Considerable work on tagging root morphological traits has been done following the pioneering work of Champoux et al. (1995). Adopting different phenotyping methodologies many researchers across the world have identified quantitative trait loci (QTL) for many root morphological traits in several mapping populations and in contrasting moisture regimes. Using these markers in breeding programs has not been very popular because of the difficulty in mapping the loci with sufficient precision. Further, the mapped regions are associated with more than one trait indicating the possibility of pleiotropy and/or tight linkage. Marker-assisted selection for traits calls for more specific markers as exemplified in monogenic traits. Liu (1997) opined that QTLs are "statistical entities" and thus dependent on several factors that can make a QTL "appear or disappear".

The importance of roots in allowing a rainfed rice plant to maintain a higher water status when subjected to low-moisture stress has been well documented (O'Toole, 1989; Hirasawa, 1995; O'Toole, 1997). Of the several components of root morphology, maximum rooting length is considered most important as it enables the plant to access moisture in deeper layers of the soil profile. Placing emphasis on this trait seemed justified as long-rooted perennial crops and trees around the rainfed lowland habitats can effectively withstand long periods without rain. Using bulked segregant analysis (Michelmore et al., 1991), we identified two polymerase chain reaction-based markers (OPBH14 and RM201) closely associated with maximum root length (MRL) in rice (Shashidhar et al., 2001). The MRL-associated DNA band amplified by RAPD primer OPBH14 was polymorphic between IR64 and Azucena (1400 and 1750bp, respectively) and mapped to two regions on the genome (short arm of chromosome 10 and long arm of chromosome 1). RM201 had already been mapped onto chromosome 9.

The MRL-associated bands amplified by OPBH14 from DNA of IR64 and Azucena were extracted from gels by the NA₄₅-DEAE cellulose membrane method. Bands were ligated into a T-tailed vector (INST/A MBI Fermentus, U.S.A) and transformed into DH5 Alpha competent cells. A clone containing the OPBH14 amplicon was used as a probe in Southern hybridization of doubled haploid lines to verify the size and authenticity of the cloned fragment. End sequences of the cloned fragment were obtained by Sangers' dideoxy method using ABI 377 prism (Avesthagen Graine Limited, Bangalore).

End sequences (5' - 3' 515 bases; 3'-5' 477 bases) of the BH14 fragments were used to design primers using Biowire Jellyfish software (http://www.biowire.com). The best among the primers was one with 18mer forward and 22mer reverse primers as indicated below. The primers designed include original RAPD (OPBH14) primer sequence in the reverse primer and one base less of the RAPD primer sequence in the forward primer from the 5' end.

These SCAR primers were used to amplify the genomic DNA of IR64 and Azucena, parents of DH population and transgressants genotypes of DH population for MRL and 40 F₄ plants of IR50/Moroberekan mapping population.

The PCR was performed in total volume of 20 microl containing 10XPCR buffer (10mM TRISCl, 50mM KCl, 1.5mM MgCl₂) 12 pmol of each primer, 50 ng of rice genomic DNA, 80 microM of each of the four dNTPs, and 1 unit of Taq polymerase (Bangalore Genei, India) with mineral oil overlay. Thermal conditions were 5min for 94C followed by 35 cycles of 94C for 1 min, 54C for 1 min, 72C for 1min and final extension of 72C for 5min. Amplified fragments were resolved in 1.4% agarose gels.

SCAR markers amplified 1400 and 1750 base pair bands in shallow and deep-rooted individuals of the double haploid mapping population of IR64/Azucena (Fig. 1) and IR50/Moroberekan F₄ population.

Single marker analysis showed significant (P<0.01) association between the SCAR and maximum root length in both F₄ population of IR50/Moroberekan and the doubled haploid population of IR64/Azucena. The BH14 SCAR mapped onto the same two loci as did its RAPD amplicon.

References

Liu, B.H. 1997. Computational tools for study of complex traits. In: A H. Paterson (ed). Molecular Dissection of Complex Traits, CRC press, Boca Raton, New York. p. 43-79.

Champoux, M.C., S. Sarkarung, D.J. Mackill, J.C. O'Toole, N. Huang and S.R. McCouch. 1995. Locating genes associated with root morphology and drought avoidance in rice via linkage to molecular markers. Theor. Appl. Genet., 90: 969-981.

Hirasawa, T. 1995. Water relations in plants. In: T. Matsuo, K. Kumazawa, R. Ishii and H. Hirata (eds). Science of the rice plant. 2. Physiology. Tokyo Food and Agriculture Policy Research Center. p. 434-460.

Michelmore, R.W., I. Paran, and R.V. Kesseli, 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci., 88: 9828-9832.

O'Toole, J.C. and T.T. Chang. 1979. Drought resistance in cereals. Rice: A case study. In: H. Mussel and R. Staples (eds), Stress Physiology in Crop Plants. Wiley Interscience, New York, U.S.A. p. 373-405.

O'Toole, J. C. 1989. Breeding for drought resistance in cereals. In F. W. G. Baker (ed), Drought resistance in cereals. CAB International, Wallingford, UK. p. 107-116.

Shashidhar, H.E, N. Sharma, R. Venuprasad. M. Toorchi, M. Chandrashekar, A. Kanbar and S. Hittalmani. 2001. Two DNA markers for maximum root length in rice validated across mapping populations and wide germplasm accessions. In: 8^th National Rice Biotechnology Network Meeting Oct 25-29^th, Aurangabad, India.