This document summarizes the presentation of Adithya P Balakrishnan on MAGIC (Multi-parent Advanced Generation Intercross) populations. It discusses how MAGIC populations are constructed using multiple parental lines that are intercrossed and selfed over multiple generations. This results in a population with increased genetic diversity and higher mapping resolution compared to biparental populations. The document provides examples of MAGIC populations developed in Arabidopsis thaliana and rice. It describes the phenotypic evaluation and genetic analysis, including QTL mapping, that has been carried out on these MAGIC populations.

1. Speaker
ADITHYA P BALAKRISHNAN
Reg. No: 04-AGRMA-01724-2018
M. Sc (Agri) III Sem
Dept. of Genetics and Plant Breeding
MASTERS SEMINAR PRESENTATION
Major Guide
Dr. N. B. PATEL
Associate Professor
Dept. of Genetics and Plant Breeding
C. P. C. A, S. D. A. U
Minor Guide
Dr. M. P. PATEL
Principal Research Scientist
Pulses Research Station
S. D. A. U
1

2. MAGIC POPULATION:
A NOVEL RESOURCE FOR CROP
IMPROVEMENT
2

3. OVERVIEW
 Introduction
 Gene mapping & era before MAGIC
 The IDEA of MAGIC
 Steps for MAGIC population construction
 Genetic analysis of MAGIC population
 Advantages and disadvantages
 How MAGIC is useful
 MAGIC and other experimental populations
 Case studies
 Conclusion
 Future prospects
3

4. M • MULTI-PARENT
A • ADVANCED
G • GENERATION
I • INTER
C • CROSS
4

5. INTRODUCTION
Breeders goal:
Improvement of yield, quality
and resistance
(Quantitative Traits)
Creation of
experimental
population
QTL Mapping
Localization of genes
responsible
5
Creation of experimental
population
QTL Mapping
Localization of genes
responsible

6. GENE MAPPING
 Gene mapping is the sequential allocation of loci to a relative position on a
chromosomes.
GENE MAPPING
Genetic Mapping Physical Mapping
6

7. REQUIREMENTS FOR QTL MAPPING
1. Suitable mapping population
2. A dense marker linkage map for the species
3. Reliable phenotypic screening methods and generation of data
4. Appropriate software packages
7 B. D. Singh & A. K. Singh, 2015

8. QTLANALYSIS
A statistical method links two
types of information:
 Phenotypic data
(Quantitative trait)
 Genotypic data (Genetic
marker)
8
© Google photos

9. MAPPING POPULATIONS
 A population that is suitable for linkage mapping of genetic markers is known as
mapping population.
 Types:
1. Conventional mapping resources
2. Second generation mapping resources
9

10. CONVENTIONAL MAPPING RESOURCES
 F₂ and BC populations
 Recombinant Inbred Lines (RILs)
 Backcross Inbred Lines (BILs)
 Doubled Haploids (DHs)
 Advanced Intercross Lines (AICLs) – Darvasi and Soller (1995)
10 Rashit et al. (2012)

11. DEVELOPMENT OF BIPARENTAL POPULATIONS
11 Collard et al. (2004)

12. LIMITATIONS OF BIPARENTAL MAPPING POPULATIONS
• Reduction of genetic heterogeneity
• Low resolution mapping
• Only two allelic variations are analysed
12 Meng et al. 2016

13. ASSOCIATION MAPPING
 Mapping based on the estimates of linkage disequilibrium (LD) obtained from
populations consisting of individuals/lines drawn from either natural or breeding
population.
Demerits of Association Mapping:
 Unknown population structure
13 B. D. Singh & A. K. Singh, 2015

14. THE IDEA OF ‘MAGIC’
 Strategy of collaborative cross to construct large set of RILs by in Mice as
heterogeneous stocks.
(Churchill et al.,2004)
 Term ‘MAGIC’ coined by Mackay and Powell (2007) and advocated by them
and Cavanagh et al., (2008).
 Concept is similar to advanced inbred lines (AILs) proposed by Darvasi and
Soller.
14 Meng et al. 2016

