Pakistan is an agricultural state with huge fertile land and other related resources. Due to the great diverseness of land and clime, Pakistan is bring forthing many harvests and veggies. Wheat is the most of import among them and is the anchor of its agribusiness based economic system. In Pakistan wheat is cultivated on about on 70 million hectares and in 2006-2007 and produced 23.7 million dozenss of wheat which is more than domestic demands ( Economic Survey of Pakistan, 2008 ) .

Pakistan has been divided into 10 production zones because of great agro ecological countries where wheat is cultivated. The zoning is chiefly based on cropping form, disease prevalence and climatic factors. Wheat histories for 37.1 % of the harvest country, 65 % of the nutrient grain land area, and 70 % of the production.A

In Pakistan, wheat is grown in different cropping systems, such as ; cotton – wheat, rice – wheat, sugar cane – wheat, maize – wheat, fallow – wheat. Of these, Cotton-Wheat and Rice-Wheat systems together account approximately 60 % of the entire wheat country whereas rain-fed wheat covers more than 1.50 thousand ha country. Rotations with Maize-Sugarcane, Pulses and fallow are besides of import

Botanically, wheat is a member of the grass household to which rice, barley, maize and several other cereal grain harvests besides belong. It ranks foremost in universe harvest production and is the national nutrient basic of 43 states. Wheat is best adapted to a cool dry clime. Wheat is one of the major agricultural harvest species in the universe. The importance of the wheat makes it inevitable to develop the most productive and profitable wheat assortments by the usage of the conventional and modern scientific methods. In this regards the usage of molecular biological science techniques for the development of the new improved assortments is the most of import facet of the modern scientific promotions. Common or bread wheat, Triticum aestivum, is hexaploid with a big genome estimated at ~17,000 Mb that is attributable to the polyploidy nature of wheat and the high content of insistent elements within the wheat genome. A figure of genomic resources have been developed or are being developed for wheat. These include a aggregation of & gt ; 500,000 wheat ESTs, bacterial unreal chromosome ringers, and & gt ; 5,000 bin-mapped EST markers.

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The development of DNA ( or molecular ) markers has irreversibly changed the subjects of works genetic sciences and works genteelness. While there are several applications of Deoxyribonucleic acid markers in genteelness, the most promising for cultivar development is called marker assisted choice ( MAS ) . MAS refers to the usage of Deoxyribonucleic acid markers that are tightly-linked to aim venue as a replacement for or to help phenotypic testing. By finding the allelomorph of a Deoxyribonucleic acid marker, workss that possess peculiar cistrons or quantitative trait venue ( QTLs ) may be identified based on their genotype instead than their phenotype.

Wheat production in Pakistan is non encouraging. Pakistan has low per acre production as compared to the wheat production in the advanced states. There are several factors responsible for the low productiveness. One of the grounds for this suffering predicament of wheat output in Pakistan is the deficiency of the usage of modern techniques for the development of new wheat assortments. This accounts for the low per acre output of the wheat, its exposure to assorted biotic and a biotic emphasiss. One of the most formidable biotic emphasiss is the diseases caused by different pathogens which non merely sabotage the wheat productiveness but spoil the attempts of the husbandmans every bit good. Among these infective diseases wheat rusts are the most destructive and unleash enormous wheat productiveness losingss yearly in Pakistan. One of them is the xanthous rust caused by a fungus, of wheat and which is the subject of this research work.

In the aftermath of this scenario it is the demand of the hr to set about the schemes based on the latest engineering and research in order to minimise the losingss perpetrated by the disease. Management and usage of the bing opposition beginning among different wheat assortments to support the harvest against this disease is best solution. This can be done by conveying rust opposition cistrons into the wheat assortments which are known for higher output and which are prone to the disease.

In order to convey immune cistrons in a certain assortment it is necessary to cognize which rust opposition cistron are carried by a peculiar wheat assortment. This can be done by working molecular markers of the rust opposition cistrons. Different types of the molecular markers can be used for this intent observing the presence of the linked rust opposition cistron in a assortment and for analyzing the diverseness among the different wheat assortments. Simple sequence repetitions ( SSR ) markers are the most suited for such intent.

More than 30 different xanthous rust opposition cistrons have been detected and SSR markers linked to many of these cistrons have been discovered. Primers of many of these markers have been published in the literature.

In this survey presence of the xanthous rust opposition cistron Yr-9 has been searched out by utilizing its SSR marker Xgwm 488 in the undermentioned Pakistani wheat assortments:

1- Bhakkar

2- 0031034

3- Inquilab-91

4- Uqab

5- Sehar

6- Shafaq

7- Kohistan

8- 4112

Purposes and Aims

This survey was undertaken with the following aims:

Turning the seeds of the proposed wheat assortments

Deoxyribonucleic acid extraction

Quality and measure measurement of the Deoxyribonucleic acid


Gel cataphoresis

Chapter 2


Scientific literature is full with the finds of priceless familial constituents in the of course happening works and carnal assortments. The survey in which different assortments of a harvest are screened for the presence or absence of these cherished genomic sections is extremely valuable. As a consequence of such attempts non merely we come to cognize which are the valuable familial characters possessed by a certain assortment but we besides become able to develop new and more productive assortments utilizing this cognition and information. This survey has focused on the observing xanthous rust immune cistrons in the selected wheat assortments of Pakistan.

