Cloning drought-related QTLs. WUEMED training course June 5-10, 2006

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1 Cloning drought-related QTLs WUEMED training course June 5-10, 2006

2 Introgression libraries QTL cloning Mapping populations High LD germplasm collections Low LD germplasm collections Functional maps Transcriptomics Proteomics Metabolomics Other omics LD-based analysis QTL mapping Candidate sequence Validation Gene/sequence Positional cloning Genome sequence, bioinformatics Reverse genetics Haplotype and allele mining Marker-assisted selection Improved genotypes Transgenics External resource Process Core genomics procedure or resource Fundamental deliverable Tuberosa and Salvi 2006, Trends Plants Science, in press

3 QTLs cloned in plants (Nov 2, 2005, association mapping not included) Species Trait QTL Refs Arabidopsis Flowering time ED1 El Assal et al, 2001 Flowering time FLW Werner et al, 2005 Glucosinolates struct. GS-elong Kroymann et al, 2001 and 2003 Root morphology BRX Mouchel et al, 2004 Maize Plant architecture Tb1 Doebley et al, 1997 Glume architecture Tga1 Wang et al, 2005 Flowering time Vgt1 Salvi et al, unpubl. (Maize Genet.Meeting 2005) Rice Heading time Hd1 Yano et al, 2000 Heading time Hd6 Takahashi et al, 2001 Heading time Hd3a Kojima et al, 2002 Heading time Ehd1 Doi et al, 2004 Heading time Hd5 Yamanouchi et al, unpubl. (PAG 2005) Grain number Gn1 Ashikari et al, 2005 UV-B resistance quvr-10 Ueda et al, 2005 Regenerability PSR1 Nishimura et al, 2005 Seed shattering qsh-1 Konishi et al, unpubl. (PAG 2005) Salt tolerance SKC1 Ren et al Sorghum Aluminum tolerance Alt SB Kochian et al, unpubl. (GCP, Rome, 2005) Tomato Fruit sugar content Brix9-2-5 Fridman et al, 2000 and 2004 Fruit shape Ovate Liu et al, 2002 Fruit weight fw2.2 Frary et al, 2000 Stigma exsertion Se2.1 Chen and Tanksley, 2004

4 Association mapping Ph, MM High LD collection QTL localized at cm Germplasm collection MM, LD Low LD collection QTL localized at < 1 cm Ph, MM Intermated population Multiparental population MM, Ph MM, Ph, Sy BAC, GS, Sy QTL localized at cm NILs cross QTL localized at < 1 cm Biparental population Coarse mapping QTL Mendelization Fine mapping Physical mapping QTL localized on BAC/genomic sequences Positional cloning Genetical genomics QTL tagging Pops for activation or inactivation tagging Ph Lines with altered phenotype Prb Candidate gene/sequence Reverse genetics Gene complementation Genetic engineering Diversity screen Gene/sequence QTL Modified from Salvi and Tuberosa, 2005, Trends in Plant Science

5 Positional cloning of QTLs in plants x Mapping population Nearly isogenic material Genetic mapping Physical mapping Sequencing and annotation

6 Positional cloning of QTLs in plants Recruitment of meiotic events in a nearly isogenic cross Recruitment of molecular markers Cross between QTL-NILs Identification of F 2 plants with crossovers Identification of homozygous F 3 plants F 4 homozygous segmental isolines x Use of species consensus map Exploitation of synteny with related model species Bulk Segregant Analysis using AFLP, RAPD, SSR, etc. Use of genome sequence and physical map Phenotyping based on replicated experimental design High resolution genetic map QTL fine genetic mapping QTL physical mapping Candidate genes Validation

7 Positional cloning of QTLs in plants: plants and markers Number of chromosomes to be screened to obtain a given resolution 600,000 Number of markers to be screened to find a marker within a given genomic region (in maize) Number of chromosomes Map resolution (cm) Number of markers 550, , , , , , , , , ,000 10% 20% 30% 40% 100% e.g. for maize: average physical/genetic = 1.5 Mb/cM. therefore, one BAC is ca. 0.1 cm. 50, Genomic interval (kb)

