MAPPING POPULATIONS

MAPPING POPULATIONS: for Linkage Mapping of
Genetic Markers

1. MAPPING POPULATIONS: for Linkage Mapping of Genetic Markers CREDIT SEMINAR Department of Plant Breeding & Genetics, RVSKVV, Gwalior RAJMATA VIJAYARAJE SCINDIA KRISHI VISHWA VIDYALAYA PRESENTED BY: SHIVANI UPADHYAY M.Sc. Ag. Final year

2. INTRODUCTION The demand being placed on food production globally require greater advances in genetic improvement of our food crops. A crucial step towards achieving these advances is the rapid identification of genetic elements that can be utilized in breeding food crops. The identification of genes underlying simply inherited traits has been very successful. Most traits of biological and economic interest in crop plants are of quantitative nature, displaying continuous variation and are under polygenic control and identification of such genes is difficult. The development of molecular marker technology has renewed interest in genetic mapping. RFLP was the first DNA marker to be developed and used in mapping experiment. The use of DNA markers has allowed construction of dense linkage maps in many important plant species and has enabled the mapping of the elusive quantitative trait loci (QTLs).

3. Genetic resources Molecular biology Crop improvement

4. Historical Aspects In 1910, Morgan provided the first experimental evidence for the chromosomal theory: he demonstrated that the inheritance pattern of white-eye gene of Drosophila indicated it to be located on the X chromosome. One year later, in 1911, Morgan described the essential features of linkage between genes. In the year 1913, Sturtevant published the first linkage map of Drosophila. Subsequently, morphological markers were used to construct linkage maps in many species. Geneticists mounted search for more abundant markers, and protein polymorphisms were the first molecular variations used to generate linkage maps. The limited number of protein polymorphisms and the environmental influence on their expression were the major drawbacks, which favored the development of DNA markers.

5. LINKAGE MAPPING Mapping is putting markers (and genes or QTL) in order, indicating the relative distances among them, and assigning them to their linkage groups on the basis of their recombination values from all pair wise combinations. A linkage map may be thought of as a ‘roadmap ’ of the chromosomes derived from two different parents (Paterson,1996a ) Linkage maps indicate the position and relative genetic distances between markers along chromosomes. Construction of linkage maps requires the following: (1) a suitable marker system, (2) an appropriate mapping population, and (3) software for proper analysis of the data.

6. The first step in mapping of markers is to select two genetically divergent parents expected to differ in lager number of markers and/or trait of interest. General Procedure for Linkage Mapping Of Molecular Markers The selected parents are crossed and a suitable mapping population is developed The parents are tested with large number of markers to identify polymorphic markers. All the individuals of the mapping population are screened with the polymorphic marker; this is called GENOTYPING . The marker genotype data are analyzed using a suitable software package to estimate recombination frequencies and genetic distances between marker pairs

7. Group the markers into linkage groups, select the most likely marker order, and prepare a marker linkage map. All individuals of the mapping population are evaluated for phenotypic expression of the trait, this is known as PHENOTYPING . The trait phenotype and marker genotype data are analyzed using a suitable computer program to identify the markers governing the target trait. Estimate the frequency of recombination between the gene and the markers and ultimately prepare a linkage map.

8. MAPPING POPULATIONS A POPULATION THAT IS SUITABLE FOR LINKAGE MAPPING OF GENETIC MARKERS IS KNOWN AS MAPPING POPULATION. Generated by crossing two or more genetically diverse lines and handling the progeny in a definite fashion. They are usually produced from controlled crosses. Mapping populations serve as basic tools needed for the identification of genomic regions harbouring genes/ QTLs and for estimating the effects of QTL.

9. Primary mapping populations are created by hybridization between two homozygous lines usually having contrasting forms for the traits of interes t. Secondary mapping populations on the other hand, are developed by crossing two lines/individuals selected from a mapping population; they are developed mainly for fine mapping of the genomic region of interest. Mortal mapping populations are short lived populations and each individual of the populations have their own unique identity. Immortal mapping populations are the population which can reproduce itself or can be reproduced without any change in genetic make up of constituent individuals. Bi-parental mapping populations are obtained by the crosses between two parents. Multi-parental mapping populations involve more than two parents. E.g. MAGIC. TYPES OF MAPPING POPULATIONS: PRIMARY AND SECONDARY MAPPING POPULATIONS. MORTAL AND IMMORTAL MAPPING POPULATIONS. BIPARENTAL AND MULTIPARENTAL MAPPING POPULATIONS.

