the end of the season or with unfavourable conditions. Warm 15° to 35°C and humid conditions exaggerate the damage by the stem rust. In such situations this disease can be most lethal and can incur up to 50% loss and in this way the stem rust is the most devastating disease among all the rusts. In the most severe attacks in the favorable conditions with susceptible varieties the disease can cause 100 percent losses.

Stripe rust which is also known as yellow rust, caused by P. striiformis West. f. sp. tritici Eriks & Henn., is typically a disease of wheat grown under the cold environmental conditions at a temperature ranging from 2° to 15°C. This rust produces a characteristic pattern of uredinia producing yellow coloured uredinospores in the form of stripes and gives its name stripe rust. As a consequence of the early attack of this disease plants remain stunted and weak and can result in production losses up to 50% which cam even exceed to 100% in the extreme conditions.

Pathogens of wheat rusts

In wheat rust disease causing pathogens belong to genus Puccinia, family Pucciniaceae, order Uredinales and class Basidiomycetes. Pathogens of the rust fungi are extremely specialized plant pathogens with narrow host ranges. Fontana and Tozzetti individually and independently are the pioneer to report the disease of wheat stem rust in 1767 (Fontana, 1932; Tozzetti, 1952). The causal organism of wheat stem rust was named P. graminis by Persoon in 1797. Chester (1946) provided one of the first detailed histories of the literature on the rust of wheat. In the early records, wheat leaf rust is not distinguished from stem rust (Chester, 1946). However, by 1815 de Candolle (1815) had shown that wheat leaf rust was caused by a distinct fungus Uredo rubigovera. The pathogen underwent a number of name changes until 1956 when Cummins and Caldwell (1956) suggested P. recondita, which has been the generally used nomenclature. Recent morphological studies by Savile (1984) and morphological and pathogen genetic studies by Anikster et al. (1997) show that P. recondita is not the incitant of wheat leaf rust. Currently P. triticina should be the preferred name as shown in by Savile (1984) and Anikster et al. (1997). This name was used by Mains and Jackson (1926) and has been used in parts of Asia and Eastern Europe for many years. In this chapter, P. triticina will be used for the leaf rust on wheat (Triticum aestivum). Although Gadd first described stripe rust of wheat in 1777, it was not until 1896 that Eriksson and Henning (1896) showed that stripe rust resulted from a separate pathogen, which they named P. glumarum. In 1953, Hylander et al. (1953) revived the name P. striiformis.

Epidemiology

Myriads of the places exist in the world where wheat rusts are know to incur devastation and inflicting tremendous economic injury (Saari and Prescott, 1985). Initial germination and infection of the pathogen takes place when the Urediniospores of the wheat rusts initiate germination. This germination commences within one to three hours of contact with free moisture over a range of temperatures depending on the rust. a characteristic property of these fungi is that the Urediniospores are produced in large numbers and can disperse to considerably large distances by the wind (Hirst and Hurst, 1967; Watson and de Sousa, 1983). Gravitational force puts break on this dispersal resulting in the deposition of these uredinospore close to the source (Roelfs and Martell, 1984). Urediniospores are very important biological entities with tremendously long life span. These spore are known for their long survival in the field even when they are away from their hosts. These spores remain viable at lower moisture content and in such circumstances they can withstand the freezing. At increasing moisture these spore tend to lose their viability especially at 50% moisture content. Topography of the land and wind attitude are the principles that govern the long distance dispersal of the urediniospores. Spread of spores from went to east takes place due to the rotational movement of the earth and at high latitude, wind is the sole agent responsible for this spreading (Roelfs, 1985; Luig, 1985 and Dubin and Stubbs, 1986). In India, spores move southward probably as a result of katabatic wind flows from the mountains into the plains (Nagarajan and Joshi, 1985). In most areas studied, spores produced in the upper levels of the crop canopy move into a geographical area where the crop phenology is less advanced.

Hot weather results in rising up of the air from inside the canopy. If higher humidity exists, spores don't tend to leave the uredinia. Low velocities of the wind tend to dry the canopy and as a consequence of this change leaves are agitated. This agitation makes the leaves to release spore from the uredinia. On the other hand speedy wind can cause accelerated release of the spores from the uredinia resulting in the dilution of the spore concentration above the canopy. However these phenomena may be reflected in the form of long distance spreading of the spores. Rain influences the spore dispersal. If can favour disease due to the fact that spore are scrubbed from the air. They are then deposited on the plants and humidity is also increased by the rains. But spore are also washed from the plant surfaces in the rains and elevated humidity levels also hamper spore movement. Rains also change the temperature which also influences the disease spreading and progress.

