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Crop Science 40:245-247 (2000)
© 2000 Crop Science Society of America

CELL BIOLOGY & MOLECULAR GENETICS

RFLP Mapping of QTLs Influencing Shoot Regeneration from Mature Seed-Derived Calli in Rice

Y. Takeuchia, T. Abea and T. Sasaharaa

a Lab. of Plant Breeding, Faculty of Agriculture, Yamagata Univ., Tsuruoka 997-8555, Japan

cyn00524{at}niftyserve.or.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
This study focused on the mapping of quantitative trait loci (QTLs) related to shoot regeneration from mature seed-derived calli of rice (Oryza sativa L.) by restriction fragment length polymorphism (RFLP) markers. The F2 population from a cross between `Norin 1' (japonica) and `Tadukan' (indica), which showed lower and higher shoot regeneration rates, respectively, was used for QTL interval mapping. The population was analyzed for 103 RFLP markers distributed over the 12 rice chromosomes. The QTL with the largest effect on shoot regeneration, which accounted for about 19% of the total variation, was mapped on chromosome 2. A minor QTL on chromosome 4 exhibited a smaller effect, accounting for about 3% of the total variation in percentage shoot regeneration. No QTLs were detected on other chromosomes with the RFLP markers that were used in the present experiments. These results indicate that at least one major gene is associated with the percentage shoot regeneration, and agree with previous genetic studies, indicating that one dominant gene is associated with shoot regeneration.


   INTRODUCTION
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 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 REFERENCES
 
EFFICIENT PLANT REGENERATION from calli is indispensable for manipulating tissue and/or protoplast cultures, and thereafter for conducting transformation experiments. The percentage of rice plants that can be successfully regenerated from calli has increased through improvements in the culture medium (Inoue and Maeda, 1981; Daigen and Abe, 1993).

Previously, genetic studies using diallel analysis have shown that the ability of rice plants to be regenerated from calli is regulated by several genes (Peng and Hodges, 1989; Abe and Futsuhara, 1991). However, the corresponding loci were not identified, and therefore not located on the linkage maps of rice.

Recently, Taguchi-Shiobara et al. (1997) detected three QTLs for regeneration from calli on rice chromosomes 1, 2, and 4 using 98 BC1F5 lines from a cross between `Nipponbare' (japonica) and `Kasalath' (indica) and 245 RFLP markers. These RFLP markers were distributed over all 12 rice chromosomes (Kurata et al., 1994). Nipponbare exhibited a relatively low ability for regeneration from calli and a recessive phenotypic expression, whereas Kasalath exhibited a relatively high regeneration ability and a dominant phenotypic expression. Taguchi-Shiobara et al. (1997) reported that the QTLs on chromosome 2 were more closely related to plant regeneration than were the QTLs on chromosomes 1 and 4. Our previous paper (Takeuchi et al., 1997) reported that the phenotypic expression of the percentage plant regeneration fit a 3 (dominant):1 (recessive) ratio on the basis of rice lines of the F2 generation from a cross between `Norin 1' (japonica) and `Tadukan' (indica), and a 1:1 ratio on the basis of a BC1F1 population.

The present experiments focused on the QTL mapping of gene loci in relation to the rate of plant regeneration from mature seed-derived calli in rice. The experiments were conducted with the same cross (Norin 1 x Tadukan) as reported in our previous paper (Takeuchi et al., 1997).


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 REFERENCES
 
Plant Materials and Shoot Regeneration
A japonica cultivar, Norin 1 was crossed with an indica cultivar, Tadikan. The ability of Norin 1 to regenerate plants from mature seed-derived calli is less than that of Tadikan (Abe and Futsuhara, 1986, 1989). Seeds of the F1 generation were harvested on 10 Sep. 1993, and grown in a greenhouse of the Shonai Branch Station of the Yamagata Prefectural Agriculture Experimental Station for generation advancement. Seventy-nine seeds of the F2 generation were harvested on 15 May 1994, and used for shoot regeneration experiments.

Brown rice seeds were prepared as described previously (Takeuchi et al., 1997). These procedures included sterilization, callus induction on a basic medium (Murashige and Skoog, 1962) as modified by Chu et al. (1975), division of single calli for preparation of replications, and subculture of calli. Percentage shoot regeneration was measured as follows. One callus that originated from one F2 seed was divided into five pieces, each of which was placed in a separate bottle and subcultured. Then, each of the five calli were subdivided into four pieces and these were subcultured in the same five bottles again. These calli were transferred into five other bottles containing regeneration medium. The regeneration medium is described by Takeuchi et al. (1997). After incubation for 45 d, the percentage shoot regeneration was calculated as the total number of regenerated (calli/20) x 100. Almost all the regeneration shoots grew well and were easily acclimatized.

