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Gene Inheritance, Mapping and Complementation

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So, let us assume that we have identified or we have isolated a particular mutant whichhas some developmental defect or some phenotype which is of our interest then how to moveahead.So, the next step what we do or what we do is that we first analyze the nature of mutants.What kind of mutants it is, what is its inheritance pattern?Because we are using genetics based approach.So, it is very important for us to know whether the mutation is heritable in nature or notthat is the first question.There are some mutations they if they are in the germ line they can be inherited tothe next generation, but if it is in the somatic tissues if they are the somatic mutationsthey will disappear in the next generation.But for us to understand more in the deTAIL to establish the proper mutants it is importantto have heritable mutants.The first thing and then second thing what we do?We try to identify, if it is heritable what is its pattern of inheritance, does it followMendelians law?So, you would have studied in your basic course of genetics that Gregor Johann Mendel hasgiven three laws of inheritance how a particular genes or genetic mutants are getting inheritedin the next generation.So, if the mutants are heritable in a fashion which can be justified or which can be explainedby the process of Mendel’s or by the Mendelian law or if they are not following then thatkind of inheritance is non Mendelian inheritance.We will be interested in having Mendelian way of inheritance which makes our life easyto study the plant developmental biology.But if it is heritable and let us assume that it is following the pattern of Mendel’sheritance, then we look and we analyze what is the pattern of segregation or what is thepattern of heritance, is the mutant dominant in nature semi dominant in nature or recessivein nature; first.Next things: if you remember the last class or if you recall the last class, what we havefound that in this technique there is a possibility to have multisite mutation.So, we will also like to know whether the mutation or the phenotype is because of asingle mutation or it is because of a higher order mutation let us assume the double mutation.Double mutation means there is a possibility that there are two mutants in two independentgenes or and the result of these two mutants might give the phenotype.And if this is the case then what happens, how they segregate, do they segregate independently?So, there is a first possibility where there is a two mutation but both the mutant or boththe locus; both the loci are segregating independently or they segregate together; co-segregation.So, first thing we would like to rule out all this possibility with our interest ormutant of interest.So, for first thing what we do as I said that heritable or non heritable.The mutants if mutant is heritable, then if you collect the seeds from the mutants andgrow to the next generation, in next generation you will get the phenotype, but if your phenotypeis disappearing from the next generation then your mutation are is non heritable.Another important thing if your mutation is inherited in Mendelian fashion, then whatare you expecting?You are expecting a result depending on the fact what is your initial mutation, if yourinitial mutation is let us assume heterozygous.Heterozygous means for every chromosome we have a pair of chromosome pair of homologouschromosome.So, if let us assume this is a gene where there is a mutation here.So, if mutation is in only one allele or one loci or in the one homologous chromosome andmutation is not present in the corresponding locus of another homologous chromosome thenthis condition is called heterozygous.In homozygous condition what happens that you can expect that mutation is present atboth the allele, here as well as here, here there is no mutation.So, if your mutation is heterozygous, then you can go to the next generation and lookthe segregation pattern and it depends on what is the nature of your mutation, if itis a dominant then you expect a particular ratio, if it is semi dominant then you expectanother genotypic and phenotypic ratio and if it is recessive then you expect a differentpattern of ratio, what would be those ratio we will see to the next slide.But another possibility is that if it does not follow the Mendelian pattern of inheritance,what is the pattern of inheritance?There are a lot of cases which has been found naturally as well as induced way which arenot following the Mendel’s pattern of inheritance.For example, if the dominance is not complete or incomplete dominance or if there is a epigeneticmutations which is not getting transmitted; which is not inherited to the next generationor the loci which are sex linked traits because sex linked there is a difference of the homozygosityis not fully maintained because one chromosome is x and other