G.Kalidasan

MAGIC, Multiparent advanced generation intercross a new genetic resource for multiple trait improvement and QTL discovery in crops

Quantitative traits

Phenotype expression

Natural variation

Experimental System

Selection or Natural Population

Experimental Population

Multiparent Advanced Generation Intercross Population

Case Studies

Selection experiments

Marker allele frequency –

Unrelated individuals

Many generations

Difference in phenotype

LD between QTL and marker

LD around a QTL

Selection and Natural populations

Comparison of allele frequency

Without phenotypic information

Loci subject to selection

Domestication

Exploits LD in diverse population

Human

Crops

Maize

Advantage

Cheaper and high density markers

Disadvantage

Spurious associations

Greater precision but low power

Mutant population

Spontaneous mutation

Induced mutation

Mutagenesis

Large resources

Poly ploidy

TILLING

Phenotyping screen

Knowledge on genes controlling trait

F2 and backcross (BC) populations

Additive effects

Few meioses

Recombinant inbred lines

RILs are advanced homozygous

lines

Increased recombination events

and improved map resolution

Epistatic interactions

Near isogenic lines(NIL)

Target trait is required for the generation of NILs.

High-resolution mapping

Double haploids

100% purity and genetic uniformity.

Genetic studies

Randomly and sequentially

intercrossed population.

Phenotypic selection to further

reduce the frequency of deleterious

alleles from the donor.

Detect QTLs with epistatic effects

Useful meiotic recombination

Linkage mapDNA Markers Position and relative genetic distance For identifying chromosomal regions that contain genes

controlling simple or complex traits using QTL analysis

QTL mapping

Advantage

High detection powerFew markers are required

Disadvantage

Large confidence interval of upto 5 to 30cMLimited resolutionOnly two alleles tested

Animals. (Mott et al., 2000) and (Yalchin et al., 2005)

Fine-mapping of multiple QTLs for multiple traits in the same population.

Advanced intercrossed lines (AILs)

Each generation reduces the extent of linkage disequilibrium (LD), thus allowing QTL to be mapped more accurately.

Lines derived from early generations can be used for QTL detection and coarse mapping

While those derived from later generations will only detect marker-trait associations if markers are located very close to the QTL.

Multiparent advanced generation intercross

Extended to plants

(Cavanagh et al., 2008)

Diverse founder lines

n/2 generations

RILs

Increased intercrossing cycles

Short generation period- Arabidopsis

Eight founder lines

G1

G2

G3

G4

G10-12

SNP genotyping platforms

SSR Markers

Statistics tools

Linear mixed effect model and Hierarchical Bayes QTL mapping - study the interrelationship between individuals MLs and founder lines and increases the precision to detect QTL

HAPPY- a software package for Multipoint QTL Mapping in Genetically Heterogeneous Animals

R/mp Map- A computational platform for the genetic analysis of

multi-parent recombinant inbred lines

Advantages

Shuffling the genes across different parents enable accurately ordering the genes

Increased recombination - novel rearrangements of alleles and greater genetic diversity.

Best combinations of genes for important traits development

1000 Magic individuals

Seeds retained - fine mapping

Epistatic and G X E interactions

Facilitate the discovery, identification and manipulation of new forms of allelic variability

Disadvantages

Extensive segregation

More time

Large scale phenotyping

(Rakshit et al., 2012)

In Arabidopsis fine mapping of QTL for germination and bolting time (Kover et al. 2009)

Studies in flowering time candidate genes

(Ehrenreich et al. 2009)

Developed computational platform R/mp Map for Genetic analysis (Huang and George, 2011)

19 ‘‘founder’’ accessions

Wide geographical distribution

Staggered planting scheme

Replanted families – randomly

assigned crosses

342 F4 outcrossed families

Each F4 family derived up to 3

MLs followed by selfing 6

generations

527 lines taken out of 1026

Developmental quantitative traits

Measured the heritability (h2)

h2 L is the proportion of variation that is due to genetic differences between lines, using the phenotypic average of the replicates within each line

h2 P is an estimate of the genetic variance if only one replicate per line were phenotyped

h2L ≥ h2P

h2L increases with the number of replicates

Mean of each line is used for QTL mapping

Phenotypic variance

Diallelic population - 0.5

Magic – 0.052

Average minor allele frequency in

founder lines is 0.22

70% of SNP shared between any pair

of founders

Increasing replication within line

reduces non- genetic variance

Improves power of QTL

A hidden Markov model (HMM) is used to make a multipoint probabilistic reconstruction of the genome of each ML as a mosaic of the founder haplotypes.

