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Journal of Heredity 2008:99(4):396–406 doi:10.1093/jhered/esn014 Advance Access publication February 28, 2008 Ó The American Genetic Association. 2008. All rights reserved. For permissions, please email: [email protected]. Construction and Analysis of 2 Reciprocal Arabidopsis Introgression Line Populations OTTÓ TÖRJÉK*, RHONDA C. MEYER*, MAIK ZEHNSDORF, MELANIE TELTOW, GEORG STROMPEN, HANNA WITUCKA-WALL, ANNA BLACHA, AND THOMAS ALTMANN From the Department of Genetics, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany (Törjék, Teltow, Strompen, Witucka-Wall, and Altmann); and the Max-PlanckInstitute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Golm, Germany (Meyer, Zehnsdorf, Blacha, and Altmann). Ottó Törjék is now at Biological Resource Center, Institute of Plant Biology, Cell Division Cycle and Stress Adaptation Group, Temesvári krt. 62, PO Box 521, H-6701 Szeged, Hungary. *These authors contributed equally to the work. Address correspondence to R. C. Meyer at the address above, or e-mail: [email protected]. Abstract Two new large reciprocal sets of introgression lines (ILs) were created between the Arabidopsis accessions Col-0 and C24. In both sets (78 ILs with Col-0 background and 62 ILs with C24 background), the donor segments cover almost the entire genome with an average substitution size of 18.3 cM. In addition to the basic sets of ILs, further subILs were developed for 2 genomic regions allowing better mapping resolution. SubILs carrying donor segments with candidate genes for flowering time and reduced fertility were used to demonstrate the usefulness of the reciprocal ILs for quantitative trait loci detection and fine mapping. For subIL development at high resolution around the reduced fertility locus, we used modified CelIbased assays in one-well format for both marker development and genotyping. This serves as a very flexible and costeffective approach. Mapping quantitative trait loci (QTL) based on segregating populations provides only rough positions of QTL (Kearsey 2002). Once QTL have been identified for a trait under study, it becomes important to characterize and confirm the contributing individual loci (Koornneef et al. 1997). One of the most promising ways to do this is the use of introgression lines (ILs). Lines of an introgression library have a common recurrent genotype, but different, short, donor segments from another line giving the ability to focus precisely on any region of the genome (Eshed and Zamir 1995). These lines have a permanent homozygous genetic constitution enabling also tests in different environments and at different time points (Eshed et al. 1992). ILs with minimized residual genetic variation allow the transformation of the individual QTL into single Mendelian factors. Using the ILs, it is possible to study nonallelic gene interactions of QTL regions with the genomic background. Furthermore, crosses between ILs each bearing nonallelic individual QTL can be combined to analyze the extent of interactions (Peleman and Rouppe van der Voort 2003). In 396 addition, creating segregating offspring for the substitution under study, ILs facilitate fine mapping and may lead toward gene discovery (Zamir 2001). The usefulness of ILs in finemapping QTL has already been demonstrated in various plant species (tomato—Frary et al. 2000; Fridman et al. 2000; rice—Takahashi et al. 2001; maize—Salvi et al. 2002; Arabidopsis—El-Assal et al. 2001; Bentsink et al. 2003). In Arabidopsis, a publicly available set of chromosome substitution strains (CSS) (Col-0 chromosomes were replaced by Ler and Nd) as well as ‘‘stepped aligned inbred recombinant strains’’ harboring donor segments of increasing length in a single chromosome or ‘‘stepped single inbred recombinant strains’’ carrying only one crossing-over event were created by Koumproglou et al. (2002). Recently Keurentjes et al. (2007) reported the development of a genome-wide (nonreciprocal) IL population between Arabidopsis accessions Ler and Cvi. In high-resolution mapping, trait complexity and number of identified recombination events, as well as sufficient marker density are key factors (Peleman