In this article we will discuss about the eight main steps that are involved in gene tagging and mapping.
Step # 1. Selection of Target Traits:
This is the first step. Traits can be qualitatively or quantitatively inherited. Qualitatively inherited traits are controlled by one or few genes which have major effect on a particular trait and follow a typical Mendelian segregation. They are not influenced by environment and genetic background.
Examples include nematode resistance in tomato, TYLCV resistance in tomato, gall midge resistance and bacterial leaf blight resistance in rice. Quantitatively inherited traits are controlled by many genes/loci. Each locus has a small effect on the trait and cumulative effect of alleles at all loci controlling the trait determines the trait expression.
These traits show a continuous variation in segregating populations and are highly influenced by the environment and the genetic background. They are difficult to tag and map. Examples include several characters of economic importance like yield, drought tolerance, quality, etc.
Step # 2. Identification of Parents Differing in the Trait of Interest:
For success of gene tagging, one needs at-least two parents differing for the alternative forms of the trait of the interest. For example, for TYLCV gene tagging and mapping, there should be two parental lines one having gene for resistance to TYLCV and the other having gene for susceptibility for TYLCV.
For quantitatively inherited traits, a single cultivar can be selected as donor line with two or more recipient lines, which should essentially not possess the target trait for example drought tolerance.
Step # 3. Development of Appropriate Population Segregating for the Trait of Interest:
In case of quantitatively inherited traits, the early generation segregating populations like F2, F3, BC1F1 can be used. Geneticists also prefer to use advanced generation materials like F6, F7, recombinant inbred lines (RILs), near isogenic lines (NILs), or double haploid lines (DHLs) since they are homozygous for all the loci analyzed. RILs are usually, at F8/F9 generation onwards.
They are developed through single seed descent method or pedigree method. NILs are developed by repeated backcrossing of the F1S, BC1F1s, etc., with the recipient line also called as recurrent parent while simultaneously selecting for the trait of interest at each backcross generation. NILs will have only one or few genomic regions from the donor line and rest of the genome will be identical to the recipient/recurrent parent.
DHLs are a special class of population which are identical to RILs in their genetic constitution. DHLs are obtained by microspore culture of F1 anthers which give rise to haploid plants followed by induced doubling of chromosomes of haploid plants to yield double haploids. For simply inherited traits, F2 population size is 150-300 plants.
The schematic illustration given below, depicts the possible Ways of developing different kinds of mapping populations. For mapping quantitatively inherited traits, advanced generation materials like NILs, RILs and DHLs are the most appropriate.
Breeders/geneticists also use a strategy called AB-QTL (advanced backcross-QTL) strategy wherein through backcrossing the population is developed and simultaneously phenotyped to identify co-segregating markers.
Step # 4. Screening the Population for the Target Trait (Phenotyping):
The method of phenotyping differs significantly between qualitatively and quantitatively inherited traits. For most of the qualitatively inherited traits like pest and disease resistance, phenotyping involves exposing the individuals of the population to a particular biotype/pathotype of the pest/disease and scoring the plants for resistance/susceptibility after a particular time interval.
Adequate care should be taken while phenotyping since the success of tagging and mapping efforts depends mainly on precise phenotyping. For quantitative traits, the process of phenotyping involves analysis of individual component characters that contribute towards the overall expression of the target trait.
For example when tagging and mapping QTLs for yield, it is necessary to phenotype individual components of yield. Performing the experiment in replicated multi-location trials helps to avoid the uncertainties induced by the environment.
Step # 5. Parental Polymorphism Survey with Markers and Identification of Markers that Co-segregate with Gene(s) of Interest in the Individuals Constituting the Population:
After developing the population and phenotyping, the next step is to identify markers that co-segregate with trait of interest. This requires analysing the polymorphism among the parental lines with molecular markers.
Usually, if mapped and co-dominant markers like SSRs are used, it is necessary to scan the parental lines with a set of uniformly spaces SSR markers (12-16 per chromosome) and identify at-least 6-8 polymorphic markers per chromosome.
Care should also be taken to ensure that the polymorphic markers on a chromosome are uniformly distributed. Just to give an example more than 20,000 SSR markers spread evenly across the rice genome are available and it is hoped that the process of tagging and mapping of agronomically important genes will become much easier due to the availability of a large number of markers and the time taken is also expected to reduce significantly. If dominant markers like RAPD and ISSRs are selected for the study, then the parental polymorphism survey should be done with as much markers as possible.
Once a set of markers polymorphic between the parental lines has been identified, the next step is to carry out co-segregation analysis for these markers. A simple strategy called ‘bulked- segregant analysis’ can be used to quickly identify markers, which co-segregate with trait of interest.