Stripe Rust

Stripe or yellow rust of wheat caused by P. striiformis f. sp. tritici can be as damaging as stem rust. However, stripe rust has a lower optimum temperature for development that limits it as a major disease in many areas of the world. Stripe rust is principally an important disease of wheat during the winter or early spring or at high elevations. Table 13.3 shows regions of the world where stripe rust has been a major or local problem.

Stripe rust of wheat may be the cause of stripe rust on barley (Stubbs, 1985). In Europe, a forma specialis of P. striiformis has evolved that is commonly found on barley and seldom on any but the most susceptible wheats (Zadoks, 1961). Puccinia striiformis f. sp. hordei was introduced into South America where it spread across the continent (Dubin and Stubbs, 1986) and was later identified in Mexico and United States (Roelfs et al., 1992).

Epidemiology of the stripe rust

The peculiarity linked with the yellow rust disease is that it is characterized by the demand of the lowest temperature for the establishment of the disease as the Puccinia striiformis has the lowest temperature among the three wheat rust pathogens. For the devastation of the yellow rust minimum, optimum and maximum temperatures are 0°, 11° and 23°C, respectively (Hogg et al., 1969; Zadoks and Bouwman 1985 and Rapilly 1979.

In Europe, P. striiformis oversummers on wheat (Zadoks, 1961). The amount of over-summering rust depends on the amount of volunteer wheat, which, in turn, is a function of moisture in the off-season. The ured-iniospores are then blown to autumn-sown wheat. In northwestern Europe, overwintering is limited to urediniomycelia in living leaf tissues as temperatures of -4°C will kill exposed sporulating lesions. Latent lesions can survive if the leaf survives. In other areas of the world, snow can insulate the sporulating lesions from the cold temperatures so air temperatures below -4°C fail to eliminate the rust lesions. The latent period for stripe rust during the winter can be up to 118 days and is suspected to be as many as 150 days under a snow cover (Zadoks, 1961).

In areas near the equator, stripe rust tends to cycle endemically from lower to higher altitudes and return following the crop phenology (Saari and Prescott, 1985). In more northern latitudes, the cycle becomes longer in distance with stripe rust moving from mountain areas to the foothills and plains.

Due to their susceptibility to ultraviolet light, urediniospores of stripe rust probably are not transported in a viable state as far as those of leaf and stem rusts. Maddison and Manners (1972) found stripe rust urediniospores three times more sensitive to ultraviolet light than those of stem rust. Still, Zadoks (1961) reports stripe rust was wind-transported in a viable state more than 800 km. The introductions of wheat stripe rust into Australia and South Africa and barley stripe rust into Colombia were probably aided by humans through jet travel (Dubin and Stubbs, 1986; O'Brien et al., 1980). However, the spread of stripe rust from Australia to New Zealand, a distance of 2 000 km, was probably through airborne urediniospores (Beresford, 1982). Perhaps an average spore of stripe rust has a lower likelihood of being airborne in a viable state over long distances than that of the other wheat rusts, but certainly some spores must be able to survive long-distance transport under special and favourable conditions. There are several examples of the sequential migration of stripe rust. Virulence for gene Yr2 (cultivars Siete Cerros, Kalyansona and Mexipak) was first recorded in Turkey and over a period of time was traced to the subcontinent of India and Pakistan (Saari and Prescott, 1985) and may be associated with the weather systems called the 'Western Disturbance'. As mentioned, barley stripe rust in South America migrated from its introduction point in Colombia to Chile over a period of a few years (Dubin and Stubbs, 1986).

Most areas of the world studied seem to have a local or nearby source of inoculum from volunteer wheat (Line et al, 1983; Stubbs, 1985; Zadoks and Bouwman, 1985). However, some evidence points to inoculum coming from non-cereal grasses (Hendrix et al., 1965; Tollenaar and Houston, 1967). Future studies of stripe rust epidemiology need to take into account not only the presence of rust on nearby grasses, but also the fact that the rust must occur on the grasses prior to its appearance on cereals. The virulence phenotype must be shown to be the same on both hosts and that it moves from the grass to wheat during the crop season. Stripe rust epidemics in the Netherlands can be generated by just a single uredinium per hectare surviving the winter if the spring season is favourable for rust development (Zadoks and Bouwman, 1985).