et al. 2005). For Törjék et al. Reciprocal Arabidopsis IL Populations species with well-analyzed genomes such as Arabidopsis, large numbers of markers are usually available if standard combinations of mapping lines (especially Col-0 and Ler) are used (Cho et al. 1999; Jander et al. 2002; Schmid et al. 2003). In case of lines with less well-characterized genomes, additional work may be necessary to develop markers for high-resolution mapping, requiring further costs and time for identification of new polymorphisms and establishment of appropriate techniques for detection. In the present work, we report the creation of a new large set of reciprocal ILs between the Arabidopsis accessions Col-0 and C24 covering the entire genome. For the top of chromosome IV and the bottom of chromosome V, additional sets of subILs were created. These latter lines were used to demonstrate the applicability of these sets of ILs to locate a major gene for flowering time (subILs for top of chromosome IV) and to fine map a locus causing reduced fertility (subILs for top of chromosome V). With respect to fine mapping, we present a highly efficient and costeffective approach based on CelI endonuclease cleavage in one-well format, which can be used for both identification of polymorphisms and genotyping at a high-resolution level. Materials and Methods DNA Isolation, Genotyping, and Primer Development All DNA samples were isolated using the procedure described in Törjék et al. (2006). Matrix-assisted laser desorption/ionisation-time of flight (MALDI-ToF) genotyping (analyzed by GAG-Bioscience GmbH, Bremen, Germany) and SNaPshot analysis were performed as described in Törjék et al. (2003) and Schmid et al. (2006). Primers were designed using Oligos 8.8 (Kalendar 2001; http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm) or Primer3 (Rozen and Skaletsky 2000). Oligonucleotides used for IL and subIL development are listed in Table 1. One-well CelI Assays for Marker Development and Genotyping Polymerase chain reaction (PCR) amplification was carried out in a volume of 12.5 ll containing 15–30 ng genomic DNA. PCR products were labeled using Cy5.5 deoxycytidine triphosphate, (dCTP; GE Healthcare, Munich, Germany). Reaction mixtures contained equal amounts of fluorescently labeled and nonlabeled dCTPs. PCR was conducted using a thermal cycler (MJ Research, Waltham, MA) as follows: 94 °C for 2 min followed by 40 polymerization cycles (94 °C for 10 s, 58 °C for 30 s, 2 °C for 3 min) and finally extension, denaturation, and reannealing steps (72 °C for 5 min, 99 °C for 10 s, 70 °C for 20 s, followed by 70 cycles of 70 °C for 20 s decreasing 0.3 °C per cycle). After PCR amplification, samples were digested with 1 ll CelI enzyme (Oleykowski et al. 1998) for 30 min at 45 °C. The digestion was stopped with 2 ll 0.5 M ethylenediaminetetraacetic acid (EDTA). Samples were precipitated with 15 ll isopropanol, washed with 20 ll of 70% ethanol and finally resuspended in 4 ll distilled water. For analysis on the LI-COR 4300 DNA analyzer (Lincoln, NE), 0.5 ll of a sample were mixed with 2 ll formamide loading buffer (33% deionized formamide, 10 mM Tris, pH 7.5, 1 mM EDTA, and 0.02% bromphenol blue) and denatured at 95 °C for 4 min and 4 °C for 5 min. Samples were loaded onto 100-tooth membrane combs (LI-COR) then electrophoresed through a polyacrylamide gel (6.5% KB Plus, LI-COR) in 1 Tris/Borate/EDTA running buffer at 1500 V, 40 mA, and 40 W. Plant Materials and SubIL Development Col-0 was initially obtained from G. Rédei (University of Missouri-Columbia, USA) and C24 from J. P. Hernalsteens (Vrije Universiteit Brussels). As a base population for IL development, we developed 2 sets of reciprocal (82 Col-0 C24/96 C24 Col-0) BC3F1 lines using the single-seed descent method. The BC3F1 lines were genotyped with a set of 111 framework markers established for MALDI-ToF analysis. Based on the genotyping data, we identified lines with favorable segments to produce substitutions in both Col-0 and C24 genomic backgrounds using marker-assisted selection. This work was organized in a stepwise procedure taking into account the genomic composition of the assayed lines: 1) 8 (1 segregating