10. Selection of parents for developing mapping populations The two lines selected as parents (P1) and (P2), should be completely homozygous. They should differ for as many qualitative and metric traits as possible. The parents should be polymorphic for as many molecular markers as possible to afford the construction of dense linkage map. The parents of mapping populations must have sufficient variation for the traits of interest at both the DNA sequence and the phenotype level.

11. F 2 POPULATION It consist of the progeny produced by selfing / sib mating of the F 1 individuals from a cross between the selected parents. Homozygous and heterogeneous population. Different plants in F 2 population differ with each other. Product of a single meiotic cycle i.e. one round of recombination. The ratios expected for dominant and co dominant markers are 3:1 and 1:2:1 respectively.

12. MERITS Appropriate population for preliminary mapping of markers and oligogenes . Useful in identifying heterotic QTLs. Requires less time and efforts for development. DEMERITS Linkage established using F 2 population is based on one cycle of meiosis. Precise evaluation of quantitative traits and their effects of G*E cannot be done. F2 populations are ephemeral and therefore cannot be maintained beyond one generation. Limited use in fine mapping and QTL mapping

13. F 2 derived F 3 (F 2:3 ) population A F2-derived F3 or F2:3 population is obtained by selfing the F 2 individuals for a single generation and harvesting the seeds from each F2 plant separately so that each F2 plant is represented as an individual plant progeny. F2:3 populations are suitable for mapping of oligogenic traits controlled by recessive genes and of QTLs . Useful for reconstitution of individual F 2 genotypes. The construction of these populations requires an extra season than that of F2 populations. The genotype and, particularly, phenotype of the F3 population do not strictly correspond to that of the F2 generation due to one more round of segregation, recombination, and inbreeding. DEMERITS MERITS

14. Doubled Haploids: Doubled haploid (DH) plants are obtained by chromosome doubling of haploid plants usually derived by culture of anthers/pollen grains produced by F1 plants. Seeds from individual DH plants are harvested separately and maintained as DH lines in the same way as RILs. The expected ratio for the genes as well as markers in a DH population is 1:1 irrespective of the marker being dominant or co dominant. DHs are similar to F2 in that they both are products of one meiotic cycle occurring in F1. But the differ in the frequency of recombinants.

15. Double Haploids:- Completely homozygous. Perpetual/ Immortal. Can be evaluated in replicated yield trials. Suitable for mapping of both qualitative as well as quantitative traits. Instant production of homozygous lines. Recombination from male side alone is accounted Non-availability of suitable haploid production techniques Anther culture induced variation. Differential regeneration response of parents. MERITS DEMERITS

16. Usually the F1 as a rule is backcrossed to the recessive parent i.e., the parent having the recessive form of the target trait. Such a backcross is called testcross, and is usually denoted by B2. Backcross populations are generated by crossing F1 plants with either of the two parents of the concerned F1. BACKCROSS POPULATION In the case of co-dominant markers, the order of backcross as well as the phase of linkage is not important when only markers are to be scored. But, in the case of dominant markers, the order of backcross is extremely important as they give difference in segregation ratio with difference in phase of linkage. The backcross populations offer one specific advantage as they can be further utilized for marker-assisted backcrossing (MABC) for introgression of the target traits as proposed in the advanced backcross QTL method ( Tanksley and Nelson 1996).

17. Limitations The construction of backcross populations, like that of F2:3 populations, requires one more generation than that of F2 populations. Cannot be evaluated in replicated trials, which makes them unsuitable for QTL mapping. They are not perpetual /they are mortal. They capture the recombination events of only one parent , i.e., the F1. It requires crossing of the F1 plants with the selected parent, which imposes additional work and may limit the population size in many crop species.

18. Recombinant Inbred Lines(RILs) Recombinant inbred lines(RILs) are a set of homozygous lines produced by sib mating/ selfing of individual F2 plants. RILs are also called F2-derived inbred lines or single seed descent (SSD) lines because they are derived from F2 populations usually by the SSD procedure. Wherever possible, F2 plants and their progeny should be selfed , and sib-mating should be resorted to only when selfing is not feasible for some reason.

19. The SSD procedure is followed for five or more (usually >8) generations, during which one seed is harvested from each plant of the F2 and the later generations and seeds from all the plants are composited and planted to raise the next generation. At the end of SSD procedure, seeds from each plant are harvested separately to obtain as many RILs as there are individual plants in the SSD population. Homozygous populations.

