Development of MAGIC lines and their Implications in Crop Improvement Doctoral Seminar –I (AGP-788) Speaker Vikas Mangal I.D. No- 39743

Multi-parent Advanced Generation Inter-Cross A second-generation mapping resource for discovery, characterization and deployment of genes responsible for complex traits.

PURPOSE: GENETIC DISSECTION OF COMPLEX TRAITS Identify genes that contribute to quantitative variation Quantitative traits are difficult to figure out Many genes + environment + other factors Most traits of biological and economic interest - quantitative nature, displaying continuous variation (polygenic control). Identification of gene-trait associations for complex traits is difficult

Mapping of genes Genetic mapping Physical mapping Determination of the relative positions of genes on a DNA molecule (or chromosome) Genetic mapping Based on the use of genetic techniques to construct maps showing the positions of genes and other sequence features on a genome Physical mapping Uses actual physical distances usually measured in number of base pairs More accurate representation of the genome

Requirements for genetic mapping A population of plants that is genetically variable for the target phenotype Marker systems allowing genotyping of the population Reproducible quantitative phenotyping methodologies Appropriate experimental and statistical Methods for detecting and locating QTL 

Mapping populations Used for gene mapping or gene tagging or for construction of genetic linkage maps Individuals of one species, or in some cases they derive from crosses among related species The type of mapping population to be used depends on the reproductive mode of the plant

Approaches to gene-trait dissection using different populations Natural Populations Selection based approaches Mapping in pedigrees Experimental populations Mutant populations Substitution lines Deletion lines Near isogenic lines Association mapping Bi-parental populations (RILs, DHs, AICs) Multi-parental populations (MAGIC)

Types of mapping populations (Biparental)

Limitations Only allows mapping of pairs of alleles for which the parents differ Needs large population size for fine mapping Lack of candidate gene identification QTLs are located with very large confidence interval (10-30 cM) The narrow genetic base Which limits the resolution for QTL detection and genetic mapping 

Association Mapping Detects and locates QTL based on the strength of the correlation between mapped genetic markers and traits. It exploits historical recombinations. It relies on decay of LD at a rate determined by the genetic distance between loci and the number of generations since it arose. Over a series of generations, in an unstructured population correlations between QTL and markers closely linked to the QTL will remain, facilitating fine mapping. (Mackay and Powell, 2007)

Demerits of Association Mapping Sensitive to population structure and substructure Lower power to detect QTLs Not suitable for coarse mapping Unable to investigate epistatic interaction (Cavanagh et al., 2008)

NESTED ASSOCIATION MAPPING Yu et. al., 2008

Content Introduction MAGIC and its meaning Steps in development of MAGIC populations Comparison between different types of populations Genetic analysis of MAGIC populations Use of MAGIC lines in breeding programme MAGReS –Another approach of MAGIC Advantages and limitations Case studies Studies in MAGIC and status in the India Summary and conclusion

Multiparent Advanced Generation Inter Cross

Advanced Intercrossed Lines (AILs) It is an extension of RILs Proposed by Darvasi and Soller in 1995 Consists of a repeatedly intermated F2 population, followed by selfing to derive AILs The additional rounds of intermating reduce the level of LD and increase the precision of QTL location Higher accuracy than RILs (Darvasi and Soller, 1995)

What is the difference between RILs and AILs? (Cavanagh et al., 2008)

Multi-parent Advanced Generation Inter-Cross (MAGIC) It is a simple extension of the Advanced intercross (Darvasi and Soller, 1995) A second-generation mapping resource for discovery, characterization and deployment of genes responsible for complex traits. It was first introduced by Mott et al. (2000) in animal as an improvement over the advanced intercrossing (AIC) and described as “Heterogenous Stock” Coined by Mackay and Powell (2008) and advocated by them. MAGIC populations were first developed and described in Arabidopsis (Kover et al., 2009)

(Cavanagh et al., 2008)

Intercrossed mapping population is created from multiple founder lines Cont’d…….. Intercrossed mapping population is created from multiple founder lines The increased recombination and diversity of MAGIC gives greater precision in QTL location and greater opportunity to detect more QTL Each generation reduces the extend 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 Reduced confidence interval.

Fig.- The theoretical decrease in confidence interval of QTL map location as a function of generation number for several initial confidence intervals in the F2 generation Fig.- The proportion of recombinant haplotypes as a function of generation number according to initial proportion of recombinant haplotypes in the F2 generation Fig. – LD decay

Why MAGIC? For both coarse and fine mapping Incorporation of multiple parents ensures the segregation of population for multiple QTL for multiple traits Negligible impact from population structure Increased mapping resolution by taking the advantages of both historical and synthetic recombination. To overcome the demerits of linkage analysis and association mapping (Cavanagh et al., 2008)

Steps in development of MAGIC line Founder selection Mixing Advanced intercrossing Inbreeding

1. Founder selection Based on genetic and phenotypic diversity Constrained set of material Elite cultivars, geographical adaptation Material of more diverse origins worldwide germplasm collections, distant relatives Each of the eight founder parents were carefully selected as a donor for at least one major trait in an improved background.