region) or 16 (2 unlinked segregating regions) descendants were analyzed for the relevant marker positions using SNaPshot assays; 2) if the segregating segment was large in the analyzed plants, the most suitable lines were backcrossed to the recurrent parent with the aim to reduce the size of the donor segment; 3) if an individual plant contained a heterozygous segment of the appropriate size, the plant was selfed for creation of homozygous substitution lines, which were then selected in the next generation; 4) finally lines carrying homozygous donor substitutions were seed bulk amplified. The complete set of ILs and their genotype data are available from the authors on request (by the time of publication) based on a simple material transfer agreement. For subIL development, we used the following initial lines: M63 (BC3F1–C24 background, heterozygous for markers between MASC04551chr.III and MASC09219chr.III and from MASC04123chr.IV to MASC04685chr.IV) and N88 (BC3F1– Col-0 background heterozygous for markers MASC03898chr.III, MASC02788chr.III and from MASC04123chr.IV to MASC02668chr.IV) for creation of subILs at the top of chromosome IV and N70 (BC3F1–Col-0 background heterozygous for markers MASC09211chr.V and MASC04350chr.V) for creation of subILs at the bottom of chromosome V. During the development of subILs, up to 300 descendants of one segregating line were involved in marker analysis and ILs were detected up to BC5F4 generation. Plants were grown in 1:1 mixture of GS 90 soil (Gebrüder Patzer, Sinntal-Jossa, Germany) and vermiculite, under a long-day regime (16 h fluorescent light at 20 °C and 60% relative humidity/8 h dark at 18 °C and 75% relative humidity. Phenotypic Analyses Flowering time was recorded as the number of days from the date of planting until the opening of the first flower. 397 Journal of Heredity 2008:99(4) Table 1. Markers used in this study Set/marker Chr. pos Primer (5#–3’) Amplicon Polymorphism size (bp) (C24/Col) Remarks a Described in Törjék et al. (2003); Schmid et al. (2006); Törjék et al. (2006) b MASC04036 103508 MASC07015 145029 uj26uj34 269893 F-CTCCACGGACACCTTGCTCC R-AGCGAGCTAGCGAAGGAACC S-TTTTTTCATGATCCGATTCAAC ACTTCTCA F-TCGAAACCCTCCGTCGCCAGC R-AATTGAAGAAGCCGAAAAGACC S-TTTTTTTTTTTTTTCTTTTCTTCAC CGGAGAATCACGACA 586 T/C 199 T/A 241/225 241/225 Johanson et al. (2000) c MASC06738, MASC07101, MASC01090, K1F13/52-53, MDD7, K9I9/43-44 MDD3 26757349 F-TGCAGCCTAATCTTCTCGTCG R-CCTTGTTTGCTTTGGTTTCTCG Described in Törjék et al. (2006) 305/298 305/298 d At5g66210b At5g66210a SGCSNP18949 26473368 F-TGTCGTTAGGCAGATGCTCAAA GTTGC R-GCTGCTCTTGACCTTAGCTGCCA TTTC 26474228 F-ACTCCCATCGATACCAAGGCCTC TACC R-CCCAAGGTTTACGGCTGAAGTC AGGTT 26492640 F-GGTAAGTCCGAACCGAACAA 1499 CelI products — 1498 CelI products — CelI products Known SNP in amplified region (Col-0/Ler); TAIR/ Polymorphism: 1005368036 18 SNPs and 1 INDEL described in the amplified region for Col-0/Wei-0 — R-AAGAAATGATTGGGCACGTC K1L20/19–21 26499030 F-GACTCCAAATTTGGCGGTGG 1350 R-AATTTGTGTTTAATTTCACGGGAC Not detected AT5g66320 26512991 F-CGTAATTGAGTTTCCCCGGTCG TCTTT R-ATGTCGCGTGACCATGAAACAA AACAT 26522800 F-CGGGATACGCTCTCTCTGTC 1749 CelI products 1115 CelI products Known SNP in amplified region (Col-0/Ler); TAIR/ Polymorphism: 1005368037 1042 CelI products Known SNP in the amplified region (Col-0, Cvi-0, Ei-2/ Nd); MASC SNP DB: MASC01070 SGCSNP18950 R-ACCGCAGGATCTAAAACACG K1F13/7–8 398 26534822 F-CCCTGTCATGGGATTGATATGA ACACG R-GAATGCGACTGCGTTACAAACC CTCAT Törjék et al. Reciprocal Arabidopsis IL Populations Table 1. Continued Set/marker At5g66400 At5g66440 SM97 Amplicon Polymorphism size (bp) (C24/Col) Remarks Chr. pos Primer (5#–3’) 26535341 F-TCAAATTCACCCAATCAACGACG ATGT R-TCTCTCCATTTCATAGGCCCAAG CATC 26548477 F-GGGCACAAGTGGTGTCTACATG AGGAA R-TCTGGATTGCCTTCTGGAAATTG TAGG 26552058 F-TCCACAAGCCCACCTACAATTTC CATT R-CAACGTGCAGTTGGTCTCATTCA CAAA 1523 CelI products 1463 CelI products 1361 CelI products AFLP in amplified region (Col-0, Cvi/ C24, Ler, Ws) a: Framework marker set; b: Additional markers for the top of chromosome IV; c: Additional markers for the bottom of chromosome IV; d: Oligonucleotides used for CelI-based assays; base positions on chromosomes: MIPS annotation. Version 171102 (Schoof et al. 2002). Chr. Pos.: chromosome position of SNP or first base of the amplicon, Polym.:Polymorphism; AFLP: amplified fragment length polymorphism. Fertility was determined visually based on the amount of pollen produced. Per line 3 flowers were analyzed under the stereomicroscope. Plants with reduced fertility had flowers with no or reduced amount of pollen in comparison to the plants with normal flowers (Törjék et al. 2006). Data Visualization and Statistical Analyses Genotypic data were visualized using the software Graphical GenoTypes (Van Berloo 1999). Statistical analyses were performed with Genstat for Windows V6.1 (Payne et al. 2002). For comparisons between genotypes, the analysis of variance procedure AUNBALANCED was used with genotype as fixed factor. Significant differences were detected using Fisher’s least significant difference at the 1% level. Means were calculated using PREDICT. Results and Discussion Basic Sets of Reciprocal ILs To obtain starting populations with a reduced amount of donor segments in individual lines but still covering the whole genome, we genotyped sets of 82 Col-0 C24 and 96 C24 Col-0 BC3F1 lines using framework single nucleotide polymorphism (SNP) markers established for MALDI-ToF analysis. Genotyping of these lines revealed a complete coverage of the genome with Col-0 and C24 donor segments in both reciprocal BC3F1 populations. For each marker position, we observed heterozygous segments in 9– 25 individual lines in Col-0 C24 BC3F1 population and 8– 38 lines in C24 Col-0 BC3F1 population. The overall proportions of heterozygosity were 18.7% for Col-0 C24 BC3F1 and 19.5% for C24 Col-0 BC3F1, which is higher (P , 0.001, chi-square test) than the expected 12.5%. Given the occurrence of heterosis observed in this cross (Meyer et al. 2004), plants with higher heterozygosity might possess higher fitness and they could have been unwittingly selected for single-seed descent breeding. For the marker-assisted development of homozygous ILs, we selected only lines with favorable genomic composition (30 C24 Col0 BC3F1 and 37 Col-0 C24 BC3F1) and processed them as described in Materials and Methods. We obtained 2 homozygous basic sets consisting of 78 ILs with C24 donor segments in Col-background and 62 ILs with Col0 introgressions in C24 background (Figure 1, Table 2). Most of the created ILs contain only 1 donor segment. A second donor segment is present in 13/78 (16.7%) ILs with Col-0 genomic background and 9/62 (14.5%) ILs with C24 background. ILs containing only 1 donor segment are suitable for ‘‘standard’’ QTL analyses. Dual ILs may be useful to separate different types of epistasis between the introgressed chromosomal segments and the genetic background (Kusterer et al. 2007). The reciprocal IL sets cover the entire Col-0 and C24 genomes with overlapping substitutions, with 2 exceptions: in the set of ILs with C24 donor segments, a gap exists between markers MASC04685 and MASC03275, which are separated by 3731 kb on chromosome IV; and in the set of ILs with Col-0 donor segments, there is no overlap for markers MASC03930 and MASC03765 on chromosome I. Because the region between these 2 markers was not analyzed with further markers, we cannot exclude a possible overlap of the donor segments for the region flanked by these 2 markers. Average donor segment sizes covered 4.47 (range: 1–14) and 4.98 (range: 1–16) marker positions in ILs with Col-0 and C24 backgrounds, respectively. According to an average distance of 3.87 cM between 2 adjacent markers (Törjék et al. 2006), these regions correspond to donor segments of 17.30 and 19.27 cM, respectively. Validation: the New ILs and SubILs Highlight Interaction between the Floral Regulators FRI and FLC To demonstrate the applicability of the ILs for (fine-) mapping of QTL, we developed additional subILs for top of 399 Journal of Heredity 2008:99(4) Figure 1. Graphical genotypes of the basic sets of ILs. 78 lines (N lines) with C24 substitutions (a) and 62 lines (M lines) with Col-0 substitutions (b) were created. ILs were developed using 111 framework SNP markers, which were evenly distributed over the 5 Arabidopsis