37. Integration of AFLP markers into an RFLP and SSLP map of rice (Oryza sativa L.)


1) National Institute of Agricultural Science and Technology, Suwon, 441-707 Korea
2) Department of Plant Breeding, Cornell University, Ithaca, NY 14853-1901, USA
3) KeyGene N.V., Agro Business Park 90, 6708 PW Wageningen, The Netherlands

The construction of saturated genetic maps requires the analysis of large numbers of DNA markers capable of sampling all types of sequence configurations. RFLP markers have been widely used to prepare framework maps based on single and low copy clones. Microsatellite markers, or simple sequence length polymorphisms (SSLP), which are derived from repetitive regions of the genome are being used to further saturate maps in several plant species (Panaud et al. 1996: Chen et al. 1997). Amplified fragment length polymorphism (AFLP) markers, which are derived by double digestion of the entire genome followed by PCR, have further expanded the repertoire of marker technologies (Vos et al. 1995; Becker et al. 1995: Maheswaran et al. 1997). A combination of molecular markers is most likely to provide the best genome coverage.

An AFLP map of rice has been constructed using F11 recombinant inbred (RI) population from Korea. The mapping population was developed by a single seed descent from an intercross between Milyang 23 (Tongil) and Gihobyeo (Japonica) and consists of 164 lines (Cho et al. 1997). The polymorphism between parents was about 80 percent based on RFLP analysis. Initially, a framework map was constructed using a subset of the RFLP markers previously mapped by Causse et al. (1994) and Kurata et al. (1994), and microsatellite markers reported by Chen et al. (1997). We used EcoRI and MseI digested DNA to generate AFLP data. A total of ten primer combinations, each with different specific 2 or 3bp overhangs, six EcoRI+2 and four MseI+3 primers, were used to amplify AFLP bands (Cho et al. 1997). Each primer combination generated between 73 and 134 bands visible on polyacylamide gels, with a mean of 101 total scorable bands and an average of 26.9 polymorphic bands (Fig.1). Of the 1011 AFLP bands visiblefrom 10 primer combinations, 269 (26.6%) were polymorphic in the MG Rl population. The best two combinations were E(AG)/M(CTA) (E for EcoRI and AG for the specific 2bp overhang, and M for MseI and CTA for the specific 3 bp overhang) and E(TG)/M(CAA), which showed over 30% polymorphism between Milyang 23 and Gihobyeo. Out of 235 segregating AFLP markers, 231 markers were integrated into the RFLP and SSLP map.

Digestion with the enzyme combination EcoRI/MseI and the use of 2 and 3 bp specific overhangs for AFLP primers proved useful for this small genome crop. More than twice as many bands were detected with this enzyme combination than in a previous report by Maheswaran et al. (1997) where the methylation sensitive enzyme, PstI was used in combination with MseI and 2 or 3bp overhangs on the primers, and 945 AFLP hands were detected from 20 primer combinations, of which 208 (21.8%) were polymorphic. The frequency of polymorphism detected by AFLP markers in a barley mapping population was 11.3% (Becker et al. 1995). Though the polymorphism rate of AFLP markers is much lower than RFLP or SSLP markers per number of bands scored, the AFLP system is efficient because it allows the simultaneous analysis of a large number of bands per gel.

The integrated MG map consists of a total of 536 markers: 231 AFLP, 212 RFLP, 86 SSLP, 5 isozyme and 2 morphological mutant loci. The AFLP markers were distributed throughout the 12 chromosomes and helped fill several gaps left by the RFLP and SSLP markers. The total map distance for this RI population is 1814cM, with an average interval size of 3.4cM (Fig. 2).

We are refining this map by adding partially-sequenced cDNA markers derived from an immature seed cDNA library developed in Korea and microsatellite markers developed at Cornell. This population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development.

Fig. 1. Autoradiogram obtained with E(AG)/M(CTA) primer combination. Marker names and alignment bands are indicated in left margin. Template identity is indicated in right margin. 10BL is the 10-base ladder: P1 and P2 indicate the parents, Milyang 23 and Gihobyeo, followed by plant numbers from the MG Rl population.

Fig. 2. The integrated map based on AFLP, SSLP, and RFLP markers. Map distances (on the left) are given in cM (Kosambi function). AFLP makers are designated as in the following example: E13M60.320-P2, which is mapped at the top of cromosome 1, indicates that the marker was amplified with the primer combination E13 (E for EcoRI, 13 for the specific 2bp overhang ) and M60 (M for MseI, and 60 for the specific 3bp overhang). AFLP makers are highlighted in italics.


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