Molecular markers

All living organisms both plants and animals are made up of cells that are programmed by genetic material called DNA. This DNA is made up of long strands of nitrogen-containing bases which are - adenine [A], cytosine [C], guanine [G] and thymine [T]. Only a small fraction of the DNA sequence typically makes up genes, i.e. that code for proteins, while the remaining and major share of the DNA represents non-coding sequences, the role of which is not yet clearly understood. The genetic material is organized into sets of chromosomes (e.g. five pairs in Arabidopsis thaliana; 30 pairs in Bos taurus [cow]), and the entire set is called the genome. In a diploid individual (i.e. where chromosomes are organized in pairs), there are two alleles of every gene - one from each parent. Molecular markers should not be considered as normal genes as they usually do not have any biological effect. Instead, they can be thought of as constant landmarks in the genome. They are identifiable DNA sequences, found at specific locations of the genome, and transmitted by the standard laws of inheritance from one generation to the next. They rely on a DNA assay, in contrast to morphological markers that are based on visible traits, and biochemical markers that are based on proteins produced by genes. Different kinds of molecular markers exist, such as restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs) markers, amplified fragment length polymorphisms (AFLPs), microsatellites and single nucleotide polymorphisms (SNPs). They may differ in a variety of ways - such as their technical requirements (e.g. whether they can be automated or require use of radioactivity); the amount of time, money and labour needed; the number of genetic markers that can be detected throughout the genome; and the amount of genetic variation found at each marker in a given population. The information provided to the breeder by the markers varies depending on the type of marker system used. Each has its advantages and disadvantages and, in the future, other systems are likely to be developed.

From markers to MAS

The molecular marker systems described above allow high-density DNA markermaps (i.e. with many markers of known location, interspersed at relatively short intervals throughout the genome) to be constructed for a range of economically important agricultural species, thus providing the framework needed for eventual applications of MAS. Using the marker map, putative genes affecting traits of interest can then be detected by testing for statistical associations between marker variants and any trait of interest. These traits might be genetically simple - for example, many traits for disease resistance in plants are controlled by one or a few genes (Young, 1999). Alternatively, they could be genetically complex quantitative traits, involving many genes (i.e. so-called quantitative trait loci [QTL]) and environmental effects. Most economically important agronomic traits tend to fall into this latter category. For example, using 280 molecular markers (comprising 134 RFLPs, 131 AFLPs and 15 microsatellites) and recording populations of rice lines for various plant water stress indicators, phenology, plant biomass, yield and yield components under irrigated and water stress conditions, Babu et al. (2003) detected a number of putative QTL for drought resistance traits. Having dentified markers physically located beside or even within genes of interest, in the next step it is now possible to carry out MAS, i.e. to select identifiable marker variants (alleles) in order to select for non-identifiable favourable variants of the genes of interest. For example, consider a hypothetical situation where a molecular marker M (with two alleles M1 and M2), identified using a DNA assay, is known to be located on a chromosome close to a gene of interest Q (with a variant Q1 that increases yield and a variant Q2 that decreases yield), that is, as yet, unknown. If a given individual in the population has the alleles M1 and Q1 on one chromosome and M2 and Q2 on the other chromosome, then any of its progeny receiving the M1 allele will have a high probability (how high depends on how close M and Q are to each other on the chromosome) of also arrying the favourable Q1 allele, and thus would be preferred for selection purposes. On the other hand, those that inherit the M2 allele will tend to have inherited the unfavourable Q2 allele, and so would not be preferred for selection. With conventional selection which relies on phenotypic values, it is not possible to use this kind of information. The success of MAS is influenced by the relationship between the markers and the genes of interest. Dekkers (2004) distinguished three kinds of relationship:

• The molecular marker is located within the gene of interest (i.e. within the gene Q, using the example above). In this situation, one can refer to gene-assisted selection (GAS). This is the most favourable situation for MAS since, by following inheritance of the M alleles, inheritance of the Q alleles is followed directly. On the other hand, these kinds of markers are the most uncommon and are thus the most difficult to find.