chromosomes. For a detailed description of the individual lines, see Supplementary Table 1a. The description of the sets is summarized in Table 2. Genotypes: yellow—homozygous Col-0, green—homozygous C24. The 5 Arabidopsis chromosomes are indicated with roman numerals. The graphical genotypes were created with Graphical GenoTypes (Van Berloo 1999) using physical genomic positions. An up-to-date, searchable file containing the names and genotypes of all ILs and subILs is available from the authors upon request. chromosome IV around the FRIGIDA gene (FRI, At4g00650), a well-known major determinant of natural variation in Arabidopsis for flowering time (Johanson et al. 2000). For better mapping resolution, we extended the framework markers with 2 additional markers (Table 1) close to the FRI gene. A third additional marker detects a deletion polymorphism in the gene itself (Johanson et al. 2000). The subILs developed are shown in Figure 2 (11 subILs with Col-0 donor segments: 2a and 8 lines with C24 donor segments: 2b). Flowering times measured in the genotyped sets of segregating populations (Table 3) revealed 4 categories depending on the genotypic composition of the lines (Table 4). Segregating sets of lines for genomic regions above and below the FRI gene (but not for FRI) such as the 400 descendants of M63/9/1/1 (BC4F4), N88/2/7 (BC5F2), and N88/2/9 (BC5F2) fell into only one category (A or C) of flowering time depending on genotypic composition (Table 3). The offspring of lines M63/9 (BC4F2), M63/9/2 (BC4F3), M63/9/34 (BC4F3), N88/2/1 (BC5F2), N88/2/14 (BC5F2), and N88/2/1/17 (BC5F3), which segregate for the FRI gene itself, always showed 2 clearly distinguishable peaks for flowering time (Table 3). The drastic changes measured in flowering time underpin the hypothesis that the single-copy gene FRI is the candidate gene for this genomic region. Functional FRI alleles cause plants to delay flowering if not subjected to vernalization. Nonfunctional FRI alleles (such as the Col-0 allele) cause plants to flower rapidly (NappZinn 1987; Johanson et al. 2000). The transgressive Törjék et al. Reciprocal Arabidopsis IL Populations Table 2. Summary of the basic IL sets No. of ILs No. of lines with double substitutions Total no. of substitutions Average segment size Coverage Col-0 background (N lines) C24 background (M lines) 78 13 91 4.47 A gap between the markers: MASC04685 and MASC03275 62 8 70 4.98 A missing overlap for adjacent markers: MASC03930 and MASC03765 segregation observed (presence of 4 highly significant groups of flowering time) suggests a strong interaction of this region with the genomic background. Functional FRI alleles enhance the expression of Flowering Locus C (FLC, At5g10140), a flowering repressor located on chromosome V (Henderson et al. 2003). Werner et al. (2005) reported differences between FLCCol-0 and FLCC24, with the ‘‘weak’’ allele assigned to C24. An important feature of the Col-0/ C24 IL population introduced here is the existence of 2 reciprocal sets. Using reciprocal ILs allowed us to determine the epistatic interactions between FRI and FLC alleles coming from the same source (Col-0/Col-0 or C24/C24) or from different sources (Col-0/C24 or C24/Col-0). The genotype-dependent flowering times measured in IL populations segregating for FRI (Table 4) corroborate published data that the interaction between FRI and the FLC plays an important role in the regulation of flowering time (Shibaike et al. 1999; Caicedo et al. 2004; Le Corre 2005; Shindo et al. 2005) and highlight the advantage of using 2 reciprocal sets of ILs. In Arabidopsis, ILs have been used successfully to verify individual QTL for flowering time (Koumproglou et al. 2002; Werner et al. 2005), carbon stable isotope ratio d13C (Juenger et al. 2005), root system size (Gerald et al. 2006), or phytate and phosphate accumulation (Bentsink et al. 2003). None of these dedicated Arabidopsis IL collections provide full genome coverage. Populations covering the full genome have been developed for tomato and rice (Eshed and Zamir 1995; Canady et al. 2005; Xi et al. 2006). Keurentjes et al. (2007) established a genome-wide (nonreciprocal) IL population between