Molecular and physical mapping of the lu-ttg region of Arabidopsis chromosome 5.

Glenn J. Thorlby, Leonid Shlumukov, Igor Y. Vizir, Cai Yun Yang , Bernard J. Mulligan and Zoe A. Wilson.

School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

Corresponding author: Dr. Zoe A. Wilson
Tel: 0115 9513235
Fax: 0115 9513251
Email: Zoe.Wilson@nottingham.ac.uk


 

Abstract

Recombinants between the makers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5 have been used to construct a molecular genetic map of the region, which encompasses a number of genes involved in floral development including Ms1, a gene required for the post-meiotic development of pollen (Dawson et al., 1993). This recessive mutation results in degeneration of pollen soon after microspore release and maps at 29.8 ± 0.8 cM on Arabidopsis chromosome 5. Clones derived from 4 YAC libraries have been used to establish a contig between the RFLP markers 4111 and 4556; this represents a genetic distance of 8.9 cM and a physical distance of approximately 1.44 Mb. In this region, 1 cM corresponds to a physical distance of approximately 160 kb.
 

Introduction

Problems have frequently been encountered when low resolution or integrated genetic maps have been used for the positional cloning of genes; these include the underestimation of physical distances between markers and the identification of a high frequency of chimeric, unstable YAC clones or clones which may map to more than one region of the genome. Therefore fine-scale molecular genetic mapping of a defined chromosomal region containing the gene of interest is an essential pre-requisite to positional cloning. As part of an effort towards the positional cloning of the male sterility 1 (Ms1) gene we have been establishing a physical map of the lu-ttg region on the top arm of Arabidopsis chromosome 5 (Thorlby et al, 1997).

The ms1 mutation is a recessive, EMS-derived mutation which results in the degeneration of pollen immediately after microspore release from the tetrad (Dawson et al., 1993). Tapetal development appears aberrant at this stage, however, the identity and function of the Ms1 gene product are unknown. A variety of map positions have been established for Ms1 on chromosome 5, ranging from 25.7 cM on the classical map (Koornneef, 1986) to 29.8 cM on the integrated genetic/RFLP map (Hauge et al., 1993). Using in excess of 400 recombinants between the genetic markers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) we have mapped Ms1 to 29.8 ± 0.8 cM (Thorlby et al., 1997).

Using a subset of these recombinants we have been conducting molecular mapping of this region; 183 recombinant plants between the ecotypes Landsberg erecta , Sn(5)-1 and Wassileskija were screened with DNA markers, which from published maps, appear to map to this region. Two groups of recombinants were generated between lu -ms1 and ms1-ttg; the use of recombinant homozygous ms1 plants for RFLP mapping was avoided because of the practical difficulties of maintaining sterile lines. Markers mapping close to ms1 were used to screen YAC libraries and a 8.9 cM contig of approximately 1.44 Mb was established between the markers 4111 (26.4 cM) and 4556 (35.3 cM) which encompasses the Ms1 locus (Thorlby et al., 1997). These YAC clones were sized and aligned with the molecular genetic markers to produce an detailed integrated map of this region.

Results

Molecular genetic mapping of the lu-ttg region

Molecular mapping has been carried out using 149 Ler x Sn(5)-1 recombinants with break points on either side of ms1 and a set of 34 Ler x Ws lines with breakpoints between ms1 -ttg (Thorlby et al., 1997). Tables 1 and 2 show the RFLP mapping data generated from the Ler x Sn(5)-1 and Ler x Ws recombinants, respectively. Map positions were established relative to the positions of lu (17.6 cM), ms1 (29.8 cM) and ttg (35.5 cM), using the approach of mapping within a defined chromosome segment (Thorlby et al., 1997). Polymorphisms in the region close to ms1 were extremely difficult to detect, particularly between the ecotypes Sn(5)-1 and Ler and for some markers, e.g. an 8 kb cosmid clone containing the TSL gene, no polymorphisms could be found between Sn(5)-1 and Ler. The map position for TSL was therefore inferred relative to flanking DNA markers by comparing available data from the RI lines (Lister and Dean, 1993), which position TSL at 0.8 cM away from RFLP marker 4560. This was found to agree with our physical mapping data of the region (Table 2).