15. MULTIPARENT ADVANCED GENERATION INTERCROSS
(MAGIC):
 Next generation emerging population for plant genetic study.
 Bridge the gap between biparental mapping and AM.
 Collection of RILs of several parental lines
15 Meng et al. 2016

16. STEPS TO BE FOLLOWED FOR DEVELOPMENT OF ‘MAGIC’
Four major steps to be followed
1. Founder Selection
2. Mixing
3. Advanced Intercrossing
4. Inbreeding
16 Huang et al. (2015)

17. (A) FOUNDER SELECTION
 Choice of founder lines
based on genetic &
phenotypic diversity
 Use of landraces for more
diversity
Huang et al. (2015)
17

18. (B) MIXING
 First stage of population
development
 Inbred founders are paired
off & inter-mated known as
Funnel to form broad
genetic base Funnel
Huang et al. (2015)18

19. (C) ADVANCED INTERCROSSING
 Mixed lines from different
funnels are randomly and
sequentially intercrossed as
in advanced intercross
 Purpose: To increase
number of recombination
 6 cycles of intercrossing for
improving power of QTL
mapping
Huang et al. (2015)19

20. (D) INBREEDING
 Individuals resulting from
AI stage progressed to
create homozygous
individuals (RILs/DHs)
Huang et al. (2015)20

21. Funnel breeding scheme for MAGIC population development modified from Cavanagh et al. (2008)
Rashit et al. (2012)
21

22. GENETIC ANALYSIS OF MAGIC POPULATION
Linkage map construction:
 Accumulation of recombination events used to achieve dense & high resolution
map
 Map construction for MAGIC may require prior filtration of markers
Haplotype mosaic reconstruction:
 A picture or pattern of a set of SNPs on one chromosome that tend to inherit
together
 Haplotype mosaic reconstruction from high-density genotypic data determines
recombination breakpoints
22 Huang et al. (2015)

23. QTL mapping approaches:
 Most common approach is to use a genome scan such as interval mapping
 Interval mapping approach based on regression first demonstrated by Xu (1996)
for a four-way cross
 This approach failed for HS of mouse
 HAPPY: an interval mapping approach based on founder probabilities
 TASSEL: GWAS software - association mapping approach
Huang et al. (2015)23

24. ADVANTAGES
 More abundant genetic diversity
 Higher allele balanced frequency
 Negligible impact from population
structure
 High mapping resolution and
detection power
 Perpetual
DISADVANTAGES
 Greater initial investment
 Time consuming
 High statistical complexity
24 Lincoln et al. 2018

25. HOW MAGIC IS USEFUL FOR CROP IMPROVEMENT
 Multiline variety: Ideal materials for breeding
Duo-Ji-Xin 3- From a 12 parent MAGIC population by Li et al.,(2014)
 Precise QTL mapping
 Linkage map construction
25
Lincoln et al. 2018

26.  Genome introgression
 Dissecting genomic structure
 Improving breeding population
Cont.…..
26 Cavanagh et al., (2008)

27. RILs, AILs and MAGIC Lines
Recombinant Inbred Lines Advanced Inter-cross Lines
Cavanagh et al., (2008)27

28. Multiparent Advanced Generation Intercross Lines
Cavanagh et al., (2008)28

29. Comparison between biparental linkage analysis, association
mapping and MAGIC.
Properties Biparental Association MAGIC
Founder parents 2 ≥100 ≥8
Crossing requirement Yes No Yes
Time to establish Moderate Low Long
Population size ⁓200 ⁓100 ⁓1000
Suitability for coarse mapping Yes No Yes
Suitability for fine mapping No Yes Yes
Amount of genotyping required Low High High
Amount of phenotyping required Low High High
Relevance of population structure No Yes No
Statistical complexity Low High High
Use of germplasm variation Low High High
Practical utility Low High High
Modified from Cavanagh et al. (2008)
Rashit et al. (2012)29

30. 30

31. CASE STUDY: 1
 Arabidopsis thaliana is a model plant for the study of plant genetics
 Identification of causal genes leads to homologous loci important for improving
crop quality & productivity
 Limitations of simple synthetic populations leads to development of first ever
MAGIC population in A. thaliana
Kover et al. (2009)Manchester, U. K 31

