Isolation and mapping of a new set of 129 RFLP markers in Arabidopsis thaliana using recombinant inbred lines

A new collection of 129 Arabidopsis thaliana RFLP markers has been established based upon DNA fragments cloned in the pUC119 plasmid vector and insert end sequences of P1 clones. Dominant/null alleles affecting low-copy number sequences account for nine of the mapped polymorphisms, suggesting that deletions are not rare in A. thaliana . Recombinant inbred (RI) lines were used for mapping these marker loci. RI line-based mapping allows integration of this set of markers with markers previously reported as well as with any markers mapped in the future using this replenishable mapping resource. These markers are useful for map-based gene isolation and genome physical mapping in A. thaliana as well as studies of chromosome colinearity (synteny) with related species.     

Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana

Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines.     

Recombinant inbred lines for genetic mapping in tomato

A cross between the cultivated tomato Lycopersicon esculentum and a related wild species L. cheesmanii yielded 97 recombinant inbred lines (RILs) which were used to construct a genetic map consisting of 132 molecular markers. Significant deviation from the expected 1:1 ratio between the two homozygous classes was found in 73% of the markers. In 98% of the deviating markers, L. esculentum alleles were present in greater frequency than the L. cheesmanii alleles. For most of the markers with skewed segregation, the direction of the deviation was maintained from F₂ to F₇ generations. The average heterozygosity in the population was 15%. This value is significantly greater than the 1.5% heterozygosity expected for RILs in the F₇ generation. On average, recombination between linked markers was twice as high in the RILs than in the F₂ population used to derive them. The utility of RILs for the mapping of qualitative and quantitative traits is discussed.     

QTL analysis of Al tolerance in recombinant inbred lines of Arabidopsis thaliana

Quantitative trait loci (QTLs) and epistasis for Arabidopsis thaliana aluminum (Al) tolerance were analyzed using a recombinant inbred (RI) population of 100 lines derived from a cross between Landsberg erecta and Columbia (Col). Root growth of the RI population was determined in hydroponics using solutions containing 0 or 4 micro M of AlCl(3 )and a series of nutrients, except P(i), at pH 5.0. Al tolerance was defined as relative root length [RRL: plus Al/minus Al (%)], and the RI lines ranged from 22.6 to 97.4% with a broad sense heritability of 0.99. Using the composite interval mapping method, two significant single factor QTLs (P<0.05) were detected by RRL on chromosomes 1 and 4, where the Col allele showed positive and negative effects on the Al tolerance. These QTLs could explain about 43% of the total variation of Al tolerance among the RI population. On the other hand, five epistatic loci pairs were identified by the complete pair-wise search method (P<0.0005). No single factor QTL and epistatic loci pairs were shared by the root length in the control and the RRL, suggesting that the loci identified by the RRL would be specific for Al treatment and controlling Al tolerance among the RI population.     

An Arabidopsis thaliana RFLP mapping set to localize mutations to chromosomal regions

Mapping of newly identified Arabidopsis thaliana mutants is an important step towards their molecular characterization and the attempt to saturate the genome by known mutations. The classical genetic analysis using phenotypic tester lines is well-established, but laborious, time-consuming and potentially ambiguous. An alternative molecular strategy was developed that is based on RFLPs. Subcloned DNA markers that detect only segregating RFLP bands distinguishing A. thaliana ecotype Landsberg from Columbia or Enkheim after EcoRI restriction digestion compose an Arabidopsis RFLP mapping set (ARMS). Up to 13 markers uniformly cover the five A. thaliana chromosomes and can be scored in only two successive Southern experiments on a single blot without mutual interference of the signals. Thus, this system allows a simple, reliable, rapid and especially inexpensive mapping of any monogenic mutant locus to the A. thaliana chromosomes. Several loci can be analysed in one experiment if the respective blots are hybridized together. This paper demonstrates the mapping of two recessive mutants affecting the development of A. thaliana leaves which had been generated in the Columbia and Enkheim ecotype by analysing less than 20 F₂ individuals. Further markers to refine or verify the result on the same blot can be chosen out of 14 additional probes detecting single segregating EcoRI polymorphic bands as well.     