• The marker is in linkage disequilibrium (LD) with Q throughout the whole population. LD is the tendency of certain combinations of alleles (e.g. M1 and Q1) to be inherited together. Populationwide LD can be found when markers and genes of interest are physically very close to each other and/or when lines or breeds have been crossed in recent generations. Selection using these markers can be called LD-MAS.

• The marker is not in linkage disequilibrium (i.e. it is in linkage equilibrium [LE]) with Q throughout the whole population. Selection using these markers can be called LE-MAS. This is the most difficult situation for applying MAS. The universal nature of DNA, molecular markers and genes means that MAS can, in theory, be applied to any agriculturally important species. Indeed, active research programmes have been devoted to building molecular marker maps and detecting QTLs for potential use in MAS programmes in a whole range of crop, livestock, forest tree and fish species. In addition, MAS can be applied to support existing conventional breeding programmes. These programmes use strategies such as: recurrent selection (i.e. using within-breed or within-line selection, important in livestock); development of crossbreds or hybrids (by crossing several improved lines or breeds) and introgression (where a target gene is introduced from, for example, a low-productive line or breed (donor) into a productive line (recipient) that lacks the target gene (a strategy especially important in plants). See Dekkers and Hospital (2002) for more details. MAS can be incorporated into any one of these strategies (e.g. for marker assisted introgression by using markers to accelerate introduction of the target gene). Alternatively, novel breeding strategies can be developed to harness the new possibilities that MAS raises.

Current Status of Applications of MA S in Agriculture

Below is a brief summary of the current status regarding application of MAS in the different agricultural sectors. For more details, a number of case studies for crops are presented in Section II of the book and for livestock, forestry and fish in Sections III, IV and V, respectively.

Crops

The promise of MAS has possibly been greeted with the most enthusiasm and expectation in this particular agricultural sector, stimulating tremendous investments in the development of molecular marker maps and research to detect associations between phenotypes and markers. Molecular marker maps have been constructed for a wide range of crop species. In a recent review, however, Dekkers and Hospital (2002) noted that "as theoretical and experimental results of QTL detection have accumulated, the initial enthusiasm for the potential genetic gains allowed by molecular genetics has been tempered by evidence for limits to the precision of the estimates of QTL effects", and that "overall, there are still few reports of successful MAS experiments or applications." They reported that marker-assisted introgression of known genes was widely used in plants, particularly by private breeding companies, whereas marker-assisted introgression of unknown genes had often proved to be less useful in practice than expected. As Young (1999) wrote: "even though marker assisted selection now plays a prominent role in the field of plant breeding, examples of successful, practical outcomes are rare. It is clear that DNA markers hold great promise, but realizing that promise remains elusive." There is also considerable divergence with respect to the applications of MAS among different crop species. For example, Koebner (2003) highlighted the relatively fast uptake of MAS in maize compared with wheat and barley, arguing that this largely reflected the breeding structure. Thus, whereas maize breeding is dominated in industrialized countries by a small number of large private companies that produce F1 hybrids, a system allowing protection from farm-saved seed and competitor use, breeding for the other major cereal species is primarily by public sector organizations and most varieties are inbred pure breeding lines, a system allowing less protection over the released varieties. Progress in arable crops is nevertheless quite advanced compared with horticultural crop species such as apples and pears, where development of molecular marker maps has been slow and only few QTL have been detected (Tartarini, 2003).

Marker-assisted selection may greatly increase the efficiency and effectiveness for breeding compared to conventional breeding. The fundamental advantages of MAS compared to conventional phenotypic selection are:

Simpler compared to phenotypic screening

Selection may be carried out at seedling stage

Single plants may be selected with high reliability.

These advantages may translate into (1) greater efficiency or (2) accelerated line development in breeding programs. Furthermore, selection based on DNA markers may be more reliable due to the influence of environmental factors on field trials. In some cases, using DNA markers may be more cost effective than the screening for the target trait. Another benefit from using MAS is that the total number of lines that need to be tested may be reduced. Since many lines can be discarded after MAS at an early generation, this permits a more effective breeding design.

The greater efficiency of target trait selection which may enable certain traits to be 'fast-tracked', since specific genotypes can be easily identified and selected. Moreover, 'background' markers may also be used to accelerate the recovery of recurrent parents during marker-assisted backcrossing (discussed later).