Arabidopsis accessions Ler and Cvi and demonstrated the complementarity of RIL and IL populations for QTL analysis. Other publicly available genome-wide sets for Arabidopsis are the CSS (Koumproglou et al. 2002) in which whole Col-0 chromosomes were replaced by Ler and Nd chromosomes and the SSRLs (Koumproglou et al. 2002) with smaller substituted segments. Fine Mapping of Reduced Fertility Locus at Bottom of Chromosome V After the successful demonstration of effects and interactions of previously characterized loci, further analyses were carried out to investigate interacting loci that cause reduced fertility. Thus, another subIL set targeting the genomic region associated with reduced fertility in RILs Total 140 21 161 4.73 Figure 2. Graphical genotypes of subILs developed for the top of chromosome IV. 11 lines (M lines) with Col-0 substitutions (a) and 8 lines (N lines) with C24 substitutions (b) were created. All M lines have an uniform homozygous C24 and all N lines an uniform homozygous Col-0 genetic background. Genotypes: yellow—homozygous Col-0, green—homozygous C24. M lines (a): 1—M63/9/1/2, 2—M63/9/1/6, 3—M63/9/34/8, 4—M63/9/38, 5—M63/9/40, 6—M63/9/51, 7—M63/9/2/5, 8—M63/9/2/8, 9—M63/9/2/12, 10—M63/9/1/1/16, 11—M63/9/1/1/18. N lines (b): 1—N88/2/14/13, 2—N88/ 2/14/21, 3—N88/2/1/10, 4—N88/2/1/31, 5—N88/2/9/15, 6—N88/2/7/1, 7—N88/2/7/27, 8—N88/2/1/17/16. 401 Journal of Heredity 2008:99(4) Table 3. Genotype-dependent flowering times measured in IL populations segregating for FRI and surrounding area: genotypic composition of the parental lines Segregating markers and their physical positions MASC02820—IV/41642 MASC04036—IV/103508 MASC07015—IV/145029 uj26uj34—IV/269893 MASC04123—IV/300135 MASC04725—IV/1091297 MASC05042—IV/2187167 MASC09212—IV/2905887 MASC04685—IV/4436613 M63/9* M63/9/1/1 M63/9/2* M63/9/34* N88/2/1* N88/2/7 N88/2/9 N88/2/14* N88/2/1/17* BC4F2 BC4F4 BC4F3 BC4F3 BC5F2 BC5F2 BC5F2 BC5F2 BC5F3 ab ab ab ab ab ab ab ab ab ab ab a a a a a a a ab ab ab ab ab b b b b ab ab ab ab ab a a a a ab ab ab ab ab ab a a a ab ab ab a a a a a a a a a a a a a a ab a a ab ab ab ab ab ab ab a a ab ab ab ab ab ab ab Selfing descendants of these lines were used for both subIL development and measurement of flowering time. The genotypes are indicated as follows: homozygous Col-0 (a); homozygous C24 (b); heterozygous C24/Col-0 (ab). Lines segregating for FRI (uj26uj34) are highlighted with asterisks. Lines with M prefix have uniform homozygous C24 genomic backgrounds and lines with N prefix homozygous Col-0 background. (Törjék et al. 2006) between MASC01090 at 26469329 bp and K1F13/52–53 at 26580794–26581516 bp was created. Due to a previous lack of known polymorphisms between Col-0 and C24 in this 112-kb genomic region, we developed additional markers by adapting assays based on CelI endonuclease cleavage. The CelI enzyme from leaf celery (Apium graveolens) has the capacity to cleave all types of single base-pair mismatches and small insertions/deletions in heteroduplex DNA molecules (Oleykowski et al. 1998; Till et al. 2004). The modified CelI-based assays for marker development were performed in one-well format incorporating Cy5.5 dCTP (Amersham, München, Germany) dyes for LI-COR gel-detection system (Supplementary Figure 1). The 10 primer pairs designed for this 112-kb region amplified on average 1260 bp in the reference Col-0 DNA (Table 1). All primer pairs were used to amplify Col-0, C24, and Col-0 C24 F1 DNA samples and after heat denaturation and reannealing subjected to CelI treatment. All amplicons lead to cleaved products in heteroduplexes (data not shown). These 10 primer pairs allowed an average marker resolution of 8.7 kb in the covered genomic region and were thereafter used for high-resolution genotyping. To this end, 23 recombinant descendants of N70-BC3F1 (homozygous Col-0 background, segregating only for the bottom of chromosome V for the last 2 framework markers: MASC09211 and MASC04350) were included in the analysis. Based on the genotypes detected by CelI assays (heterozygous or homozygous), we created a high-resolution physical map for the 23 individual plants. The CelI approach does not distinguish between