Quantitative trait locus mapping for seed mineral concentrations in two Arabidopsis thaliana recombinant inbred populations

Biofortification of foods, achieved by increasing the concentrations of minerals such as iron (Fe) and zinc (Zn), is a goal of plant scientists. Understanding genes that influence seed mineral concentration in a model plant such as Arabidopsis could help in the development of nutritionally enhanced crop cultivars. Quantitative trait locus (QTL) mapping for seed concentrations of calcium (Ca), copper (Cu), Fe, potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sulfur (S), and Zn was performed using two recombinant inbred line (RIL) populations, Columbia (Col) x Landsberg erecta (Ler) and Cape Verde Islands (Cvi) x Ler, grown on multiple occasions. QTL mapping was also performed using data from silique hulls and the ratio of seed:hull mineral concentration of the Cvi x Ler population. Over 100 QTLs that affected seed mineral concentration were identified. Twenty-nine seed QTLs were found in more than one experiment, and several QTLs were found for both seed and hull mineral traits. A number of candidate genes affecting seed mineral concentration are discussed. These results indicate that A. thaliana is a suitable and convenient model for discovery of genes that affect seed mineral concentration. Some strong QTLs had no obvious candidate genes, offering the possibility of identifying unknown genes that affect mineral uptake and translocation to seeds.     

Variance for water-use efficiency among ecotypes and recombinant inbred lines of Arabidopsis thaliana (Brassicaceae)

Knowledge of natural variation among ecotypes and recombinant inbred lines of Arabidopsis thaliana L. Heynh for season-long water-use efficiency (WUE, moles of carbon accumulated per mole of water used) is useful in the design of experiments to understand the genetic control of this phenomenon. Water-use efficiency among 31 container-grown Arabidopsis ecotypes ranged from a high of 2.40 to a low of 1.86 mg/g (grams of dry aerial biomass per gram of water used). Genetic variance for WUE was observed among 80 F₅ recombinant inbred lines (RILs) derived from a cross between the highest (Lip-0) by the lowest (Edi-0) ecotype. The heritability of WUE was calculated as 0.18 ± 0.07. The mean WUE for the ten highest and ten lowest RILs was 2.42 and 1.05 mg/g, respectively. A significant difference was observed in the composition of stable isotopes of plant carbon (δ¹³C) between the mean of the ten highest (-31.23±) and the mean of the ten lowest (-31.96±) RILs based on WUE. Characterization of the 80 RILs provides a structured population for further genetic and physiologic study of WUE.     

Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers

Quantitative approaches conducted in a single mapping population are limited by the extent of genetic variation distinguishing the parental genotypes. To overcome this limitation and allow a more complete dissection of the genetic architecture of complex traits, we built an integrated set of 15 new large Arabidopsis thaliana recombinant inbred line (RIL) populations optimized for quantitative trait loci (QTL) mapping, having Columbia as a common parent crossed to distant accessions. Here we present 5 of these populations that were validated by investigating three traits: flowering time, rosette size, and seed production as an estimate of fitness. The large number of RILs in each population (between 319 and 377 lines) and the high density of evenly spaced genetic markers scored ensure high power and precision in QTL mapping even under a minimal phenotyping framework. Moreover, the use of common markers across the different maps allows a direct comparison of the QTL detected within the different RIL sets. In addition, we show that following a selective phenotyping strategy by performing QTL analyses on genotypically chosen subsets of 164 RILs (core populations) does not impair the power of detection of QTL with phenotypic contributions >7%.     