Importance of QTL mapping for MAS

The identification of genes and quantitative trait loci (QTLs) and DNA markers that are linked to them is accomplished via QTL mapping experiments. QTL mapping thus represents the foundation of the development of markers for MAS. Previously, it was generally assumed that markers could be directly used in MAS. However, there are many factors that influence the accuracy of QTL mapping such as population size and type, level of replication of phenotypic data, environmental effects and genotyping errors. These factors are particularly important for more complex quantitative traits with many QTLs each with relatively small effects (e.g. drought tolerance, yield). Therefore, in recent years it has become widely-accepted that QTL confirmation, validation and/or additional marker testing steps may be required after QTL mapping and prior to MAS. These steps may include:

Marker conversion - may be required such that the marker genotyping method is technically simpler for MAS or so that the reliability is improved.

QTL confirmation - testing the accuracy of results from the primary QTL mapping study

QTL validation - generally refers to the verification that a QTL is effective in different genetic backgrounds

Marker validation - testing the level of polymorphism of most tightly-linked markers within a narrow window (say 5 - 10 cM) spanning a target locus and also testing the reliability of markers to predict phenotype.

MAS schemes in plant breeding

Marker assisted backcrossing

There are three levels of selection in which markers may be applied in backcross breeding. In the first level, markers may be used to screen for the target trait, which may be useful for traits that have laborious phenotypic screening procedures or recessive alleles. The second level of selection involves selecting backcross progeny with the target gene and tightly-linked flanking markers in order to minimize linkage drag. We refer to this as 'recombinant selection'. The third level of MAB involves selecting backcross progeny (that have already been selected for the target trait) with 'background' markers.  In other words, markers can be used to select against the donor genome, which may accelerate the recovery of the recurrent parent genome. With conventional backcrossing, it takes a minimum of five to six generations to recover the recurrent parent. Data from simulation studies suggests that at least two but possibly three or even four backcross generations can be saved by using markers.

Marker assisted pyramiding

Pyramiding is the process of simultaneously combining multiple genes/QTLs together into a single genotype. This is possible through conventional breeding but extremely difficult or impossible at early generations. Using conventional phenotypic selection, individual plants must be phenotypically screened for all traits tested. Therefore, it may be very difficult to assess plants from certain population types (e.g. F2) or for traits with destructive bioassays. DNA markers may facilitate selection because DNA marker assays are non-destructive and markers for multiple specific genes/QTLs can be tested using a single DNA sample without phenotyping. The most widespread application for pyramiding has been for combining multiple disease resistance genes in order to develop durable disease resistance.

Early generation marker assisted selection

One of the most intuitive stages to use markers to select plants is at an early generation (especially F2 or F3). The main advantage is that many plants with unwanted gene combinations, especially those that lack essential disease resistance traits and plant height, can be simply discarded. This has important consequences in the later stages of the breeding program because the evaluation for other traits can be more efficiently and cheaply designed for fewer breeding lines (especially in terms of field space).

Current obstacles for the adoption of MAS

There are many barriers to the adoption of MAS in plant breeding. Currently, one of the most important barriers for MAS in rice today is the prohibitive cost. Although there are only a small number of reports analyzing the economics of MAS versus conventional breeding in the literature, the cost-effectiveness of using MAS compared to conventional plant breeding varies considerably between studies. Two additional factors need to be considered for cost-analysis: (1) the equipment and consumables required to establish and maintain a marker lab is considerable; and (2) there is a large initial cost in the development of markers which is seldom reported. For marker assisted backcrossing, the initial cost of using markers would be more expensive compared to conventional breeding in the short term however time savings could lead to an accelerated variety release which could translate into greater profits in the medium to long term.

Another important factor obstructing the successful application of markers for line development is the low reliability of markers to determine phenotype. This is often attributable to the 'thoroughness' of the primary QTL mapping study. Even QTLs that are detected with high LOD scores and explain a large proportion of the phenotype may be affected by sampling bias (especially in small populations), and therefore may not be useful for MAS. Furthermore, the effect of a QTL may depend on the genetic background. This emphasizes the importance of testing the QTL effects and the reliability of markers (i.e. QTL/marker validation) before MAS is undertaken.

Finally the level of integration between molecular geneticists and plant breeders (and scientists from other disciplines) may not be adequate to ensure that markers are effectively applied for line development.

Chapter 3

MATERIALS AND METHODS

Chemicals and reagents

Molecular biology grade chemicals were used in this study for preparing different buffers and solutions. The solutions and buffers used in this study were prepared according to standard protocols of the molecular biology.

