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Tagging QTLs for maximum root length in rainfed lowland rice by combined selective genotyping and STMs markers

Mahmoud Toorchi1,*, H.E. Shashidhar2, Naveen Sharma2 and Shailaja Hittalmani2

1. Molecular Breeding Lab, Faculty of Agriculture, The University of Tabriz, Tabriz, Iran
2
.Marker–Assisted Selection Lab, Department of Genetics and Plant Breeding, College of Agriculture, University of Agricultural Sciences, GKVK, Bangalore, 560 065, India. Email: masuas@satyam.net.in
*Corresponding author Email: mtoorchi@yahoo.com FAX + (98 411) 334 5332

Abstract

A dynamic root system is fine-tuned to soil moisture status and is known to regulate the amount of water available to the plant depending on its distribution in the soil. Recent advances in genome research, particularly in the field of molecular marker technology, have generated opportunity to dissect the variation in quantitative traits in a more meaningful way. We adapted selective genotyping strategy and STMs markers for mapping QTLs for maximum root length in rainfed lowland rice. Total of 69 extreme plants were selected from P124 x IR64 mapping population for selective genotyping because of good combination of genetic factors conferring drought tolerance and yielding ability. Forty-two pairs of STMS primer pairs that were earlier found to be polymorphic between the IR64 and Azucena were selected and used in the present study. A total of 4 QTLs were found for maximum root length under well watered and low moisture stress and mean conditions.

Introduction

Root system and stress-induced response form important components of drought resistance. A dynamic root system is fine-tuned to soil moisture status and is known to regulate the amount of water available to the plant depending on its distribution in the soil. Among the root morphological traits, maximum root length, root diameter and root: shoot dry weight ratio were found to be associated with drought resistance in upland conditions (Toorchi, et al., 2002). Despite ample genetic variability for many root morphological traits and other components (primary traits) of drought resistance, genetic improvement of root characteristics in rice, using conventional selection has been difficult (Thanh et al., 1999) and progress towards improvement of grain yield in rainfed lowlands has been hampered. This could be because of the differences in intensity of drought (timing and severity), the intrinsic (sometimes contrasting) potential of the crop to tolerate stress (like sorghum and rice) and the constraints of the study itself as, standardized protocols for assessing drought resistance are conspicuously absent (Blum, 1999).

Recent advances in genome research, particularly in the field of molecular marker technology, have generated opportunity to dissect the variation in quantitative traits in a more meaningful way. Selection based on molecular marker genotypes may yield a correlated response to selection with regard to traits controlled by marker-linked loci. Especially for complex quantitative traits, the expectations with regards to possibilities of this form of indirect selection are high. Such traits often have a low heritability, implying that direct conventional selection is ineffective. Nandi et al. (1997) used selective genotyping strategy and AFLP markers for mapping major gene and QTLs for submergence tolerance in rainfed lowland rice. This study was carried out to identify STMS (Sequence Tagged Micro Satellite) markers linked to maximum root length in succeeding backcross generations.

Material and methods

Five deep rooted and four shallow rooted transgressants for maximum root length were chosen from rice doubled haploid mapping population of IR64/Azucena and backcrossed to IR64. Among the nine backcrosses, P124 x IR64 was selected to map QTLs for maximum root length because of good combination of genetic factors conferring drought tolerance and yielding ability (Toorchi, et al. 2003). Three and four hundred plants from backcross population involving P124 x IR64 was staggered, 100 plants each under well watered (WW) and low moisture stress (LMS) conditions. Ten extreme plants from each stagger were selected for genotyping using STMS primers. The experiment was carried out at Hebbal campus of the University of Agricultural Sciences, Bangalore, India, during 1999-2000. Phenotyping procedure for maximum root length were as explained by Shashidhar et al. (1999).