Sheep linkage mapping: RFLP markers for comparative mapping studies

Restriction fragment length polymorphisms (RFLPs) detected using cDNA probes for conserved genes provide an important set of markers that anchor or link syntenic groups in a range of divergent mammalian species. DNA probes from sheep, cattle, pig, human and mouse were screened against sheep DNA samples and 24 new RFLP markers for sheep were identified. Among the loci tested, 22 had a homologue that has been mapped in humans. An RFLP for fibronectin (FN1) was linked to α-inhibin (INHA) at a distance of 5cM. The FN1 locus has been assigned to sheep chromosome 2q41–q44 and linkage between FN1 and INHA assigns INHA to the same chromosome in sheep. In addition to the new loci reported here, 28 RFLPs have been published previously by this group and these are collated together with RFLPs published from other laboratories. RFLPs have been reported for 86 loci in sheep. Fifty-four loci have been mapped to 16 different chromosomes.     

High-throughput genetic mapping in Arabidopsis thaliana

To facilitate rapid determination of the chromosomal location of novel mutations, we have improved current approaches to gene mapping using microsatellite length polymorphisms. The high-throughput linkage analysis method described here allows a novel gene to be tested for linkage against the whole genome of a multicellular eukaryote, Arabidopsis thaliana, in a single polyacrylamide gel. The procedure is based on the simultaneous co-amplification of 21 microsatellites in a single tube, using a multiplex PCR mix containing 21 primer pairs, each including one oligonucleotide labeled with one of three fluorescent dyes that have different emission wavelengths. The amplification products, which range in number from 21 to 42, depending on the genotype of the individual being tested, are electrophoresed in a single lane on a polyacrylamide gel. The use of an automated fragment analyzer makes it possible to perform linkage analysis on a one gel-one gene basis using DNA samples from 19 F₂ individuals obtained from an outcross involving a mutant and a wild-type that is genetically polymorphic with respect to the ecotype in which the mutant was generated. Discrimination of the amplification products is facilitated not only by labeling with different fluorochromes, but also by prior testing of different sequences for the ability to prime the amplification of each microsatellite, in order to ensure that multiplex PCR yields compatible amplification products of non-overlapping size. The method is particularly useful in large-scale mutagenesis projects, as well as for routine mapping of single mutants, since it reveals the map position of a gene less than 24 h after the F₂ individuals to be analyzed have become available. The concepts employed here can easily be extended to other biological systems. Received: 24 September 1998 / Accepted: 9 December 1998     

Comparison between marker-assisted selection and phenotypical selection in a set of Arabidopsis thaliana recombinant inbred lines

 Parents were selected from a well-characterised Arabidopsis recombinant inbred line (RIL) population based on (1) their phenotype for flowering time or (2) marker and QTL information that had been assessed previously. The F₂ offspring obtained from pairs of selected RILs was analysed for these traits, and the results obtained with these two methods of selection were compared. Selection based on marker and QTL information gave approximately the same result as selection based on phenotype. The relative high heritability of flowering time in Arabidopsis facilitated successful phenotypical selection. The difference in selection result that was anticipated to be in favour of the marker-assisted approach was therefore not observed. Received: 29 November 1997 / Accepted: 8 June 1998     

Relationships among maize inbred lines and populations from European and North-American origins as estimated using RFLP markers

RFLP markers have proven to be a reliable and highly informative tool for characterizing genetic diversity in maize. Joint analysis of inbred lines and populations should provide valuable information with respect to (1) a better understanding of the genetic basis of present elite germplasm and (2) the identification of populations that may prove to be useful sources of genetic diversity for breeding programs. Sixty-two inbred lines of known heterotic groups and ten maize populations, some of them significant contributors to the genetic basis of the heterotic groups, were assayed at 28 RFLP loci. Joint data analyses first underlined that the populations displayed a large number of alleles that were absent in the set of inbred lines. Associations among inbreds and populations further proved consistent with pedigree data of the inbreds and provided new information on the genetical basis of heterotic groups. In particular, European flint inbreds were revealed to be as close to the Northeastern U.S. flint population studied as to the typical European populations. These results advocate the analysis of larger sets of populations by means of molecular markers in order to (1) gain insight into the history of maize germplasm and (2) set up appropriate strategies for the use of genetic resources in breeding programs. Received: 23 February 1998 / Accepted: 5 February 1999     