Wheat rust diseases

Wheat rust diseases are an disposed illustration of the fact how works infective Fungis have been commiting the unbelievable devastation of the agricultural economic system all over the universe. The diseases which are studied under the rubric of wheat rusts, the most common these yearss is called foliage or brown rust and is caused by P. triticina Eriks. This malignant disease traverses the foliage blades, but it besides invades the leaf sheaths peculiarly when it inflicts in the extremely favourable environmental conditions which entail high inoculants densenesss and enormously susceptible cultivars. In such fortunes most normally the disease lacks the abundant teliospore production of root rust at the terminal of the season. As a effect of the state of affairs brown leaf lesions are produced instead than black root lesions that are the features of the root rust. Leaf rust teliospores are produced at the lower surfaces of the foliages and they are originated from the telia. The other distinctive feature associated with them is that cuticular cells maintain on covering them. At a temperature 10A° to 30A°C the disease prevails really quickly and disease issues anyplace the wheat is cultivated all over the universe. Losses in grain output are chiefly attributed to cut down floret set and grain shriveling. The disease inflicts hurt to the flowerets, tillers and whole workss in highly susceptible genotypes. These losingss can be interpreted in the economic footings runing between 10 to 30 per centum.

An other foliage rust disease is caused by. recondita composite ( P. triticiduri V. Bourgin ) in the Mediterranean Sea and infects both hard wheat and bread wheat ( Ezzahiri et al. , 1992 ) . The pathogen seldom produces urediniospores. These urediniospores are by and large on the lower surface of the foliage. Epidemics produced by this disease are largely local because urediniospores are missing here. A batch of telia are given rise in a ring around the initial uredinium.

An other rust disease known as root rust, is caused by P. graminis Pers. f. sp. tritici Eriks. & A ; E. Henn. This disease is besides known as black rust or summer rust as it produces a larger figure of glistening black teliospores. These teliospores are produced in the uredinium at the terminal of the season or with unfavorable conditions. Warm 15A° to 35A°C and humid conditions exaggerate the harm by the root rust. In such state of affairss this disease can be most deadly and can incur up to 50 % loss and in this manner the root rust is the most annihilating disease among all the rusts. In the most terrible onslaughts in the favourable conditions with susceptible assortments the disease can do 100 per centum losingss.

Stripe rust which is besides known as xanthous rust, caused by P. striiformis West. f. sp. tritici Eriks & A ; Henn. , is typically a disease of wheat grown under the cold environmental conditions at a temperature runing from 2A° to 15A°C. This rust produces a characteristic form of uredinia bring forthing xanthous colored uredinospores in the signifier of chevrons and gives its name band rust. As a effect of the early onslaught of this disease workss remain scrawny and weak and can ensue in production losingss up to 50 % which cam even exceed to 100 % in the utmost conditions.

Pathogens of wheat rusts

In wheat rust disease doing pathogens belong to genus Puccinia, household Pucciniaceae, order Uredinales and category Basidiomycetes. Pathogens of the rust Fungis are highly specialised works pathogens with narrow host scopes. Fontana and Tozzetti separately and independently are the innovator to describe the disease of wheat root rust in 1767 ( Fontana, 1932 ; Tozzetti, 1952 ) . The causal being of wheat root rust was named P. graminis by Persoon in 1797. Chester ( 1946 ) provided one of the first elaborate histories of the literature on the rust of wheat. In the early records, wheat foliage rust is non distinguished from root rust ( Chester, 1946 ) . However, by 1815 de Candolle ( 1815 ) had shown that wheat foliage rust was caused by a distinguishable fungus Uredo rubigovera. The pathogen underwent a figure of name alterations until 1956 when Cummins and Caldwell ( 1956 ) suggested P. recondita, which has been the by and large used terminology. Recent morphological surveies by Savile ( 1984 ) and morphological and pathogen familial surveies by Anikster et Al. ( 1997 ) show that P. recondita is non the incitant of wheat foliage rust. Currently P. triticina should be the preferable 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 old ages. In this chapter, P. triticina will be used for the foliage rust on wheat ( Triticum aestivum ) . Although Gadd foremost described stripe rust of wheat in 1777, it was non until 1896 that Eriksson and Henning ( 1896 ) showed that band rust resulted from a separate pathogen, which they named P. glumarum. In 1953, Hylander et Al. ( 1953 ) revived the name P. striiformis.