chromosome is y ok.So, let us assume that the mutant or we will be more interested or we will be more happyto have a mutant which is following the Mendelian pattern of inheritance.If it is following the Mendelian pattern of inheritance, then what could be the natureof mutants?So, first thing is that if your mutant is dominant and if you have a heterozygous locifor the mutants you can expect that you can expect a phenotype even in the heterozygouscondition, but if your mutation is recessive in nature then the just it disruption of oneloci is not sufficient to give the phenotype.In this case you have to have mutation in both the genetic loci and if this is the casethen you will see the phenotype.So, your phenotype will be only if you have mutation in both the homologous chromosomeand this is the condition called homozygous condition.So, what happens?So, how to study this?So, you take this plant mutant plant and go to the next generation.In next generation let us assume your mutant plant was heterozygous.And if it is heterozygous if your mute if mutation is dominant it will already havea phenotype, but if it is recessive then your mutant plant will not have phenotype, butif you go to the next generation you are going to get wild type heterozygous and homozygouspopulation in the ratio of 1 is to 2 is to 1 and if as I said if it is dominant thenyou expect these 3 to be the mutant.So, your phenotypic ratio is going to be 3 mutants and 1 wild type, but if your mutationis recessive in nature then only this homozygous which is like 25 percent of the total populationis going to have the mutant phenotype.So, if you look the segregation pattern and if you quantify the number of mutants in thenext generation you can tell whether your mutant is dominant or recessive in nature.Another condition what happens is the semi dominant.So, in semi dominant mutant condition what happens that you have wild type, heterozygousand homozygous.Now, the heterozygous and homozygous in case of dominant mutant purely dominant mutantthey have a same phenotype.In case of recessive mutants only homozygous will have a phenotype, but in case of semidominant mutants the heterozygous and homozygous both will have a phenotype, but the severityof the phenotype will be more in case a homozygous than the heterozygous.So, you have a range of phenotype in this case.Another case how to rule out: let us assume that your original plant or the mutant plantis homozygous.If it is homozygous which means that both the loci is mutated.If both the loci are mutated, then next generation if you go to the next generation all the plantsare going to give the phenotype; all mutants.Here it is very difficult to tell what is the nature of your mutants, whether it isdominant or recessive it does not matter because this both the loci are mutated, so they willnot segregate.So, to rule out the possibility you will have to make a back cross to generate heterozygous.So, you will take this homozygous mutant plant cross with the wild type plant and generatea F 1 which will have only one locus mutated another locus as a wild type.This is heterozygous plant and then take this heterozygous plant for the next generationand look the phenotype.If it is dominant, if your mutation is dominant you expect 3 is to 1, 3 mutant 1 wild type,if it is recessive then you expect same ratio 3 is to 1, but here mutant will be 1 and dominantmutant will be 3, but if it is a semi dominant case you are expecting 1 is to 2 is to 1.So, 1 will be pure wild type absolutely normal, 2 will have some phenotype, but 1 fractionthis is going to be more severe than this one.So, essentially this both population will have the phenotype, but severity will be different.So, look here this is a case of semi dominant mutants.So, this is wild type and this is homozygous.If you compare wild type and homozygous look the phenotypic severity this plant is verysmall, the leaves are very small shape are very defect, but if you look the F1 populationwhich is basically heterozygous; heterozygous is the severity of phenotype is intermediate.So, it is less than the wild type, the growth is less than the wild type, but more thanthe mutant.So, this is a kind of semi dominant mutant phenotype.So, next thing what we would analyze with this mutant is whether mutation is singlesite mutation or multi site mutation.How we can do that?We can do this by simple doing the Mendelian cross.So, let us assume that you have a two mutations; one is a and another mutants is b in geneb and you will have wild type and then you have mutant.Let us assume the phenotype is because of the double mutant phenotype and you if youmake this kind of cross go to the F1 generation and look the phenotype.So, this is wild type this is a mutant one and go and look the F1 plants, this will tellan idea whether your mutant is dominant or recessive.If your F1 plants they already have the phenotype which means that your mutants are dominant,if your F1 plant do not display any phenotype