Principles of QTL Mapping. M.Imtiaz

Transcription

1 Principles of QTL Mapping M.Imtiaz

2 Introduction Definitions of terminology Reasons for QTL mapping Principles of QTL mapping Requirements For QTL Mapping Demonstration with experimental data Merit of QTL mapping Limitations of QTL mapping

3 Definitions: Qualitative trait: A trait which segregates in discrete Mendelian Manner (e.g. 3:1; single gene; diseases; resistant or susceptible); single locus, few alleles Quantitative trait: A trait which does not segregates in discrete Mendelian Manner (e.g. 3:1; many genes; preharvest sprouting (PHS), plant height); multiple allele, multilocus QTL (quantitative trait locus): site on a chromosome containing a gene(s) affecting a quantitative trait

4 Examples of traits Developmental traits Flowering time, days to maturity seed size, plant height Physiological traits stomatal conductance, seed dormancy, photosynthesis rate, nitrogen uptake, phosphate uptake, Biochemical traits enzymatic activities, oil content, Resistance biotic and abiotic stresses

5 Quantitative trait Features... Continuous distribution Involvement of multiple loci (polygenic) Effect of each can be small Environmental effects can be large

6 Reasons for QTL mapping Understanding the genetic basis of a trait(s) QTLs affecting differences in plant height Detecting genes for enzymes and /or regulators of biochemical pathways Genetic variation in secondary metabolism in Arabidopsis Develop molecular markers tags for specific QTLs for use in breeding Preharvest sprouting and ascochyta blight markers To isolate the gene(s) controlling complex phenotypes Functional genomics Vernalization, rust genes in wheat

7 Flow Chart explaining QTL analyses Create a Linkage map with molecular markers Parent A Recombinant Inbred Lines (RILs, F2, F3, Double Haploid Lines) Parent B Genotyping with molecular markers Phenotyping segregating population trait data analysis for each line Linking trait data with marker data Mapping software QTL Trait QTL mapped to a particular chromosome

8 Principles of QTL Mapping Parent allele A Parent allele B Plant height:8cm Plant height:8cm M QTL: Parent B allele increases height by 2.0 cm Plant height:10cm

9 Requirements For QTL Mapping Segregating Population for measurable trait F2, F3, Recombinant Inbred Lines (RILs), Double Haploid Lines Reliable method for measuring the quantitative trait Molecular Markers Restriction Fragment Length Polymorphism (RFLP) Amplified Fragment Length Polymorphism (AFLP) Cleaved Amplified Polymorphic Sequences (CAPS) Simple Sequence repeats (SSR) SNP single nucleotide polymorphisms QTL Mapping Software (Statistical methods) Mapmanager QTX Win Cartographer

10 Development of a mapping population Parent A X Parent B F1 F2 F3 F8 RILs

11 Case Study Preharvest Sprouting Wheat Preharvest sprouting (PHS) is the germination of physiologically mature grains in the wheat spike when excessively humid environments persist prior or during harvest time Francis et al 2008 Imtiaz et al 2008

12 Economic Importance of PHS Major cause of wheat downgrading Yield losses, Decrease in test weight Activation of the enzyme complex in the seed, in particular αamylase Low falling numbers Cost estimated at about $80/tonne Average loss is about $3040 m pa 1

13 Level of PHS Resistance in Wheat Varieties No. of wheat varieties SUN325 Tolerant >0.8 Germination index <2% of 65 wheat varieties evaluated possess PHS tolerance

14 QTLs mapping Develop molecular markers linked to genes conferring PHS resistance (e.g. seed dormancy) in Ae. tauschii / other sources Mapping populations: Synthetic 37 (Syn37) = Altar84*AUS18836 Synthetic 36 (Syn36) = Altar84*AUS18905 Phenotypic assessment Seed dormancy GI Whole spike assay Visual sprouting

15 Phenotyping Need to be Good Germination Index (GI) Germinated seeds are counted daily and expressed as weighted germination index: GI= (7xn1+6xn2+ 1xn7) / total days of test x number of grains in test

16 Phenotyping Continued Sprouting Index (SI) 1. Misting for 48 hours with 30 mins of wetting every 6 hours 2. Score the spikes on the basis of visible sprouting from 1 (no sprouting) to 6 (>90% sprouted)

17 SI Scores: Sprouted/Nonsprouted Score: 1 6

18 Phenotyping Continued Visible sprouted (VI) Dry at 40 0 C, threshed carefully and record percent sprouted seeds.

19 Quantitative Trait Distribution of RILs for Seed Dormancy GI 2 GI 7 GI 14 Syn36 Janz Number of RILs Germination index (GI) Transgressive segregants outside the range of the parental values are evident

20 Related issues can be explored Seed Color Sodium hydroxide (NaOH) Chromameter testing L* refers to the brightness a* is the redgreen component b* is the yellowblue component Red White Diagrammatic representation of the L*a*b (CIE 1976) colour space

21 Distribution of RILs for Seed Colour Janz Syn Syn37 Janz number of BC1RILs number of BC1RILs a/l ratio L Brightness White Red Red White Locus XRC R 2 (%) 40 Mapped at 3DL White grained PHS resistant germplasm identified

22 Genotyping Linked Markers QTLs Mapping Bulk segregant analysis (BSA) Whole genome mapping Syn37 Janz Resistant bulk Susceptible bulk

23 Interval Mapping 4A: Mapping Software

24 Chromosome 3D QTLs on chromosomes 1D, 2D, 5D and 6D were also detected. XRC is red grain colour

25 QTL Identification Imtiaz et al 2008 Epistatic interactions. QTL for whole spike assay QTL for seed dormancy QTL for VI

26 Graphical Genotyping Seed Color Janz Susceptible Syn37 Resistant White Red

27 Ascochyta Blight

28 Maps under Construction

29 Merits of QTL Mapping Identification of novel genes) Can identify New functional alleles of known function genes QTL mapping can help in answering questions like: How many QTLs are responsible for a specific trait? On what chromosome(s) are these QTL located? Are the QTLs for one trait affecting QTL for another (height and yield)? What is the gene action (dominant, recessive) What is the Individual contribution of each gene to the character (additive)? Does epistasis among QTLs play a significant role? Are these QTLs expressed across environments?

30 Limitations of QTL Mapping Mainly identifies loci with large effects Less strong ones can be hard to pursue No. of QTLs detected, their position and effects are subjected to statistical error Small additive effects / epistatic loci are not detected and may require further analyses Need many, polymorphic markers for dense linkage map Cloning can be challenging but not impossible

31 Any Question & Thank you