Myriads of the topographic points exist in the universe where wheat rusts are know to incur desolation and bring downing enormous economic hurt ( Saari and Prescott, 1985 ) . Initial sprouting and infection of the pathogen takes topographic point when the Urediniospores of the wheat rusts initiate sprouting. This sprouting commences within one to three hours of contact with free wet over a scope of temperatures depending on the rust. a characteristic belongings of these Fungis is that the Urediniospores are produced in big Numberss and can scatter to well big distances by the air current ( Hirst and Hurst, 1967 ; Watson and de Sousa, 1983 ) . Gravitational force puts interruption on this dispersion ensuing in the deposition of these uredinospore stopping point to the beginning ( Roelfs and Martell, 1984 ) . Urediniospores are really of import biological entities with enormously long life span. These spore are known for their long endurance in the field even when they are off from their hosts. These spores remain feasible at lower wet content and in such fortunes they can defy the freeze. At increasing wet these spore tend to lose their viability particularly at 50 % wet content. Topography of the land and weave attitude are the rules that govern the long distance dispersion of the urediniospores. Spread of spores from went to east takes topographic point due to the rotational motion of the Earth and at high latitude, air current is the exclusive agent responsible for this spreading ( Roelfs, 1985 ; Luig, 1985 and Dubin and Stubbs, 1986 ) . In India, spores move southerly likely as a consequence of catabatic air current flows from the mountains into the fields ( Nagarajan and Joshi, 1985 ) . In most countries studied, spores produced in the upper degrees of the harvest canopy move into a geographical country where the harvest phenology is less advanced.

Hot conditions consequences in lifting up of the air from inside the canopy. If higher humidness exists, spores do n’t be given to go forth the uredinia. Low speeds of the air current tend to dry the canopy and as a effect of this alteration leaves are agitated. This agitation makes the foliages to let go of spore from the uredinia. On the other manus speedy air current can do 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 signifier of long distance spreading of the spores. Rain influences the spore dispersion. If can favor disease due to the fact that spore are scrubbed from the air. They are so deposited on the workss and humidness is besides increased by the rains. But spore are besides washed from the works surfaces in the rains and elevated humidness degrees besides hamper spore motion. Rains besides change the temperature which besides influences the disease spreading and advancement.

Stripe Rust

Stripe or xanthous rust of wheat caused by P. striiformis f. sp. tritici can be every bit detrimental as root rust. However, stripe rust has a lower optimal temperature for development that limits it as a major disease in many countries of the universe. Stripe rust is chiefly an of import disease of wheat during the winter or early spring or at high lifts. Table 13.3 shows parts of the universe where band rust has been a major or local job.

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 normally 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 subsequently identified in Mexico and United States ( Roelfs et al. , 1992 ) .

Epidemiology of the band rust

The distinctive feature linked with the xanthous rust disease is that it is characterized by the demand of the lowest temperature for the constitution of the disease as the Puccinia striiformis has the lowest temperature among the three wheat rust pathogens. For the desolation of the xanthous rust lower limit, optimal and maximal temperatures are 0A° , 11A° and 23A°C, severally ( Hogg et al. , 1969 ; Zadoks and Bouwman 1985 and Rapilly 1979.

In Europe, P. striiformis oversummers on wheat ( Zadoks, 1961 ) . The sum of over-summering rust depends on the sum of voluntary wheat, which, in bend, is a map of wet in the off-season. The ured-iniospores are so blown to autumn-sown wheat. In northwesterly Europe, overwintering is limited to urediniomycelia in life foliage tissues as temperatures of -4A°C will kill exposed sporulating lesions. Latent lesions can last if the foliage survives. In other countries of the universe, snow can insulate the sporulating lesions from the cold temperatures so air temperatures below -4A°C fail to extinguish the rust lesions. The latent period for stripe rust during the winter can be up to 118 yearss and is suspected to be every bit many as 150 yearss under a snow screen ( Zadoks, 1961 ) .

In countries near the equator, stripe rust tends to rhythm endemically from lower to higher heights and return following the harvest phenology ( Saari and Prescott, 1985 ) . In more northern latitudes, the rhythm becomes longer in distance with stripe rust traveling from mountain countries to the foothills and fields.

Due to their susceptibleness to ultraviolet visible radiation, urediniospores of band rust likely are non transported in a feasible province every bit far as those of foliage and root rusts. Maddison and Manners ( 1972 ) found stripe rust urediniospores three times more sensitive to ultraviolet visible radiation than those of root rust. Still, Zadoks ( 1961 ) studies stripe rust was wind-transported in a feasible province more than 800 kilometer. The debuts of wheat band rust into Australia and South Africa and barley band rust into Colombia were likely aided by worlds 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 kilometer, was likely through airborne urediniospores ( Beresford, 1982 ) . Possibly an mean spore of band rust has a lower likeliness of being airborne in a feasible province over long distances than that of the other wheat rusts, but surely some spores must be able to last long-distance conveyance under particular and favorable conditions. There are several illustrations of the consecutive migration of stripe rust. Virulence for cistron Yr2 ( cultivars Siete Cerros, Kalyansona and Mexipak ) was foremost recorded in Turkey and over a period of clip was traced to the subcontinent of India and Pakistan ( Saari and Prescott, 1985 ) and may be associated with the conditions systems called the ‘Western Disturbance ‘ . As mentioned, barley band rust in South America migrated from its debut point in Colombia to Chile over a period of a few old ages ( Dubin and Stubbs, 1986 ) .

