Marker assisted selection

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  • (Genotyping means using laboratory methods to determine the sequence of nucleotides in the DNA from an individual, usually at one particular gene or piece of a gene.)

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  • 1. DEPARTMENT OF ANIMAL BREEDING & GENETICS (MAS) MARKER ASSISTED SELECTION SUBMITTED BY : SUNIL KUMAR BHAKAR
  • 2. OVERVIEW  DEFINITION  MARKER TYPES  PROPERTY OF IDEAL MARKER FOR MAS  POSITIVE AND NEGATIVE SELECTABLE MARKERS  GENE VS MARKER  RELATION B/W MARKER AND ASSOCIATED GENE  STEPS FOR MAS  FACTOR FOR SUCCESS  MAS WORK BEST FOR TRAITS  HIGH THROUGHPUT GENOTYPING TECHNIQUES  CONCLUSION
  • 3. What is MAS  Marker assisted selection or marker aided selection (MAS) is a process whereby a marker (morphological, biochemical molecular etc.) is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g. productivity, disease resistance, abiotic stress tolerance etc..).  This process is used in plant and animal breeding.
  • 4. POSITIVE AND NEGATIVE SELECTABLE MARKER  Positive selectable markers are selectable markers that confers selective advantage to its host organism. An example would be antibiotics resistance, which allows the host organism to survive antibiotics selection.  Negative selectable markers are selectable markers that would eliminate its host organism upon selection. An example would be thymidine kinase, which would make the host sensitive to ganciclovir selection. MARKER TYPES
  • 5. Biological markers Different pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites.
  • 6. Morphological markers  Animals are selected based on appearance.  Disadvantage: lack of polymorphism
  • 7. Biochemical markers Animal are selected on the basis of biochemical properties. Eg. Hb, AMYLASE, BLOOD GROUPS ETC. Disadvantage: Sex limited Age dependent Influenced by environment It covers less than 10% of genome
  • 8. Cytological markers The chromosomal banding produced by different stains; for example, G banding. structural and numerical variations are identified. Structural- Deletions, Insertions etc. Numerical- Trisomy , Monosomy , Nullysomy Disadvantage : low polymorphism
  • 9. DNA-based / molecular  To avoid problems specific to morphological markers, the DNA-based markers have been developed.  A unique (DNA sequence), occurring in proximity to the gene or locus of interest, can be identified by a range of molecular techniques.  Three common technologies used as molecular markers are: RFLP, SSR (microsatellites) and SNP .  ADVANTASES 1. Highly polymorphic. 2. Simple inheritance. 3. Abundantly occur throughout the genome. 4. Easy and fast to detect, 5. Minimum pleiotropic effect. 6. Detection is not dependent on the developmental stage of the organism.
  • 10. Important Properties of Ideal Markers for MAS  Easy recognition of all possible phenotypes(homo- and heterozygote's) from all different alleles.  Demonstrates measurable differences in expression between trait types and/or gene of interest alleles, early in the development of the organism.  Low or null interaction among the markers allowing the use of many at the same time in a segregating population.  Abundant in number.  Polymorphic.
  • 11. Gene Vs Marker  The gene of interest is directly related with production of protein(s) that produce certain phenotypes whereas markers not influence the trait of interest but are genetically linked .  In many traits, genes are discovered and can be directly assayed for their presence with a high level of confidence. However, if a gene is not isolated, markers help is taken to tag a gene of interest. In such case there may be some inaccurate (even false) positive results due to recombination between the marker of interest and gene (or QTL). A perfect marker would elicit no false positive results.
  • 12. Relationships b/w markers and respective gene Three kinds of relationships between the markers and respective genes could be distinguished : (1) The molecular marker located within the gene of interest, which is the most favourable situation for MAS and in this case, it could be ideally referred to as gene-assisted selection. Such markers are called as direct marker. (2) The marker in linkage disequilibrium (LD) with the gene of interest throughout the population. LD is the tendency of certain combination of alleles to be inherited together. Population-wide LD can be found when markers and genes of interest are physically close to each other. Selection using these markers can be called as LD-MAS. (3) The marker in linkage equilibrium (LE) with the gene of interest throughout the population, which is the most difficult and challenging situation for applying MAS.
  • 13. Cont…  Direct markers are preferred for effective implementation of marker-assisted selection, followed by LD and LE markers, the latter requiring within-family analysis and selection.  Ease of application and potential for extra-genetic gain is greatest for direct markers, followed by LD markers, but is antagonistic to ease of detection, which is greatest for LE markers.