Worldwide germplasm collections Founder lines Worldwide germplasm collections Huang et al., 2015

Agronomic relevance of the eight founder lines used in developing the Indica MAGIC populations Sl. No. Germplasm / variety Origin  Agronomic relevance 1. Fedearroz 50 Colombia Popular variety in several countries, with stay green/ delayed senescence and quality traits, disease tolerance, progenitor of many breeding lines  2. Shan-Huang Zhan-2 (SHZ-2) China Blast resistant, high yielding  3. IR64633-87-2-2-3-3 (PSBRc82) IRRI High yielding variety 4. IR77186-122-2-2-3 (PSBRc 158)   5.  IR77298-14-1-2-10   Drought tolerant in lowlands with IR64 background and tungro resistance 6. IR4630-22-2-5-1-3 Salt tolerant at seedling and reproductive stages  7.  IR45427-2B-2-2B-1-1 Fe toxicity tolerant 8. Sambha Mahsuri + Sub1 Mega variety with wide compatibility, good grain quality and submergence tolerance 

2. Mixing Multiple parents are intercrossed to form a broad genetic base The inbred founders are paired off and inter-mated, known as funnel The result of this stage is a set of lines whose genomes comprised contributions from each of the founders

Funnel-1 Huang et al., 2015

3. Advanced intercrossing Mixed lines from different funnels are randomly and sequentially intercrossed as in the advanced intercross The main goal is to increase the number of recombinations in the populations Yamamoto et al. (2014) concluded that at least six cycles of intercrossing is required for large improvements in QTL mapping power

Funnels AIL 1 AIL 2 Huang et al., 2015

4. Inbreeding Development of homozygous individuals RILs in plants can be created by single seed descent method The multiple generation of selfing will introduce additional recombination but less than during the mixing and advanced intercrossing stages.

Crossing scheme

MAGIC : EFFECTIVE POPULATION SIZE 1000 MAGIC individuals are adequate to map a single additive locus that accounts for 5% of the phenotypic variation to within 0.96 cM distance (Valder et al. 2006). 500 lines, sufficient resolution may be obtained even in presence of high epistasis

QTL MAPPING Power to detect a QTL depends on the phenotypic variance it explains, which ultimately depends on the frequency of the minor allele frequency at the QTL. QTL minor allele frequency in MAGIC (1/8) 0.125 0.5 in diallelic populations

Comparison B/W Biparental Linkage Analysis, Association Mapping And MAGIC Cavanagh et al. (2008)

Genetic analysis of MAGIC population 1. Linkage map construction The large number of polymorphic markers across all founders and accumulation of recombination events through many generations of the MAGIC pedigree can be used to achieve dense and high-resolution mapping of the genome The higher levels of recombination in the MAGIC population can be seen most clearly in the region around centromeres, where there is very little separation between markers in biparental populations The first linkage map from a MAIC population was constructed in wheat (Huang et al., 2012)

Fig. 3 Comparison of wheat 9K SNP maps of Chr 3A in MAGIC four-way population with those from two biparental populations- Gladius × Drysdale (GD, orange) and Synthetic × Opata (SO, green).

founder probabilities 2. Haplotype mosaic reconstruction: A picture or pattern of a set of single-nucleotide polymorphisms (SNPs) on one chromosome that tend to always occur together. MAGIC populations, as with high level of recombination, can be use for this purpose. For haplotype recostruction three types of genetic data can be used – marker scores founder probabilities mosaics This supports identification of recombination hotspots and QTL for recombination events.

(A) (B) Founder lines RILs Fig: Comparison of different representations of simulated genetic data in MAGIC populations. a Example haplotype mosaics (colored lines) and SNP data (black and white lines) for founders (first eight) and five RILs (last five); b construction of RIL haplotype mosaics by finding best path through estimated founder probabilities. Colored segments indicate most likely founders in that region

3. QTL mapping approach The use of heterogenous stock (HS) improves the power to detect and localize QTL The large number of parental accession increases the  allelic and phenotypic diversity The larger number of accumulated recombination events increase the mapping accuracy of the detected QTL compared to an F2 cross

Use of MAGIC lines in breeding programs MAGIC populations may be used directly as source materials for the extraction and development of breeding lines and varieties Development of variety with several agronomically beneficial traits Variety which can adopt to several diverse regions of the world and suitable for diverse climatic conditions Can provide solutions to a range of production constraints (particularly stress tolerance) An assessment and understanding of the potential of enhanced recombination in generating novel diversity

Multi‑parent Advanced Generation Recurrent Selection (MAGReS) Development of MAGIC line QTL analysis Selection of plants 2 to 3 generations of intercrossing Development of homozygous inbred line MAGReS populations Huang et al., 2015

Case studies

MAGIC line in Arabidopsis thaliana Heterogeneous stock of 19 intermated accessions of the plant OUTPUT QTL affecting natural variation in flowering time, identified on chromosome 5 (~3.5 Mb) . Detected two QTLs on chromosomes 3 and 4 for the number of days to germination. QTL on chromosome 3(~15.9 Mb) for nitrilase gene cluster