Diallelic SNPs cannot distinguish between all founders so information from neighboring SNPs is used to compute the posterior probability Pis

(L) is that at a given locus L, the ML i is descended from founder s.

Locus is defined to be the interval between two adjacent genotyped SNPs, labeled by the name of the left-hand SNP.

Used fixed-effects QTL models but to accommodate population structure, in different ways used multiple-QTL modeling or random effects to explain the correlations introduced in population structure

Checked with hierarchical Bayesian random effects model

All approaches model the mosaic structure of the MAGIC genomes as described in and implemented in the R package HAPPY

Detected two QTLs on chromosomes 3 and 4 for the number of days to germination and bolting time.

Constructed a linkage map from a four parent MAGIC population and validate it against a comprehensive DArT consensus map drawing together maps from over 100 biparental populations

Incorporated the alien introgressions in to the linkage map

Level of LD across the genome and compare it with previous estimates for LD from previous studies

The power and precision of MAGIC for QTL mapping for plant height , an important trait for yield potential

Selected four elite wheat cultivar , A- Yitpi , B- Baxter, C- Chara, D- Westonia

Genetic diversity based on genetic survey of international wheat samples

Diverse geographical distribution

Phenotypic diversity for a range of traits

Genotyping

Used 1285 DArT markers, 57 SSR markers and 1536 SNPs

384 SNPs observed to be polymorphic among the parental lines

were selected for genome wide coverage

Phenotyping

1100 RILs – Plant height was recorded

R package mpMap

Filtered with monomorphic markers

Estimated the recombination fraction all pairs of loci with function ‘mpestrf’

Grouped the markers based on estimated recombination fractions and LOD scores with the function ‘mpgroup’

For map resolution computed recombination events for all lines using the function ‘mpprob’

Which calculates the multipoint probablity at each locus that the observed genotype is inherited from each of the four founder

Both internal and external comparisons was done

Examined a series of diagnostic plots to propose changes to ordering which were then tested through ‘ compare orders’ function in R/mpMap

Used heatmaps based on both recombination fractions and LD using R/Ldheatmap

The tool provided visualization of the relationships between all pairs of markers

External comparison

Diagnostic checks are compared it to an external DArT consensus wheat map

Each individual consensus map was based on genotyping involving the analyis of between 206 and 1525markers, with an average density of 582 markers

Test the introgression in magic population

Sr36 is an introgression from Triticum timopheevii for stem rust resistance – carried in variety Baxter on chromosome 2B cause segregation distortion

Aimed to identify markers associated with it and identify lines containing it

Computed the degree of segregation distortion for which i) the Baxter allele differed from all other founder alleles

ii) mutual recombination of < 0.05 was observed were tagged as potential markers in the introgression

Estimated the probablity of a line having inherited an allele from the founder Baxter for the identified markers(‘mpprob’)

Linkage disequilbrium

Multipoint probablities were computed using the function ‘mpprob’ in R/mpMap and LD was computed using the function ‘mpcalcld’

QTL Mapping

For all analyses used the ‘mplMmm’ function in the R package mpMap, which performs the interval mapping in the context of a linear mixed model

Three QTL for plant height were detected near known genes on chromosomes 2D, 4B, 4D.

(MAGIC) populations combine the advantages of linkage analysis and association studies.

The increased recombination in MAGIC populations leads to novel

rearrangements of alleles and greater genetic diversity

Can facilitate the discovery, identification and manipulation of new forms of allelic variability

They require longer time and more resource to be generated and they are likely to show extensive segregation for developmental traits.

MAGIC populations are likely to bring paradigm shift towards QTL analysis in plant species

The experimental method was underway since it has to be studied in many crops

The tools used for QTL mapping are very complex, so simplified models has to be developed in near future for understanding.

In near future the method will bring success in finding our economic interest of traits in plants.