8 Candidate gene validation (when you have found some) Overexpression or down-regulation by genetic engineering or RNAi. Use of reverse genetics tools (T-DNA or transposone tagging, TILLING) Genic complementation or mutant characterization Gene replacement (!) Correlation among data of linkage and association mapping (!) Correlation among phenotype and pattern (time, tissue, genotype) of gene expression at either the RNA and/or protein level (!) Correlate with association mapping studies (see below)

9 Association mapping Search of statistical association between allelic variants at markers/genes and phenotype, across germplasm (e.g. cultivars, wild accessions, etc). Relies on historic linkage disequilibrium (LD) instead of experimentally created LD Two approaches: Whole-genome association mapping Association analysis at candidate genes You need to know the level of LD in your species, population, genomic region

10 Detection of LD for association mapping Mod. from Rafalski A., 2002, Curr Opin Plant Biol

11 LD extent and resolution of association mapping LD declines slowly Fewer markers are needed for QTL detection Lower mapping resolution is allowed LD declines rapidly Many markers are needed for QTL detection High mapping resolution is allowed Mod. from Rafalski A., 2002, Curr Opin Plant Biol

12 LD in plants LD values can vary tremendously among species, intraspecific germplasm collections and genomic regions LD (r 2 ) decay over bp in six maize genes (Wilson et al Plant Cell) Maize: up to 2 kb (collection including landraces) or > 600 kb (collection of elite inbred lines) Arabidopsis: from 100 kb to cm distances Rice: up to 100 kb in cultivated rice Barley: up to tens of kb in wild accessions and landraces; >200 kb in cultivated varieties.

13 Comparison of mapping power among approaches da: Flint-Garcia et al., 2003, Ann Rev Plant Biol

14 QTL mapping vs Whole-genome association mapping Experimental cross required Phenotype to be collected Limited mapping resolution (exceptions) Essentially two alleles tested Constraint to segregating loci between parental lines High detection power No experimental crosses are required, works with existing germplasm (Potentially) high mapping resolution More than two alleles tested Many loci for a single trait are concurrently analysed Phenotypic data can be already available Statistical and analytical problems (e.g. no gold-standard for significance tests; presence of hidden population structure, etc) When LD is low, too many markers would be required

15 General considerations QTLs positionally cloned almost invariabily had r 2 >15% of total phenotypic variability in primary mapping (but genetic effect can change during isogenization due to epistasis!). So be ready to validate the QTL effect into QTL-NILs. Only in a few cases an early candidate gene was available (maize Tb1, Arabidopsis FLW and GS-elong). More commonly a candidate become apparent at physical mapping. Any type of gene have been found behing QTLs (from metabolic enzymes to transcription factors). At least three examples (tomato fw2.2, maize tb1 and Vgt1) of regulatory regions involved (both promotore-like and enhancer like). Usually fewer recombinants have been utilized than predicted by theory (most cloned QTLs reside in highly recombinogenic region, although you cannot tell from the beginning: maize Tga1 is 2 cm from centromere!) QTN (Quantitative trait nucleotide) are mapped so far extremely close to the original QTL likelihood-profile peak observed in primary mapping.

16 Positional cloning of Vgt1 (Vegetative to generative transition 1), a QTL for flowering time in maize

17 Relatioships between developmental stages and drought-stress events in maize F M Yield decrease (%) per day of drought-stress Days from sowing F: Flowering M: Maturity

18 Vgt1: Vegetative to Generative Transition 1 C22-4 N C22-4 N28 C22-4 N28

19 Positional cloning of Vgt1 Production and characterization of meiotic events Identification of molecular markers N28 NIL-1 NIL-2 x C22-4 Ca F 2 plants Selected F 3 plants High throughput marker analysis BSA with AFLPs F 4 homozygous segmental isolines Replicated field trials High resolution genetic map High resolution QTL analysis Physical mapping Candidate genes Validation

