The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:78-81
Brief Communication |
A Linkage Map for CRINKLED PETAL: A Homeotic Gene of Clarkia tembloriensis (Onagraceae)
From the Department of Botany, Miami University, Oxford, Ohio.
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Abstract |
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Homeotic mutations in flowers lead to the development of floral organs in abnormal locations. In most laboratory-induced examples of this type of mutation, two adjacent whorls of organs are affected, resulting in two whorls of abnormal organ formation. However, the crinkled petal mutant of Clarkia tembloriensis is interesting because it is a naturally occurring mutation and it affects only the second whorl of organs, producing sepaloid petals. In this study one wild-type population (Cantua Creek-2) and one crinkled petal mutant population (Red Rocks) were compared using 181 different primers in random amplified polymorphic DNA (RAPD) analysis. Bulk DNA from each parent population and their subsequent crosses were used to compare the genetic differences between the two populations and to search for molecular markers linked with the CRINKLED PETAL locus. A linkage map was developed for the CRINKLED PETAL gene, and markers were discovered which flanked both sides of the locus.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clarkia tembloriensis (Onagraceae) is a wildflower found growing in discrete populations in the Temblor region of southern California. Within these populations, flowers with two distinct petal phenotypes have been observed (Vasek 1964). The typical wild-type flowers of C. tembloriensis possess four petals composed of a smooth-textured deltoid limb with a slender claw. These petals are bright pink in color, with a maroon spot at the base of the limb. All four petals are expanded and very prominently displayed. In striking contrast are the flowers from the homeotic mutant called crinkled petal (cp) (Smith-Huerta 1992, 1996; Vasek 1964). The cp mutant flowers also possess four "petals," but these organs are small and linear-lanceolate in shape, with abundant trichomes. They have a greenish-pink, wrinkled appearance, lack the maroon spot at the base, and are reflexed backward. In previous developmental studies, Smith-Huerta (1992) showed that the second whorl organs of the cp mutant are initiated in the proper time and location for wild-type petals, but in terms of color, morphology, and growth rate, they closely resemble wild-type sepals.
The crinkled petal phenotype occurs in at least 10 natural populations in California (Vasek 1964) and is controlled by a single recessive gene (Vasek 1966). In some populations both the wild-type and crinkled petal phenotypes occur together; in others, only one of the phenotypes is present (Vasek 1964). In this study, plants from the wholly wild-type population at Cantua Creek-2 (CC-2) were compared with plants from the cp mutant population at Red Rocks (RR). These two populations show minor differences in germination time, growth rate, and branching pattern (Leong L, personal observation), as well as the obvious major difference in floral morphology.
In this study we attempted to locate the CRINKLED PETAL gene using random amplified polymorphic DNA analysis (Welsh and McClelland 1990; Williams et al. 1990) to examine the genetic differences between two populations of C. tembloriensis that display different petal phenotypes. We used RAPDs with template DNA from parental wild-type and mutant plants, and DNA from subsequent crosses [F3 for mutants; F3 and selfed backcross (BC2) for wild type] to examine the genetic variation between the CC-2 wild-type petal and RR crinkled petal populations. We then used the observed differences to search for markers linked with the gene responsible for the petal phenotype. Furthermore, by comparing the linkage of polymorphic bands with the segregation of the wild-type and cp mutant petal phenotypes in homozygous individuals (cp+/cp+ or cp/cp) of the F3 generation, a linkage map was developed for the CRINKLED PETAL gene of C. tembloriensis.
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Materials and Methods |
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Clarkia tembloriensis seeds from the wild-type CC-2 population and the RR mutant population were sown on vermiculite and germinated in a growth chamber under a 12-h light/12-h dark cycle with a constant temperature of 13°C (Smith-Huerta 1992). Seedlings were transplanted into Metro Mix (Scott's) and grown in a growth chamber under a 16-h light/8-h dark cycle at a constant temperature of 23°C. Plants were watered as necessary with a dilute fertilizer solution (Liquid Miracle Gro, Scott's). To produce F1, F2, F3, and backcross generations, flowers were emasculated 1 day prior to anthesis and hand pollinations were performed. F3 populations were used to determine the genotype of the wild-type F2 parents relative to the cp locus.
Healthy bud, stem, and shoot tissues were collected from plants of each type and generation. DNA extraction was performed using a CTAB method based on Doyle and Doyle (1987) with a 4x CTAB extraction buffer plus 5% PVP and 1% sodium bisulfite (dissolved in the buffer before tissue was added). DNA was resuspended in sterile deionized water. Salt concentrations were adjusted to 0.25 M, and then the protocol according to Michaels et al. (1994) was used to remove RNA and clean up polysaccharide contaminants. DNA was stored in sterile water at -20°C.
Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR; Welsh and McClelland 1990; Williams et al. 1990) was performed in a thermocycler (Perkin-Elmer GeneAmp PCR System 2400) using the following components for each 12.5 µl reaction: 0.2 µM primer, 0.2 mM of each dNTP, 4 mM MgCl2, 10x Stoffel Fragment PCR Buffer (final concentration 10 mM KCl, 10 mM Tris-HCl pH 8.3), 2% DMSO, 2x BSA, and 2 units of AmpliTaq DNA Polymerase Stoffel Fragment (Perkin-Elmer, Foster City, CA). The following program was used: 94°C for 3 min, followed by 50 cycles of 94°C for 1 min, 36°C for 1 min, and 72°C for 2 min. The reactions were then terminated by an extension of 72°C for 7 min and maintained at 4°C until loading for gel electrophoresis. PCR products were run on 1.3% agarose in 1x TAE buffer, and gels were then stained with ethidium bromide and photographed under ultraviolet (UV) light. Photography and analyses were performed using the AlphaImager camera system (Alpha Innotech, San Leandro, CA). Gels were compared visually for presence or absence of polymorphic bands. A total of 181 different random 10-mer primers (UBC-120–UBC-300; Nucleic Acid Protein Service, University of British Columbia, Vancouver, BC, Canada) were used in RAPD-PCR to compare the banding patterns of the two populations.
For initial population comparisons, DNAs from nine plants of the parental CC-2 population were combined together, as were DNAs from nine plants from the RR population (called CC-2 bulk and RR bulk, respectively). The purpose of bulking the DNAs from each population was to provide an estimate of the variation between the two populations and to disregard variation between individuals within each population (based on the idea of "bulked segregant analysis" from Michelmore et al. 1991). An ethanol precipitation from Michaels et al. (1994) was used to clean the bulked DNAs again. Bulk CC-2 and bulk RR DNAs were used in RAPD-PCR with each of the 181 primers. Primers that resulted in polymorphisms between the two population bulks were checked at least twice to confirm the polymorphism. Those primers producing a confirmed polymorphism were tested for linkage with the petal phenotype by running PCRs with DNA from various F3 individuals bearing the mutant phenotype (genotype cp/cp). Primers resulting in a majority of mutant F3 individuals showing the same band phenotype as one of the parental types were used again on F3 and BC2 (selfed backcross) individuals known to be homozygous for the wild-type cp+ allele (genotype cp+/cp+).
Segregation data for RAPD markers associated with the petal phenotype were analyzed using the genetic analysis program MapMaker/Exp version 3.0 (Lander et al. 1987; Lincoln et al. 1992).
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Results |
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One hundred eighty-one 10-mer primers were screened through PCR with bulk DNAs from the CC-2 and RR populations, resulting in a total of 1060 scorable bands; 1018 bands were present in both populations (96.0%) and 42 were present in only one population (4.0%). To check further for linkage with the petal phenotype, PCR was performed on template DNA from individuals of the F3 generation using those primers that produced polymorphic bands. Of the bands that were found to be polymorphic between the two populations, 30 showed segregation of the band in the F3 population and were used to create a linkage map for the cp gene. Those bands that did not segregate at all in the F3 were considered uninformative for mapping purposes.
Two linkage groups were determined using MapMaker/Exp version 3.0 (Table 1). Group 1 is significant because it demonstrates linkage between the cp locus and three different molecular markers. The linkage map for this group is shown in Figure 1. Primer sequences for the markers used in mapping were as follows: 5'-CCC CCC AGA T-3' (primer UBC-163); 5'-CAT ATC AGG G-3' (primer UBC-207); 5'-CTT GAC GGG G-3' (primer UBC-251). The marker UBC-1631040 correlated especially strongly with the wild-type allele, mapping only 4.6 cM apart. This band occurs in the wild-type CC-2 bulk and most of the F3 and BC2 individuals homozygous for the wild-type allele. It does not occur in the mutant RR bulk nor in any of the F3 mutant individuals except F3-25-08, indicating a tight linkage with the wild-type allele.
