Microsatellites are tandemly repeated short nucleotide sequences found in all eukaryotic and some prokaryotic genomes (Tautz et al., Nature 22:652- 656, 1986). Allele-specific length polymorphisms of microsatellites are known to exist in maize (Calderon-Urrea and Dellaporta, MNL 68:70, 1994). In barley the number of alleles of ribulose-1,5-bisphosphate carboxylase activase distinguished by microsatellite repeat variation far exceeds the number detected by analysis of other molecular markers such as chloroplast DNA, ribosomal DNA or RFLPs (Maroof et al., Proc. Natl. Acad. Sci. USA 91:5466-5470, 1994). Although little is known about the function of microsatellites within or near plant genes, their prevalence, high degree of repeat number variability and Mendelian inheritance have made them useful in genome mapping and as potential markers for plant breeding studies.
The Y1 gene codes for phytoene synthase, an enzyme that condenses two geranyl-geranyl pyrophosphate molecules into one molecule of phytoene during the biosynthesis of carotenoids (Buckner et al., MNL 67:65, 1993). The dominant allele of Y1 cloned by Buckner et al. (Plant Cell 2:867-876, 1990) has been sequenced (unpublished data) and shown to contain a CCA trinucleotide repeated eleven times upstream of the coding sequence. However, it is not known if this microsatellite is present in other alleles of Y1. The purpose of this study was to determine if the CCA microsatellite exists in other alleles of Y1, if it is polymorphic in length and if the number of trinucleotide repeats correlates with the expression of the Y1 gene.
DNA was isolated from individual etiolated shoots grown for three to five days at 30C on germination paper saturated with 1 mM CaCl2. PCR reactions were performed in 50 µl with approximately 1 µg of genomic maize DNA. The reaction mix contained 1x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.01% gelatin), 200 µM of each dNTP, and 1.25 units of Taq Polymerase (Perkin Elmer Co., Norwalk, CT). The reaction mix was incubated in a HybaidTM thermal cycler (National Labnet Co., Woodbridge, NJ) according to the following parameters: 94C for 1 min, 59C for 1 min, 72C for 30 sec. After 30 cycles the samples remained at 72C for 5 min. The PCR products were resolved on 2.5% MetaphorTM agarose (FMC Bio Products, Rockland, ME) gels made in 0.09 M Tris, 0.09 M boric acid and 2 mM EDTA. PCR products to be sequenced were excised from 1.2% Seakem Low-Gelling agarose (FMC Bio Products, Rockland, ME) gels made in 40 mM Tris-acetate and 1 mM EDTA, pH 7.8 and purified using WizardTM; PCR Preps DNA Purification System (Promega, Madison, WI). Approximately 12 ng of purified PCR products were sequenced using an Applied Biosystems Inc. (ABI, Forrest City, CA) Taq DyeDeoxyTM; Terminator Cycle Sequencing Kit as described by the manufacturer.
Electrophoresis of the PCR products indicated that allele-specific length polymorphisms exist in this region of Y1. In order to determine if these length polymorphisms were due to the alleles having different numbers of the CCA repeat, the PCR product of each allele analyzed was gel purified and sequenced. These sequence data (Fig.1) show that the alleles studied contain between 5 and 11 CCA repeats.
In addition to the CCA trinucleotide repeat variation, we have also identified that a TCATC sequence directly 5' of the CCA repeat is duplicated in four of the nine alleles analyzed (Fig. 1A,B,E and F). Seven of the alleles also have the sequence CCATC directly 5' of the TCATC sequence (A,B,C,D,G,H, and I). It is interesting to note that the last CCA repeat is part of a second CCATC sequence. It appears that the CCATC and TCATC sequences are imperfect repeats of each other and that together they constitute a pentanucleotide repeat. Only two of the alleles analyzed (A and B) contained both the CCATC and TCATC sequence duplicated. These two alleles also contain the trinucleotide CTG repeated 33 bp 5' of the CCA repeat.
Many alleles of Y1 have been previously described (Buckner et al., Plant Cell 2:867-876, 1990; Robertson and Anderson, J. Hered. 52:53-60, 1961), however, the DNA sequence has been determined for only one dominant allele of Y1 (unpublished data). Therefore, the sequence variation that distinguishes recessive and dominant alleles of Y1 has not been described. Trinucleotide repeat expansion has been correlated with disease-associated alleles in several human diseases (Stallings, Genomics 21:116-121, 1994). However, our study has shown that no correlation exists in the number of CCA repeats within this region of the Y1 gene and the expression patterns of the different alleles of Y1. For example, the sequence of this region in the dominant allele of Y1 from the inbred line M14 is identical to that of the recessive alleles y1-wmut and y1-8549 (Fig. 1). In addition, this region in the dominant allele found in inbred line B73 is identical in sequence to the standard recessive allele of Y1 (Fig. 1). The recessive teosinte and y1-lemon yellow alleles have 5 and 6 CCA repeats, respectively, and both lack the CCATC repeat which is 5' of the CCA repeat in all other alleles of Y1 analyzed. It is not clear from these data whether the number of repeats influences the expression of these two alleles.
The teosinte allele analyzed in this study was derived from Zea luxurians. However, it should be noted that since maize and teosinte readily hybridize, this allele could be the result of a previous introgression of a maize Y1 allele into this accession of Zea luxurians. Teosinte is thought by many to be the progenitor to maize (Beadle, Sci. Am. 242:112-119, 1980; Galinat, Corn and Corn Improvement, G.F. Sprague and J.W. Dudley, eds., pp 1- 31, 1988). Because the teosinte allele has five CCA repeats it is likely that the microsatellite repeat has expanded in teosinte and may have done so before the evolutionary event that gave rise to maize. Several species and varieties of teosinte in addition to Zea luxurians have been described. These forms of teosinte differ in their growth pattern (annual and perennial) and ploidy. Therefore, it would be interesting to determine the extent of CCA repeat number variation at this locus in these other teosintes, and to determine if any alleles of teosinte or maize contain a single copy of this CCA.
Fig.1. Sequence comparison of the CCA microsatellite region of the Y1 alleles analyzed. The CCA microsatellite repeat is double underlined and the pentanucleotide and other trinucleotide repeats are single underlined. Sequence A: allele cloned from the hybrid of inbred lines Q66 and Q67 (Plant Cell 2:867-876, 1990), B: allele in inbred line H99, C: allele in inbred line M14, D: allele in inbred line B73, E: y1-lemon yellow allele (received from G.F. Sprague), F: Zea luxurians allele, G: standard recessive allele of Y1 (Plant Cell 2:867-876, 1990), H: y1-wmut allele and I: y1-8549 allele (J. Hered. 52:53-60).
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