Probes pCIT718, 21503 and KG10 were mapped using both the Ler x Sn(5)-1 and the Ler x Ws recombinant lines and slight positional differences were observed depending on the recombinant ecotypes used. However, a number of individual lines from both recombinant populations gave anomalous results with these probes and an additional SSLP marker, CDPK.9. The results suggest variable marker orders implying that there may be a high level of recombination or rearrangement occurring in this region of chromosome 5 (manuscript in preparation). The average data showing the consensus map positions for these markers is presented in Table 1 and these map positions agree with the data obtained from physical mapping of the ms1 to ttg region.
 

Physical mapping of ms1 region

YAC clones were identified by screening the Grill, Ward and Ecker YAC libraries, and by limited hybridisations to the CIC library, with molecular markers from this region (Table 3). Three end-probes (RI3F12, RI9B3, RI8B11) (Thorlby et al., 1997) were used to orientate the YAC islands and to screen the libraries to assist in contig assembly. When used to probe the CIC library the clones RI9B3 and RI8B11 linked two previously isolated YAC islands, spanning the chromosomal regions between the markers 4111-TSL and pCIT718-4556, respectively. Comparisons between our genetic and physical maps of the region indicates that 1 cM equates on average to 160 kb.
 

Discussion

We have established a map position of 29.8 ± 0.75 cM for ms1, which agrees with the integrated map position of this gene (Hauge et al., 1993), by measuring recombination frequency relative to two flanking markers, lu and ttg (Thorlby et al., 1997).

We have conducted fine-scale molecular genetic mapping of the region between the markers lu and ttg relative to the genetic distance between these markers, which by consensus is 17.9 cM (Hauge et al., 1993; Meinke and Koornneef, 1994; Meinke, 1995). Our molecular map shows differences between the statistically derived integrated map (Hauge et al., 1993; Meinke and Koornneef, 1994; Meinke, 1995) and the recombinant inbred map (Lister and Dean, 1993), which has a lower density of markers and which was constructed on the basis of significantly fewer recombinants in this region. Polymorphisms were difficult to detect in the Ms1 region and in some cases larger probes were required for RFLP screening; for example, a lambda clone was identified corresponding to the RI9B3 YAC endprobe since polymorphisms could not be detected with the endprobe itself.

DNA markers mapped to this region were used to identify clones from four YAC libraries and establish a 8.9 cM contig. Two contig "islands" were initially established by library screening and these were subsequently linked by screening the CIC library with the RI9B3 and RI8B11 YAC end-probes. Polymorphisms could not be detected in the recombinant populations using any of the three YAC end-probes. However, a larger genomic lambda clone (lambda 9B3) encompassing the YAC RI9B3 endprobe identified a Ler x Sn(5)-1 polymorphism which co-segregated with the ms1 mutation.

The established contig contains 71 YACs and corresponds well with the YAC clones from contigs 6 and 7 on chromosome 5 identified by Schmidt et al. (1997). Thirteen of the clones have been previously identified as possibly chimeric (Schmidt et al., 1994; Dunn, 1995; Morroll and Wilson, in prep). These YACs may be due to multiple cloning events or alternatively to genomic rearrangements such as deletions, recombinations or rearrangements. It may also be that they represent duplicated regions in the genome and may correspond to alternative chromosome locations.

Our data indicates that the physical distance between the RFLP markers 4111 and 4556 corresponds to approximately 1.44 Mb and a genetic distance of 8.9 cM. Therefore in this region 1 cM equates to approximately 160 kb. Our fine-scale map and the future availability of the recombinant mapping population (deposited with the Nottingham Arabidopsis Stock Centre)(Thorlby et al., 1997), together provide a detailed resource for the positional cloning and mapping of genes which lie in the lu-ms1-ttg region of chromosome 5.
 