32. Parental lines and construction of the MAGIC lines
Accession Origin
Bur-0 Ireland
Can-0 Canary Isles
Col-0 USA
Ct-1 Italy
Edi-0 Scotland
Hi-0 Netherland
Kn-0 Lithuania
Ler-0 Germany
Mt-0 Libia
No-0 Germany
Oy-0 Norway
Po-0 Germany
Rsch-4 Russia
Sf-2 Spain
Manchester, U. K Kover et al. (2009)32

33. Tsu-0 Japan
Wil-2 Russia
Ws-0 Russia
Wu-0 Germany
Zu-0 Germany
 Criteria for selection of 19 founder lines: Wide geographical distribution
Common/Popular in use
 Construction of MAGIC Lines (MLs):
1. Intermating of 19 lines to produce 342 F₄ families
2. Selfing up to 6 generations
3. Up to 3 MLs from each F₄ family
 ‘Cousins’: Lines derived from same F₄ family
Manchester, U. K Kover et al. (2009)33

34. Phenotyping
 Done on 459 MLs + 19 parental accession grown in pots in greenhouse of FIRS
Botanical experimental grounds (Manchester)
 Traits considered:
Days to germination
Growth rate
Days to bolt
Days to bolt to flower
Days to Flower (SD) and (LD)
RLN (SD) and (LD)
Erecta
Glabrous
Manchester, U. K Kover et al. (2009)34

35. Genotyping
 MLs + founder lines genotyped using Illumina Golden gate assay with SNP
markers
QTL Mapping:
 By two alternative methods: 1.) Empirical Bayes linear mixed effects model
2.) Hierarchical Bayesian method
 Mapping of known QTLs with higher precision
 Finding novel QTLs for germination data and bolting time
Manchester, U. K Kover et al. (2009)35

36. List of QTL identified and their location
Phenotype SNP logP Chr
Days to germination MN3_15977654 6.46 3
MN4_1553589 3.04 4
Days to bolt MASC00497 0.26 1
NMSNP1-24738247 0.89 1
FRI_2343 1 4
MN5_3491425 1.02 5
logP-2log10(ANOVA P-value) at the QTL peak; chr-chromosome at the 90% confidence interval (CI) for the QTL
Table modified
from Kover et al. (2009)
Trait Range nP nL h²P h²L nQTL h²QTL
Days to
germination
4-31 2227 433 0.50 0.84 1.94 27.34
Days to bolt 13-85 2202 433 0.72 0.93 3.63 63.70
Range in measured phenotypes and heritability for the traits measured
nP is the number of plants phenotyped for the trait, nL is the number of MLs. h2 P is the estimated heritability between
plants and h2 L the estimated heritability between lines. nQTL is the average number of QTL found in multiple QTL
models fitted to 500 resampled data sets. h2QTL is the average fraction of variance accounted for by the multiple QTL
models. 36

37. Results
 QTL on chromosome 4 is likely to be caused by FRIGIDA gene (affects flowering
time)
Haplotypes with a deletion at this locus bolt earlier
 Developed MAGIC is useful for high precision mapping
 Results supports similar efforts to produce MAGIC lines in other organisms
Manchester, U. K Kover et al. (2009)37

38. CASE STUDY: 2
 Bandillo et al. (2013) released MAGIC populations in Rice for QTL mapping and
varietal development.
 Use of two major rice ecotypes: indica, japonica
 MAGIC populations developed: 1. indica MAGIC
2. japonica MAGIC
3. MAGIC plus
4. Global MAGIC
Bandillo et al. (2013)IRRI, Phillipines 38