which means your mutants are recessive.And then you go and make a cross go to the F2 generation and now it depends on what isthe segregation factor pattern of both the loci.If the segregation of both the loci are totally independent and if mutation is because ofthe disruption of both the loci because of the mutation in the both the loci, then youare expecting a typical ratio 3; 9 is to 3 is to 3 is to 1, the phenotypic ratio whichwe always get when you make a dihybrid cross; Mendelian dihybrid cross.And in this case this population the 1 this is the population which will be homo doublehomozygous; double homozygous for the mutant.So, this will be a small a small; a small b; small b.So, if we get 1 out of 16 as a mutant in F2 generation, this tells that your this is aresult of double mutant both the mutants are recessive in nature and they are not co segregatingthey are basically segregating independently that is why we have got this particular ratio.But if they are genetically linked, if they are co segregating the ratio will be totallychanged depending on the strength of linkage ok.So, this is the preliminary genetic analysis one can do just after identify the mutants.Then the next step will be that you have identified the mutant, you know what is the pattern ofits segregation, now the next important thing is to identify what are the genetic loci associatedwith this mutants which means that mapping the mutations mapping the gene.How to do that?So, there are two approach depending on what is that what type of mutants you have generated,if you have generated point mutation for example, by EMS then we normally take map based cloningbased approaches and if you have generated mutation through the insertional mutationusing T-DNA or transposon, then you can do gene mapping by gene tagging.So, now we will take the first approach map based cloning.So, this is done by some molecular marker.So, this is very important for you to understand and what happens here we do by chromosomewalking.So, there are certain things, so there are molecular markers already reported or peoplehave identified molecular markers, what are molecular markers may be we will see to thenext slide, but this can be either PCR based marker or restriction digestion based markers.For quick analysis we prefer PCR based marker because this is very fast and then what wedo, we try to analyze linkage and segregation pattern between these marker, molecular markersand the phenotype ok.One very important marker which is preferably being used are INDEL markers.These markers are produced at the site of insertion and deletion.So, basically in markers you can have two kinds of marker; one is called again dominantmarkers and another are co-dominant markers.So, co-dominant marker; so in case of dominant marker what happens that this marker willgive a pattern in one particular genetic background and it will not give a band in another geneticbackground.For example, if you are using PCR based, but in co dominant in both the condition or boththe genetic background, it will give a band, but the size of bands will be different.So, for our use or for most important thing co dominant markers are very good markersbecause in case of dominant marker when you see the band you are sure that this is present,but if you do not see the band you are not 100 percent sure that absence of band is actuallybecause this is a marker, it could be simply a technical problem of the PCR.So, this that is why in co dominant markers you see both the bands in both the condition,but based on this different size you can clearly tell this is this genetic background, thisis that genetic background ok.So, for example, in most of the plants these markers has been well identified, well establishedand now people know if you take some example of Arabidopsis thaliana model plants.So, this is chromosome number III of Arabidopsis thaliana these are different varieties ofArabidopsis Columbia, Landsberg all this kind of C 24.So, different variety and what people have tried to identify, what are the markers?Markers basically are the genetic elements which is different in two variety.So, for example, if you look this is Columbia, so Columbia has been taken as a referencebackground and if you look that LER, so, here if you look this is, so there is a differencehere.So, Columbia has a different difference in the genetic loci with the LER and you canchoose this region and if you design a specific primer for this region, then that primer canreally distinguish whether it is Columbia background or it is LER background and thatkind of markers you could use to identify whether a particular region of a chromosomeis from the Columbia background or from the LER background.These are some names of these markers.For example, if you look because of some insertion some deletions there is a difference, so thisis NGA59.So, this is the name of one marker in this case you have identified a region where thereis a difference.So, for