Most countries of the universe studied seem to hold a local or nearby beginning of inoculant from voluntary wheat ( Line et al, 1983 ; Stubbs, 1985 ; Zadoks and Bouwman, 1985 ) . However, some grounds points to inoculum coming from non-cereal grasses ( Hendrix et al. , 1965 ; Tollenaar and Houston, 1967 ) . Future surveies of stripe rust epidemiology demand to take into history non merely the presence of rust on nearby grasses, but besides the fact that the rust must happen on the grasses prior to its visual aspect on cereals. The virulency phenotype must be shown to be the same on both hosts and that it moves from the grass to wheat during the harvest season. Stripe rust epidemics in the Netherlands can be generated by merely a individual uredinium per hectare lasting the winter if the spring season is favorable for rust development ( Zadoks and Bouwman, 1985 ) .

Molecular markers

All life organisms both workss and animate beings are made up of cells that are programmed by familial stuff called DNA. This Deoxyribonucleic acid is made up of long strands of nitrogen-containing bases which are – A [ A ] , C [ C ] , G [ G ] and thymine [ T ] . Merely a little fraction of the DNA sequence typically makes up cistrons, i.e. that codification for proteins, while the staying and major portion of the Deoxyribonucleic acid represents non-coding sequences, the function of which is non yet clearly understood. The familial stuff is organized into sets of chromosomes ( e.g. five braces in Arabidopsis thaliana ; 30 braces in Bos Sanchez [ cow ] ) , and the full set is called the genome. In a diploid person ( i.e. where chromosomes are organized in brace ) , there are two allelomorphs of every cistron – one from each parent. Molecular markers should non be considered as normal cistrons as they normally do non hold any biological consequence. Alternatively, they can be thought of as changeless landmarks in the genome. They are identifiable DNA sequences, found at specific locations of the genome, and transmitted by the standard Torahs of heritage from one coevals to the following. They rely on a DNA check, in contrast to morphological markers that are based on seeable traits, and biochemical markers that are based on proteins produced by cistrons. Different sorts of molecular markers exist, such as limitation fragment length polymorphisms ( RFLPs ) , random amplified polymorphous DNA ( RAPDs ) markers, amplified fragment length polymorphisms ( AFLPs ) , microsatellites and individual base polymorphisms ( SNPs ) . They may differ in a assortment of ways – such as their proficient demands ( e.g. whether they can be automated or require usage of radiation ) ; the sum of clip, money and labor needed ; the figure of familial markers that can be detected throughout the genome ; and the sum of familial fluctuation 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 hereafter, 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 comparatively short intervals throughout the genome ) to be constructed for a scope of economically of import agricultural species, therefore supplying the model needed for eventual applications of MAS. Using the marker map, putative cistrons impacting traits of involvement can so be detected by proving for statistical associations between marker discrepancies and any trait of involvement. These traits might be genetically simple – for illustration, many traits for disease opposition in workss are controlled by one or a few cistrons ( Young, 1999 ) . Alternatively, they could be genetically complex quantitative traits, affecting many cistrons ( i.e. alleged quantitative trait venue [ QTL ] ) and environmental effects. Most economically of import agronomic traits tend to fall into this latter class. For illustration, utilizing 280 molecular markers ( consisting 134 RFLPs, 131 AFLPs and 15 microsatellites ) and entering populations of rice lines for assorted works H2O emphasis indexs, phenology, works biomass, output and output constituents under irrigated and H2O emphasis conditions, Babu et Al. ( 2003 ) detected a figure of putative QTL for drouth opposition traits. Having dentified markers physically located beside or even within cistrons of involvement, in the following measure it is now possible to transport out MAS, i.e. to choose identifiable marker discrepancies ( allelomorphs ) in order to choose for non-identifiable favorable discrepancies of the cistrons of involvement. For illustration, see a conjectural state of affairs where a molecular marker M ( with two allelomorphs M1 and M2 ) , identified utilizing a Deoxyribonucleic acid check, is known to be located on a chromosome near to a cistron of involvement Q ( with a variant Q1 that increases output and a variant Q2 that decreases output ) , that is, as yet, unknown. If a given person in the population has the allelomorphs M1 and Q1 on one chromosome and M2 and Q2 on the other chromosome, so any of its offspring having the M1 allelomorph will hold a high chance ( how high depends on how close M and Q are to each other on the chromosome ) of besides arrying the favorable Q1 allelomorph, and therefore would be preferred for choice intents. On the other manus, those that inherit the M2 allelomorph will be given to hold inherited the unfavorable Q2 allelomorph, and so would non be preferred for choice. With conventional choice which relies on phenotypic values, it is non possible to utilize this sort of information. The success of MAS is influenced by the relationship between the markers and the cistrons of involvement. Deckers ( 2004 ) distinguished three sorts of relationship:

aˆ? The molecular marker is located within the cistron of involvement ( i.e. within the cistron Q, utilizing the illustration above ) . In this state of affairs, one can mention to gene-assisted choice ( GAS ) . This is the most favorable state of affairs for MAS since, by following heritage of the M allelomorphs, heritage of the Q allelomorphs is followed straight. On the other manus, these sorts of markers are the most uncommon and are therefore the most hard to happen.

aˆ? The marker is in linkage disequilibrium ( LD ) with Q throughout the whole population. LD is the inclination of certain combinations of allelomorphs ( e.g. M1 and Q1 ) to be inherited together. Populationwide LD can be found when markers and cistrons of involvement are physically really near to each other and/or when lines or strains have been crossed in recent coevalss. Choice utilizing these markers can be called LD-MAS.

aˆ? The marker is non in linkage disequilibrium ( i.e. it is in linkage equilibrium [ LE ] ) with Q throughout the whole population. Choice utilizing these markers can be called LE-MAS. This is the most hard state of affairs for using MAS. The cosmopolitan nature of DNA, molecular markers and cistrons means that MAS can, in theory, be applied to any agriculturally of import species. Indeed, active research programmes have been devoted to constructing molecular marker maps and observing QTLs for possible usage in MAS programmes in a whole scope of harvest, farm animal, forest tree and fish species. In add-on, MAS can be applied to back up bing conventional genteelness programmes. These programmes use schemes such as: recurrent choice ( i.e. utilizing within-breed or within-line choice, of import in farm animal ) ; development of crossbreds or loanblends ( by traversing several improved lines or strains ) and introgression ( where a mark cistron is introduced from, for illustration, a low-productive line or strain ( giver ) into a productive line ( receiver ) that lacks the mark cistron ( a scheme particularly of import in workss ) . See Dekkers and Hospital ( 2002 ) for more inside informations. MAS can be incorporated into any one of these schemes ( e.g. for marker assisted introgression by utilizing markers to speed up debut of the mark cistron ) . Alternatively, fresh genteelness schemes can be developed to tackle the new possibilities that MAS raises.

Current Status of Applications of MA S in Agriculture

Below is a brief sum-up of the current position sing application of MAS in the different agricultural sectors. For more inside informations, a figure of instance surveies for harvests are presented in Section II of the book and for farm animal, forestry and fish in Sections III, IV and V, severally.


The promise of MAS has perchance been greeted with the most enthusiasm and outlook in this peculiar agricultural sector, exciting enormous investings in the development of molecular marker maps and research to observe associations between phenotypes and markers. Molecular marker maps have been constructed for a broad scope of harvest species. In a recent reappraisal, nevertheless, Deckers and Hospital ( 2002 ) noted that “ as theoretical and experimental consequences of QTL sensing have accumulated, the initial enthusiasm for the possible familial additions allowed by molecular genetic sciences has been tempered by grounds for bounds to the preciseness of the estimations of QTL effects ” , and that “ overall, there are still few studies of successful MAS experiments or applications. ” They reported that marker-assisted introgression of known cistrons was widely used in workss, peculiarly by private genteelness companies, whereas marker-assisted introgression of unknown cistrons had frequently proved to be less utile in pattern than expected. As Young ( 1999 ) wrote: “ even though marker assisted choice now plays a outstanding function in the field of works genteelness, illustrations of successful, practical results are rare. It is clear that Deoxyribonucleic acid markers hold great promise, but recognizing that promise remains elusive. ” There is besides considerable divergency with regard to the applications of MAS among different harvest species. For illustration, Koebner ( 2003 ) highlighted the comparatively fast consumption of MAS in corn compared with wheat and barley, reasoning that this mostly reflected the genteelness construction. Therefore, whereas maize genteelness is dominated in industrialised states by a little figure of big private companies that produce F1 loanblends, a system leting protection from farm-saved seed and rival usage, engendering for the other major cereal species is chiefly by public sector organisations and most assortments are inbred pure genteelness lines, a system leting less protection over the released assortments. Advancement in cultivable harvests is however rather advanced compared with horticultural harvest species such as apples and pears, where development of molecular marker maps has been slow and merely few QTL have been detected ( Tartarini, 2003 ) .

Marker-assisted choice may greatly increase the efficiency and effectivity for engendering compared to conventional genteelness. The cardinal advantages of MAS compared to conventional phenotypic choice are:

Simpler compared to phenotypic testing

Choice may be carried out at seedling phase

Individual workss may be selected with high dependability.