20 Mendelization of Vgt1 C22-4 N28 DPS h 2 = 0.83 Node no. h 2 = 0.97 Salvi et al., 2002 Plant Mol Biol 48:

21 Physical mapping of Vgt1 AFLP13 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 Vgt1 Node number AFLP N28 C22-4 R43 R48 R6 R5 R40 R41 R31 R66 R70 R38 R58 R22 R19 R67 R50 R33 R42 Vgt1: Ca. 2 kb (non-coding) No hits in Genbank using standard BLASTs 27 SNPs and indels Salvi et al., 2006, unpublished

22 CMV3 A619 A6 Gt112 Salvi et al., 2006, unpublished

23 Genotyping of inbreds at Vgt1 and nearby genomic regions 192 markers N inbreds Vgt1 C kb kb Salvi et al., 2006, unpublished

24 Positional cloning of Root-ABA1 a QTL controlling root architecture and concentration of L-ABA in maize

25 QTL analysis for L-ABA in the F3:4 cross Os420 x IABO78 Root-ABA1 Tuberosa et al., 1998, TAG

26 7 6 Os420 x IABO78, S3/94 S4/94 S3/95 S4/95 MEAN Chr. 2 LOD ((!"#$%%&''

27 01& 1! # ) "!&*&# + %,-!.*.# + / ) "!.*.# + / %,-!&*&# +

28 01& 2 &)/ 1!2 )#! # ) "!&*&# + %,-!.*.# + / ) "!.*.# + / %,-!&*&# + Landi et al., (2005) Mol Breed.

29 2 &)/ 3 41!22 )3 # ) "!&*&# + %,-!.*.# + / ) "!.*.# + / %,-!&*&# + Landi et al., (2005) Mol Breed.

30 L-ABA +/+ L-ABA -/-

31 ! 41#!/ 4# 6 0/48 %,- ) "!/ 41#! 4# /! 56 7#!/ 56 7# 1// / 1// / 1 /.*. &*&.*. &*& 5 / %,- + )

32 A maize introgression library from the cross Gaspé Flint x B73

33 B73 F 1 (B73xGaspé) Gaspé

34 Differences in seminal root morphology in a simple hydroponic system (paper roll) Salvi et al., 2006, unpublished

35 IL 1-1 Introgression library Chrom n IL IL n-4

36 B73 Gaspé Flint X BC 3 SSR analyses BC 4 Backcross (winter 2001/02) B73 X F 1 SSR analyses BC 5 x Chrom. 1 BC 5 F 2 plants ¼ ½ ¼ 88 BC 1 (2004) (summer 2002) (BC 5 F 3 ) IL Chrom

37 Towards the completion of an introgression library in maize Summer 2006: set of 70 homozygous introgression library lines in the field (4 reps) for evaluation of flowering time and related traits and analysis of seminal roots in paper roll. Chromosomes/markers BC5 lines

38 Masle et al., 2005, Nature, 436, 866 QTL supporting interval covered a region of 37 genes, including the Erecta gene, a Leucine-rich repeat receptor like kinase (LRR-RLK) = (candidate gene) Lod score profile for rosette Delta (Carbon isotope discrimination) collected on a RIL population Col (ERECTA) x Ler (erecta). R 2 : 21-64%

39 Complementation analysis Lower SC Higher SC

40 Genetical genomics Jansen and Nap, 2001, TIG

41 Diversity screen A population genetics approach to the identification of genes underlying traits of agronomic importance Based on the assumption that genes important for domestication and breeding have experienced reduction of genetic diversity due to artificial selection Such genes would be missed by standard QTL or association mapping because they are expected to be little polymorphic or fixed in cultivated germplasm Applied in maize only (availability of wild ancestor, clear subdivision in landraces and elite material, high standards of genomics tools)

42 Effect of domestication and plant breeding on genetic diversity of maize genes From Yamasaid et al, 2005, Plant Cell