20. RILs are immortal. Most commonly used for QTL mapping. Suitable for fine mapping &map saturation. RILs are twice as efficient as an F 2 population. Multilocation trials can be conducted as they are homozygous lines. Require 6-10 seasons to develop. Development difficult in crops which show non tolerance to inbreeding depression. Only used for Additive and AXA type of interactions. Genetically variable over time due to mechanical mixture, mutations etc.. Smaller confidence interval . MERITS DEMERITS

21. Immortalized F2 population Immortalized F2 populations can be developed by paired crossing of the randomly chosen RILs derived from a cross in all possible combinations excluding reciprocals. The set of RILs used for crossing along with the F1s produced, provide a true representation of all possible genotype combinations (including the heterozygotes) expected in the F2 of the cross from which the RILs are derived. The RILs can be maintained by selfing and required quantity of F1 seed can be produced at will by fresh hybridization. This population therefore provides an opportunity to map heterotic QTLs and interaction effects from multilocation data.

22. ADVANTAGES Population identical to the conventional F2 population can be produced and replicated “n” number of times . Individual F2 genotypes can be evaluated over the years and locations. Eliminates the need of genotyping the immortalized F2s. Their genotype can be deduced based on their parental RILs genotypes. Thus economizing the cost of mapping. Can be used for mapping all the traits for which the parents of the RILs and the RILs show contrasting phenotype. It is possible to eliminate the additive X dominance(J) and dominance X dominance ( i ) effects .

23. Near Isogenic Lines.(NILs) Near-isogenic lines (NILs) are pairs of homozygous lines that are identical in genotype, except for a single gene/locus. NILs are generally produced by backcross procedure. But they can also be produced by selfing . The first strategy used for molecular mapping was based on NIL itself.

24. Immortal populations Su i t a b l e for gene tagging Us e ful i n fu n ct i o n al genomics Requ i re ma n y g e n e rat i ons to develop Not useful for linkage mapping. Linkage drag MERITS DEMERITS

25. Segregation Ratios I n M apping Populations

26. Marker type F2 RILs DHs NILs B1 B2 Codominant 1:2:1 1:1 1:1 1:1 1:1 1:1 Dominant 3:1 1:1 1:1 1:1 1:0 1:1 Segregation ratios at dominant and codominant marker loci in different mapping populations Codominant markers: RFLPs, SSRs, CAPSs Dominant markers: RAPDs, AFLPs, most SCARs, IISRs, SNPs, DArT , SFPs, RAD markers.

27. Other Bi-parental Mapping Populations Chromosomal segment substitution lines (CSSLs). Backcross inbreed lines(BILs) Advanced Intercrossed lines.(AILs) Interconnected mapping populations.

28. Multi-parental Population (MAGIC) Multi-parent Advanced Generation Intercross (MAGIC) populations are a collection of RILs produced from a complex cross/ outbreed population involving several parental lines. The parental lines may be inbred lines, clones, or individuals selected on the basis of their origin or use. This concept was first used in mice as “ heterogeneous stocks” and later extended to plants by Mackay and Powell (2007), who also proposed the name MAGIC. MAGIC populations are perpetual, lack population structure, can be used for both linkage and association analysis . They are an ideal resource for construction of high-density maps . Parents of the MAGIC population may be selected to represent a large part of variation present in the elite germplasm of a crop species.

29. Steps for development of MAGIC lines Inbreeding Advanced intercrosses Mixing of parents Founder selection

30. This complex cross is handled as per the SSD procedure to develop the required number of RILs, which together constitute the MAGIC population. A simple approach to generate a MAGIC population is to produce a complex cross involving multiple, typically eight, parental lines and to isolate RILs from this cross. The 8 parental lines are crossed in pairs to produce four different single crosses, and these single crosses are crossed in pairs to generate two double crosses Finally, the two double crosses are mated together to produce an eight-parent complex cross.

31. DEVELOPMENT OF MAGIC LINES

32. APPLICATIONS: The short term mapping populations like F 2 , BC, can be a good starting point in molecular mapping. Long term mapping populations like RILs, DHs, NILs, CSSLs, Immortalized F2, MAGIC can be developed for precision phenotyping of the traits of importance. RILs, NILs, DHs are homozygous lines and can be used for studying of additive gene actions or additive QTLs. Immortalized F2 population, F2 population, BC poulstion can be used for studying heterotic QTLs.

33. Parent 1 Parent 2 F 1 Back crossing Anther Culture Selfing F 2 Pedigree Method Bulk Method SSD RILs Immortal mapping populations NILs DHs Characterization for target traits Molecular genotyping Identification of markers linked to target traits MAS F 2 population Backcross population X

34. CONCLUSION:- Development and characterization of mapping populations should become an integral part of the ongoing breeding programs in important crops. The role of geneticists and plant breeders become crucial for reaping the full benefits of molecular plant breeding. MARKER MAPPING POPULATION SOFTWARE

35. REFERENCES Marker- assisted plant breeding: Principles and Practices by BD Singh and AK Singh http://bioinformatics.iasri.res.in www.ndsu.edu