These advantages may interpret into ( 1 ) greater efficiency or ( 2 ) accelerated line development in engendering plans. Furthermore, choice based on DNA markers may be more dependable due to the influence of environmental factors on field tests. In some instances, utilizing DNA markers may be more cost effectual than the showing for the mark trait. Another benefit from utilizing MAS is that the entire figure of lines that need to be tested may be reduced. Since many lines can be discarded after MAS at an early coevals, this permits a more effectual genteelness design.

The greater efficiency of mark trait choice which may enable certain traits to be ‘fast-tracked ‘ , since specific genotypes can be easy identified and selected. Furthermore, ‘background ‘ markers may besides be used to speed up the recovery of perennial parents during marker-assisted backcrossing ( discussed subsequently ) .

Importance of QTL mapping for MAS

The designation of cistrons and quantitative trait venue ( QTLs ) and DNA markers that are linked to them is accomplished via QTL mapping experiments. QTL mapping therefore represents the foundation of the development of markers for MAS. Previously, it was by and large assumed that markers could be straight used in MAS. However, there are many factors that influence the truth of QTL mapping such as population size and type, degree of reproduction of phenotypic informations, environmental effects and genotyping mistakes. These factors are peculiarly of import for more complex quantitative traits with many QTLs each with comparatively little effects ( e.g. drought tolerance, output ) . Therefore, in recent old ages it has become widely-accepted that QTL verification, proof and/or extra marker proving stairss may be required after QTL function and anterior to MAS. These stairss may include:

Marker transition – may be required such that the marker genotyping method is technically simpler for MAS or so that the dependability is improved.

QTL verification – proving the truth of consequences from the primary QTL function survey

QTL proof – by and large refers to the confirmation that a QTL is effectual in different familial backgrounds

Marker proof – proving the degree of polymorphism of most tightly-linked markers within a narrow window ( say 5 – 10 centimeter ) crossing a mark venue and besides proving the dependability of markers to foretell phenotype.

MAS strategies in works genteelness

Marker assisted backcrossing

There are three degrees of choice in which markers may be applied in backcross genteelness. In the first degree, markers may be used to test for the mark trait, which may be utile for traits that have arduous phenotypic showing processs or recessionary allelomorphs. The 2nd degree of choice involves choosing backcross offspring with the mark cistron and tightly-linked flanking markers in order to minimise linkage retarding force. We refer to this as ‘recombinant choice ‘ . The 3rd degree of MAB involves choosing backcross offspring ( that have already been selected for the mark trait ) with ‘background ‘ markers.A In other words, markers can be used to choose against the donor genome, which may speed up the recovery of the perennial parent genome. With conventional backcrossing, it takes a lower limit of five to six coevalss to retrieve the recurrent parent. Datas from simulation surveies suggests that at least two but perchance three or even four backcross coevalss can be saved by utilizing markers.

Marker assisted pyramiding

Pyramiding is the procedure of at the same time uniting multiple genes/QTLs together into a individual genotype. This is possible through conventional genteelness but highly hard or impossible at early coevalss. Using conventional phenotypic choice, single workss must be phenotypically screened for all traits tested. Therefore, it may be really hard to measure workss from certain population types ( e.g. F2 ) or for traits with destructive bio-assaies. Deoxyribonucleic acid markers may ease choice because DNA marker checks are non-destructive and markers for multiple specific genes/QTLs can be tested utilizing a individual DNA sample without phenotyping. The most widespread application for pyramiding has been for uniting multiple disease opposition cistrons in order to develop lasting disease opposition.

Early coevals marker assisted choice

One of the most intuitive phases to utilize markers to choose workss is at an early coevals ( particularly F2 or F3 ) . The chief advantage is that many workss with unwanted cistron combinations, particularly those that lack indispensable disease opposition traits and works tallness, can be merely discarded. This has of import effects in the ulterior phases of the genteelness plan because the rating for other traits can be more expeditiously and cheaply designed for fewer genteelness lines ( particularly in footings of field infinite ) .

Current obstructions for the acceptance of MAS

There are many barriers to the acceptance of MAS in works genteelness. Presently, one of the most of import barriers for MAS in rice today is the prohibitory cost. Although there are merely a little figure of studies analysing the economic sciences of MAS versus conventional genteelness in the literature, the cost-effectiveness of utilizing MAS compared to conventional works genteelness varies well between surveies. Two extra factors need to be considered for cost-analysis: ( 1 ) the equipment and consumables required to set up and keep a marker lab is considerable ; and ( 2 ) there is a big initial cost in the development of markers which is rarely reported. For marker assisted backcrossing, the initial cost of utilizing markers would be more expensive compared to conventional genteelness in the short term nevertheless clip nest eggs could take to an accelerated assortment release which could interpret into greater net incomes in the medium to long term.