S.No. Traits No. of QTL Chromosome no. Gene 1. Bolting time 4 1 ETHYLENE INSENSITIVE 5 & FLOWERING LOCUS T FRIGIDA 5 FLOWERING LOCUS C 2. Flowering time (Long days) 2 Short days PHYTOCHOME E 3. Days to germination 3 NITRILASE

MAGIC population in Rice  Bandillo et al. (2013) have developed- Indica MAGIC 8 indica parents MAGIC plus 8 indica parents with two additional rounds of 8-way intercrossing Japonica MAGIC 8 japonica parents Global MAGIC 16 parents – 8 indica and 8 japonica Founder exhibit high yield potential, good grain quality, and tolerance to a range of biotic and abiotic stresses 

Result of the study Blast disease Salinity and submergence tolerance The populations were phenotyped for multiple traits , includes: Blast and bacterial blight resistance Salinity and submergence tolerance Grain quality Blast disease Detected three contributing QTLs on chromosomes 2, 6 and 9. Out of the 8 founders, 6 were resistant to blast disease under natural conditions in the blast nursery at IRRI.

Bacterial blight Salt tolerance GWAS results from this study also detected the xa5 gene on chromosome 5 in the population. The xa5 gene is a recessive gene and confers resistance to Philippines races 1, 2, 3, 5, 7, 8, 9 and 10. The subset of indica MAGIC lines and the 8 founders were screened at maximum tillering stage in the screen house at IRRI. Results indicate that majority of the 200 lines carried the Xa4 gene. Salt tolerance Presence of lines with good levels of tolerance, presumably from the salt tolerant parents IR4630-22-2-5-1-3 or IR45427-2B-2-2B-1-1. MLM analysis detected significant markers on chromosome 1 near the previously detected QTLs SALTSN, salt sensitivity, qSKC-1 and near the Saltol QTL.

Submergence tolerance Detected 54 associated markers 49 of which were detected on chromosome 9 in the region of the Sub1 locus. Grain quality GWAS detected known major effect QTLs along with several potentially novel QTL for grain quality and grain shape

MAGIC population in Wheat  Huang et al. (2012) have developed two MAGIC populations One with 4 founders and another with eight founder lines They have constructed a linkage map with 1162 Diversity Arrays Technology ( DArT ) 

Result of the study They described the creation and use of an ‘Elite’ eight-parent MAGIC population consisting of 1,091 F7 lines of winter-sown wheat. Wheat yellow or stripe rust resistance controlled by transgressively inherited three QTL on the long arms of chromosomes 2D, 2A and 1A. Marker for awn (presence/absence) is on chromosome 5BL, which is also associated with fusarium resistance.

MAGIC population in Maize They identified three suggestive QTL for Grain Yield. The major QTL for GY is a locus on the short arm of Chr 6 pleiotropic to PH (plant height) and EH (ear height). They found a pleiotropic QTL on Chr 8 explaining 19% of flowering time variance and having effects on PH and EH.

MAGIC population in Tomato They grew the population in two greenhouse trials and detected QTLs for fruit weight. We mapped three stable QTLs and six specific of a location. The linkage map obtained showed an 87% increase in recombination frequencies compared to biparental populations.

The goal of this project is to develop at least 1,000 Recombinant Inbred Lines (RIL) derived from MAGIC population that will be used for locating QTLs associated with drought tolerance related traits. Highly recombined MAGIC population will be used directly as source materials for development of breeding lines and varieties adapted to different drier regions of West and Central Africa

Status of MAGIC populations in India ICRISAT Crop Founder lines used Stage of development Chickpea Eight kabuli varieties - Pigeonpea Eight 7 funnels at S1 stage Peanut 14 funnels at S1

Development of MAGIC lines in Chickpea

These lines will be phenotyped for disease screening and yield and yield related traits under multi-location trials. The phenotypic data together with genotypic data will be used to identify the marker trait associations (MTA) for the targeted traits and can be further used in marker assisted selection for pigeonpea improvement.

MAGIC breeding programme (crossing among better genotypes for most of the traits) like WH 1105, HD 2967 and DBW 17 followed by selection in segregating generations which may lead to identification of physiologically efficient and high yielding genotypes coupled with better nutrient use under low as well as optimum input conditions. Genotypes resistant to both low and optimum input conditions may be identified.

Advantages of MAGIC populations A population can be established containing lines that capture the majority of the variation available in the gene pool Shuffling the genes across different parents enable accurately ordering the genes Increased recombination Best combinations of genes for important traits development Study gene – gene interactions A QTL may not be effective in a single background (one of the eight parents), but may be effective in the MAGIC populations (a mixed background of the 8 parents) MAGIC population can be used for extraction of good combination and directly release as a variety

Limitations Extensive segregation More time Large scale phenotyping Incompatibility between the parents Require more inputs Better marker system is necessary to identify QTLs

Conclusion

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