  • 14. Steps for MAS  Generally the first step is to map the gene or quantitative trait locus (QTL) of interest by using different techniques and then use this information for marker assisted selection.  Generally, the markers to be used should be close to gene of interest (<5 recombination unit or cM) in order to ensure that only minor fraction of the selected individuals will be recombinants.  Generally, not only a single marker but rather two markers are used in order to reduce the chances of an error due to homologous recombination. For example, if two flanking markers are used at same time with an interval between them of approximately 20cM, there is higher probability (99%) for recovery of the target gene.
  • 15. CONT….  Three common technologies used as molecular markers are: restriction fragment length polymorphisms, simple sequence repeats, and single nucleotide polymorphisms.  Genetic variants often differ from each other by the sequence of a single nucleotide so single nucleotide polymorphisms SNPs (pronounced “snips”),is commonly the basis of genotyping tests.  It is therefore possible to genotype an animal using a DNA- based genotyping test and select individuals carrying the preferred genetic variant.
  • 16. Factor for success of MAS In general , the success of MAS depends on following factor :- (i) Availability of genetic map with an adequate number of uniformly-spaced polymorphic markers to accurately locate desired QTLs or major gene(s). (ii) Close linkage between the QTL or a major gene of interest and adjacent markers. (iii) Ideally markers should be <5 cM from a gene or QTL Marker A QTL5 cM RELIABILITY FOR SELECTION Using marker A only: 1 – rA = ~95% Marker A QTL Marker B 5 cM 5 cM Using markers A and B: 1 - 2 rArB = ~99.5%
  • 17. Cont… Note- Using a pair of flanking markers can greatly improve reliability but increases time and cost. (iv) Adequate recombination between the markers and rest of the genome. (v) Ability to analyze a larger number of Animals in a time and cost effective manner.
  • 18. Fundament Advantages of MAS 1. Greater efficiency. 2. Accelerated line development in breeding programs. For example, time and labour savings may arise from the substitution of difficult or time-consuming field trials (that need to be conducted at particular times of year or at specific locations, or are technically complicated) with DNA marker tests. 3 .Selection based on DNA markers may be more reliable due to the influence of environmental factors on field trials.
  • 19. CONT… 4. Another benefit from using MAS is that the total number of lines that need to be tested may be reduced considerably. 5. Since many lines can be discarded after MAS at an early generation, this permits a more effective breeding design. 6. Greater efficiency of target trait selection may enable certain traits to be fast-tracked, thus specific genotypes can be easily identified and selected.
  • 20. MAS work best for traits  1. Low h2  2. Are difficult or expensive to measure. e.g. disease resistance.  3. Can not be measured untill after selection has occurred e.g. Carcass data.  4 .Are currently not selected for due to lack of available phenotypic data. e.g.(tenderness).
  • 21. Traits most likely to benefit for MAS (descending order ). 1. Disease resistance . 2. Carcass quality and palatability attributes. 3. Fertility and reproductive efficiency. 4. Maintenance requirements. 5. Carcass quantity and yields. 6. Milk production and maternal ability. 7. Growth performance.
  • 22. High throughput genotyping techniques  Recently high-throughput genotyping techniques are developed which allows marker aided screening of many genotypes. This will help breeders in shifting traditional breeding to marker aided selection. One of example of such automation is using DNA isolation robots, capillary electrophoresis and pipetting robots.  One of recent example of capillary system is Applied Bio systems 3130 Genetic Analyser.
  • 23. CONCLUSIONS  In context of MAS, markers can be effectively utilized for two basic purposes :- (i) tracing favourable allele(s) (dominant or recessive) across generations. (ii) Identifying the most suitable individual(s) among the segregating progeny, based on allelic composition across a part or the entire genome.  It is important to combine DNA results (which look at single genes) with other criteria, such as EPDs (which look at numerous genes) and the animal’s actual phenotype for the trait (if available), to ensure that selection is distributed over all the genes that contribute towards the trait of interest.
  • 24. CONT….  Don't ignore animals that have good EPDs for a given trait and yet are not carrying the favorable form of a gene for that trait. These animals are likely to be a source of good alleles for the many other genes that contribute towards that trait.  Ideally the information from genetic tests should be integrated into a genetic evaluation system that weighs all the information about an animal. Combining information from both EPDs and genetic tests into a selection decision will be superior to selection on either EPDs or markers alone.