39. Agronomic relevance of the 8 founder lines used in developing the indica MAGIC
populations
Germplasm/variety GID Varietal type Origin Agronomic relevance
Indica type
Fedearroz 50 1846419 Indica Colombia Popular variety in several countries, with stay
green/ delayed senescence & quality traits, disease
tolerance, progenitor of many breeding lines
Shan-Huang Zhan-2
(SHZ-2)
402862 Indica China Blast resistant, high yielding; in the pedigrees of
many varieties in south China
IR64633-87-2-2-3-3
(PSBRc82)
94801 Indica IRRI High yielding and most popular variety of the
Philippines
IR77186-122-2-2-3
(PSBRc 158)
1111266 Indica/tropical japonica
background
IRRI High yielding variety in New Plant Type II
background
IR77298-14-1-2-10 2154106 Indica IRRI Drought tolerant in lowlands with IR64 background
and tungro resistance
IR4630-22-2-5-1-3 56023 Indica IRRI Good plant type, salt tolerant at seedling and
reproductive stages
IR45427-2B-2-2B-1-3 1935108 Indica IRRI Fe toxicity tolerant
Sambha Mahsuri+Sub 2254836 Indica IRRI Mega variety with wide compatibility, good grain
quality and submergence tolerance
Bandillo et al. (2013)IRRI, Phillipines 39

40. Crossing scheme for development of indica MAGIC population
Bandillo et al. (2013)
 ⁓60 seeds were
advanced by selfing
from each of 35 8-
way crosses
 SPS for advancing
to next generation
 Population size
targeted was 35×60
= 2100 lines
IRRI, Phillipines 40

41. Japonica group
CSR 30 1158955 Basmati group India Sodicity tolerance; Basmati type long
aromatic grain
Cypress 417083 Tropical japonica USA High yielding, good grain quality and
cold tolerant
IAC 165 599974 Tropical japonica Latin America Aerobic rice adaptation
Jinbubyeo 312160 Temperate japonica Korea High yielding and cold tolerant
WAB 56-125 94428 O. glaberrima in indica
background
WARDA NERICA background (O. glaberrima);
heat tolerant and early flowering
IR73571-3B-11-3-K2 2007669 Cross between tropical
japonica and indica
IRRI-Korea
project
Tongil type, salinity tolerant
Inia Tacuari 1846418 Tropical japonica Uruguay With earliness, wide adaptation, &
good grain quality
Colombia XXI 2351848 Tropical japonica Colombia High yielding and delayed senescence
Germplasm/variety GID Varietal type Origin Agronomic relevance
Agronomic relevance of the 8 founder lines used in developing the japonica MAGIC
populations
Bandillo et al. (2013)
IRRI, Phillipines 41

42. Crossing scheme for development of MAGIC-Global and MAGIC-Plus
Bandillo et al. (2013)
MAGIC-Plus:
 Two extra rounds of
intercross prior of indica
8-way crosses prior to
selfing
Global-MAGIC:
 Representative of 16
parents (8 indica type
and 8 japonica type)
 16-way crosses further
advanced by selfing
IRRI, Phillipines 42

43.  At S₄ stage of SSD, subset (200 lines) of indica MAGIC was phenotyped for:
1. Biotic stress
Blast and blight resistance
2. Abiotic stress
Salt and submergence tolerance
3. Grain quality
 Genotyping by 96-plex ApeKI GBS protocol
Marker used: SNP
Trait Analysis by aSSociation Evolution and Linkage (TASSEL) programme to
perform GWAS
 Cladogram of 200 lines constructed
 MLM analysis to overcome negligible structure of cladogram
IRRI, Phillipines Bandillo et al. (2013)43

44. Cladogram (neighbor joining) of the 200 S4 indica MAGIC lines and 8 founders using 634 SNP marker sites
Bandillo et al. (2013)IRRI, Phillipines
44

45. Results
 GWA mapping identified several known major genes and QTLs
1. Sub1- submergence tolerance
2. Xa4 and Xa5- resistance to bacterial blight
 GWAS detected major effect QTLs for grain quality and shape
IRRI, Phillipines Bandillo et al. (2013)45

46. Grain quality - Manhattan plots (MLM) showing GWA for (a) amylose content – waxy chromosome 6 (b) grain
length GS3 on chromosome 3. x axis – position on chromosomes 1 to 12; y-axis (−) Log p-value of markers
Bandillo et al. (2013)IRRI, Phillipines 46

47. MAGIC Global Selected
MAGIC Plus
© IRRI photos 47

48. CASE STUDY:3
 Tomato (Solanum lycopersicum): one of the most important vegetables consumed
world wide and model species for studying fleshy fruit development
 This work presents first MAGIC population in Tomato and describes its potential
for
(i) intraspecific variation exploitation
(ii) QTL mapping
(iii) causal polymorphism identification
Pascual et al. (2014)France 48

