Fig. 1. Bulked segregant analysis with RAPD marker OPM6 M. Marker, RP. Resistant parent, SP. Susceptible parent, RB. Resistant bulk and SB. Susceptible bulk. Arrow. Resistant specific RAPD M06.
1) Plant Molecular Biology Laboratory, Division of Plant Breeding, Genetics and Biochemistry, International Rice Research Institute, Manila,Philippines
2) Plant Protection Research Institute, GAAS, Guangzhou, China.
3) Rice Research Institute, GAAS, China.
Much interest has recently arisen in the PCR-based RAPD method of DNA fingerprinting (Williams et al. 1990). One variant of this technique, Bulk Segregant Analysis, developed by Michelmore et at. (1991) is currently receiving particular attention because of its simplicity. It is used to screen for differences between two pooled DNA samples derived from a segregating population and is a poweful tool for gene mapping. We report now the use of Bulk Segregant Analysis to identify a RAPD marker linked to a gall midge resistant gene in cv. Duokang # 1 from China.
Gall midge (Orseolia oryzae) is the major dipteran pest of rice. It causes sporadic and occasionally catastrophic losses in areas of South China, Cambodia, Northeast Thailand, Sri Lanka and India, and is found in several other Asian countries. Orseolia oryzivora, the African rice gall midge, is a problem in Nigeria and several other African rice growing countries. Six biotypes of gall midge are known in India, four in China, two in Sri Lanka and others in Thailand, Indonesia and Cambodia. Recently, Tan et al. (1993) reported that cv. Daqiuqi provides resistance against all four biotypes of gall midge identified in China. Its single dominant gall midge resistance gene is now being used in breeding programs in Guangdong and is present in the derived line Duokang # 1.
An F`3` mapping population was prepared from a cross between Duokang # 1 (gall midge resistant) and Feng Ying Zhan # 1 (susceptible) and 160 lines were phenotyped for all midge resistance at Guanzho Acad. Agric. Sci., China in July, 1994. Plants were evaluated in the screen house using Chinese biotype I (from Huaxian) and the standard evaluation system of IRRI. Table 1 summarizes the phenotypic data. Out of 160 lines, 29 showed complete resistance and 13 showed complete susceptibility.
Table 1. Phenotypic testing of 160 F3 families from the cross between
Duokang#1 X Feng Ying Zhan #1
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Group Plants infested No. of F`3` families
in a family
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1. 0 29
2. 1-10 05
3. 11-20 24
4. 21-80 60
5. 81-90 08
6. 91-99 21
7. 100 13
Reaction of check varieties
Duokang #1 0
Feng Ying Zhan 76.2-100
77.8-100
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Phenotypic screening was carried out against Biotype I of Huaxian.
Seeds from the 160 F`3` lines were also grown at IRRI. Leaves were harvested
after six weeks and DNA was extracted by the method of Tai and Tanksley
(1990). DNA (2ug) from each of 12 lines with phenotypic score 0 and from
each of 12 lines with phenotypic score 100 were pooled to make the resistant
and susceptible bulks, respectively. These bulks were used as target DNAs
for RAPD analysis along with DNA from the parental cultivars Duokang #1 and
Feng Ying Zhan #1. The two bulked samples and both the parents were screened
with random 10-mer oligonucleotide primers obtained from Operon Technologies
Inc., Alameda, Calif., to generate RAPD products. The amplification reaction
conditions were as described by Williams et al. (1990) with the
modifications of 45 cycles of 1 min. at 94 deg, 1 min. at 34 deg, 2 min. at
72 deg using the fastest available transition between each temperature in a
Perkin-Elmer Cetus Thermal Cycler. Amplification products were analysed by
agarose gel electrophoresis and staining with ethidium bromide.A RAPD marker OPM6 was identified as being tightly linked to gall midge resistance gene of Duokang # 1. Fig. 1 shows the amplification products for the two parental lines and the two bulks with this primer. Several bands are evident in each lane but the 1400 bp product (arrowed) has the important property of being present in the resistant parent lane and the resistant bulk lane but absent from the other two lanes. This is preliminary evidence that the locus corresponding to the 1400 bp band cosegregates with gall midge resistance. To confirm that the polymorphism in the DNA bulks was not an artefact, both the parents and the individuals used to construct the bulks were examined with the same primer. All 12 resistant lines, like the resistant parent, gave the 1400 bp band, but only 2 out of 12 susceptible lines gave this band. Finally, the 1400 bp product was monitored in 26 resistant (0% infestation) and 26 susceptible lines (91-100% infestation). RAPD analysis of these lines revealed that the 1400 bp marker was amplified in all 26 resistant lines but amplified in only 5 out of 26 susceptible lines. We estimate that the RAPD marker identified a locus 5-10 cM distant from the gall midge resistance gene.
The 1400 bp RAPD product is now being mapped on the rice genome. It will be interesting to see whether it is close to the Gm2 gene recently mapped on
Fig. 1. Bulked segregant analysis with RAPD marker OPM6 M. Marker, RP.
Resistant parent, SP. Susceptible parent, RB. Resistant bulk and SB.
Susceptible bulk. Arrow. Resistant specific RAPD M06.
to chromosome 4 by Mohan et al. (1994). Mapping should enable us to identify close flanking markers for use in marker-aided selection. At present, the rice breeding program in South China calls for transfer of the gall midge resistant gene from "sunshine sensitive" low land cultivars to "temperature sensitive" upland cultivars but this program has a major drawback in that the screening for resistance to this insect is possible only once in three breeding generations per year. Marker-aided selection will accelerate this program.
We thank L. Estenor for assistance and gratefully acknowledge finanical support for this work from the Deutsche Gesellschaft far Technische Zusammen- arbeit under the Asian Rice Biotechnology Network. SKK was supported on a postdoctoral fellowship from the Rockfeller Foundation.
References
Michelmore, R. W., I. Paran and R. V. Kessell, 1991. Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations. Proc. Nati. Acad. Sci. USA 88: 9828-9832.
Mohan, M., S. Nair, J. S. Bentur, U. P. Rao and J. Bennett, 1994. RFLP and RAPD mapping of the rice Gm2 gene that confers resistance to biotype 1 of gall midge (Orseolia oryzae). Theor. Appl. Genet. 87: 782-788.
Tai, T. H. and S. D. Tanksley, 1990. A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue. Plant Mot. Biol. Rep. 8(4): 297-303.
Tan, Y., Y. Pan, Y. Zhang, Z. Lixia and Y. Xu, 1993. Resistance to gall midge (GM) Orseolia oryzae in Chinese rice varieties compared with varieties from other countries. IRRN 18(4): 13-14.
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