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        Zi-Xuan Wang, Akio Kojima¹, Nori Kurata, Yuzo Minobe and Takuji Sasaki
        Rice Genome Research Program, National Institute of Agrobiological Resources, 2-1-2 Kannondai.
        Tsukuba 305, Japan / STAFF (Society for Techno-innovation of Agriculture, Forestry and Fisheries)
        Institute, 446-Ippaizuka, Kamiyokoba, Tsukuba, 305, Japan
        1) Present address: Kyushu National Agricultural Experimental Station, Kirishima 5-2, Miyazaki 880, Japan

    In our rice genome mapping project, we could construct a high-density linkage map by using molecular markers (Kurata et al. 1994). However, there were a number of genomic clones that could not be assigned to respective chromosomes, because of lack of RFLPs between the parent varieties using the given enzymes. Those clones are likely to contain a genomic fragment with a specific function and a specific chromosomal position. We are interested in assigning those clones to their respective chromosomes. We describe here a method to assign the non-RFLP producing genomic clones rapidly by using rice aneuhaploids, tetrasomics and trisomics.
    Three types of aneuhaploid (Aneuhaplo 5, 9 and 11, 2n=13), 4 types of tetrasomics (Tetra 6, 7, 10 and 12; 2n=26) and 4 types of trisomics (Triple 1, 2, 4 and 8; 2n=25), which cover 11 chromosomes of rice (Iwata and Omura 1984; Wang and Iwata 1995), were used to assign clones to specific chromosomes. The aneuploid for rice chromosome 3 was not available in the experiment. Assignments of DNA clones to specific chromosomes were performed by dosage comparison among genomic Southern hybridization of all aneuploids. These aneuploids were in the background of a japonica variety of "Nipponbare". The diploid "Nipponbare" was used as a control. Eleven aneuploids and "Nipponbare" were grown in the greenhouse. DNA extraction and Southern blots were performed according to Kurata et al. (1994).
    A total of 33 genomic clones from a Pst I library (pNP, 11 clones) and a Hind III library (pNH, 22 clones), made using a japonica variety "Nipponbare" and Bluesript II SK+ vector, were used for chromosome assignment. All these clones were single copy in the genome and showed monomorphism in Southern analysis between the two parent varieties "Nipponbare" and "Kasalath" with the 8 enzymes: Apa I, BamH I, Bgl II, Dra I, EcoR I, EcoR V, Hind III and Kpn I used in our mapping project (Kurata et al. 1994).
    Chromosomal assignment of clones was determined by using the dosage effects of the extra chromosomes of aneuploids, as has been described in tomato (Young el al. 1987) and in rice (Wang et al. 1995, Yu et al. 1995). In such an analysis, the most probable source of error is variation in the quantity of DNAs among aneuploids loaded on individual lanes of the gel used for the Southern analysis. To avoid this problem, another clone having a different size was used together in the hybridization experiment as an internal control probe of each lane. This could help adjust signal intensities of each lane and made it possible to confirm an increased hybridization signal of a clone in a given aneuploid. Two clones having different sizes were mixed and digoxigenin-labeled to hybridize to DNAs from aneuhaploids, tetrasomic, trisomics and "Nipponbare". As shown in Figs. 1 and 2, hybridization signals obtained as autoradiograms could clearly show which aneuploid had an extra copy or copies of the cloned DNA. By looking at the increase in dosage of a DNA clone in one of the aneuhaploids, tetrasomics, or trisomics, the chromosomal location of the DNA clone was determined.
    Fig. 1 shows an autoradiogram of two clones, pNH93 and G37, probed onto Hind III-digested DNAs from 3 aneuhaploids, 4 tetrasomics and "Nipponbare". pNH93 could be easily assigned on chromosome 7 and G37 on chromosome 10, as indicated by the increased signals in the DNAs from Tetra7 and Tetra10, respectively. In this case, both clones hybridized to the tetrasomics with high intensity signals were unequivocally determined to be located on these chromosomes. G37 was used as an internal control. The assignment of G37 to chromosome 10 determined in this study consistent with the previous mapping data reported by Saito et al. (1991).
    Because aneuhaploids and tetrasomics only cover 7 rice chromosomes, we also used 4 types trisomics to increase the coverage of chromosomes and to facilitate the effectiveness of the assignment. Fig. 2 shows an autoradiogram of two clones, pNH 146 and pNH221, probed onto Hind III-digested DNAs from 3 aneuhaploids, 4 tetrasomics, 4 trisomics and "Nipponbare". The clone pNH146 was also easily assigned to chromosome 1, as indicated by the increased signals in the DNA from Triple 1. pNH221 could not be assigned, because of lack of dosage effect in the aneuploids used in the experment. This clone may belong to chromosome 3, which is only missing chromosome in this study. We found trisomics also useful in this kind of analysis for dosage comparison. However, when trisomics were used alone in the analysis, it is difficult to determine the dosage effect with certainty, since the difference in 3 versus 2 dosages is not so great. By using rice aneuhaploids and tetrasomics, it becomes easier to determine the dosage effect, because the dosage difference is 4 versus 2 in tetrasomics and 2 versus 1 in aneuhaploids. This is easily seen by comparing the signal differences between Fig. 1 and Fig, 2. Aneuhaploids and tetrasomics are thus more effective than trisomics in this kind of studies.
    Out of the 33 clones tested, we have clearly assigned 24 clones to 8 rice chromosomes (Table 1). Among them, a few clones could be mapped on our high-density

We are grateful to Professor N. Iwata. Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University for supporting this work, and Dr. 1. Havukkala, STAFF Institute, for comments on the manuscript.

Chromosome	Clones
1	pNP42, pNH6, pNH69, pNH 104, pNH 146, pNH 166
2	pNP 101, pNH21, pNH72, pNH88
5	pNH32
6	pNP63, pNP25, pNH77
7	pNH68, pNH93, pNH129, pNH139, pNH263
9	pNP 103
10	pNP 167, pNH84, pNH245
12	pNH 123