49. Founder lines selection and population construction
Construction of a tomato 8-way MAGIC population. Large fruited founders noted as L1 Levovil, L2 Stupicke PR, L3 LA0147,
L4 Ferum. Small fruited founders noted as C1 Cervil, C2 Criollo, C3 Plovdiv24A, C4 LA1420. DCF1Hy: double cross F1 hybrid
 Total number of founder lines: 8
 Founder selection based on:
molecular characterization data
of 360 tomato accessions
 Four founders of S.
lycopersicum: Levovil, Stupicke
PR, LA0147 and Ferum
 Four founders of S.
lycopersicum var. cerasiforme
Cervil, Criollo, Plovdiv24A and
LA1420
France Pascual et al. (2014)49

50. Genotyping
 DNA was isolated from young leaves of each funder line and 397 MLs
 Marker used: SNP
 Genotyping performed by Fluidigm 96.96 Dynamic Arrays according to the
manufacturer’s protocol
 Construction of genetic map
Phenotyping
 Trial conduction at South France: Location A- Avignon
Location B- La Costière
 Fruit weight (FW) was evaluated from a minimum of 10 ripe fruits per genotype
harvested
Pascual et al. (2014)50

51. Distribution of fruit weight (gr) in the MAGIC lines grown in (a).
Avignon. (b). La Costière
 Large range of phenotypic variation in
the population, including transgressive
lines
 Difference in average FW among
locations
 Analyzed the data separately and then
compared the QTLs obtained in each
location
Founder trait values are indicated with vertical lines (A Cervil, B
Levovil, C Criollo, D Stupicke PR, E Plovdiv24A, F LA1420, G Ferum,
H LA0147). Pascual et al. (2014)
51

52. QTL detection in the MAGIC population for FW: Manhattan plot
(MLM) showing corrected p-values
location A (Avignon) Location B- La Costière
Pascual et al. (2014)France 52

53. Results
 The population developed represents a new permanent resource for tomato
genetic community
 Illustrated power of MAGIC for future fine mapping experiments by locating
QTLs for FW on chromosome number 2, 3, 5, 11 and 12
 Tomato MAGIC was developed after S₃ generation, hence retain residual
heterozygosity
Pascual et al. (2014)France 53

54. CASE STUDY: 4
 This study is about creation of Soybean MAGIC population and its potential
utilities
 Eight founder parents selected: 4 popular soybean varieties
4 promising exotic collections
Shivakumar et al. (2017)MP, India 54

55. Details of the parents used in development of MAGIC population
Name of parent Characteristic feature
JS 335 Wider adaptable variety with resistance to bacterial pustule
JS 95-60 A popular variety of central India resistance to girdle and blue beetle, root rot, bacterial
pustule
NRC 37 A popular variety of central India having moderately resistance to collar rot, bacterial pustule,
pod and bud blight
NRC 86 A new variety of central India with high degree of resistance to bacterial pustule and pod
blight
EC333901 A promising line collected from USA for higher yield and its attributing traits
EC546882 A promising line collected from Brazil for higher yield and its attributing traits
EC572136 A diverse and high yielding line collected from China
EC572109 Promising line collected from China for higher yield and its attributing traits
Shivakumar et al. (2017)MP, India 55

56. Details of the 2-way and 4-way intercrosses (DCHs) performed
during the kharif 2013 and 2014 respectively
Type of intercross Genotypic combinations No. of pods harvested No. of seeds
2-way intercross EC572109 × JS 95-60 71 102
EC572136 × JS 335 64 75
EC546882 × NRC 37 53 105
EC333901 × NRC 86 66 115
Total 254 417
4-way intercrosses [EC546882 × NRC37] × [EC572136 × JS335] 39 58
[EC546882 × NRC37] × [EC333901 × NRC86] 64 105
[EC546882 × NRC37] × [EC572109 × JS9560] 49 76
[EC333901 × NRC86] × [EC572136 × JS335] 75 135
[EC572109 × JS9560] × [EC572136 × JS335] 31 52
[EC572109 × JS9560] × [EC333901 × NRC86] 145 211
Total 405 637
Shivakumar et al. (2017)MP, India
56

