Division of Plant Breeding, Genetics and Biochemistry, International Rice Research Institute, P. 0. Box 933, 1099 Manila, Philippines
Restriction fragment length polymorphisms (RFLPs) have been used to construct detalied genetic maps of rice (McCouch et al. 1988; Saito et al. 1991) and other plants (Paterson et al. 1991; Moore et al. 1993). Because of intensive studies at Cornell University (USA) and the National Institute of Agrobiological Resources (Japan), maps containing in total over 2000 markers are now avalilable for rice (Causse et al. 1994; Kurata et al. 1994). This marker density gives a high probability that any gene of interest can be placed on the map through linkage to one or more markers. It also improves the efficiency of the mapping of quantitative trait loci (Tanksley 1993).
Compared with morphological and isozyme markers, RFLP markers have the advantage of being numerous, neutral and co-dominant, but because of Southern hybridization they are difficult and expensive to use. For this reason, it is now common to tag a gene of interest with RAPD (Williams et al. 1990) or DAF (Caetano-Anolles et al. 1992) markers and then to use Southern hybridization to place the tag on the genetic map. Southern hybridization is regarded also as the method of choice in molecular marker-aided selection, but the polymerase chain reaction (PCR) (Saiki et al. 1988) may offer an economical and convenient alternative, provided DNA sequence information is available for each mapped locus (Willams et al. 1993). We have therefore embarked on a DNA sequencing program to convert about 300 clones of the Cornell RFLP marker collection from Southern hybridization probes to sequence tagged sites (STS) suitable for PCR. To date, more than 280 clones have been partially sequenced from each end, and we have begun to synthesize and validate the corresponding PCR primer pairs. We report now the conversion of 44 markers on chromosome 1 of rice to STS and provide evidence that about 78% of these sites are suitable for use in PCR.
The 1994 Cornell map of the rice genome (Causse et al. 1994) shows the locations of 112 RFLP markers on chromosome 1. We sequenced the ends of 43 of these mapped clones (Fig. 1) using the universal forward and reverse sequencing primers for pUC-based plasmids. The 43 clones included 25 RG clones, 16 RZ clones, 1 CDO clone and 1 BCD clone. The RG clones were derived from a random Pst1 genomic library for rice, whereas the RZ, CDO and BCD clones were derived from cDNA libraries of rice, oats and barley, respectively. In general, cycle sequencing (US Biochemicals, TAQuence Cycle-Sequencing Kit, product number 71075) yielded about 150-250 bases of sequence from each end.
Fig. 1. Sequence tagged sites of chromosome #1 of rice. Mapped RFLP probes
(Causse et al., 1994) were partially sequenced from each end and the sequence
data were used to design pairs of PCR primers. The primer pairs were
validated as described in the text. Loci in brackets failed this validation
test. Underlined loci showed evidence of introns in the PCR products from
genomic DNA. Asterisks indicate loci showing polymorphism between O. sativa
and O. longistaminata.
Cycle sequencing was particulary effective in sequencing through the poly T tracts at the 3'-end of cDNA clones.
The PCRPlan component of the PCGene software package was used to design primer pairs (19-to-23-mers) for PCR amplification of the 43 loci. in addition, we made a pair of primers for the Sal T locus based on the cDNA sequence (Clacs et al. 1990). The oligonucleotides were synthesized on a Beckman Oligo 1000 DNA symthesizer at the 30 nmole scale. The primer pairs were validated by checking whether they amplified (i) a single PCR product of the correct size when the corresponding clone was used as template and (ii) a single PCR product when IR36 DNA was used as template. Of the 44 primer pairs, 34 passed this validation test. The 10 primer pairs that failed validation did so for one or more of the following reasons:
(i) they failed to amplify a product of the expected size from plasmid DNA, (ii) they failed to amplify a band from IR36 DNA, (iii) they amplified more than one band from plasmid and/or IR36 DNA. In Fig. 1 the failed loci are enclosed in brackets.
For each of 44 primer pairs we compared the products obtained with four different DNA templates: plasmid DNA and DNAs extracted from IR36 (indica), Taichung 65 Uaponica) and O. longistaminata (wild species with AA genome). We found several different types of result and illustrate some of them in Fig. 2 by reference to 5 particular loci. RG532 represents the failed loci. It failed validation because two bands were produced from IR36 DNA.
Fig. 2. Validation of PCR primer pairs for five representative loci on
chromosome 1 of rice. Each primer pair (20 ng of each primer) was tested
with four DNA templates: (i. to r.) plasmid DNA (1 ng), IR36 DNA (160 ng),
Taichung 65 DNA (160 ng) and O. longistaminata DNA (160 ng). PCR temperature
profile: 35 cycles of 94 deg C for 1 min, 55 deg C for 1 min, 72 deg C for 2
min; final elongation step of 72 deg C for 5 min. Gel electrophoresis: 150V
for 2 h in 2% agarose with 0.5 mg/L ethidium bromide.
The other 4 loci in Fig. 2 represent results obtained with validated loci. The most common result with the validated loci was that all four DNA templates gave the same size of product (e.g., RG811). This type of pattern was seen at 13 of the 19 validated RG loci and at 4 of the 14 validated RZ loci. At another 9 RZ loci and at the SAIT locus (underlined in Fig. 1), the PCR products from the 3 rice genomic DNAs were larger than the product obtained for the plasmid, consistent with the presence of introns in the genomic DNAs (e.g., RZ19). The intron sizes vaired from 0.1 to 2.0 kbp. RZ382 was exceptional in that the PCR product obtained from genomic DNA was smaller than that obtained from the corresponding plasmid; we are uncertain as to the explanation for this result. At 6 RG loci and at 4 RZ loci (with asterisks in Fig. 1), the O. longistaminata product was either absent or was different in size from those obtained with the other two genomic templates (e.g., RG810 and RZ527).
We are now attempting to detect RFLPs between O. longistaminata and O. sativa and between IR36 and Taichung 65 at the validated loci by digesting the PCR products with 4-base cutters. The ease with which such polymorphisms can be found will to a large extent determine the utility of STS in molecular marker-aided selection.
We thank Dr. Susan McCouch and Dr. Ning Huang for their kind gift of clones, Dr. G. S. Khush for his encouragement of this work, and the members of the Plant Molecular Biology Laboratory and the Genome Mapping Laboratory for their assistance. We gratefully acknowledge the Rockefeller Foundation for financial support (grant no. 92002 # 14).
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