Another of import factor blockading the successful application of markers for line development is the low dependability of markers to find phenotype. This is frequently attributable to the ‘thoroughness ‘ of the primary QTL function survey. Even QTLs that are detected with high LOD tonss and explicate a big proportion of the phenotype may be affected by trying prejudice ( particularly in little populations ) , and hence may non be utile for MAS. Furthermore, the consequence of a QTL may depend on the familial background. This emphasizes the importance of proving the QTL effects and the dependability of markers ( i.e. QTL/marker proof ) before MAS is undertaken.

Finally the degree of integrating between molecular geneticists and works breeders ( and scientists from other subjects ) may non be equal to guarantee that markers are efficaciously applied for line development.

Chapter 3


Chemicals and reagents

Molecular biological science class chemicals were used in this survey for fixing different buffers and solutions. The solutions and buffers used in this survey were prepared harmonizing to standard protocols of the molecular biological science.

Following buffers and solutions were prepared harmonizing to the undermentioned methods:

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

Tris solution with pH 8 was used in the readying of 2 % CTAB buffer, TAE buffer and TE buffer. In order to fix a liter solution 121 gm tris-base was dissolved in 800 ml H2O and its pH was adjusted at 8 utilizing concentrated HCl. After the accommodation of the pH the volume was raised to one litre with the distilled H2O. Then the solution was taken in a reagent bottle ( maintaining one 3rd volume of the bottle empty ) and autoclaved this.

Preparation of 0.5 molar EDTA solution ( pH 8 )

EDTA solution has been used for the readying of 2 % CTAB buffer and TE buffer. In order to fix this solution 800ml distilled H2O was taken in a beaker and kept it on the magnetic scaremonger. In this beaker 186 gm Na salt of EDTA was added. Then 20 gram pellets of Na OH were besides added and stirred it till it all dissolved and so the pH was adjusted at 8 with the aid of NaOH and HCl. The solution was so taken in a reagent bottle and autoclaved.

Preparation of 2 % CTAB buffer.

This is the buffer which was used for the extraction of entire genomic Deoxyribonucleic acid. To fix this buffer 600ml distilled H2O was taken in a beaker and maintain on magnetic scaremonger. To this H2O 20 gm CTAB, 10 gm PVP, 83 gm Na Cl, 40 milliliter 0.5M EDTA ( pH8 ) and 100 milliliters Tris ( pH8 ) were added and mixed by magnetic stirring. Then this buffer was autoclaved before the farther usage.

Preparation of TE buffer

TE buffer has been used for the concluding disintegration of the extracted DNA. This buffer was prepared by utilizing antecedently prepared and autoclaved tris and EDTA solutions. In order to fix 100 ml TE buffer 90 milliliter two-base hit distilled autoclaved H2O was taken in a glass cylinder and to this 1 milliliter I M tris solution and 0.2 milliliter 0.5 M EDTA solutions were added and volume was made up with dual distilled autoclaved H2O to 100 milliliters.

Seeds and Assortments:

For this research work seeds of the selected 10 assortments were gotten from Wheat Research Institute, Ayub Agriculture Research Institute, Jhang Road, Faisalabad. Name callings and the sequence with which these assortments have been given and discussed subsequently on is the followers:

1- 2192

2- 0031034

3- Inquilab-91

4- Uqab

5- Sehar

6- Shafaq

7- Kohistan

8- 4112

9- Bhakkar

Seed Sterilization and Plant growing

Seeds of these selected wheat assortments were surface sterilized by soaking in 15 % bleach for 20 proceedingss, washed thrice with sterilised H2O and placed on sterilised cotton in the glass home base for a hebdomad and so the healthy seedlings were transferred to the dirt in the pots for the farther growing for a twosome of hebdomads and so their foliages were used for DNA extraction.

Isolation of Genomic DNA from Wheat Plants

Entire works DNA was isolated from immature wheat foliages 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 agitating H2O bath. Young leaves ( 1gram ) were excised from the workss and froze in liquid N. Then these foliages were land into all right pulverization with stamp and howitzers in liquid N. This pulverization was transferred to 50ml falcon tubing and suspended in 15ml hot extraction buffer and placed in H2O bath for 30 proceedingss at 65-700C with soft shacking. After 30 proceedingss 15ml trichloromethane: isoamyl intoxicant ( 24:1 ) was added, assorted gently by inverting the tubings and centrifuged 10 proceedingss at 9000rpm at room temperature. Transparent supernatant was transferred to a fresh 50ml. To this supernatant 0.6 volume isopropyl alcohol was added, the contents were assorted by inverting the tubes several times and incubated them at -200C for 30 proceedingss. Then the tubings were centrifuged 10 proceedingss at 5000rpm, supernatant was discarded, pellet was washed with 70 % ethyl alcohol, air dried and dissolved in 1ml TE ( 10mM Tris and 1mM, EDTA ) . To acquire rid of RNA 2ul RNAase ( 5mg/ul ) was added to each tubing and incubated at 370C for one hr. After one hr to each tubing 100ul 3M Na ethanoate pH 5.2 and 2.5ml absolute ethyl alcohol were added. The contents were assorted, incubated at -200C for 30 proceedingss, centrifuged at5 proceedingss at 12000rpm, supernatant was discarded, pellet was air dried and dissolved in 1ml TE. Quality and concentration of the Deoxyribonucleic acid was found by fractionating it on 1 % agarose gel and its concentration was found out by taking OD260.