For example, A set of resistant and susceptible F2 lines (usually 10-15 lines in each case) are bulked separately and analysed with parental polymorphic markers. If a fragment (hereafter called as marker) is present in the resistant donor, absent in the susceptible recipient, present in the resistant bulk and absent in the susceptible bulk, then the marker is most probably associated with resistance.
The marker is then analysed individually in all the lines constituting the resistant and susceptible bulks. If the marker is present in a majority (>70%) of individuals constituting resistant bulk and absent in a majority of individuals constituting the susceptible bulk, then it can be assumed that the marker is linked to the resistance.
The next step is to perform co-segregation analysis with all the individuals constituting the population and then determine linkage distances based on the extent of resistant individuals showing amplification of the resistance linked marker. In a similar way, markers co-segregating with susceptibility can also be identified.
Once a fragment/band (marker) is observed to co-segregate with resistance or susceptibility in the individuals of the mapping population, then the next step is to undertake co-segregation analysis in all the individuals constituting the population, (i.e. to check for presence of the marker in the resistant or susceptible individuals of the population).
Once a marker is confirmed to clearly co-segregate with trait phenotype in a majority of individuals of the population, the next step is to identify its chromosomal location.
Thus, if an SSR marker is identified to tightly co- segregate with trait phenotype in a population, then the tentative chromosomal location of the gene controlling the trait can be easily identified and more SSR markers in the vicinity of the co-segregating marker can be used for locating the exact position of the gene.
Step # 6. Construction of Linkage Map:
Linkage maps are basically, a kind of “road map” of the chromosomes drawn based on segregation pattern of markers. They indicate the position and relative genetic distances between markers along chromosomes, which is quite analogous to signs or landmarks along a highway. A clear co-segregation data is required to draw linkage maps.
Based on the co-segregation patterns, percentage recombination is calculated for each pair of markers in terms of centimorgans (cM), which is the unit for linkage distance. Statistical software’s like ‘Mapmaker’, ‘Map manager’, ‘Join Map’, ‘Cartographer’ and ‘Linkage’ can be used for construction of linkage maps. The co-segregation data has to be fed to the computer to excel format and the software automatically, constructs linkage map after calculating ‘LOD’ score for each pair of markers.
For constructing linkage maps for quantitative traits, usually marker intervals showing association with trait phenotype are identified and based on the extent to which these intervals are showing association, linkage distances are calculated using the software ‘Mapmaker-QTL’ after taking into consideration the LOD scores.
However, reports of QTL mapping have tended to be based on individual small to moderately sized mapping populations screened with a relatively small number of markers providing relatively low resolution of marker-trait association. Very few of the QTLs reported have been used for MAS in plant breeding.
Step # 7. Test for Reliability of the Identified Markers in Predicting the Trait in Alternate Population (Marker Validation):
Once a marker or sets of markers are identified to be tightly linked to a particular gene, the next step is to validate the markers and their linkage distances in alternate populations.
Alternate populations can be developed by selecting another donor line possessing the same resistance gene and crossing it with a susceptible parent. Before deploying markers in practical breeding, it is always ideal to validate the same in 1-2 alternate populations.
Step # 8. Utilisation of the Marker in Breeding Programmes:
Once closely linked markers (which are <2 cM from gene of interest) and/or flanking markers (which are <5 cM on either side of the gene) are identified and validated in alternate populations, they are ready for use in marker assisted breeding programmes. Flanking markers have distinct advantage compared to single marker since selection based on flanking markers will eliminate all false positives.
For e.g. if a single marker is ~2 cM away from the target gene ~2 out of 100 segregating plants will be false positives. But if flanking markers which are ~5 cM apart from the target gene their combined recombinational values would be minimum (since their combined recombination will be 5/100 x 5/100 = 25/10000 = 0.0025 cM).
Finally, when a marker or a set of markers are to be deployed in practical breeding, it is necessary to verify whether they are polymorphic between the parental lines used in the particular breeding programme. If the markers are polymorphic, then they can be reliably deployed to track the introgression of the gene in breeding programmes.
It should be realised that the genetic basis of complex traits and the interaction between all related traits will become much better understood. This will allow accurate modeling of gent networks and development of robust simulation tools for designing target genomic ideotypes. With the availability of such knowledge and tools, early stages of plant breeding programmes will become much more efficient in a design-led way.
However, there will continue to be no substitute for multi-locational replicated evaluation trials for screening elite breeding lines for selection and validation of finished products before distribution to local breeding institutions, companies and farmer’s fields.