Total of 69 extreme plants were selected for selective genotyping. DNA extraction was done as per modified CTAB (Cetyltrimethylammonium bromide) method (Cao and Oard 1997). Forty-two pairs of STMS primer pairs (Rice Map Pairs, Research Genetics Inc., Alabama, USA) that were earlier found to be polymorphic between the IR64 and Azucena were selected from the map reported by Temnykh et al. (2000) and used in the present study. PCR products using STMS primer pairs were separated on 10% denaturing polyacrylamide gel. The electrophoresis was carried out at 1500V for three hours and stained with silver staining procedure as described by Panaud et al (1996). Linkage or Recombination frequencies between markers were calculated using MAPMAKER/ EXP Version 3.0 (Lander and Botstein, 1989). QTLs linked to markers for maximum root length were determined in WW and LMS conditions as well as for the mean environment using single point analysis.

Results and discussion

Of the 42 SSR markers screened between the parents, 20 STMS markers revealed polymorphism between parents P124 and IR64, respectively. These primers were screened across 69 BC1F2 extreme individual selected from backcrossed population involving P124. Plants selected from population size of 700 plants which have been evaluated for MRL under WW or LMS conditions. Since selective genotyping approach has been recommended to target a single trait (Paterson, 1996), co-segregation of polymorphic markers with phenotypic expression was considered only for MRL. The result of analysis for tagging of QTLs contributing in performance of MRL is presented in Table 1.

RM215 on chromosome 9 showed co-segregation with QTL controlling MRL under both WW and LMS conditions. This marker explained 2.5 % of the total variation in MRL under WW condition while; contribution of this QTL in total variability of MRL under LMS condition was 16 %. RM278 another STMS marker on chromosome 9 showed linkage with MRL QTLs under WW condition. Contribution of this locus to phenotypic expression of MRL was 21.7 % as revealed by regression analysis. RM222 on chromosome 10 resides in vicinity of a locus controlling MRL only under WW condition.

Table 1: Microsatellite markers linked to QTLs controling maximum root length under well watered (WW), low moisture stress (LMS) and mean conditions (MC).

Microsatellite markers

Chrom. No.

Condition

R2 (%)

Pr>F

RM215

9

WW

2.5

0.012

   

LMS

16

0.054

   

MC

6.1

0.005

RM222

10

WW

22.6

0.058

RM278

9

WW

21.7

0.009

RM6

2

WW

1.1

0.062

MC

1.5

0.05

Contribution of this QTL to total variability of MRL was 22.7 %, i.e. the ratio of sum of squares due to regression to the total sum of squares. In addition, a QTL on chromosome 2 contributing to expression of MRL showed a strong linkage to RM6 under WW condition. Among STMS markers studied, RM222, RM278, and RM6 showed co-segregation with QTLs contributing to MRL only under WW condition and explained 27 % of the total variation. Under LMS condition, RM215 was the only microsatellite marker co-segregating with a QTL contributing to MRL. This QTL explained a high proportion (16 %) of the variation in MRL. When mean environment was taken into account as phenotype expression of MRL, only RM215 and RM6 showed a significant linkage with QTLs of interest.

A total of 10 QTLs were observed for maximum root length in F2 population of rice evaluated in hydroponic under greenhouse conditions (Price and Tomos, 1997b). Hemamalini et al. (2000) have found 1 to many QTLs for root length, total root number, root volume, root thickness, respectively under well-watered condition but failed to find common QTL for root traits under well-watered and low moisture stress conditions. Toorchi et al (2002) have reported two different RAPD markers linked to maximum root length under well- watered and low moisture stress conditions, respectively, of which one QTL was constant under both the conditions.

References

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Toorchi, M., H.E. Shashidhar, T. M. Gireesha and S. Hittalmani, 2003. Performance of backcrosses involving transgressant doubled haploid lines in rice under contrasting moisture regimes: Yield component and marker heterozygosity. Crop Sci. 43:1448-1456.

Toorchi, M., H.E. Shashidhar, N. Sharma, and S. Hittalmani, 2002. Tagging QTLs for maximum root length in rainfed lowland rice (Oryza sativa L.) using molecular markers. Cellular & Molecular Biology Letters 7:771-776.

Toorchi, M., H.E. Shashidhar, S. Hittalmani & T. M. Gireesha, 2002. Rice root morphology under contrasting moisture regimes and contribution of molecular marker heterozygosity. Euphytica 126: 251-257.

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