Polymerase concatenation reaction ( PCR ) .

All the chemicals used in this survey were of molecular biological science class.

Optimization of concentration of ingredients

Concentrations of the chemical ingredients for polymerase concatenation reaction were optimized by transporting out the reaction utilizing assorted concentrations of these ingredients. Finally following composing of the 20 ul reaction mixture was optimized and used for this survey.

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

Entire 50ul

PCR profile was optimized as follows:

One rhythm

Denaturation at 950C 5 proceedingss

Forty rhythms

Denaturation at 950C for one minute,

Annealing at 500C for one minute

Extension at 720C for one minute

One rhythm

Extension of 10 proceedingss at 720C.

Keep at 220C

Analysis of the PCR Products ( Gel Electrophoresis )

For the analysis of the PCR merchandise 1 % agarose gel was prepared in 0.5X TAE buffer. In a clean glass flask 100 milliliter 0.5X TAE buffer was taken and 1 gm agarose was added to this and heated in an oven boulder clay boiling and to this homogenous solution 10ug ethidium bromide was added and poured in a tray holding specific combs and allowed it to settle till the hardening at room temperature. Then the gel was placed in the buffer and DNA was loaded in the Wellss after blending with 6X gel lading buffer. Then the gel cataphoresis system was connected with the power supply and fractional process of the PCR merchandises was performed at 100 Vs for 40 proceedingss and so the consequences were observed in UV visible radiation and recorded in the computerized gel certification system.

Chapter 4


Quality and Quantity Analysis

Before running the PCR reaction the Deoxyribonucleic acid was analyzed for its quality trial. DNA quality was checked by fixing 0.8 % agarose gel carry 10ug/ml ethydium bormide. To this gel 1 ul of the eventually dissolved DNA sample was taken in a microfuge tubing. This sample was so dilute with TE buffer to 10 ul. Then 2 ul 6X gel lading buffer was added and assorted and whole stuff was loaded to the gel and electrophoresed at low electromotive force. From this gel the DNA quality was analyzed by detecting it under the UV visible radiation and exposure was taken for the consequence digest. This is shown in fgure 1 which shows and confirms fine DNA extraction. Deoxyribonucleic acid of all the eight varieities is of good quality with crisp distinct sets and no debasement was seen. This gel exposure besides showed that the solution is extremely concentrated and it was diluted for the concluding usage in the PCR reaction. From the gel exposure it was concluded that the concentration of the DNA is same in all the samples and it is about 25ng/ul Deoxyribonucleic acid for each assortment. PCR analysis performed on the footing of this information of the DNA concentration indicated that this appraisal was right.

PCR Analysis

Harmonizing to the method described in Materials and Methods PCR reaction was performed and the consequences are shown in figure 2. For the PCR reaction optimal conditions and optimal concentrations of the ingredients were determined by utilizing running PCR at different tempering temperatures and utilizing different concentrations of the templet and other ingredients. The PCR reaction was performed with the optimal measures as are given in the stuffs and methods. These consequences show that the assortments show elaboration of the Xwgm-SSR marker.

Chapter 5


So this research has found that Blue Silver, Era, Bakhtawar, Perula and Fareed 2000 wheat assortments are holding Xgwm-16 SSR marker and likely the linked Yr 5 cistron every bit good.

This is interesting and valuable information for the scientist who are involved in bring forthing new wheat assortments by engendering. Harnessing the latest techniques and engineerings is inevitable in order to run into the demands and demands of of all time increasing population of the universe. One of the best applications of the molecular biological science is to familial characters of the best assortments of different harvests and brings best traits together in the best assortments.

In order to convey immune cistrons in a certain assortment it is necessary to cognize which rust opposition cistron are carried by a peculiar wheat assortment. This can be done by working molecular markers of the rust opposition cistrons. Different types of the molecular markers can be used for this intent observing the presence of the linked rust opposition cistron in a assortment and for analyzing the diverseness among the different wheat assortments. Simple sequence repetitions ( SSR ) markers are the most suited for such intent.

More than 30 different xanthous rust opposition cistrons have been detected and SSR markers linked to many of these cistrons have been discovered. Primers of many of these markers have been published in the literature and there are legion studies in celebrated scientific diaries of profiting from this information. This research work is besides such a little enterprise.


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