example, in this region if you compare this and this is base pair.So, this is 110, this is 141, so difference is 30 nucleotide which means that in Columbiaeither there is a deletion of 30 nucleotides in this particular region or in Ws4 thereis this insertion of 30 nucleotide.So, if you look here, so what happen?If you take Columbia genome so, there is a region here and if you take correspondingWs4 genome here is a region, but there is a small insertion.So, if you design a primer here, if you design a primer here and if you say primer here andhere, what you are going to get from Columbia background?You are going to get this band size which is 111 and from this background you are goingto get this.So, this is 111 plus this 30 nucleotide insertion which is 141.So, if you take these and if you run the gel, what you can expect?Another important thing you can expect here.So, let us assume if a genetic loci is only Columbia, then if use this marker you aregoing to get 111 base pair band.If this loci is heterozygous one loci is Columbia and one loci is Ws and if you use here thenwhat are you going from the Columbia loci you are going to get a band of 111 from thisWs4 loci you are going to get a band of 41 and if you have a genetic loci which is forthis region which is homozygous for Ws4 and if use the same primer you are only goingto get a band of 141 nucleotide.So, if you run on the gell you can clearly see this kind of pattern, if it is homozygousfor Columbia then you are going to get a band of this size, if it is heterozygous for Columbiaand Ws4 you are going to get a band of this size and a band of this size.So, this is coming from one locus, this is coming from another locus, but if it is homozygousfor Ws4 then you are going to get only one band of 141 base pair.So, just looking this banding pattern and looking this set of primers you can clearlydistinguish what is the genetic loci on the chromosome number 3 or somewhere here whereverthis marker is located.So, this marker for example, is located on the chromosome.So, this way the entire genetic map has been prepared for all the chromosomes and largenumber of molecular markers has been identified.If we have to map a gene, then what you can do?You can simply choose these genes and show association and look their segregation patternwith your mutant.So, what you do basically?Let us assume I have a mutant plant which is in Columbia background and I have I knowthat this plant this mutant is recessive in nature, it follows Mendelian pattern of segregationwhich means that if I take heterozygous of this and go to the next generation in F2 generationI am expecting that 3 is to 1 ratio and 1 this ratio 25 percents plants are going tobe mutant.So, if I take this plant and mutant plant and cross with wild type, mutant is Columbiabackground if I take Ws4 background.And if I cross them what I am going to expect?In F1 generation I am generating heterozygous.Heterozygous means half set of genomes are coming from Columbia background another halfset of genomes are coming from Ws4 background this is heterozygous.And then if I allow them for selfing and go to the next generation there will be a lotof crossing over in some chromosome 1, some chromosome 2 and basically what it is doingthat there is merging of the genome.So, if you go to the next generation and go to the F2 generation the genomes of Columbiaand Ws4 will be totally merge and you may find a region different kind of possibilityyou can find a chromosome like this, you can find the chromosome like this.So, any possibility whatever you can imagine you can find here in the F2 generation, butwhat one thing we are doing that, then we take this plant.So, let us assume I have taken 100 plants from here from F2 population and here as well;so from this F2 population.So, the 25 percent of the plant which will be like let us assume approximately 25 plantsthey will be the mutant, they will show the phenotype.So, what it means that for this mutant since my mutation is coming from the Columbia backgroundand this mutation is totally absent in the Ws4 background which means that in these 25plants the locus where this mutation is it will be homozygous for Columbia.Why this will be homozygous for Columbia?Because it will if it will be homozygous then and only then it will have the phenotype.Since it has a phenotype it means that it is a homozygous; homozygous for the mutation.And since mutation is only coming from the Columbia which means that region where thereis a mutation will be only from the Columbia and there will be no region from the Ws inthat one.Now, if you take all these 25 mutant plants with the mutant phenotype and check for theirdifferent chromosome, let us assume there are 5 chromosomes typically in Arabidopsisand if I measure a chromosome here a marker here; a marker here; a marker here I do usethis PCR based molecular marker for different region.And let us assume that my site of mutation is somewhere here then what I am expecting?If