57. Traits Targeted
 Higher yield and its attributing traits
 Wider adaptability
 Resistance to bacterial pustule, pod blight, blue beetle, collar rot
 Hybridity of F₁s was confirmed by morphological markers:
Stem pigmentation
Flower colour
Pubescence
 True hybrid seeds used to make 764 eight-way hybrids and evaluated for the traits
under consideration
MP, India Shivakumar et al. (2017)57

58. Results:
 The developed MAGIC population can be
used for multilocation tests to exploit
diversity and variability.
 Genetic base of Soybean got broadened
by bringing diversity from China, Brazil
and USA in genetic background of Indian
cultivars.
 Source to develop better plant types for
changing environment Soybean MAGIC population developed at ICAR-Indian
Institute of Soybean Research Indore. The plants derived from 8-
way F1 were grown under net house condition during kharif
2016.MP, India Shivakumar et al. (2017)58

59. CASE STUDY: 5
 This study presents a genetic linkage map of an elite but highly diverse eight-
founder MAGIC population in common wheat (Triticum aestivum L.)
 MAGIC population design that involved a greatly reduced number of overall
crossings
Germany Stadlmeier. M. (2018)59

60. Founder lines
 Criteria: (i) variation for disease resistance, quality, and agronomic traits
(ii) derivation from diverse breeding programs
(iii)Importance within the respective quality group
 Eight winter wheat lines: ‘Event’ (A)
‘Format’ (B)
‘BAYP4535’ (C)
‘Potenzial’ (D)
‘Ambition’ (E)
‘Bussard’ (F)
‘Firl3565’ (G)
‘Julius’(H)
Germany Stadlmeier. M. (2018)60

61. Construction of MAGIC Population
 Among 516 lines
advanced to F₆:₇
394 lines were
selected based on
sufficient seed
availability for field
trials and suitability
for experiments in
the agricultural
environment
Crossing scheme based on Cavanagh et al. (2008) of the 8-founder BMWpop
Stadlmeier. M. (2018)Germany
61

62. DNA extraction, Genotyping
 Genomic DNA was extracted according to the procedure described by Plaschke et
al. (1995)
 Genotyping with a functional PCR marker for powdery mildew resistance allele
Pm3a (parent ‘BAYP4535’ is known to carry that gene)
 Linkage map constructed and validated
Germany Stadlmeier. M. (2018)62

63. Powdery Mildew QTL
 Five QTLs were detected in simple interval mapping explaining 72.5% of the total
phenotypic variance
Trait QTL Chr. R²
PM [1-9] QPm.lfl-1A 1A 34.1
QPm.lfl-1B 1B 4.5
QPm.lfl-4A 4A 6.6
QPm.lfl-6B 6B 17.4
QPm.lfl-7A 7A 18.3
Chromosome (Chr.)proportion of phenotypic variance explained (R2),
Table modified from Stadlmeier. M. (2018)
QTL for seedling resistance to powdery mildew (PM) in BMWpop
Germany 63

64. Results
 A successful QTL mapping for seedling resistance to powdery mildew from
simplified eight-founder MAGIC design
 The eight-founder Bavarian MAGIC Wheat Population (BMWpop) and its genetic
linkage map is a valuable genetic resource
Germany Stadlmeier. M. (2018)64

65. CONCLUSION
 The MAGIC population serves as an alternative to conventional mapping
population with a great increment in precision of gene mapping.
 It acts as a resource for gene discovery and deployment of new genes and there by
plays a key role in crop improvement.
 Moreover, this population have high potential for multiline variety development
and fine mapping.
65

66. Future Prospects
 The idea of MAGIC can be extended to more crops in association with the
advances in genomics
 Real longevity of the results to be tested
 Multivariate analysis
 Epistasis detection
 Multi-parent advanced generation recurrent selection (MAGeS)
66

67. STATUS OF MAGIC IN INDIA
Crop Number of founder lines Design
Chickpea Eight kabuli (in progress) -
Pigeonpea Eight (in progress) 7 funnels
Peanut Eight (in progress) 14 funnels
Location: ICRISAT, Hyderabad
Huang et al. (2015)67

68. Always
Seek
Knowledge
68