Following buffers and solutions were prepared according to the following methods:

Preparation of 1 molar (1M) Tris solution (pH 8)

Tris solution with pH 8 was used in the preparation of 2%CTAB buffer, TAE buffer and TE buffer. In order to prepare a liter solution 121 gram tris-base was dissolved in 800 ml water and its pH was adjusted at 8 using concentrated HCl. After the adjustment of the pH the volume was raised to one liter with the distilled water. Then the solution was taken in a reagent bottle (keeping one third volume of the bottle empty) and autoclaved this.

Preparation of 0.5 molar EDTA solution (pH 8)

EDTA solution has been used for the preparation of 2% CTAB buffer and TE buffer. In order to prepare this solution 800ml distilled water was taken in a beaker and kept it on the magnetic stirrer. In this beaker 186 gram sodium salt of EDTA was added. Then 20 gram pellets of Na OH were also added and stirred it till it all dissolved and then the pH was adjusted at 8 with the help of NaOH and HCl. The solution was then taken in a reagent bottle and autoclaved.

Preparation of 2% CTAB buffer.

This is the buffer which was used for the extraction of total genomic DNA. To prepare this buffer 600ml distilled water was taken in a beaker and kept on magnetic stirrer. To this water 20 gram CTAB, 10 gram PVP, 83 gram Na Cl, 40 ml 0.5M EDTA (pH8) and 100 ml Tris (pH8) were added and mixed by magnetic stirring. Then this buffer was autoclaved before the further use.

Preparation of TE buffer

TE buffer has been used for the final dissolution of the extracted DNA. This buffer was prepared by using previously prepared and autoclaved tris and EDTA solutions. In order to prepare 100 ml TE buffer 90 ml double distilled autoclaved water was taken in a glass cylinder and to this 1 ml I M tris solution and 0.2 ml 0.5 M EDTA solutions were added and volume was made up with double distilled autoclaved water to 100 ml.

Seeds and Varieties:

For this research work seeds of the selected ten varieties were gotten from Wheat Research Institute, Ayub Agriculture Research Institute, Jhang Road, Faisalabad. Names and the sequence with which these varieties have been given and discussed later on is the following:

1- 2192

2- 0031034

3- Inquilab-91

4- Uqab

5- Sehar

6- Shafaq

7- Kohistan

8- 4112

9- Bhakkar

Seed Sterilization and Plant growth

Seeds of these selected wheat varieties were surface sterilized by soaking in 15% bleach for 20 minutes, washed thrice with sterilized water and placed on sterilized cotton in the glass plate for a week and then the healthy seedlings were transferred to the soil in the pots for the further growth for a couple of weeks and then their leaves were used for DNA extraction.

Isolation of Genomic DNA from Wheat Plants

Total plant DNA was isolated from young wheat leaves by the method described by (Doyle and Doyle, 1990). Extraction buffer (2%CTAB, 1%PVP, 1.4M NaCl, 100mM Tris, 20mM, EDTA and 1%2-Mercaptoethanol) was heated to 65-700C in shaking water bath. Young leaves (1gram) were excised from the plants and froze in liquid nitrogen. Then these leaves were ground into fine powder with pestle and mortars in liquid nitrogen. This powder was transferred to 50ml falcon tube and suspended in 15ml hot extraction buffer and placed in water bath for 30 minutes at 65-700C with gentle shacking. After 30 minutes 15ml chloroform: isoamyl alcohol (24:1) was added, mixed gently by inverting the tubes and centrifuged 10 minutes at 9000rpm at room temperature. Transparent supernatant was transferred to a fresh 50ml. To this supernatant 0.6 volume isopropanol was added, the contents were mixed by inverting the tubes several times and incubated them at -200C for 30 minutes. Then the tubes were centrifuged 10 minutes at 5000rpm, supernatant was discarded, pellet was washed with 70% ethanol, air dried and dissolved in 1ml TE (10mM Tris and 1mM, EDTA). To get rid of RNA 2ul RNAase (5mg/ul) was added to each tube and incubated at 370C for one hour. After one hour to each tube 100ul 3M sodium acetate pH 5.2 and 2.5ml absolute ethanol were added. The contents were mixed, incubated at -200C for 30 minutes, centrifuged at5 minutes at 12000rpm, supernatant was discarded, pellet was air dried and dissolved in 1ml TE. Quality and concentration of the DNA was found by fractionating it on 1%agarose gel and its concentration was found out by taking OD260.