homozygous Col-0 and homozygous C24 genotypes; therefore, we determined the genotypes iteratively, based on the genotype of the adjacent marker, Table 4. Genotype-dependent flowering times measured in IL populations segregating for FRI and surrounding area: flowering time measured in the genotyped segregating populations revealed 4 categories depending on the genotypic composition of the lines Group Genotypic composition Flowering time ± standard error No. of analyzed plants (lines?) A C24 background with FRICol-0/FRICol-0 30.125 ± 0.740a 96 B C24 background with FRICol-0/FRIC24 or FRIC24/FRIC24 Col-0 background with FRICol-0/FRICol-0 49.2306 ± 0.725b 100 38.416 ± 0.600c 146 Col-0 background with FRICol-0/FRIC24 or FRIC24/FRIC24 107.016 ± 0.906d 64 C D Homozygous subILs developed and their allocation to the different flowering time groups SM(MASC02820–MASC04685)—M63/9/38 BC4F3 SM(MASC02820–MASC04123)—M63/9/2/5 BC4F4 SM(MASC02820–MASC04036)—M63/9/1/1/16 BC4F5 SM(MASC04725–MASC04685)a—M63/9/34/8 BC4F4 SM(MASC04725–MASC04685)b—M63/9/1/2 BC4F4 SN(MASC04725)—N88/2/1/31 BC5F3 SN(MASC02820–MASC07015)a—N88/2/7/1 BC5F3 SN(MASC04685)—N88/2/9/15 BC5F3 SN(MASC02820–MASC07015)b—N88/2/1/17/2 BC5F4 SN(uj26uj34–MASC04685)—N88/2/14/13 BC5F3 SN(MASC02820-MASC04725)—N88/2/1/10 BC5F3 Flowering times are means calculated in analysis of variance and given in days to flowering ± standard error of means. Significantly (least significant difference at 1% level) different means are indicated by different letters. 402 Törjék et al. Reciprocal Arabidopsis IL Populations Figure 3. Graphical genotypes of subILs developed for the bottom of chromosome V. Five lines (N lines) with C24 substitutions (a) were created. All N lines have an uniform homozygous Col-0 genetic background. The lines N70/106/20, N70/ 118/23, N70/70/11, and N70/110/4 show normal fertility, whereas line N70/106/17 shows reduced fertility. The fertile lines share a 56-kb segment not covered by C24 introgressions, indicating the location of the gene causing reduced fertility. The lines were genotyped using CelI. The homozygous Col-0 and C24 genotypes were determined iteratively using an additional check for genotypes: DNA samples of the subILs were analyzed either unmixed, mixed with Col-0 DNA, or mixed with C24 DNA, amplified, and CelI cleaved for the markers At5g66210a (b), K1F13/7-8 (c), and At5g66440 (d). Comparing the 3 fingerprints, we could confirm the genotypes determined in the previous steps. Genotypes: yellow—homozygous Col-0, green—homozygous C24. N lines (a–d): 1—N70/106/20, 2—N70/118/23, 3—N70/70/11, 4—N70/110/4, 5—N70/106/17, 6—Col C24 F1 (b–d). The mixed DNA samples and the heterozygous control DNA of Col C24 F1 showing CelI cleavage are indicated with asterisks. Cleavage products are indicated with arrowheads. taking advantage of the close physical distances (Supplementary Figure 2). With the high marker resolution present in the ILs, the average marker resolution of 8.7 kb genomic region corresponds to 0.032 cM based on the average recombination frequency of 274.23 kb cM 1 (measured in C24/Col-0 RILs previously, Törjék et al. 2006). Assuming independent crossovers and no interference, double crossover (DCO) events occur at very low frequencies (;10 5 cM 1). Even allowing for a DCO frequency of 10 3 cM 1 in the mapping population, the iterative approach could be applied for genomic regions of up to 82 kb. For subIL development, 4 BC3F2 lines (N70/70, N70/ 106, N70/118, and N70/110) and their selfed progenies were analyzed using the same assays. This allowed us to identify and select 5 subILs (Figure 3). The line N70/106/ 17 (BC3F3) contains a C24 introgression covering the entire region under study and has a reduced fertility phenotype. The fertile BC3F3 lines N70/106/20, N70/ 118/23, N70/70/11, und N70/110/4 have differently placed substitutions, but with a 56-kb gap between SGCSNP18949 and At5g66440. Use of the subILs therefore allowed us to confirm the presence of the QTL causing reduced fertility in the marker interval defined by the RIL analysis and to rapidly narrow down the region associated with reduced fertility from initially 112 kb to 56 kb. 403 Journal of Heredity 