Combined mapping of AFLP and RFLP markers in barley

AFLP marker technology allows efficient DNA fingerprinting and the analysis of large numbers of polymorphic restriction fragments on polyacrylamide gels. Using the doubled haploids from the F₁ of the cross Proctor × Nudinka, 118 AFLP markers were mapped onto a barley (Hordeum vulgare L.) RFLP map, also including five microsatellite and four protein marker loci. The AFLP markers mapped to all parts of the barley chromosomes and filled in the gaps on barley chromosomes 2L, 4L and 6 in which no RFLP loci had been mapped. Interestingly, the AFLP markers seldom interrupted RFLP clusters, but grouped next to them. The combined map covers 1873 cM, with a total of 282 markers. The merging of AFLP and RFLP markers increased the total map length; 402 cM were added to the map at the tips of chromosomes or in regions corresponding to earlier gaps. Another 375 cM resulted from mapping AFLP markers near to RFLP clusters or in between non-clustered RFLP markers.     

Combined mapping of DALP and AFLP markers in cultivated sunflower using F9 recombinant inbred lines

A genetic map was constructed with specific PCRs, DALPs and AFLPs using F8-generation sunflower recombinant inbred lines. RI lines generated from a F2 population of one cross between the two cultivated inbred lines HA89 (maintainer for Pet1 CMS) and LR4 (restorer for Pet1 CMS) were used. A total of 305 markers were located using seven sPCR, 64 DALP and 301 AFLP loci. They were generated with one, seven and 14 primer pairs, respectively. The map construction consisted of a two-step strategy using 6 and 3.1 LOD scores revealed by a simulation file. Mapped markers were assembled into 18 linkage groups covering 2,168.6 cM with an average of 6.1 cM. The distribution of DALPs and AFLPs revealed that both markers tagged different regions to enable covering most of the sunflower genome. This leads to the longest map published so far for sunflower.     

New phenotypic characteristics of three tmm alleles in Arabidopsis thaliana

Key message

Three new tmm mutants were isolated and showed differential phenotypes from tmm - 1 , and TMM overexpression led to abnormal leaf trichomes.

Abstract

TOO MANY MOUTH (TMM) plays a significant role in the stomatal signal transduction pathway, which involves in the regulation of stomatal distribution and patterning. Three mutants with clustered stomata were isolated and identified as new alleles of tmm. tmm-4 mutation included a base transversion from adenine to thymidine in position 1,033 of the TMM coding region and resulted in premature termination of translation at position 345 of TMM. tmm-5 had a base transition from cytosine to thymidine in 244 of TMM and translated 82 amino acids before premature termination. tmm-6 mutation took a base transition from guanine to adenine in 463 of TMM and changed a glycine (Gly) to an arginine (Arg) in position 155 of the protein. tmm-6 had an evident reduction of stomatal clusters and fewer stomata in cluster compared with other tmm alleles, possibly due to decreased level of entry divisions in cells next to two stomata or their precursors. tmm-5 and tmm-6 were hypersensitive to abscisic acid (ABA) in seedling growth and seed germination, while tmm-4 was defective in response to ABA during seed dormancy, suggesting that TMM was involved in ABA signaling transduction. Interestingly, overexpression of TMM resulted in the reduction of leaf trichomes and their branches, and this might reveal a new function of TMM in trichome development.     

An integrated genetic/RFLP map of the Arabidopsis thaliana genome

We have assembled an integrated genetic/restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the flowering plant Arabidopsis thaliana . The map is based on two independent sets of RFLP data, RFLP data for 123 new markers, and pair-wise segregation data of 125 classical genetic markers. Mathematical integration of the independent data sets was performed using the joinmap computer package. Sixty-two markers common to two or more data sets were exploited to facilitate integration of the individual maps. The current map, which encompasses a total genetic distance of 520 cM, contains 125 classical genetic markers and 306 RFLP markers. Comparison of the integrated consensus map with the individual maps demonstrates that the overall linear order of the integrated map is in good agreement with the component maps. It must be emphasized, however, that the integrated map represents the 'best fit' which is clearly subject to the statistical limitations of the available data. We present several examples where local differences in map order are observed between the integrated and component maps. It is likely, given the problems associated with statistical integration of mapping data from different populations, that the integrated map will contain additional local inconsistencies and problematic regions. None the less, the unified map provides a framework for building an increasingly accurate and useful map. Subsequent refinements of the map will be available electronically end researchers are invited to submit revised map data to the corresponding author for inclusion in future updates (see Appendix 1).     