I take this 25 plant, so my mutants are fixed here.So, this locus is homozygous for Columbia in these 25 plants I am not talking aboutremaining 75 plants there might be something different, but in this 25 plants which clearlyshows the phenotype this locus is homozygous for Columbia.Now, if I look; so in these 25 plants this will be this region will be Columbia, butall other region can be anything as per the Mendelian pattern.So, if I look this region or if I check this region in this 25 plant, what I am going toexpect?I am going to expect a pattern of this.So, out of these 25 plants 25 percent of this population will be wild type for Columbia;25 percent of this could be wild type for Ws and 50 percent of these can be heterozygousfor Columbia and Ws, this is a typical pattern Mendelian pattern here.So, what I do?I check different regions of the chromosome and if I found on the chromosome let us assumeon chromosome number 1 all three marker shows independent assortment with respect to thephenotype, if they are giving 1 is to 2 is to 1 ratio for these marker which means thatmy mutation is not present in this gene in this chromosome.Similarly, you rule out this chromosome; you rule out this chromosome; you rule out thischromosome; you rule out this chromosome.But once you will reach to this chromosome and if your mutation is here, if you lookhere a marker which is present here since this is far from the site of mutation thereis still possibility that there is a crossing over events here.But once you start moving this is called chromosome walking, if you check for here you might stillget 1 is to 2 is to 1 segregation for this marker in this 25 plants, but once you willcome closer to the site of mutation either from this direction or from this direction,what you are expecting.The wild type Ws start decreasing, then heterozygous or homozygous Columbia will start increasing.So, then there will be a deviation you cannot expect 1 is to 2 is to 1 and then if you comecloser; closer; closer and let us assume if you look this marker this might be presentvery close or very near to the sight of mutation.In all these 25 percent there is a possibility that it might be either only heterozygousor only homozygous for the Columbia and then if you come down; come down there will bea point where all the plants will have only Columbia genome.So, then you might get a condition where 100 percent of this 25 plants will have homozygousColumbia and they do not have any genotype for Ws this is called your mapping window.So, now, we do from this direction we keep on mapping and we found that here is a markerwhich is 100 percent Columbia, here is the marker which is 100 percent Columbia, nowthis is my window.Now, I know that the mutation is somewhere in this window.If you want to further go and fine tune this mapping, then you will have to increase thenumber of mutant plants this is not possible with 25 plant, you can go 1000 plants more,more.And then you can do the fine mapping and you can do, but these days since we are in thepost genomic era, most of the plant model plants their entire genome has been sequenced,now in the different varieties of those plants genomes has been given sequenced and now sequencingis no longer very costly or very time consuming process.So, we can take this mutant and go and sequence whole genome sequencing.And then why we have done this is called rough mapping, why we have done this rough mappingbecause this entire plant was exposed with the EMS.So, there is a possibility that there is a large number of point mutation in the genome,but we have to find out which mutation is actually responsible for the phenotype, allmutation might not be responsible for the phenotype.And then if we have already defined this window, then we can only look what are the mutationsin this window and then one by one you can rule out depending on your knowledge if somethingis in the inter in the intergenic region we can rule out.Similarly you can point out and you may find may be one point mutation or two point mutationin this gene which might have the relevant phenotype.Then we can take in the next step and validate that actually this mutant is responsible forthe phenotype.So, this is to map a point mutation whereas mapping T-DNA insertion site is relativelyeasy and it has been done with the several way.One way of doing it that this is called TAIL PCR.In TAIL PCR what you do?Since, this T-DNA region which you have engineered, so, you know the sequence, you can designsome primers for this T-DNA.For example, SP 1 primer, SP 2 primer, SP 3 primer and let us assume this is integratedin the genome and this might be the plant genome and then you can design an arbitraryprimers here.So, this primer basically this is not very specific this is more kind of random primers.So, it can bind in a very or you have a kind of pool of primers.So, you might find a primer which might bind here and this primer if you run first primaryPCR with SP 1 you can enrich