Polymerase chain reaction (PCR).

All the chemicals used in this study were of molecular biology grade.

Optimization of concentration of ingredients

Concentrations of the chemical ingredients for polymerase chain reaction were optimized by carrying out the reaction using various concentrations of these ingredients. Finally following composition of the 20 ul reaction mixture was optimized and used for this study.

Chemical Stock Quantity

PCR buffer 10X 5ul

MgCl2 20mM 3ul

dNTPs 10mM 1ul

Primer 1 30ng 1ul

Primer 2 30ng 1ul

Taq polymerase 5units/ul 0.5ul

H2O 37.5ul

Template DNA 50ng/ul 2ul

Total 50ul

PCR profile was optimized as follows:

One cycle

Denaturation at 950C 5 minutes

Forty cycles

Denaturation at 950C for one minute,

Annealing at 500C for one minute

Extension at 720C for one minute

One cycle

Extension of 10 minutes at 720C.

Hold at 220C

Analysis of the PCR Products (Gel Electrophoresis)

For the analysis of the PCR product 1% agarose gel was prepared in 0.5X TAE buffer. In a clean glass flask 100 ml 0.5X TAE buffer was taken and 1 gram agarose was added to this and heated in an oven till boiling and to this homogeneous solution 10ug ethidium bromide was added and poured in a tray having specific combs and allowed it to settle till the solidification at room temperature. Then the gel was placed in the buffer and DNA was loaded in the wells after mixing with 6X gel loading buffer. Then the gel electrophoresis system was connected with the power supply and fractionation of the PCR products was performed at 100 volts for 40 minutes and then the results were observed in UV light and recorded in the computerized gel documentation system.

Chapter 4

RESULTS

Quality and Quantity Analysis

Before running the PCR reaction the DNA was analyzed for its quality test. DNA quality was checked by preparing 0.8% agarose gel carry 10ug/ml ethydium bormide. To this gel 1 ul of the finally dissolved DNA sample was taken in a microfuge tube. This sample was then dilute with TE buffer to 10 ul. Then 2 ul 6X gel loading buffer was added and mixed and whole material was loaded to the gel and electrophoresed at low voltage. From this gel the DNA quality was analyzed by observing it under the UV light and photograph was taken for the result compilation. This is shown in fgure 1 which shows and confirms fine DNA extraction. DNA of all the eight varieities is of good quality with sharp discrete bands and no degradation was seen. This gel photograph also showed that the solution is highly concentrated and it was diluted for the final use in the PCR reaction. From the gel photograph it was concluded that the concentration of the DNA is same in all the samples and it is about 25ng/ul DNA for each variety. PCR analysis performed on the basis of this information of the DNA concentration indicated that this assessment was right.

PCR Analysis

According to the method described in Materials and Methods PCR reaction was performed and the results are shown in figure 2. For the PCR reaction optimum conditions and optimum concentrations of the ingredients were determined by using running PCR at different annealing temperatures and using different concentrations of the template and other ingredients. The PCR reaction was performed with the optimum quantities as are given in the materials and methods. These results show that the varieties show amplification of the Xwgm-SSR marker.

Chapter 5

DISCUSSION

So this research has found that Blue Silver, Era, Bakhtawar, Perula and Fareed 2000 wheat varieties are having Xgwm-16 SSR marker and probably the linked Yr 5 gene as well.

This is interesting and valuable information for the scientist who are involved in producing new wheat varieties by breeding. Harnessing the latest techniques and technologies is inevitable in order to meet the demands and requirements of ever increasing population of the world. One of the best applications of the molecular biology is to genetic characters of the best varieties of different crops and brings best traits together in the best varieties.

In order to bring resistant genes in a certain variety it is necessary to know which rust resistance gene are carried by a particular wheat variety. This can be done by exploiting molecular markers of the rust resistance genes. Different types of the molecular markers can be used for this purpose detecting the presence of the linked rust resistance gene in a variety and for studying the diversity among the different wheat varieties. Simple sequence repeats (SSR) markers are the most suitable for such purpose.

More than thirty different yellow rust resistance genes have been detected and SSR markers linked to many of these genes have been discovered. Primers of many of these markers have been published in the literature and there are numerous reports in renowned scientific journals of benefiting from this information. This research work is also such a small endeavor.

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