Materials and Methods

Arabidopsis stocks; Plant growth; Generation of recombinants

Arabidopsis thaliana Ler ( NW20), Sn(5)-1 (N930) and Ws (N1601), the chromosome 5 marker line (lucoms1ttg in a Ler background, N240) were obtained from the Nottingham Arabidopsis Stock Centre (NASC), Nottingham.

Three groups of recombinants (Thorlby et al., 1997) were used in this study i) Ler/Ws recombinants (provided by David Marks, University of Minnesota) which were homozygous Ler at the ttg locus and heterozygous Ws/Ler (or homozygous Ws) at the ms1 locus, ii) lu/ms1 recombinants which were homozygous Ler at the lu locus and heterozygous Ler/Sn(5)-1 (or homozygous Sn(5)-1) at the ms1 locus, and iii) ms1/ttg recombinants which were homozygous Ler at the ttg locus and heterozygous Ler/Sn(5)-1 (or homozygous Sn(5)-1) at the ms1 locus.
 

In vitro growth of plants for DNA isolation

Plants were grown in vitro for DNA isolation based on Czako and Marton (1992). DNA was isolated from 1-3g of plant material by a method based on that of Dellaporta et al. (1983). DNA (1-2 µg) from the parental lines and recombinants was digested using a range of restriction enzymes and hybridised using random-primed radiolabelled probes. CAPS primers for NIT4 were as described by Bartel and Fink (1994). PCR for CAPS analysis was conducted using 25 cycles of 94oC for 1 min; 55oC for 1 min.; 72oC for 2 min (Techne PHC3 temperature cycler). SSLP primers for CDPK.9 were as described by Dunn (1996). Amplification was conducted using 40 cycles of 94oC for 15 sec; 55oC for 15 sec; 72oC for 30 sec (Techne PHC3 temperature cycler). Protocols are described in full in Thorlby et al (1997).
 

YAC maintenance and DNA preparation

YAC libraries (Ecker, 1990; Ward and Jen, 1990; Grill and Somerville, 1991) were provided by the ABRC, Ohio State University. Selected clones from the CIC library (Creusot et al., 1995) were kindly provided by Dr. Renate Schmidt (JI Centre, Norwich) and from the ABRC. YAC clones were grown on selective medium (Ausubel et al., 1988) at 30oC and DNA isolated by the Puragene extraction kit (Flowgen). High molecular weight YAC DNA was prepared using a CHEF yeast genomic DNA plug kit (Bio-Rad). YAC hybridisations and PFGE was conducted as described in Thorlby et al. (1997).
 

Lambda library screening

The Ler genomic l library constructed by Voytas et al. (1990) (ABRC CD4-8) was screened using radioactively labelled RI9B3 YAC endprobe DNA. Putative positive signals were re-screened and positive clones selected and confirmed following DNA isolation and Southern blotting.
 

Acknowledgements.

We are grateful to Chris Somerville (Carnegie Institute, Stanford, CA), Eric Ward (Ciba-Geigy, NC), Joe Ecker (University of Pennsylvania, Philadelphia, PA) and Francine Creusot (URA CNRS, Orsay, Cedex, France) for use of their YAC libraries, to Brian Hauge and Howard Goodman (MGH, Boston) for cosmid clones, to Elliot Meyerowitz (CalTech, Pasadena) for RFLP markers and the PI clone and to Judy Roe (University of California, Berkeley) for the TSL clone. Thanks are also due to David Marks (University of Minnesota, Minneapolis) for the Ler x Ws recombinants. We would like to acknowledge the ABRC, Ohio State University for clones and libraries. We are indebted to Drs. Renate Schmidt (Max Delbrück Laboratory, Cologne) and Caroline Dean (JI, Norwich) for continual collaboration and assistance on contig establishment and for initial screening using the RI9B3 and RI8B11 end-probes. This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) as part of the Plant Molecular Biology Programmes I and II under grants PG42/536 and PG42/556 to BJM and ZAW.

 
 

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