this amplification, then you use another primer keep on enrichingand if you do a combination of primer you might amplify this region, the region fromhere to here.And then you take this PCR amplified products, clone in a vector how to do cloning you wouldhave studied somewhere, take this PCR fragment and clone in a cloning vector and now if youhave clone in a cloning vector this is your fragment, in fragment you have some regionof plant genomic DNA and some region of T-DNA.Now, you have some primer binding site here sequence from here and identify what nucleotidesequence is coming from the plant and then you can do, you can check this go to the websiteyou just try out what is the sequence and where this is positioned in the plant thisway you can map that what is the site of insertion.Another way of mapping is plasmid rescue.Here what you do?When you are engineering your T-DNA you put a origin of replication for bacteria.You know that if origin of replication is present, then this DNA if it is circular innature it can propagate in the bacteria it can replicate in the bacteria.Take this let us assume that this T-DNA is integrated in the plant genome use some enzymesite.So, let us, but ensure that this enzyme site is not cutting in the T-DNA.So, if you use a restriction site it might come here; here; here; here anywhere in theplant genome and then when it will digest use this fragment and you can re-ligate it.So, re ligation will generate a this kind of molecule where you will have T-DNA as wellas a fraction of plant genomic DNA.Since, you have origin of replication this is a kind of your plasmid you transform tothe bacteria E.coli bacteria and in bacteria it can propagate, extract the plasmid andsequence using T-DNA specific primer and then you can identify what are the sequences whichare just flanking the T-DNA and you can basically position or you can identify the locus ofintegration.Third way of doing identifying the site of insertion is inverse PCR.In inverse PCR what you do?Basically you choose a site restriction site which cut at least once in the T-DNA and thensomewhere in the genomic DNA.When you will digest it you know the sequence where it cut, you have designed a primer whichis specific to the T-DNA another primer which is specific to this region and then you re-ligateit; when you re-ligate it this will.So, you have a primer positioned here; you have a primer positioned here do the PCR.And this will amplify this region and clone this region in a particular cloning vectorsequence them and identify what are the flanking sequence in the T-DNA.In another technique which we are using to map the site of an insertion is adaptor PCR.Adaptor PCR is quite similar, but what we do that you digest with the restriction enzymeand then in the end what you do fill this end depending on what kind of enzyme you areusing are you using blunt end enzyme, sticky enzyme, but then additionally what you do?You add some adaptor molecules at the both the ends of this molecules.And when you add these adaptor molecules since you know the adapter sequence you can havea primer specific to the adaptor sequence, you know the T-DNA sequence, you can designanother set of primer and you can do the PCR amplification, take this fragment, clone andsequence.So, you can identify what sequence this is and depend based on this you can positionit in the genome; in the plant genome ok.As I said that this has been very extensively used and lot of transgenic lines has beengenerated in Arabidopsis it is deposited in the database.Now, simply based on your mutant phenotype or if you want to identify genes or if youknow the genes you want to identify the phenotype you just simply order those mutants and ifyou order the mutants you know that there is a mutation and you know that there is aT-DNA insertion, you can simply go and do the genotyping and validate that actuallythere is insertion.So, what you do for doing this?So, let us assume this is the gene of interest and you know the site of insertion becausethis is already established, sequenced, mapped, everything is done.Now, you design a primer in the gene, forward primer and reverse primer just outside theflanking region of the T-DNA and use another primer in the T-DNA and if you do the combinationof PCR.So, one PCR you do using gene specific forward and reverse primer and another PCR you dousing T-DNA specific primer.Either with this depending on the orientation or this with the, it depends on what orientationthis T-DNA got inserted.If T-DNA is inserted in this orientation then you would like to use this primer with thisto have amplification, if it is in opposite orientation in this orientation then yourprimer position will change and then you would like to check with this, but what is importanthere.Thus this band you are going to get only if your genomic copy is wild type and there isno insertion and this band you are going to get only if there is a T-DNA insertion youwill not get on the wild type genome.