Extreme QTL mapping of germination speed in Arabidopsis thaliana

Seed germination is a key life history transition for annual plants and partly determines lifetime performance and fitness. Germination speed, the elapsed time for a nondormant seed to germinate, is a poorly understood trait important for plants’ competitiveness and fitness in fluctuating environments. Germination speed varied by 30% among 18 Arabidopsis thaliana populations measured, and exhibited weak negative correlation with flowering time and seed weight, with significant genotype effect (P < 0.005). To dissect the genetic architecture of germination speed, we developed the extreme QTL (X‐QTL) mapping method in A. thaliana. The method has been shown in yeast to increase QTL mapping power by integrating selective screening and bulk‐segregant analysis in a very large mapping population. By pooled genotyping of top 5% of rapid germinants from ~100 000 F₃ individuals, three X‐QTL regions were identified on chromosomes 1, 3 and 4. All regions were confirmed as QTL regions by sequencing 192 rapid germinants from an independent F₃ selection experiment. Positional overlaps were found between X‐QTLs and previously identified seed, life history and fitness QTLs. Our method provides a rapid mapping platform in A. thaliana with potentially greater power. One can also relate identified X‐QTLs to the A. thaliana physical map, facilitating candidate gene identification.     

RFLP mapping of Brassica napus using doubled haploid lines

The combined use of doubled haploid lines and molecular markers can provide new genetic information for use in breeding programs. An F₁-derived doubled haploid (DH) population of Brassica napus obtained from a cross between an annual canola cultivar (Stellar) and a biennial rapeseed (Major) was used to construct a linkage map of 132 restriction fragment length polymorphism loci. The marker loci were arranged into 22 linkage groups and six pairs of linked loci covering 1016 cM. The DH map was compared to a partial map constructed with a common set of markers for an F₂ population derived from the same F₁ plant, and the overall maps were not significantly different. Comparisons of maps in Brassica species suggest that less recombination occurs in B. napus (n = 19) than expected from the combined map distances of the two hypothesized diploid progenitors, B. oleracea (n = 9) and B. rapa (n=10). A high percentage (32%) of segregating marker loci were duplicated in the DH map, and conserved linkage arrangements of some duplicated loci indicated possible intergenome homoeology in the amphidiploid or intragenome duplications from the diploid progenitors. Deviation from Mendelian segregation ratios (P < 0.05) was observed for 30% of the marker loci in the DH population and for 24% in the F₂ population. Deviation towards each parent occurred at equal frequencies in both populations and marker loci that showed deviation clustered in specific linkage groups. The DH lines and molecular marker map generated for this study can be used to map loci for agronomic traits segregating in this population. Present address Embrapa/Cenargen, C.P. 0.2372, CEP 70.770, Brasilia DF, Brazil     

Isolation of new promoter-mediated co-suppressed lines in Arabidopsis thaliana

Four new independent lines that exhibit co-suppression of an introduced cab140::tms2 gene and the native cab140 gene have been isolated in Arabidopsis thaliana. These lines are of particular interest because the homology shared between the introduced and native genes is 1.3 kb of promoter DNA that only contains 14 bp of transcribed region. Most other reported examples of co-suppression involve homologies between transcribed portions of genes. A similar line, lct, had been isolated previously from EMS-mutagenized seeds, and we concluded that this example of co-suppression was probably due to a mutation that mapped at or near the introduced cab140::tms2 gene [Brusslan JA, Karlin-Neumann GA, Huang L, Tobin EM: Plant Cell 5: 667–677 (1993)]. Our observations with these four new lines, however, suggest that an epigenetic event(s) rather than a mutation might be the cause of co-suppression in these and the lct line.