2008:99(4) To verify the results of the initial CelI analyses, the DNA samples of the subILs were mixed with either Col-0 or C24 reference DNAs. This analysis allowed the detection of all homozygotes as well as heterozygotes (Figure 3) and confirmed the genotypes determined in the earlier steps. Currently, plant endonucleases such as the CelI enzyme are mostly used for large-scale identification of point mutations in mutagenized plant populations termed as Targeted Identification of Local Lesions IN Genomes (TILLING) (McCallum et al. 2000). In analogy to TILLING, this technique can also be applied to detection and distinction of natural variation in genes, termed ecoTILLING (Comai et al. 2004; Gilchrist et al. 2006) where creation of heteroduplexes will be allowed by mixing of ecotype DNAs to a reference DNA sample. The use of this procedure in genotyping and mapping is limited because it allows only the separation of heterozygous from homozygous data points, whereas the homozygous parental genotypes are not directly distinguishable. A further limitation for this technique are the high costs associated with the fluorescent primer labeling required for the most efficient platforms. Over short physical distances where there is only a very small chance for 2 recombination breakpoints, the CelI-based assays provide a time- and cost-effective method for high-resolution genotyping. Using codominant flanking markers, the distinction between the homozygous parental CelI genotypes can be inferred from the adjacent marker genotypes. If necessary, the CelI assays enable an indirect determination of all 3 genotypes mixing sample DNAs with both parental lines separately and subjecting the amplicons of these DNA mixes to CelI treatment. For a small number of lines, the incorporation of fluorescently labeled dNTPs (such as Cy5.5 dCTP for LI-COR) into the PCR products, the costs for detection will only slightly increase in comparison to the reaction costs without labeling. This procedure can be applied to any fully sequenced genome, which thus can be screened rapidly for polymorphisms. If a polymorphic CelI-based marker was identified, the same procedure can be used also for high-resolution genotyping of the relevant plants. Furthermore, this approach can also be applied to scan genomic regions for mutations using mixed DNA samples from mutant and wild-type plants. influence QTL detection differentially in RILs and ILs. QTL detected in only one type of population should not be generally dismissed and the 2 approaches be considered complementary (Keurentjes et al. 2007). Thus, the new reciprocal IL set complements the large reciprocal RIL set available for the same combination Col-0/C24 (Törjek et al. 2006). In addition to the basic sets of ILs, further subILs were developed for 2 genomic regions allowing better mapping resolution. These subILs carrying donor segments with candidate genes for flowering time and reduced fertility were used to demonstrate the applicability of the ILs. In the latter case, the previously defined interval of 112 kb harboring the responsible gene for reduced fertility has been refined to a 56-kb region. In addition, stable homozygous subILs were developed around this locus avoiding the reduced fertility phenotype. During the development of these lines at high physical resolution, we used a modified CelI-based assays in one-well format for both marker development and genotyping. This improved procedure offers a very flexible and cost-effective approach. Supplementary Material Supplementary material can be found at http://www. jhered.oxfordjournals.org/. Funding The Bundesministerium für Bildung und Forschung (#0312275A) to T.A.; the Deutsche Forschungsgemeinschaft (AL 387/6-1, 6-2) to T.A. and R.C.M.; the European Community (QLG2-CT-2001-01097) to T.A.; the MaxPlanck-Society. Acknowledgments We thank B. Till and S. Hennikoff for the opportunity to participate in the TILLING training program (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). Conclusion In this study, we present 2 new large reciprocal sets of ILs created between the Arabidopsis accessions Col-0 and C24. 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