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Discussion |
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When no sequence information is available for an organism, one way to determine the location of a gene is to identify markers that are physically close to it on a chromosome. The closer the proximity of two markers on a chromosome, the more likely they will segregate together, and the less likely that they will cross over separately during meiosis. Three bands were determined to be linked with the wild-type petal allele: UBC-1631040, UBC-207425, and UBC-251410. In fact, the marker UBC-1631040 occurs in all scorable instances of the wild-type phenotype in the F3 and BC2 generations, with a very low frequency of occurrence in the F3 mutant phenotype plants. Preliminary attempts to clone this marker were unsuccessful, but this may be a very useful marker for future attempts to locate the position of the cp locus. The likelihood of finding the cp locus in the future is enhanced by the fact that linked markers have been located on both sides of this locus: UBC-1631040 on one side and UBC-251410 on the other. As a demonstration of the potential usefulness of this finding, Thorlby et al. (1997) were able to determine a fine-scale molecular map position for the Arabidopsis male sterility gene ms1 by measuring the recombination frequency of two molecular markers which flank both sides of the ms1 gene. A similar mapping study could be performed on the cp gene with its flanking markers. Furthermore, by creating and searching a genomic DNA library for C. tembloriensis, it might be possible to find contiguous DNA fragments that span the region between the two flanking markers. A chromosome walk across this region could then lead to a more accurate mapping of the cp locus and would make sequencing the cp gene a viable option.
To explain the mechanism of floral organ development, Bowman et al. (1991) have suggested a series of three overlapping floral factors or functions called A, B, and C which, when expressed in different combinations, specify the development of the four different floral whorls. The expression of factor A alone results in sepals, factors A and B together produce petals, B and C make stamens, and C alone stimulates the production of carpels. According to this ‘ABC model,’ a mutation in one of these factors should have an impact on two adjacent whorls of organs since each factor is required for the development of two whorls. Indeed, this prediction is consistent with many studies describing homeotic mutations that disrupt the development of two adjacent floral whorls in a variety of plant systems (Coen and Meyerowitz 1991). However, not all homeotic mutants behave exactly as this model predicts. Exceptions such as the superman mutation of Arabidopsis, which affects the carpel whorl only (Bowman et al. 1992; Sakai et al. 1995), and the green petals mutation of Petunia which affects the petals only (Angenent et al. 1992; Halfter et al. 1994; van der Krol and Chua 1993; van Tunen and Angenent 1991), do not follow the pattern of the basic ABC model. In the same way, the cp mutant does not fit the normal model, as it produces a mutant petal whorl, but otherwise appears to be unaffected anywhere else. However, this deviance from the model does not necessarily imply that the ABC model is not relevant for C. tembloriensis or other one-whorl mutants. Although the cp mutant is considered a class B mutant (Smith-Huerta 1992), it may be that the CRINKLED PETAL gene is not responsible for the production of the "B" factor of C. tembloriensis, but is involved instead in the response of the plant to the presence of the A + B factor product.
The crinkled petal mutant of C. tembloriensis is significant not only because it represents a deviation from the more common two-whorl mutant effect, but also because it is not a laboratory-induced mutation as is the case of most of the commonly studied floral mutants. The cp mutation has been observed in at least 10 populations in the wild (Vasek 1964). Other studies have reported apparently homologous sepaloid petal phenotypes in other species of Clarkia (crinkled petal form in C. exilis, Vasek 1966; bicalyx mutant in C. concinna, Ford and Gottlieb 1992) and in other genera of the Onagraceae including Epilobium and Oenothera (cruciata mutants; Renner 1959). All of these mutants are fully fertile and appear to be unaffected in the other three floral whorls. The existence of these mutations suggests that the single-whorl mutant condition, while by no means a common occurrence, is not as unusual in nature as might be anticipated, at least in the Onagraceae. Further pursuit of the cp locus in C. tembloriensis could thus prove very useful to other studies. Although it was shown by Vasek (1966) that the CRINKLED PETAL genes of C. tembloriensis and C. exilis are homologous, it is not known whether the same is true for the rest of the sepaloid petal mutations in the Onagraceae, or for other sepaloid petal mutants in other plant families. Continued investigation of the crinkled petal mutant of C. tembloriensis and other similar mutants will help us to compare and contrast the relationships between the different floral whorls in different plant systems and may help to provide insight into the evolution of the flower itself.
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Acknowledgments |
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We thank A. Huerta for assistance in preparation of the figures, and N. Money and two anonymous reviewers for helpful comments on the manuscript. This project was supported by Academic Challenge grants from the Botany Department of Miami University, and by a grant-in-aid of research from Sigma Xi, the Scientific Research Society. This article represents a portion of a master's thesis submitted by L. L. Y. Leong to the Department of Botany, Miami University, Oxford, Ohio.
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Footnotes |
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Address correspondence to Laurel Leong, Department of Botany, Miami University, Oxford, OH 45056, or e-mail: Leongll{at}aol.com.
Corresponding Editor: Susan Gabay-Laughnan
Received February 21, 2000
Accepted November 3, 2000
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