DNA Extraction, Probe, and Southern Analysis
Total genomic DNA from the non-regenerated calli and leaves of regenerated plants was extracted according to the CTAB method of Murray and Thompson (1980) and digested with the restriction enzymes ApaI, BamHI, BglII, EcoRI, EcoRV, and HindIII. The DNA fragments were electrophoretically separated on a 0.7% (w/v) agarose gel, stained with ethidium bromide, and transferred to a nylon membrane according to the method of Southern (1975). Southern hybridization was carried out with a non-radioactive (digoxigenin) DNA labeling and detection kit (Boehringer Mannheim Biochemica, Germany). RFLP markers with intervals of 15 to 25 cM in each of 12 chromosomes were selected from the list published from the list by Kurata et al. (1994). After detection of QTLs on chromosomes 2 and 4, more RFLP markers specific to these chromosomes were used to more accurately determine the locations of these QTLs. A total of 103 RFLP markers that were provided by the Rice Genome Research Program in Japan (RGP) were used. A computer program (MAPL Ver. 3.0)(Ukai et al., 1995) based on the Kosambi function (Kosambi, 1944) was used to calculate the mapping distances on the chromosomes and to map QTLs by the interval mapping method.


   Results and discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
REFERENCES
 
The percentage plant regeneration (mean ± standard deviation) of callus pieces derived from Norin 1, Tadukan, and F1 seeds were 21.6 ± 16.0 (n = 34 seed), 91.1 ± 12.1 (n = 27 seed), and 81.1 ± 17.6% (n = 10 seed), respectively. Two QTLs, one on chromosome 2 and one on chromosome 4, were detected in the interval mapping of the QTL by RFLP markers (Fig. 1) , and no QTLs were detected on other chromosomes. The highest LOD score of 9.9 was recorded between the RFLP markers C424 and G45 (Fig. 1). The QTL on chromosome 2 accounted for about 19% of the total variation in the percentage shoot regeneration (Table 1) . A LOD score of 2.1 on chromosome 4 was observed between the RFLP markers G271 and C975. This value was negligible compared with that on chromosome 2 (Fig. 1). The QTL on chromosome 4 accounted for about 3% of the total variation in the percentage shoot regeneration (Table 1).



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  		Fig. 1 Linkage maps of RFLP markers on chromosomes 2 and 4 (abscissa) and LOD scores detected by an interval mapping analysis (ordinate). *, Significant at the 0.05 probability level in a one-way ANOVA

 

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  		Table 1 Putative QTLs affecting plant regeneration from mature seed-derived calli

 
A one-way ANOVA confirmed that the QTL on chromosome 2 was significant at the 0.05 probability level, but the other QTL on chromosome 4 was not significant. Furthermore, a two-factor model in the ANOVA showed that digenic epistasis between these two QTLs was also not significant. Thus, the QTL analysis showed that the shoot regeneration ability from mature seed-derived calli was regulated by one major gene on chromosome 2. These results are consistent with our previous finding that the higher plant regeneration of Tadukan is probably regulated by a single gene (Takeuchi et al., 1997). In addition, our results regarding the location of the major QTL on chromosome 2 and the percentage variation, appear to agree with those of Taguchi-Shiobara et al. (1997). Furthermore, the cultivars showing the positive QTLs in these two studies (Kasalath in the experiments by Taguchi-Shiobara et al. (1997) and Tadukan in the present experiments) were indica cultivars with high regeneration rates.

To clarify the relationship of the QTL on chromosome 2 to the rate of shoot regeneration, it may be necessary to develop isogenic rice lines with respect to plant regeneration. Mano et al. (1996) conducted QTL mapping related to shoot regeneration in barley (Hordeum vulgare L.) using doubled haploid lines. Interestingly, the QTLs that they detected accounted for a much greater percentage of the total variation (about 50%) than was found in the present study with rice.


   ACKNOWLEDGMENTS
 
We thank Dr. Y. Ukai, The University of Tokyo, for his valuable suggestions for the statistical analyses.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 




This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Nishimura, M. Ashikari, S. Lin, T. Takashi, E. R. Angeles, T. Yamamoto, and M. Matsuoka
Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems
PNAS, August 16, 2005; 102(33): 11940 - 11944.
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This Article
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